Genotype-independent de novo regeneration protocol in Cannabis sativa L. through direct organogenesis from cotyledonary nodes

Genotype-independent de novo regeneration protocol in Cannabis sativa L. through direct organogenesis from cotyledonary nodes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genotype-independent de novo regeneration protocol in Cannabis sativa L. through direct organogenesis from cotyledonary nodes Praveen Lakshman Bennur, Martin O’Brien, Shyama C. Fernando, Monika S. Doblin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7237557/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Nov, 2025 Read the published version in Plant Methods → Version 1 posted 9 You are reading this latest preprint version Abstract Efficient regeneration protocols are essential for large-scale propagation and genetic manipulation of recalcitrant medicinal species such as Cannabis sativa . Existing direct and indirect regeneration methods are highly genotype and explant-dependent, limiting broader applicability. Here, we report a five-stage (S 0 -S 4 ) optimised protocol that is reproducible and achieves high-efficiency direct de novo regeneration using cotyledonary node explants from both hemp and medicinal cannabis genotypes. A 1% (v/v) H₂O₂-based sterilisation method significantly improved seed germination and reduced endophyte contamination. Among embryo-derived explants, the cotyledonary node attached to the cotyledon showed superior regeneration efficiency through two distinct pathways: axillary shoot initiation and de novo regeneration, the latter achieving ~ 70–90% efficiency in six hemp cultivars and three medicinal cannabis lines on TDZ and NAA containing shoot regeneration medium. Histological analysis confirmed true de novo shoot formation from peripheral cortical cells, independent of pre-existing meristems or callus. De novo shoots were initiated within 2 d of shoot regeneration medium treatment, indicating rapid cellular commitment to organogenesis, with optimal regeneration between 7–14 d. Prolonged exposure proved detrimental, causing excessive callusing and vitrification. Repeated subculturing during proliferation stage enabled scalable shoot multiplication, yielding an average of 7 shoots per responding explant (~ 11.4 shoots per seed), outperforming previously published cotyledon-based (~ 2-fold) and hypocotyl-based (~ 5-fold) methods under comparable conditions. Regenerated plantlets developed healthy roots (with IAA or IBA) and acclimatised readily, exhibiting normal vegetative and reproductive growth. The protocol’s reproducibility across diverse cannabis genotypes and its applicability to other medicinal angiosperm species in this study highlights its value for both research and commercial applications. cannabis recalcitrance de novo regeneration genotype-independent cotyledonary node TDZ Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Plant biotechnology has significantly advanced over the past few decades, driven by the ability to manipulate plant systems through tissue culture and genetic modification techniques. The success of genetic engineering in most plant species fundamentally relies on efficient tissue culture, as most stable transformation protocols require regeneration, a process involving the formation of new shoots and/or roots through de novo organogenesis ( 1 ) from, in this case, transformed tissues, organs, unorganised calli or even single cells. Tissue culture serves as a foundational tool for various other biotechnological techniques, including micropropagation for rapid plant multiplication ( 2 ), double haploid production for breeding programs ( 3 ), protoplast fusion for somatic hybridisation ( 4 ), and gene editing for plant improvement ( 5 ), which enables applications ranging from germplasm conservation ( 6 ) to large-scale commercial plant production. These methods offer immense potential for enhancing plant traits, improving yields, and introducing novel characteristics. However, the effectiveness of tissue culture-based techniques is often constrained by the inherent recalcitrance in certain species. Recalcitrance includes regeneration recalcitrance, referring to the failure to regenerate tissues such as embryos or shoots through standard protocols, and genetic transformation recalcitrance, defined as the inability of a plant to incorporate foreign DNA into its genome ( 7 ). This poses significant challenges in developing efficient, reliable, and reproducible regeneration protocols, ultimately impacting the consistency of plant biotechnology outcomes. Among medicinal plants, Cannabis sativa L. (commonly referred to as cannabis) is a multi-purpose crop with both medicinal and industrial applications, renowned for its therapeutic compounds, high-quality stem fibre, nutrient-rich seeds, and its potential in phytoremediation ( 8 ). This crop is classified into industrial hemp and medicinal cannabis (MC), with legal distinction in many countries based on the concentration of Δ 9 -tetrahydrocannabinol (THC). Typically, genotypes with < 1.0% THC are designated as hemp, while those exceeding this threshold are classified as MC ( 8 ). Despite its economic significance and diverse utility, regeneration and genetic transformation of cannabis pose substantial challenges due to its recalcitrant nature ( 8 ). While cannabis can be readily propagated through conventional methods such as stem cuttings ( 9 , 10 ) or in vitro micropropagation through shoot tips ( 11 , 12 ), nodal segments ( 13 , 14 , 15 ) and inflorescences ( 16 , 17 ), these clonal approaches do not enable genetic manipulation studies necessary to rapidly improve traits such as disease resistance, yield, or cannabinoid profile. Therefore, developing efficient regeneration protocols is critical for advancing biotechnological research in this important crop. Plant regeneration through organogenesis, which facilitates the recovery of whole plants from a single cell or tissue, is an essential prerequisite for genetic transformation and genome editing ( 18 , 19 ). Despite significant advances in cannabis micropropagation and shoot proliferation, reliable de novo plant regeneration protocols remain limited ( 20 ). Previous studies have demonstrated indirect (with an intermediate callus phase) or direct (from preexisting meristems) regeneration from explants such as leaves ( 21 , 22 ) and petioles ( 23 ), epicotyls ( 24 ), cotyledons ( 25 , 26 , 27 ), and hypocotyls ( 27 , 28 ), typically using combinations of cytokinins and auxins to induce shoot organogenesis. However, these methods have shown high genotype dependency, which limits the applicability of transgenic studies to other cannabis germplasm ( 29 , 30 , 31 ). For example, Zhang et al. ( 31 ) screened 100 hemp cultivars and found regeneration rates ranging from 0-7.09%, with significant variation between genotypes, where most were generally on the low end of regeneration efficiency. Protocols leveraging Agrobacterium -mediated transformation have achieved transgenic hairy root cultures ( 32 ) (i.e., autonomous root systems devoid of other plant organs), yet the regeneration of fully developed transgenic plants remains inconsistent. Recent efforts to overcome these limitations have included exploring different explant types (e.g. embryonic/meristematic tissues), plant growth regulators (e.g. synthetic cytokinins), novel approaches such as the introduction of morphogenic genes (e.g., GRF/GIF chimeras), nanoparticle delivery systems and integration of cutting-edge techniques such as CRISPR-Cas9 for targeted gene editing ( 7 , 31 , 33 ). While progress has been made with some transformation protocols showing success within specific research settings ( 34 ), the development of standardised protocols that work consistently across diverse cannabis germplasm remains an ongoing challenge for the field. Given the rising global demand for cannabis in various applications, there is an urgent need to develop robust and scalable regeneration methods to unlock its full potential. In this study, we sought to develop a high-efficiency shoot regeneration protocol through direct organogenesis using various seed explants, including cotyledons, hypocotyls, and particularly the cotyledonary node (CN) across a range of cannabis lines. By systematically optimising key stages (S 0 -S 4 ) of the regeneration process and with careful attention to both explant type and the optimal duration of plant growth hormone treatments, we have established a reliable and scalable de novo regeneration protocol for cannabis. Additionally, we assessed the effectiveness of our protocol in a selection of other medicinal species with limited or no published methods, demonstrating its potential broader applicability beyond cannabis. Materials and Methods Plant material Industrial hemp seeds of cultivars Han FNH, Han FNQ, Bama, Puma, Yuma, Han Cold, Han NE and Si-1 were sourced from The Hemp Corporation Pty Ltd (Vacy, NSW, Australia) while CRS-1, CFX-2, Katani, Futura-75, and Ferimon-12 were procured from Midlands Seed Pty Ltd (Richmond, Tasmania, Australia). Seeds from all cultivars were stored at 4°C in sealed containers with silica bead desiccant until use. Fresh seeds of Han FNQ were produced in-house. Briefly, Han FNQ seedlings were initially cultivated following the method described by Welling et al. ( 35 ). At reproductive maturity, pollen was collected from dehiscing anthers of male plants and dusted onto the stigmas of flowers within each inflorescence of female plants. Successful pollination was confirmed by stigma senescence and bract swelling. Seed maturity was determined by yellowing leaves and bract drying. Once mature, the seeds were harvested manually and any underdeveloped seeds were discarded. Seeds from three MC lines-MC-1 (7.5% total THC, 8.6% total CBD), MC-2 (4.2% total THC, 8.1% total CBD), and MC-3 (2.6% total THC, 4.5% total CBD) were supplied by Cann Group Ltd (Port Melbourne, Victoria, Australia). These MC seeds were ~ 8 yrs old and originated from a controlled glasshouse environment. Each line was derived from a feminised population pollinated by a single pollen donor. Seeds of eight medicinal species, Aloe ferox , Artemisia absinthium , Ar. annua, Ar. vulgaris , Borago officinalis , Caesalpinia pulcherrima, Delonix regia and Echinacea purpurea , were purchased from The Seed Vine (Tenterfield, NSW, Australia). S 0 - Seed sterilisation and germination Batches of 40 seeds were initially surface-sterilised by soaking in 45 mL 1% (v/v) H₂O₂ (hydrogen peroxide, 7.5%, Milestone Chemicals Australia Pty Ltd, Heidelberg West, VIC) in a sterile 50 mL tube and incubated in the dark at 24°C for 24 h to initiate germination. The solution was then replaced with fresh 1% (v/v) H₂O₂, and seeds were incubated for an additional 24 h under identical conditions and germination (seeds displaying radicle emergence) scored at 48 h. After 48 h, germinated seeds were dissected to remove both the outer pericarp (hull) and seed coat. The dissected embryos underwent secondary sterilisation in 45 mL 1% (v/v) H₂O₂ with shaking at 150 rpm, 24°C for 1 h. Sterilised embryos were transferred (12 per plate) to 90 mm diameter plastic Petri dishes containing germination medium (GM; 0.5x Murashige and Skoog (MS) salts and vitamins ( 36 ) (M519, PhytoTech Labs, Kansas, USA), 1.5% (w/v) commercial-grade sucrose, 0.65% (w/v) agar (A296, PhytoTech Labs), pH 5.7) and grown under 16/8 h L/D photoperiod (140–170 µmol m − 2 s − 1 PAR) conditions at 24°C for 5 d. Each embryo was then scored for both endophyte contamination and responsiveness, i.e. whether it grew beyond radicle emergence. S 1 - Explant preparation After 5 d on GM, various explants were prepared from 7 d-old seedlings and assessed for regeneration potential. Using sterile forceps and a scalpel (blade no. 11), cotyledons were excised from the seedling by making cuts at the distal end to remove the cotyledon tip and at the proximal end where the cotyledon attaches to the primary shoot, to create wound sites at both ends. For hypocotyls, cuts were made at both the proximal end below the CN and the distal end above the radicle. For the CN with cotyledon explant, the hypocotyl and radicle were excised just below the CN, the region where the two cotyledons converge, followed by removal of the primary shoot. The cotyledons were then separated through a complete vertical cut. The prepared explant pairs were subsequently used for regeneration. For comparative analysis, additional CN-based explants were also prepared: half CN with half-cotyledon (CN with cotyledon explant halved longitudinally), whole CN (intact CN without cotyledons) and CN with hypocotyl (CN with top portion of hypocotyl). S 1 - Shoot regeneration To evaluate regeneration potential, a maximum of 10 cotyledons (abaxial side down) or hypocotyls (oriented horizontally) were placed per 90 mm Petri dish containing shoot regeneration medium (SRM; 1x MS, 0.4 mg/L thidiazuron (TDZ, T888, PhytoTech Labs), 0.2 mg/L α-naphthaleneacetic acid (NAA, N600, PhytoTech Labs), 3% (w/v) commercial-grade sucrose, 0.8% (w/v) agar, pH 5.7). Similarly, up to four CN with cotyledon explant pairs (8 explants in total) were placed per dish with the abaxial side down and the proximal end embedded in the medium. Control plates containing the same medium without growth hormones were used for comparison. All plates were maintained in a Conviron GEN1000 growth incubator (Controlled Environments Ltd, Winnipeg, Canada) at 24°C, 16/8 h L/D photoperiod with white light from broad-spectrum 12W T5 LEDs (140–170 µmol m − 2 s − 1 PAR). After 7 d, the primary shoot (identified as the central emerging shoot) from each explant was re-excised to promote multiple shoot initiation, and explants returned to control or SRM plates for an additional 7 d. After 14 d, the overall regeneration efficiency was calculated as the percentage of explants forming one or more shoots. This included both axillary shoot growth (observed on both control and SRM plates) and de novo shoot formation (exclusively observed on SRM). De novo regeneration efficiency was calculated as the percentage of explants exhibiting de novo shoot organogenesis on SRM after 14 d. To determine minimum SRM exposure time for de novo shoot induction, CN with cotyledon explants were subjected to varying SRM durations (1, 2, 4, 6, 8, 10, 12, and 14 d) including a control (no SRM exposure, direct transfer to proliferation medium) and 5 min vacuum infiltration in liquid SRM (to enhance medium penetration into explant tissues). After treatment, explants were transferred to proliferation medium for the remainder of the 14 d period to standardise total culture duration across all treatments and then evaluated for de novo regeneration. Shoot proliferation (S 2 ), elongation and rooting (S 3 ) Multiple shoot clumps were excised from regenerating CN with cotyledon explants after 14 d on SRM or control medium and transferred to shoot proliferation medium (SPM; 1x MS, 0.175 mg/L indole-3-acetic acid (IAA, I885, PhytoTech Labs), 1% (w/v) commercial-grade sucrose, 0.8% (w/v) agar, pH 5.7). During the excision process, the axillary shoot, cotyledon and callus tissue were removed. Plates were maintained under the same growth conditions as the shoot regeneration phase. After 14 d on SPM, elongated shoots (~ 2 cm in length) were separated from shoot clumps and transferred to fresh SPM in 500 mL screw-cap polycarbonate culture vessels (84 x 102 mm, Thermo Fisher Scientific NZ Ltd, Auckland, New Zealand) and incubated under the same conditions for 3–4 weeks to enable further elongation and rooting. The remaining shoot clumps were divided and subcultured at 14 d intervals for three cycles on fresh SPM plates. At each subculture, elongated shoots were counted and transferred to culture vessels. For MC lines, while the shoot proliferation phase utilised standard SPM plates, a modified SPM containing 0.5 mg/L indole-3-butyric acid (IBA, I538, PhytoTech Labs) instead of IAA was used specifically for rooting the elongated shoots to allow a direct comparison with rooting in vegetative nodal cuttings treated with Clonex rooting hormone gel (Yates, Australia), which contains IBA as the active ingredient. S 4 - Acclimatisation Elongated plantlets with developed roots were gently removed from SPM culture vessels and thoroughly rinsed with reverse osmosis (RO) water to eliminate any residual medium. They were then transplanted into 500 mL plastic pots filled with coco perlite (70:30) medium (Epping Hydroponics, Epping, VIC, Australia) saturated with RO water and supplemented with half-strength CANNA Classic Vega A and Vega B nutrient solutions (4 mL of each Vega/L of RO water) (CANNA Australasia, Subiaco, WA, Australia). The pots were placed inside a plastic humidity chamber where the plantlets acclimatised for 2 d under 100% relative humidity at 24°C, 18/6 h L/D photoperiod with white fluorescent lights (140–160 µmol m − 2 s − 1 PAR) in a controlled environment room. After 2 d, plantlets were removed from the humidity chamber and grown for another 14 d before being transferred to 1.5 L pots for two weeks and then 8 L pots containing coco perlite medium to support further growth and development till seed harvest. Microscopy Stereomicroscopy images were captured using a Leica M80 stereo microscope equipped with a Leica DMS4500 digital microscope camera. Image acquisition and processing were performed with the Leica Application Suite (version 4.12.0), using the Image Builder application. Photographs of cultures and plants were captured on a Canon 90D camera fitted with a 24–105 mm f/4-7.1 IS Canon lens. To examine internal tissue structure, CN with cotyledon explants exposed to SRM for 7 d were embedded in Super Cryoembedding Medium (SCEM, Section-Lab, Japan). Transverse sections of 30 µm were then taken with a Leica CM3050 S cryostat and visualised using an Olympus BX63 compound microscope. Images were captured with an Olympus DP60 camera. Schematic diagrams were hand-drawn on an iPad using the Procreate app (version 5.3.15). Statistical analysis All experiments followed a randomised design. Data were analysed using Microsoft Excel (version 2410). Graphs were prepared using Microsoft Excel and BioRender 2024. Results The availability of an efficient plant regeneration protocol is a prerequisite for large-scale in vitro propagation and/or genetic manipulation of any plant. This study aimed to develop a reproducible, genotype-independent regeneration protocol for cannabis for both applications. To ensure systematic protocol optimisation, the process was divided into distinct ordered stages: S 0 , seed sterilisation and germination; S 1 , explant excision and shoot induction; S 2 , shoot proliferation; S 3 , elongation and rooting; and S 4 , acclimatisation (Fig. S1 ). The optimisation of each stage focused on refining key parameters that critically influence regeneration efficiency, building on previous findings by Chaohua et al. ( 25 ), Galán-Ávila et al. ( 27 ) and Ahsan et al. ( 37 ). In S 0 , we modified an established H 2 O 2 sterilisation protocol to significantly enhance seed germination rates and reduce endophyte contamination, thereby increasing the availability of viable explants ( 37 ). S 1 involved a systematic evaluation of embryonic explants, including cotyledons ( 25 , 27 ) and hypocotyls ( 27 ), which had been previously explored for regeneration, in addition to the cotyledonary node (CN). Rather than extensively testing different media formulations and hormonal combinations, we specifically optimised exposure time to a previously validated shoot regeneration medium (SRM) containing TDZ and NAA ( 25 , 27 ). For S 2 , we used a shoot proliferation medium (SPM) containing IAA in conjunction with subculturing intervals to maximise shoot production. In S 3 , we used IAA ( 23 , 26 ) and IBA ( 21 , 24 , 25 ) for shoot elongation and root induction, aligning with published protocols. Finally, in S 4 , the acclimatisation process was optimised by creating an optimal ex vitro environment to enhance survival rates and facilitate the successful establishment of regenerated cannabis plants. The following sections provide a detailed account of the optimisation process of each stage. S 0 - Effect of H 2 O 2 on seed germination and endophyte contamination Plant tissue sterilisation is an essential first step to obtain aseptic, healthy explants as donor material for shoot regeneration. Standardising a sterilisation protocol is especially important when starting with wild collections or field-grown seed populations arising from open pollination, as is the case with hemp. Such seeds often exhibit phenotypic variability, reflecting their genetic diversity as a result of outcrossing. Also, exposure to external environmental conditions can lead to a range of biological contaminants that accumulate on the external surface of the seed as well as the presence of non-pathogenic microbes (endophytes) within the seed. This necessitates the optimisation of sterilisation protocols to address microbial contamination without compromising seed viability. In cannabis, seed-associated endophytes are known to vary between genotypes, cultivation environment ( 38 , 39 ) and post-harvest storage conditions ( 38 ). While endophytes can benefit plant growth in field conditions, they severely hinder tissue culture by introducing persistent contamination ( 40 ). Our early efforts focused on hemp cultivars due to larger seed volumes being readily available. Published protocols have successfully used ethanol and sodium hypochlorite for seed sterilisation ( 25 , 27 ). However, when applied to field-grown seeds of hemp cultivars Han FNH (Fig. 1 a, b) and Han FNQ, these methods resulted in poor germination and persistent bacterial, fungal and endophyte contamination. The radicle was observed to emerge from the rigid outer pericarp (hull) (Fig. 1 c), but often this layer remained attached to the embryo together with the membranous seed coat (Fig. 1 d), raising the possibility that microbes may persist in parts of the embryo surface less accessible to these sterilisation agents. In an attempt to improve germination rate and reduce contamination, we explored alternative approaches and drawing from previous studies ( 37 , 41 ), adopted a 1% (v/v) H₂O₂ treatment with modifications to enhance the efficacy of the sterilisation solution. Treatment of hemp seeds with fresh 1% (v/v) H₂O₂ twice for 24 h followed by an additional 1 h wash with fresh 1% (v/v) H₂O₂ after manually removing both the pericarp and seed coat (Fig. 1 e) significantly improved germination rates from ~ 20% when seeds are treated with ethanol and sodium hypochlorite to an average of 78.2% in Han FNH and 63.3% in Han FNQ (Table 1 ). Furthermore, this modified seed sterilisation regime was found to completely eliminate surface-derived bacterial and fungal contamination. Table 1 Effect of 1% (v/v) H₂O₂ sterilisation treatment on seed germination and endophyte contamination in hemp cultivars Han FNH and Han FNQ Hemp cultivar After 48 h in 1% (v/v) H 2 O 2 After 5 d on GM Germination rate (%) Endophyte-free seeds (%) Endophyte-contaminated seeds (%) Unresponsive seeds (%) Han FNH 78.2 ± 0.6 60.4 ± 3.8 6.3 ± 2.4 10.8 ± 2.0 Han FNQ 63.3 ± 10.0 29.6 ± 11.3 28.2 ± 8.2 5.4 ± 1.0 Han FNQ (fresh seeds) 99.9 ± 0.2 95.7 ± 2.1 2.7 ± 1.1 1.5 ± 1.3 Values show mean ± SD from three independent replicates (Han FNH-230, 160, 75; Han FNQ-116, 100, 200; Han FNQ fresh seeds-50, 50, 240). Data presented as a percentage of total seeds used Following sterilisation, seeds were placed on germination medium (GM) ( 27 ) for 5 d (Fig. 1 f) to determine which were endophyte-free and capable of further growth, thus suitable for explant excision for regeneration purposes. After 5 d on GM, an average of 60.4% of total Han FNH and 29.6% of Han FNQ seeds were found to be endophyte-free (Table 1 and Fig. 1 g), while 6.3% and 28.2%, respectively, showed endophyte contamination (Table 1 and Fig. 1 h). Unresponsive seeds where the embryo had germinated but exhibited minimal growth (i.e., had poor viability) accounted for 10.8% of total seeds in Han FNH and 5.4% in Han FNQ on average (Table 1 and Fig. 1 i). Therefore, the proportion of seeds identified as responsive and endophyte-free on GM (Fig. 1 j) differed considerably between the two hemp cultivars. The standardised 1% (v/v) H 2 O 2 sterilisation protocol was applied to an additional five hemp cultivars and three MC lines to determine if this variability in endophyte-free seed amongst cannabis genotypes is typical, as this impacts the total number of seeds required per experiment to achieve successful regeneration. Among the additional hemp cultivars, germination rates again varied widely, ranging from 33.8% in Futura-75 to 81.3% in CFX-2 (Table S1 ). The proportion of total seeds free from endophyte contamination followed a similar trend, with Futura-75 displaying the lowest at 20.6%, while CFX-2 showed the highest at 63.5% after 5 d on GM (Table S1 ). Seeds from the MC lines, produced in an indoor facility, exhibited notably higher germination rates of 86–96% after 1% (v/v) H 2 O 2 treatment (Table S1 ). These lines also demonstrated a high proportion of seeds free from endophyte contamination, ranging between 68–76%, indicating that the sterilisation process can effectively eliminate endophyte contaminants. It was noted that a relatively high proportion of MC seeds were unresponsive, between 14–28% (Table S1 ), indicating a loss in seed viability that was likely due to a prolonged storage period. The hemp results suggested that poor seed germination and high endophyte contamination may be associated traits. To determine whether improving germination could assist in reducing both endophyte contamination and seed unresponsiveness, we generated fresh seeds of Han FNQ under controlled indoor conditions and evaluated them using our 1% (v/v) H 2 O 2 sterilisation protocol. Fresh seeds exhibited significant improvements compared to the original seed batch (Table 1 ). The germination rate increased to 99.9%, seeds free from endophyte contamination rose to 95.7%, and unresponsive seeds decreased to 1.5% (Table 1 ). These data suggest that undertaking a growth cycle to generate fresh seed is advantageous to boost responsive explant numbers and thereby enhance regeneration efficiency in the long term. S 1 - Effect of different explant types and SRM exposure time on direct regeneration Comparative evaluation of embryo-derived explants The shoot induction step (S 1 ) typically involves exposing explants to a specific regime of plant growth hormones that either activate pre-existing (sometimes dormant) meristematic cells or stimulate de novo shoot organogenesis in responsive tissue. To systematically evaluate the regeneration potential of seed explants, we first replicated the published protocols with cotyledons ( 25 ) and hypocotyls ( 27 ) excised from 7 d-old seedlings subjected to SRM containing TDZ and NAA, the hormone combination optimised in previous cannabis regeneration studies ( 25 , 27 ). Among the hemp cultivars previously tested, we selected CRS-1, CFX-2, Katani, Han FNH and Han FNQ due to the availability of more endophyte-free seeds compared to other cultivars (Table 1 and Table S1 ). Despite meticulously replicating the published methods, including SRM composition and exposure time of 2–5 weeks, cotyledons and hypocotyls repeatedly failed to regenerate shoots at the previously observed rates across all five hemp cultivars tested. Instead, they predominantly formed callus at their cut surfaces, particularly at their proximal end (Fig. S2 ). While previous studies have reported regeneration rates of 4–55% from cotyledons ( 25 ) and ~ 50% from hypocotyls ( 27 ), our rates were dramatically lower. Only two of 2,358 cotyledon explants from CRS-1 (0.1%), two of 100 from Han FNH (2%) and a single cotyledon explant from 60 Han FNQ (1.7%) regenerated shoots, all at the proximal end of the cotyledon (Table S2 ). No regeneration was observed with hypocotyl explants of any of the five hemp cultivars. The consistency of negative results across multiple hemp cultivars supports the notion that non-meristematic seed explants are unable to undergo direct regeneration on SRM ( 42 ). Closer examination of the regenerating cotyledon explants suggested that these rare cases may have resulted from inadvertent retention of a portion of the CN, the region at the point of insertion of the cotyledon into the embryonic axis (near the primary shoot) (Fig. 2 a and b). Hence, we hypothesised that successful direct regeneration in cannabis may require an explant with inherent meristematic activity. We therefore explored the CN, which retains such characteristics, as a potentially more effective explant alternative. Anatomically, in most epigeal species in which cotyledons emerge above-ground during seedling growth, the CN is a critical junction ( 43 ). Formed during early embryogenesis, this region houses both dormant axillary meristems at each cotyledon base and undifferentiated cells with significant regenerative potential that can be activated by exogenous hormones ( 44 ) (Fig. 2 a). In contrast, hypogeal species where cotyledons remain underground during seedling growth often lack clearly defined or functionally competent axillary nodes at this junction ( 45 ). This anatomical distinction between germination types further supports the use of CN explants in cannabis, an epigeal species, for efficient direct shoot regeneration. Eleven hemp cultivars were used to investigate the regenerative potential of CN explants and evaluate possible genotype dependency. The CN was excised along with the attached cotyledon from 7 d-old seedlings (Fig. 2 b-e) following protocols developed in other epigeal species ( 44 , 46 , 47 ). Using the same SRM, regrowth of the primary shoot was observed in most explants after 7 d and was removed to reduce apical dominance (i.e., the more vigorous growth of the primary shoot due to endogenous auxin accumulation that suppresses axillary bud growth). After 4–5 weeks, shoot regeneration was observed at the proximal end of the cotyledon in all 11 cultivars, typically in the form of multiple shoots with one dominant central shoot surrounded by numerous smaller shoots (Fig. S3a). The overall regeneration efficiency ranged from 3.2% in Yuma to 22.2% in Han FNQ (Table S3). This indicates that the CN with cotyledon explant is far superior to cotyledon or hypocotyl in producing shoot regeneration in a seemingly genotype-independent manner. However, while shoot induction was successful, prolonged exposure to TDZ led to extensive callus formation (Fig. S3a). Optimising exposure time to SRM to enhance shoot regeneration To mitigate TDZ-induced limitations on shoot regeneration, we investigated whether shortening the SRM exposure time from 5 to 3 weeks could improve regeneration outcomes. We selected four hemp cultivars, CFX-2 and Katani, which showed moderate regeneration efficiency, along with Futura-75 and Ferimon-12, previously reported to regenerate successfully ( 27 , 30 ). With the shortened exposure time, a reduction in callus proliferation was observed, though still persistent, together with a substantial improvement in the overall regeneration efficiency, increasing to 67.5%-90% across cultivars (Table S4). Next, we proceeded to test whether reducing SRM exposure to 14 d could further enhance shoot regeneration while mitigating persistent callus formation. Han FNH was selected for this experiment due to its relatively high proportion of endophyte-free seeds (Table 1 ). To better assess the effects of TDZ and NAA on shoot regeneration, we included a control medium (MS without hormones) to enable a detailed comparison of regeneration efficiency and shoot development on SRM. Unlike previous experiments where explants were evaluated individually, CN explants with attached cotyledons were tracked in pairs to capture the full regenerative potential of the CN from individual seeds (Fig. 2 b). As expected, regrowth of the primary shoot was observed within 4–7 d of exposing the explants to either medium. After 7 d, it was excised at the epicotyl region to reduce apical dominance. On both media, this excision led to the elongation of the axillary shoot (Fig. 3 a, b), the dominant central shoot identified in earlier experiments. This response mirrors the natural hormonal regulation in seedlings where removal of the primary shoot disrupts basipetal auxin flow from the shoot apex while allowing cytokinin synthesis, shifting the hormonal balance to favour axillary shoot outgrowth (Fig. S4a) ( 48 ). In contrast, on SRM, elongation of the axillary shoot was accompanied by additional shoot regeneration at the base of the excised primary shoot and on both sides of the axillary shoot in a symmetrical pattern (Fig. 3 b). We have termed this direct de novo regeneration. Consequently, the overall regeneration efficiency varied significantly between the two treatments. On control medium, regeneration reached 24.3% after 14 d, consisting of axillary shoot elongation only (Fig. 3 c). However, SRM significantly enhanced regeneration due to the additional de novo shoot formation, increasing overall efficiency to 77.8% over the same period (Student T-test, p = 0.01, Fig. 3 c), with de novo regeneration accounting for 70.1% (Fig. 3 c). Axillary shoot elongation occurred to a similar extent in both treatments, but de novo shoot initiation was exclusive to SRM-treated explants following removal of the primary shoot (compare Fig. 3 a and b). Axillary shoot elongation may be supported by endogenous cytokinin, potentially present in the cotyledons, along with nutritional cues such as sugars (Fig. S4b). In contrast, explants cultured on SRM were exposed to exogenous cytokinin (TDZ), which may act in conjunction with endogenous signals to facilitate de novo shoot formation (Fig. S4b). Furthermore, minimal callus formation was observed at the proximal end of explants compared to those maintained for 3 weeks on SRM, suggesting the reduction in TDZ and NAA exposure was beneficial. Another noteworthy observation was that the paired explants exhibited distinct responses under different media conditions. On control medium, an average of 8.3% of seeds produced shoots from both explants, whereas this increased substantially to 54.2% on SRM (Table S5). However, the percentage of seeds with at least one regenerating explant was similar between control and SRM (~ 32%), suggesting that SRM primarily promoted regeneration in both explants rather than affecting the likelihood of at least one explant regenerating (Table S5). Additionally, the proportion of seeds where neither explant regenerated was significantly reduced on SRM, decreasing from 59.7% in the control to 13.9% (Table S5). These results highlight the strong stimulatory effect of TDZ and NAA in SRM on direct shoot regeneration. Importantly, the ability to obtain two regenerative explants from a single seed demonstrates that our excision method preserves the zone(s) critical for shoot regeneration. To determine whether 14 d of SRM exposure was optimal across multiple cannabis genotypes, we tested its efficacy in the Han FNQ hemp cultivar and three MC lines. In Han FNQ, SRM exposure for 14 d resulted in a de novo regeneration efficiency of 78% (Table S6) with minimal callus growth, similar to the observations made in Han FNH. The protocol also successfully induced regeneration in all three tested MC lines, with de novo shoot formation comparable to that observed in hemp cultivars (Fig. S5a). Notably, MC lines exhibited even higher de novo regeneration efficiencies (84-91.7%) than the hemp cultivars tested, highlighting the robustness of the 14 d SRM treatment across genotypes (Table S6). Evaluating variants of CN explants to define regeneration competence With the aim of maximising regeneration potential, we also evaluated how different excisions of the CN region affect shoot regeneration. Using Han FNH due to its strong regeneration response (Fig. 3 b), we excised and compared the performance of three other CN-based explant types from 7 d-old seedlings relative to CN with cotyledon by culturing them on SRM for 14 d (Fig. S6). The three CN variants, half CN with half-cotyledon (CN with cotyledon explant halved longitudinally, 4 per seed), whole CN (intact CN without cotyledons, 1 per seed), and CN with hypocotyl (whole CN with the top portion of hypocotyl, 1 per seed), allowed us to evaluate if there is a trade-off between explant quantity and regeneration efficiency. CN with cotyledon explants exhibited efficient de novo shoot formation (57.5%, Table S7) with minimal callus growth, consistent with our previous trials (Fig. S6a-c). Half-CN with half-cotyledon explants were hypothesised to increase shoot regeneration due to the separation of the axillary meristematic zone by longitudinal bisection of the CN and cotyledon, thereby potentially enhancing axillary meristem activation (Fig. S6d and e). Instead, these explants exhibited a greatly reduced de novo regeneration efficiency of 23.8% (Table S7). The high degree of wounding led to excessive callusing at the cut edges of the cotyledons but mainly at the axillary node region, probably compromising the structural integrity of the CN and impairing de novo shoot formation (Fig. S6f). Axillary shoot regeneration was reduced to < 2%, likely due to the damage caused to the axillary node from the vertical bisection (Table S7). Whole CN explants without attached cotyledons retained intact CN structure and avoided damage to the axillary nodes (Fig. S6g and h), enabling assessment of cotyledon necessity for efficient de novo regeneration. Axillary shoot growth was observed in 24 of 40 explants (60%) (Table S7), approximately twice the amount of the CN with cotyledon explants, as expected, due to two axillary meristem regions being present rather than one (Fig. S4b). However, the de novo regeneration was severely compromised, often delayed and accompanied by substantial callus proliferation at the base of the explant (Fig. S6i). Moreover, since only one explant could be obtained per seed and regeneration was both slower and less prolific, whole CN explants exhibited the lowest de novo regeneration efficiency at 12.5% (Table S7). When CN with hypocotyl explants were tested, regeneration was exclusively observed at the CN region, while the remaining hypocotyl sections developed callus (Fig. S6j-l). Despite differences in efficiency, all CN-based explants exhibited some level of regeneration, indicating that the CN plays an indispensable role in direct shoot organogenesis. Among them, CN with cotyledon emerged as the most efficient and reliable explant, demonstrating superior regeneration capacity. Determining the onset and cellular origin of regeneration from CN explant To further examine the timing and progression of de novo shoot formation, we conducted a time-course analysis over 14 d on SRM using CN with cotyledon explants from Han FNH and Han FNQ cultivars. Morphological changes of representative explants were documented daily on both control medium (Han FNH) and SRM (both cultivars). As previously observed (Fig. 3 ), regrowth of the primary shoot and axillary shoot development occurred within 4–7 d across all conditions (Fig. S7). However, only axillary shoot elongation was noted in control explants throughout the 14 d period (Fig. S7a–e). For SRM-treated explants, by day 10 in Han FNH (Fig. S7f-j) and as early as day 7 in Han FNQ (Fig. S7k-o), multiple de novo shoots emerged around the axillary shoot and at the base of the excised primary shoot (Fig. S7h and k). These findings suggested that 7–10 d SRM exposure represents a suitable timeframe to capture early organogenic events. In a separate experiment using Han FNH CN with cotyledon explants, regeneration was observed at 7 d on SRM, similar to Han FNQ in the earlier time-course study (Fig. 4 a). These explants were then used for histological analysis. Microscopic observations revealed distinct origins of the two types of shoot regeneration in these explants (Fig. 4 a). Transverse sections showed that the axillary shoot emerged from the vascular ring of the primary shoot, confirming its origin from pre-existing meristematic tissue (Fig. 4 b). Concurrently, multiple de novo shoots differentiated from peripheral cortical cells at the junction between the base of the excised primary shoot and the cotyledon attachment site. These de novo shoots developed independently of the vasculature and without intervening callus formation, indicating true de novo organogenesis induced by exogenous TDZ and NAA (Fig. 4 b). Given the emergence of de novo shoots by 7–10 d, we investigated whether the standard 14 d SRM exposure could be shortened further without compromising efficient regeneration. Brief exposures (< 2 d) failed to induce de novo shoots in Han FNH CN with cotyledon explants, but exhibited only axillary shoot elongation, similar to the control (Table S8), suggesting these were insufficient to activate de novo shoot formation. However, regeneration efficiency steadily increased with longer exposures, reaching 6.7% at 2 d, 30% at 6 d, and peaking at 50% at 10 d before slightly declining to 43% at 12 d and 14 d (Table S8). These findings indicate that a minimum of 2 d is required to trigger de novo regeneration, while exposures at 10–14 d are optimal for maximising shoot induction. S 2 - Shoot proliferation in response to IAA Following shoot induction on SRM, the developing multiple shoots must undergo further proliferation to allow the separation of individual shoots. Without timely separation and regular subculturing, apical dominance results in the elongation of only one or two shoots while suppressing the growth of adjacent de novo regenerated shoots ( 48 ), limiting overall shoot yield per explant and increasing resource competition. Shoot proliferation is achieved on an auxin-only medium (SPM) with reduced sucrose concentration (1% w/v) relative to SRM, which gradually supports the transition to autotrophy, a critical step for subsequent rooting and acclimatisation. Our preliminary work in MC compared different auxins such as IAA and IBA in SPM, revealing that IBA-supplemented medium caused excessive callusing at the primary shoot excision site. Given the small size of the shoot clumps and the negative impact of callusing we observed on shoot proliferation, IAA was selected as the preferred auxin for this process. We initially assessed shoot proliferation from explants maintained on SRM for long durations. Five weeks of SRM exposure followed by 4–5 weeks of proliferation on the same medium resulted in restricted proliferation (≤ 4 shoots per explant) across cultivars (Table S3), accompanied by excessive callus formation and shoot vitrification (translucent, brittle, and water-soaked tissues) (Fig. S3b). Reducing SRM exposure to 3 weeks mitigated, but did not eliminate, TDZ-induced callusing. However, when explants were subsequently transferred to TDZ-free SPM, shoot proliferation improved substantially, with yields increasing to 3-7.2 shoots per explant depending on cultivar (Table S4). Although callusing persisted, vitrification was notably absent (Fig. S3c). To further optimise proliferation efficiency, Han FNH was assessed for shoot proliferation response following a shortened 14 d SRM exposure. Shoot clumps formed after 14 d on either control media or SRM were excised from residual tissues (cotyledons, callus, and axillary shoot) and transferred to SPM where they began to proliferate within 14 d (Fig. 5 a). Clumps from the control medium exhibited limited shoot proliferation either from the nodal region of the excised primary shoot or from the regrowth of axillary shoot (Fig. 5 b). In contrast, clumps from SRM displayed active proliferation on SPM, with multiple shoots emerging from most of the explant pairs with less callus growth at the base of the shoot clump compared to 3 weeks on SRM (Fig. 5 c and Fig. S3c). Over the course of three fortnightly subcultures on SPM, control-derived clumps showed a marked decline in proliferation, producing fewer shoots in the first and second subcultures and none by the third (Fig. S8a). However, SRM-derived clumps exhibited significantly enhanced and sustained shoot proliferation across all three subcultures, with the highest shoot numbers observed in the third round (Fig. S8a). The mean number of shoots per responding explant (determined by averaging the total number of shoots observed across all subcultures and the remainder on the original explant) increased from 1.4 shoots per explant (1.7 per seed) exposed to control medium to 7 (11.4 per seed) in the SRM-exposed explants, with the highest observed response being 28 shoots from a single explant (50 shoots from a single seed) (Student’s T-test, p = 0.02, Fig. 5 d). To further assess the reproducibility of shoot proliferation, the protocol was extended to the Han FNQ cultivar and three MC lines. Similar trends were observed in Han FNQ, which produced an average of 7.1 shoots per explant (11.6 per seed), closely aligning with the results obtained for Han FNH (Table S6). Likewise, all three MC lines demonstrated similarly consistent shoot proliferation, with shoot numbers ranging between 6-7.6 shoots per responding explant (~ 13 per seed) (Fig. S5b-e and Table S6). S 3 - Shoot elongation and rooting in response to IAA and IBA Following shoot proliferation, the shoots continuously removed from shoot clumps during each subculture require elongation and root formation to develop into individual plants. While the proliferation stage maximises shoot production, these detached shoots need further growth before ex vitro establishment to complete the regeneration process. Their transfer from Petri dishes to larger culture vessels provides increased space, allowing them to elongate rapidly and develop sufficiently to support independent growth. Different auxins, particularly IAA, are widely used for both shoot elongation and rooting in cannabis regeneration protocols. Building on our findings from the proliferation stage (S 2 ), we maintained IAA in SPM as the primary auxin for elongation and rooting, creating a streamlined protocol that minimises media changes while effectively supporting both processes. The shoots of the Han FNH cultivar derived from SRM and measuring ~ 2 cm in length were transferred to SPM for elongation and rooting (Fig. 6 a). Within four weeks, these shoots successfully elongated and simultaneously developed root systems (Fig. 6 b and c). The rooting efficiency of this cultivar was evaluated across the three successive subcultures on SPM. A progressive increase in mean rooting efficiency was observed, from 18.1% in subculture 1 to 29% in subculture 2 and 34.7% in subculture 3 (Fig. S8b). Interestingly, in vitro flowering was also observed in this cultivar, with no flowering occurring during subculture 1, followed by 34.2% during subculture 2, and 12.4% during subculture 3 (Fig. S8c). This precocious flowering phenomenon, while potentially reducing the number of shoots, indicates the capacity of regenerated plantlets to complete their life cycle and may offer insights into the cultivar's response to in vitro conditions. Given the relatively low rooting efficiency observed with IAA in Han FNH and other hemp cultivars (Han FNQ) used in this study, we explored IBA as an alternative auxin for root induction in MC. IBA has been widely documented for its effectiveness in cannabis regeneration protocols ( 21 , 24 , 25 , 28 ). It has significant commercial relevance in the cannabis industry, particularly for MC production, where it serves as the active ingredient in Clonex, a commonly used rooting agent for vegetative nodal cuttings ( 49 ). When evaluated on SPM substituted with IBA instead of IAA, the shoots of MC lines exhibited substantially higher rooting efficiencies compared to IAA-based rooting in hemp. MC-1, MC-2, and MC-3 achieved 66.7%, 81%, and 75% rooting success, respectively, with no signs of reproductive growth (Fig. S8d). The marked difference in flowering response between Han FNH and the MC lines suggests that in vitro flowering may be genotype-dependent, though comprehensive comparisons across additional cultivars are necessary to confirm this observation. S 4 - Acclimatisation The final step in a successful regeneration protocol is the acclimatisation of regenerated plantlets to ex vitro conditions. Well-developed shoots with established roots, as obtained in the previous step (S 3 ), are crucial for effective deflasking and successful transition to non-sterile environments. Given the robust rooting achieved in our protocol, we tested the acclimatisation potential, a relatively straightforward step, of regenerated plantlets to confirm they could complete their life cycle. Among the 173 rooted shoots of Han FNH, 15 were randomly selected and transferred from the culture vessels containing SPM supplemented with IAA to coco perlite medium for acclimatisation (Fig. 6 d and e). The plantlets acclimatised exceptionally well inside a humidity chamber for 2 d, with a 100% survival rate observed (Fig. 6 e). Following this initial phase, the plantlets were removed and grown for an additional 14 d under standard conditions. Throughout this period, the plantlets demonstrated normal vegetative growth, comparable to nodal cuttings, the conventional method for commercial propagation (Fig. 6 f and g). Over time, they exhibited significant vegetative and reproductive growth, with both male and female plants observed (Fig. 6 h), the latter producing viable seeds, confirming that plants regenerated via tissue culture are capable of completing their growth and reproduction cycles. De novo shoot regeneration in other medicinal species Having established an efficient protocol for de novo shoot regeneration from the CN with cotyledon explants of cannabis, we sought to investigate whether this approach could be applied to other medicinally important plant species that have traditionally been considered recalcitrant to tissue culture methods. We selected eight angiosperm medicinal species representing diverse growth habits, taxonomic classifications, morphological characteristics and germination patterns to assess the broader applicability of our protocol. This selection allowed us to evaluate whether the regenerative capacity of the CN region observed in cannabis was a species-specific phenomenon or potentially a more universal characteristic that could be exploited for regeneration across various medicinal plants. A limited number of seeds ( 10 – 30 ) of the woody and herbaceous eudicot and monocot medicinal species (Table S9) were subjected to the identical regeneration procedure outlined in Fig. S1 . De novo shoot regeneration was observed in five out of eight species: Artemisia absinthium and A. vulgaris (wormwoods), Borago officinalis (borage), Aloe ferox (bitter aloe) and Delonix regia (royal poinciana) after exposure to at least 14 d on SRM (Fig. 7 ). Notably, four of these species ( Artemisia spp., B. officinalis , and D. regia ) are epigeal, with regeneration originating from the CN region, similar to cannabis (Fig. 7 a-c, e). Interestingly, A. ferox , a hypogeal species, also regenerated shoots but with an altered pattern and unclear origin (Fig. 7 d). This suggests that the CN is a worthy explant to trial for de novo shoot regeneration, particularly in epigeal species. Discussion This study aimed to establish an efficient and reproducible shoot regeneration protocol in cannabis. Our findings provide critical insights into the spatial origin and mechanisms of direct shoot organogenesis, revealing the CN region as the principal site of de novo shoot regeneration in the young seedling, an aspect largely overlooked or ambiguously addressed in previous cannabis protocols ( 22 , 25 , 27 ). The CN region is the site of dual regeneration pathways in cannabis We identified two distinct regeneration pathways from CN explants: 1) direct shoot initiation from pre-existing meristematic tissue (axillary buds) with vascular connectivity to the primary shoot (Fig. 4 ) and 2) de novo organogenesis from peripheral cortical cells, independent of vasculature, induced by TDZ and NAA (Fig. 4 ). Histological analysis confirmed that de novo shoots occurred without intervening callus and pre-existing meristem, as observed in other epigeal species ( 43 , 47 , 50 , 51 ). These findings challenge earlier reports attributing regeneration to callus-mediated pathways without considering underlying meristems in the cotyledon axils ( 22 , 25 , 27 ). TDZ and NAA alone were insufficient to induce shoot regeneration in explants lacking meristems (Fig. S2 and Table S2 ), instead promoting only callus formation in cotyledon and hypocotyl explants. This limitation aligns with previous studies in hemp, which attributed the lack of organogenic potential in these tissues to the absence of meristems ( 42 ). Moreover, similar de novo regeneration systems have enabled successful transformation in other recalcitrant species ( 52 , 53 , 54 ), underscoring the value of targeting accessible organogenic cells for both regeneration and transformation purposes. The low shoot regeneration (< 2 shoots per explant) in previous hypocotyl-based cannabis protocols is likely due to the retention of axillary meristems rather than true de novo organogenesis, exemplified by the typical initiation pattern of two shoots per explant ( 27 , 30 ). This is consistent with our control treatment, where CN with cotyledon explants produced few shoots (avg. 1.4 per explant, Fig. 5 d), primarily from axillary nodes. Another study ( 28 ) also reported improved regeneration when the CN region was retained in hypocotyl explants, reinforcing the importance of meristem inclusion, though without distinguishing between axillary and de novo origins. While axillary meristems are inherently regeneration-competent and have been used in cannabis regeneration ( 13 ), our identification of de novo organogenesis provides a complementary pathway that expands regeneration potential. Among the CN-based explants tested, cotyledon presence proved critical for efficient de novo regeneration, with CN with cotyledon explants demonstrating the highest regeneration potential (Table S7). This likely reflects the role of cotyledon-derived morphogenic signals, nutritional support, and hormone homeostasis, as reported in legumes such as Phaseolus vulgaris (green bean) ( 47 ), Cajanus cajan (pigeonpea) ( 50 ), Vigna radiata (mungbean) ( 55 ) and V. mungo (blackgram) ( 56 ). Detection of endogenous cytokinins in cannabis cotyledons supports this contributory role when retained alongside the CN region ( 42 ). Additionally, the partial or complete removal of the cotyledons, as in half-CN and half-cotyledon and whole CN explants, respectively, may have disrupted the cotyledon-derived hormonal and nutritional signals, further limiting regenerative competence (Table S7). Structural integrity of the node region also proved to be critical, exhibiting differential responses to TDZ and NAA. Significant damage to axillary nodes, as seen in half-CN and half-cotyledon explants, substantially reduced axillary shoot formation and increased callusing, thereby decreasing de novo regeneration (Fig. S6d-f and Table S7). This is consistent with previous reports in Phaseolus vulgaris ( 47 ) and Glycine max ( 57 ), emphasising the importance of intact axillary buds for successful regeneration. In contrast, while structurally intact, whole CN explants exhibited low and delayed regeneration, likely due to the apical dominance exerted by the two axillary shoots and profuse callusing, along with cotyledon absence (Fig. S6g-i and Table S7). Similar suppression of lateral shoot development due to apical dominance has been reported in cannabis tissue culture ( 42 ). However, the relative contribution of each of these factors remains unclear and warrants further investigation to refine explant selection for maximal regeneration. Understanding the precise cellular origin of shoot regeneration is critical for advancing cannabis transformation. Our histological evidence (Fig. 4 ) shows that de novo shoots originate from outer cortical cells, making them ideal targets for transformation due to their greater accessibility compared to deeper cell layers ( 47 ). This may enhance DNA delivery and reduce chimerism, a limitation in previous hypocotyl-based transformations ( 30 ) where regeneration likely arose from deeper, more mature meristematic tissues that cannot be uniformly transformed. Additionally, bypassing the callus phase reduces the risk of somaclonal variation, enhancing the genetic stability of regenerated lines ( 47 ). Clarifying the distinct regeneration pathways, therefore, provides a mechanistic foundation for improving transformation efficiency in cannabis, an area that continues to present technical challenges despite recent advances ( 58 ). Optimising exogenous hormone exposure time is essential for efficient de novo regeneration Successful shoot organogenesis depends on optimal auxin:cytokinin balance in conjunction with the explant’s endogenous hormonal status ( 42 ). In our study, the strategic combination of the synthetic cytokinin TDZ and auxin NAA proved essential for inducing de novo regeneration from CN explants, while hormone-free medium failed to elicit the same. This combination has consistently outperformed other tested cytokinin-auxin combinations in cannabis (BAP, zeatin, kinetin, and meta-topolin coupled with NAA and IBA) in promoting shoot induction ( 25 , 26 , 27 , 28 ). TDZ's high morphogenic potential stimulates shoot initiation and early developmental processes ( 59 ), while NAA supports cell differentiation and elongation ( 60 ). However, our results highlight a critical nuance: the duration of hormone exposure significantly affects regeneration outcomes. While short-term TDZ + NAA treatments (2–6 d) were effective, 7–14 d provided optimal conditions for maximum shoot induction with minimal callusing (Fig. S7). Prolonged exposure (3–5 weeks) led to excessive callusing, vitrification and shoot inhibition, effects primarily attributed to TDZ ( 61 ), with a marked decline in regenerative potential (Fig. S3). This sensitivity is consistent with reports in legumes, where short-term exposure (typically 7 d) induced maximal regeneration while avoiding cytokinin dominance effects ( 62 ). Previous cannabis protocols using higher TDZ concentrations or extended exposure often achieved regeneration at the cost of increased callusing ( 22 ), reduced shoot proliferation and lengthy protocols ( 22 , 25 ), further supporting our optimised approach. Notably, de novo regeneration was observed in 6.7% of explants in 2 d of SRM treatment (Table S8), and histological studies confirmed that by 7 d, most peripheral cells were already committed to the organogenesis (Fig. 4 b). This indicates hormonal signalling required for de novo regeneration primes cortical cells within the first 1–2 d of exposure, suggesting optimal timing for infection with Agrobacterium or other DNA delivery methods is before or very early in this window, while cells remain uncommitted and accessible for transformation. To our knowledge, no previous cannabis regeneration study has reported such an early onset of de novo organogenesis or considered its relevance for transformation efficiency. Cytokinin profiling studies in hemp ( 42 ) showed hypocotyl-derived callus, even when cultured on cytokinin-rich medium (BAP + Kinetin), failed to accumulate active cytokinins, indicating a limited endogenous-exogenous hormonal interaction and response capacity. This underscores the importance of both appropriate hormonal treatment and biological competence of the explant, in our case, the CN's unique combination of meristematic structure and hormone responsiveness that enables de novo regeneration. Multiple subculturing of de novo shoots enables scalable regeneration Our CN-based approach uniquely enables large-scale shoot production from a single seed through continuous proliferation achieved by periodic subculturing. Up to three subcultures were performed, with shoot numbers peaking in the third culturing; although a slight decline was observed at the fourth (reminder shoots on the original explant), further proliferation remained possible (Fig. S8a). This scalable regeneration system achieved a maximum of 50 shoots per seed in the Han FNH cultivar, with performance being genotype-dependent across the tested varieties. The protocol significantly reduces the cost per shoot while generating numerous genetically identical plants, making it valuable for both breeding programs and commercial propagation. The protocol’s efficiency is further enhanced by the timely transfer of regenerated shoots from cytokinin-rich to cytokinin-free medium, which is critical to prevent the inhibitory effects of prolonged cytokinin (TDZ) exposure on elongation and rooting, a limitation commonly reported in similar protocols ( 52 , 63 ). Moreover, in transformation workflows, iterative subculturing aids in the recovery and selection of uniformly transformed tissues while progressively eliminating chimeric shoots ( 64 ), making our protocol a robust platform for downstream applications. Our rooting experiments revealed genotype-specific challenges requiring further optimisation. While both IAA and IBA induced roots, efficiency with IAA remained suboptimal in hemp (Fig. S8b), likely attributed to the relatively low concentration used (0.175 mg/L) compared to the higher IBA concentration (0.5 mg/L) employed in MC lines, where rooting responses were more favourable (Fig. S8d) and consistent with previous protocols ( 14 , 21 , 24 , 25 ). However, IBA’s tendency to promote callus formation highlights the need for tailored rooting strategies across different cannabis genotypes ( 29 ). The occurrence of in vitro flowering during the rooting stage, despite strict long-day photoperiod conditions, may reflect genotype-specific responses and light intensity stress. This aligns with previous findings that certain cannabis genotypes can initiate flowering in vitro even under extended photoperiods, demonstrating the species' unique capacity for photoperiod-insensitive floral induction ( 65 ). While reproductive development may redirect metabolic energy from root development, it presents opportunities for accelerating breeding cycles ( 65 ), studying the regulation of secondary metabolites ( 66 ) and production of floral tissue for regeneration ( 16 ). Such an approach could facilitate multiple crosses within a limited space and time, offering a scalable tool for both research and commercial breeding programs. CN explants enable efficient genotype-independent regeneration Compared to existing protocols, our CN-based regeneration system delivers significantly higher efficiency and consistent shoot proliferation across diverse cannabis genotypes. Cotyledon-based methods have reported highly variable efficiencies (4–55%) and modest shoots per explant (1.3-8) ( 22 , 25 ), while hypocotyl-based approaches typically achieved ~ 50% efficiency with only 1.7 shoots per explant ( 27 , 30 ). Furthermore, recent protocols using CN with hypocotyl, though reporting higher efficiencies, demonstrated substantial variability (26–87% efficiency; 2.6–8.6 shoots per explant) across cultivars ( 28 ). Critically, these earlier studies failed to distinguish shoots arising from pre-existing meristems from true de novo regeneration, likely inflating their reported efficiencies. In contrast, our protocol applied stringent criteria to exclude primary and axillary shoots, quantifying only those initiated de novo . Using this conservative approach, we consistently achieved 70–90% efficiency with an average of 7 de novo shoots per responding explant (~ 11.4 shoots per seed), representing approximately a 2-fold improvement over cotyledon-based and 5-fold over hypocotyl-based methods under comparable culture conditions. Moreover, by using paired CN with cotyledon explants, our protocol effectively doubled explant yield per seed, enhancing scalability for high-throughput applications. This positions the CN as a universally responsive explant type that circumvents the genotype dependency barriers that have long constrained cannabis biotechnology, with implications extending beyond cannabis ( 67 ). Successful regeneration in five out of eight medicinal plant species spanning multiple families and growth forms demonstrates the broad applicability of our protocol across taxonomically diverse angiosperms (Fig. 7 and Table S9). Notably, four of the responsive species ( Artemisia spp., B. officinalis and D. regia ) exhibit epigeal germination similar to cannabis, supporting previous observations in Vigna species where regeneration from CN was consistently observed in epigeal but not in hypogeal types, unless modified by genomic factors such as allotetraploidy ( 43 ). This observation suggests that epigeal species, characterised by photosynthetically active cotyledons and the presence of axillary buds, exhibit greater regenerative competence than hypogeal species, where cotyledons remain underground and axillary buds are often less developed or absent ( 43 ). These physiological and anatomical differences likely influence the availability and transport of morphogenic signals, nutrients, and hormones, thereby affecting regeneration potential. Interestingly, Aloe ferox , a hypogeal monocot, also exhibited de novo shoot formation, although its precise anatomical origin remains unclear. This suggests that CN-based regeneration may extend to at least some hypogeal taxa, potentially due to variation in nodal architecture or hormone responsiveness, but further analysis is needed to clarify this. While our observations are derived from a limited number of species, they support the hypothesis that the CN region harbours a conserved, regeneration-competent meristematic architecture that can be reliably activated across epigeal species, similar to legumes ( 43 , 45 , 68 ) and possibly beyond, offering a high-efficiency, transformation-compatible platform for genetic improvement in cannabis and other recalcitrant species. However, while the regeneration phase itself demonstrated genotype independence, upstream challenges remain. Seed germination and endophyte contamination varied significantly across cannabis cultivars (Table 1 and Table S1 ), influenced by genetic background, storage conditions, and seed age ( 69 ). Freshly harvested seeds achieved near-complete germination and minimal contamination, underscoring their advantages ( 70 ). However, producing fresh seeds under controlled conditions is often impractical, particularly for perennial species or in low-resource settings and is disadvantageous when the genetic eliteness of a line is to be retained. Although the 1% (v/v) H₂O₂ protocol significantly improved explant availability across genotypes, these findings highlight that the full potential of this genotype-independent regeneration system is contingent upon the availability of healthy explants, particularly when working with long-stored or field-grown material common in cannabis and other medicinal species. Conclusion In this study, we developed a high-efficiency regeneration protocol for cannabis by optimising five stages (S 0 -S 4 ) of tissue culture and identifying CN with attached cotyledon as the optimal explant for de novo shoot regeneration. Optimising hormone exposure time significantly enhanced regeneration efficiency, while the protocol's genotype-independent nature enabled reliable performance across diverse hemp cultivars and high-THC MC lines. This approach addresses long-standing recalcitrance barriers in cannabis tissue culture and establishes a foundation for large-scale propagation of genetically identical shoots, genetic transformation, and germplasm conservation. Its successful extension to other medicinal plants and its prior validation in legumes suggests broader applicability to a range of recalcitrant species. Overall, this work offers a valuable platform for advanced biotechnological applications that could enhance sustainable cultivation practices, improve desirable traits, and unlock the therapeutic and industrial potential of cannabis and other medicinal plants. Declarations Acknowledgements The authors would like to acknowledge the funding provided by the Australian Research Council and the Australian Department of Education Regional Research Collaboration Program. We would like to thank La Trobe University and the University of Melbourne for their support on this project and Cann Group Ltd for providing access to their facilities and plant materials. Further thanks are extended to A/Prof Berin Boughton for training and use of the cryostat and Prof Phil Brewer for invaluable discussions. Author contributions PLB, MOB, SCF and MSD: conceptualisation and experiment design; PLB, MOB and SCF: performed the experiments; PLB: writing the original draft, figure preparation and data analysis; MOB and MSD: supervision and manuscript revision; PLB, MOB, and MSD: review and editing. All authors reviewed the manuscript. Funding This project was funded by the Australian Research Council to the Industrial Transformation Research Hub for Medicinal Agriculture (ARC MedAg Hub, IH180100006) and the Australian Department of Education Regional Research Collaboration Program – Next Generation Protected Cropping in a Regional Manufacturing Facility. Data availability All data generated or analysed during this study are included in this published article and its supplementary information files. Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests The authors have no competing interests to declare that are relevant to the content of this article. References Altpeter F, Springer NM, Bartley LE, Blechl AE, Brutnell TP, Citovsky V, et al. Advancing crop transformation in the era of genome editing. Plant Cell. 2016;28(7):1510–20. https://doi.org/10.1105/tpc.16.00196 . Moraes RM, Cerdeira AL, Lourenço MV. Using micropropagation to develop medicinal plants into crops. Molecules. 2021;26(6):1752. https://doi.org/10.3390/molecules26061752 . Dwivedi SL, Britt AB, Tripathi L, Sharma S, Upadhyaya HD, Ortiz R, Haploids. Constraints and opportunities in plant breeding. Biotechnol Adv. 2015;33(6):812–29. https://doi.org/10.1016/j.biotechadv.2015.07.001 . Dutt M, Mahmoud LM, Chamusco K, Stanton D, Chase CD, Nielsen E, et al. Utilization of somatic fusion techniques for the development of HLB tolerant breeding resources employing the Australian finger lime ( Citrus australasica ). PLoS ONE. 2021;16(8):e0255842. https://doi.org/10.1371/journal.pone.0255842 . Mohammadi MA, Wai MH, Rizwan HM, Qarluq AQ, Xu M, Wang L, et al. Advances in micropropagation, somatic embryogenesis, somatic hybridizations, genetic transformation and cryopreservation for Passiflora improvement. Plant Methods. 2023;19:50. https://doi.org/10.1186/s13007-023-01030-0 . Richins M, Montes C, Merkle S. Conservation of green and white ash germplasm using the cryopreservation of embryogenic cultures. Plants. 2024;13(3):352. https://doi.org/10.3390/plants13030352 . Bennur PL, O'Brien M, Fernando SC, Doblin MS. Improving transformation and regeneration efficiency in medicinal plants: Insights from other recalcitrant species. J Exp Bot. 2024;76(1):52–75. https://doi.org/10.1093/jxb/erae189 . Monthony AS, Page SR, Hesami M, Jones AMP. The past, present and future of Cannabis sativa tissue culture. Plants. 2021;10(1):185. https://doi.org/10.3390/plants10010185 . Caplan D, Stemeroff J, Dixon MA, Zheng Y. Vegetative propagation of cannabis by stem cuttings: effects of leaf number, cutting position, rooting hormone, and leaf tip removal. Can J Plant Sci. 2018;98(5):1126–32. https://doi.org/10.1139/cjps-2018-0038 . Chandra S, Lata H, ElSohly MA, Walker LA, Potter D. Cannabis cultivation: Methodological issues for obtaining medical-grade product. Epilepsy Behav. 2017;70:302–12. https://doi.org/10.1016/j.yebeh.2016.11.029 . Stephen C, Zayas VA, Galic A, Bridgen MP. Micropropagation of hemp ( Cannabis sativa L). HortScience. 2023;58(3):307–16. https://doi.org/10.21273/hortsci16969-22 . Wróbel T, Dreger M, Wielgus K, Słomski R. Modified nodal cuttings and shoot tips protocol for rapid regeneration of Cannabis sativa L. J Nat Fibers. 2020;19(2):536–45. https://doi.org/10.1080/15440478.2020.1748160 . Lata H, Chandra S, Khan I, ElSohly MA. Thidiazuron-induced high-frequency direct shoot organogenesis of Cannabis sativa L. Vitro Cell Dev Biol Plant. 2009;45:12–9. https://doi.org/10.1007/s11627-008-9167-5 . Lata H, Chandra S, Khan IA, ElSohly MA. Propagation through alginate encapsulation of axillary buds of Cannabis sativa L. - an important medicinal plant. Physiol Mol Biol Plants. 2009;15:79–86. https://doi.org/10.1007/s12298-009-0008-8 . Lata H, Chandra S, Techen N, Khan IA, ElSohly MA. In vitro mass propagation of Cannabis sativa L.: A protocol refinement using novel aromatic cytokinin meta-topolin and the assessment of eco-physiological, biochemical and genetic fidelity of micropropagated plants. J Appl Res Med Aromat Plants. 2016;3(1):18–26. https://doi.org/10.1016/j.jarmap.2015.12.001 Piunno KF, Golenia G, Boudko EA, Downey C, Jones AMP. Regeneration of shoots from immature and mature inflorescences of Cannabis sativa . Can J Plant Sci. 2019;99(4):556–9. https://doi.org/10.1139/cjps-2018-0308 . Monthony AS, Bagheri S, Zheng Y, Jones AMP. Flower power: floral reversion as a viable alternative to nodal micropropagation in Cannabis sativa . Vitro Cell Dev Biol Plant. 2021;57:1018–30. https://doi.org/10.1007/s11627-021-10181-5 . Hesami M, Baiton A, Alizadeh M, Pepe M, Torkamaneh D, Jones AMP. Advances and perspectives in tissue culture and genetic engineering of cannabis. Int J Mol Sci. 2021;22(11):5671. https://doi.org/10.3390/ijms22115671 . Monthony AS, Kyne ST, Grainger CM, Jones AMP. Recalcitrance of Cannabis sativa to de novo regeneration; a multi-genotype replication study. PLoS ONE. 2021;16(8):e0235525. https://doi.org/10.1371/journal.pone.0235525 . Adhikary D, Kulkarni M, El-Mezawy A, Mobini S, Elhiti M, Gjuric R, et al. Medical cannabis and industrial hemp tissue culture: Present status and future potential. Front Plant Sci. 2021;12:627240. https://doi.org/10.3389/fpls.2021.627240 . Lata H, Chandra S, Khan IA, Elsohly MA. High frequency plant regeneration from leaf derived callus of high ∆ 9 -tetrahydrocannabinol yielding Cannabis sativa L. Planta Med. 2010;76(14):1629–33. http://dx.doi.org/10.1055/s-0030-1249773 . Ahsan SM, Kwon DB, Injamum-Ul-Hoque M, Rahman MM, Yeam I, Choi HW. De novo regeneration of Cannabis sativa cv. Cheungsam and evaluation of secondary metabolites of its callus. Horticulturae. 2024;10(12):1331. https://doi.org/10.3390/horticulturae10121331 . Slusarkiewicz-Jarzina A, Ponitka A, Kaczmarek Z. Influence of cultivar, explant source and plant growth regulator on callus induction and plant regeneration of Cannabis sativa L. Acta Biol Crac Ser Bot. 2005;47(2):145–51. https://abcbot.pl/pdf/47_2/145-151.pdf . Movahedi M, Ghasemi-Omran V, Torabi S. The effect of different concentrations of TDZ and BA on in vitro regeneration of Iranian cannabis ( Cannabis sativa ) using cotyledon and epicotyl explants. J Plant Mol Breed. 2015;3(2):20–7. https://doi.org/10.22058/jpmb.2015.15371 . Chaohua C, Gonggu Z, Lining Z, Chunsheng G, Qing T, Jianhua C, et al. A rapid shoot regeneration protocol from the cotyledons of hemp ( Cannabis sativa L). Ind Crops Prod. 2016;83:61–5. https://doi.org/10.1016/j.indcrop.2015.12.035 . Wielgus K, Luwanska A, Lassocinski W, Kaczmarek Z. Estimation of Cannabis sativa L. tissue culture conditions essential for callus induction and plant regeneration. J Nat Fibers. 2008;5(3):199–207. https://doi.org/10.1080/15440470801976045 . Galán-Ávila A, García-Fortea E, Prohens J, Herraiz FJ. Development of a direct in vitro plant regeneration protocol from Cannabis sativa L. seedling explants: Developmental morphology of shoot regeneration and ploidy level of regenerated plants. Front Plant Sci. 2020;11:645. https://doi.org/10.3389/fpls.2020.00645 . Sorokin A, Kovalchuk I. Development of efficient and scalable regeneration tissue culture method for Cannabis sativa . Plant Sci. 2025;350:112296. https://doi.org/10.1016/j.plantsci.2024.112296 . Feeney M, Punja ZK. Tissue culture and Agrobacterium -mediated transformation of hemp ( Cannabis sativa L). Vitro Cell Dev Biol Plant. 2003;39:578–85. https://doi.org/10.1079/IVP2003454 . Galán-Ávila A, Gramazio P, Ron M, Prohens J, Herraiz FJ. A novel and rapid method for Agrobacterium-mediated production of stably transformed Cannabis sativa L. plants. Ind Crops Prod. 2021;170:113691. https://doi.org/10.1016/j.indcrop.2021.113691 . Zhang X, Xu G, Cheng C, Lei L, Sun J, Xu Y, et al. Establishment of an Agrobacterium -mediated genetic transformation and CRISPR/Cas9-mediated targeted mutagenesis in Hemp ( Cannabis Sativa L). Plant Biotechnol J. 2021;19(10):1979–87. https://doi.org/10.1111/pbi.13611 . Wahby I, Caba JM, Ligero F. Agrobacterium infection of hemp ( Cannabis sativa L.): establishment of hairy root cultures. J Plant Interact. 2013;8(4):312–20. https://doi.org/10.1080/17429145.2012.746399 . Ahmed S, Gao X, Jahan MA, Adams M, Wu N, Kovinich N. Nanoparticle-based genetic transformation of Cannabis sativa . J Biotechnol. 2021;326:48–51. https://doi.org/10.1016/j.jbiotec.2020.12.014 . Xu G, Liu Y, Yu S, Kong D, Tang K, Dai Z, et al. CsMIKC1 regulates inflorescence development and grain production in Cannabis sativa plants. Hortic Res. 2024;11(8):uhae161. https://doi.org/10.1093/hr/uhae161 . Welling MT, Deseo MA, Bacic A, Doblin MS. Untargeted metabolomic analyses reveal chemical complexity of dioecious cannabis flowers. Aust J Chem. 2021;74(6):463–79. https://doi.org/10.1071/CH21033 . Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962;15(3):473–97. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x . Ahsan SM, Shin JH, Choi HW. Availability of hydrogen peroxide solutions as a germination liquid medium for contamination-free in vitro seedling development of Cannabis sativa . Hortic Sci Technol. 2022;40(6):605–13. https://doi.org/10.7235/HORT.20220055 . Lobato C, de Freitas JM, Habich D, Kögl I, Berg G, Cernava T. Wild again: recovery of a beneficial Cannabis seed endophyte from low domestication genotypes. Microbiome. 2024;12:239. https://doi.org/10.1186/s40168-024-01951-5 . Buirs L, Punja ZK. Endophytes in Cannabis sativa : Identifying and characterizing microbes with beneficial and detrimental effects on plant health. Plants. 2025;14(8):1247. https://doi.org/10.3390/plants14081247 . Quambusch M, Winkelmann T. Bacterial endophytes in plant tissue culture: Mode of action, detection, and control. In: Loyola-Vargas VM, Ochoa-Alejo N, editors. Plant cell culture protocols. 4th ed. New York: Springer; 2018. pp. 69–88. https://doi.org/10.1007/978-1-4939-8594-4_4 . Sorokin A, Yadav NS, Gaudet D, Kovalchuk I. Development and standardization of rapid and efficient seed germination protocol for Cannabis sativa . Bio Protoc. 2021;11(1):e3875. https://doi.org/10.21769/BioProtoc.3875 . Smýkalová I, Vrbová M, Cvečková M, Plačková L, Žukauskaitė A, Zatloukal M, et al. The effects of novel synthetic cytokinin derivatives and endogenous cytokinins on the in vitro growth responses of hemp ( Cannabis sativa L.) explants. Plant Cell Tissue Organ Cult. 2019;139:381–94. https://doi.org/10.1007/s11240-019-01693-5 . Avenido RA, Hattori K. Differences in shoot regeneration response from cotyledonary node explants in Asiatic Vigna species support genomic grouping within subgenus Ceratotropis (Piper) Verdc. Plant Cell Tissue Organ Cult. 1999;58:99–110. https://doi.org/10.1023/A:1006325327624 . Xu H, Guo Y, Qiu L, Ran Y. Progress in soybean genetic transformation over the last decade. Front Plant Sci. 2022;13:900318. https://doi.org/10.3389/fpls.2022.900318 . Avenido R, Motoda J, Hattori K. Direct shoot regeneration from cotyledonary nodes as a marker for genomic groupings within the Asiatic Vigna (subgenus Ceratotropis {Piper} Verdc.) species. Plant Growth Regul. 2001;35:59–67. https://doi.org/10.1023/A:1013874902530 . Hinchee MAW, Connor-Ward DV, Newell CA, McDonnell RE, Sato SJ, Gasser CS, et al. Production of transgenic soybean plants using Agrobacterium -mediated DNA transfer. Nat Biotechnol. 1988;6:915–22. https://doi.org/10.1038/nbt0888-915 . Franklin CI, Trieu TN, Gonzales RA, Dixon RA. Plant regeneration from seedling explants of green bean ( Phaseolus vulgaris L) via organogenesis. Plant Cell Tissue Organ Cult. 1991;24:199–206. https://doi.org/10.1007/BF00033477 . Kebrom TH. A growing stem inhibits bud outgrowth – The overlooked theory of apical dominance. Front Plant Sci. 2017;8:1874. https://doi.org/10.3389/fpls.2017.01874 . Kurtz LE, Borbas LN, Brand MH, Lubell-Brand JD. Ex vitro rooting of Cannabis sativa microcuttings and their performance compared to retip and stem cuttings. HortScience. 2022;57(12):1576–9. https://doi.org/10.21273/HORTSCI16890-22 . Shiva prakash N, Pental D, Bhalla-Sarin N. Regeneration of pigeonpea ( Cajanus cajan ) from cotyledonary node via multiple shoot formation. Plant Cell Rep. 1994;13:623–7. https://doi.org/10.1007/BF00232933 . Jackson JA, Hobbs SLA. Rapid multiple shoot production from cotyledonary node explants of pea ( Pisum sativum L). Vitro Cell Dev Biol. 1990;26:835–8. https://doi.org/10.1007/BF02623626 . Hsieh Y-F, Jain M, Wang J, Gallo M. Direct organogenesis from cotyledonary node explants suitable for Agrobacterium -mediated transformation in peanut ( Arachis hypogaea L). Plant Cell Tissue Organ Cult. 2017;128:161–75. https://doi.org/10.1007/s11240-016-1095-1 . Patial V, Krishna R, Arya G, Singh VK, Agarwal M, Goel S, et al. Development of an efficient, genotype independent plant regeneration and transformation protocol using cotyledonary nodes in safflower ( Carthamus tinctorius L). J Plant Biochem Biotechnol. 2016;25(4):421–32. https://doi.org/10.1007/s13562-016-0354-x . Paz MM, Martinez JC, Kalvig AB, Fonger TM, Wang K. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium -mediated soybean transformation. Plant Cell Rep. 2006;25:206–13. https://doi.org/10.1007/s00299-005-0048-7 . Gulati A, Jaiwal PK. Plant regeneration from cotyledonary node explants of mungbean ( Vigna radiata (L.) Wilczek). Plant Cell Rep. 1994;13:523–7. https://doi.org/10.1007/BF00232949 . Sen J, Guha-Mukherjee S. vitro induction of multiple shoots and plant regeneration in Vigna . Vitro Cell Dev Biol Plant. 1998;34:276–80. https://doi.org/10.1007/BF02822733 . Shan Z, Raemakers K, Tzitzikas EN, Ma Z, Visser RGF. Development of a highly efficient, repetitive system of organogenesis in soybean ( Glycine max (L.) Merr). Plant Cell Rep. 2005;24:507–12. https://doi.org/10.1007/s00299-005-0971-7 . Shujat S, Robinson GI, Norouzkhani F, Kovalchuk I. Using advanced biotechnological techniques to improve cannabis cultivars. Biocatal Agric Biotechnol. 2024;60:103250. https://doi.org/10.1016/j.bcab.2024.103250 . Erland LAE, Giebelhaus RT, Victor JMR, Murch SJ, Saxena PK. The morphoregulatory role of Thidiazuron: Metabolomics-guided hypothesis generation for mechanisms of activity. Biomol. 2020;10(9):1253. https://doi.org/10.3390/biom10091253 . Campanoni P, Nick P. Auxin-dependent cell division and cell elongation. 1-Naphthaleneacetic Acid and 2,4-Dichlorophenoxyacetic Acid activate different pathways. Plant Physiol. 2005;137(3):939–48. https://doi.org/10.1104/pp.104.053843 . Dewir YH, Nurmansyah, Naidoo Y, Teixeira da Silva JA. Thidiazuron-induced abnormalities in plant tissue cultures. Plant Cell Rep. 2018;37:1451–70. https://doi.org/10.1007/s00299-018-2326-1 . Malik KA, Saxena PK. Regeneration in Phaseolus vulgaris L.: High-frequency induction of direct shoot formation in intact seedlings by N(6)-benzylaminopurine and thidiazuron. Planta. 1992;186:384–9. https://doi.org/10.1007/BF00195319 . Huang H, Wei Y, Zhai Y, Ouyang K, Chen X, Bai L. High frequency regeneration of plants via callus-mediated organogenesis from cotyledon and hypocotyl cultures in a multipurpose tropical tree ( Neolamarkia Cadamba ). Sci Rep. 2020;10:4558. https://doi.org/10.1038/s41598-020-61612-z . Mathews H, Dewey V, Wagoner W, Bestwick RK. Molecular and cellular evidence of chimaeric tissues in primary transgenics and elimination of chimaerism through improved selection protocols. Transgenic Res. 1998;7:123–9. https://doi.org/10.1023/A:1008872425917 . Moher M, Jones M, Zheng Y. Photoperiodic response of in vitro Cannabis sativa plants. HortScience. 2021;56(1):108–13. https://doi.org/10.21273/HORTSCI15452-20 . Lavie O, Buxdorf K, Eshed Williams L. Optimizing cannabis cultivation: an efficient in vitro system for flowering induction. Plant Methods. 2024;20:141. https://doi.org/10.1186/s13007-024-01265-5 . Maren NA, Duan H, Da K, Yencho GC, Ranney TG, Liu W. Genotype-independent plant transformation. Hortic Res. 2022;9:uhac047. https://doi.org/10.1093/hr/uhac047 . Avenido RA, Hattori K. Benzyladenine-preconditioning in germinating mungbean seedlings stimulates axillary buds in cotyledonary nodes resulting in multiple shoot regeneration. Breed Sci. 2001;51(2):137–42. https://doi.org/10.1270/jsbbs.51.137 . Scott M, Rani M, Samsatly J, Charron JB, Jabaji S. Endophytes of industrial hemp ( Cannabis sativa L.) cultivars: identification of culturable bacteria and fungi in leaves, petioles, and seeds. Can J Microbiol. 2018;64(10):664–80. https://doi.org/10.1139/cjm-2018-0108 . Collado R, Veitía N, Bermúdez-Caraballoso I, García LR, Torres D, Romero C, et al. Efficient in vitro plant regeneration via indirect organogenesis for different common bean cultivars. Sci Hortic. 2013;153:109–16. https://doi.org/10.1016/j.scienta.2013.02.007 . Additional Declarations No competing interests reported. Supplementary Files AdditionalFile1.docx Additional file 1 File format: Microsoft Word (.docx) Title of data: Supplementary Figures 1-8 Description: This file contains Supplementary Figures 1 to 8, which provide additional visual data supporting the main findings. Each figure is labelled and accompanied by a figure legend. AdditionalFile2.docx Additional file 2 File format: Microsoft Word (.docx) Title of data: Supplementary Tables 1-9 Description: This file contains Supplementary Tables 1 to 9, which provide additional statistical data supporting the main findings. Each table has a title and legend. 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Fernando","email":"","orcid":"","institution":"La Trobe University","correspondingAuthor":false,"prefix":"","firstName":"Shyama","middleName":"C.","lastName":"Fernando","suffix":""},{"id":501402328,"identity":"f5320f60-b982-43fd-b3a3-e61b1ab78222","order_by":3,"name":"Monika S. Doblin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYBACgwP8B5gZCiQY+NnB/ANgQbxaLA/wJDAzGEgwSDYzMDYQpcX+AI8BM0iNwWFitZgd4DF8XGBgkWd8mPn544qKO3IM7M3bJBhqDuPTYmw8w0Ci2Owwm2HjmTPPjBl4jpVJMBzDq8VMmsdAInHbYQbDxsa2w4kNEjlmEgxsuLUYwLRsbmb/CNJS3yD/BqjlHxFaNjDzgG1JYJDgMZNgbMOj5TBPMsgviTMO8xTObDjzzLCNJ63YIrEvHbeW4/0HHxdU1CX2t7dv+NhQcUeen/3wxhsfvlnj1MLAjC7ABiIScGsYBaNgFIyCUUAEAAC9bFOdqyZDTAAAAABJRU5ErkJggg==","orcid":"","institution":"La Trobe University","correspondingAuthor":true,"prefix":"","firstName":"Monika","middleName":"S.","lastName":"Doblin","suffix":""}],"badges":[],"createdAt":"2025-07-28 23:38:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7237557/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7237557/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13007-025-01468-4","type":"published","date":"2025-11-08T15:57:49+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89506669,"identity":"0cb6c790-639c-464b-8fc2-323b4d28dcf3","added_by":"auto","created_at":"2025-08-20 17:19:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6649597,"visible":true,"origin":"","legend":"\u003cp\u003eS\u003csub\u003e0 \u003c/sub\u003e- Sterilisation and germination of Han FNH seeds. \u003cstrong\u003ea, b\u003c/strong\u003e Seeds exhibit a red colouration due to fungicide treatment. \u003cstrong\u003ec\u003c/strong\u003e Germinated seed after 48 h of soaking in 1% (v/v) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2.\u003c/sub\u003e \u003cstrong\u003ed\u003c/strong\u003e After removing pericarp (hull). \u003cstrong\u003ee\u003c/strong\u003e Without seed coat, exposing cotyledons, hypocotyl and radicle before a 1 h wash with 1% (v/v) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. \u003cstrong\u003ef\u003c/strong\u003e Sterilised seeds on GM after 2 d sterilisation treatment. \u003cstrong\u003eg\u003c/strong\u003e Germinated (green arrow), endophyte-contaminated (red arrow) and unresponsive (blue arrow) seeds after 5 d on GM. \u003cstrong\u003eh\u003c/strong\u003e Enlarged view of an endophyte-contaminated seed (image captured from below the Petri dish). \u003cstrong\u003ei\u003c/strong\u003e Enlarged view of an unresponsive seed, showing the geminated embryo with minimal growth. \u003cstrong\u003ej\u003c/strong\u003e Enlarged view of a 7 d-old seedling.\u003c/p\u003e","description":"","filename":"Figure14.png","url":"https://assets-eu.researchsquare.com/files/rs-7237557/v1/27471fc783eeaf23b922ff39.png"},{"id":89506667,"identity":"40ff094b-0bf8-4d99-a149-048745204464","added_by":"auto","created_at":"2025-08-20 17:19:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5286123,"visible":true,"origin":"","legend":"\u003cp\u003eS\u003csub\u003e1 \u003c/sub\u003e- Generation of CN with cotyledon explant from a 7 d-old Han FNH seedling. \u003cstrong\u003ea\u003c/strong\u003e Diagram illustrating whole seedling structure (left) and magnified view (right) of the CN region of a seedling, where dormant axillary meristems and undifferentiated cells are located at the junction between the cotyledons and the primary shoot. The vertical dotted line indicates the excision plane for bisecting the seedling through the primary shoot, effectively separating the cotyledons such that each retains the CN region, enabling the preparation of two explants per seedling. \u003cstrong\u003eb\u003c/strong\u003e Excised cotyledons showing the retained CN region at their base. \u003cstrong\u003ec-e\u003c/strong\u003e Progressive steps of explant preparation. \u003cstrong\u003ec\u003c/strong\u003e Seedling after hypocotyl removal with the primary shoot and cotyledons intact. \u003cstrong\u003ed\u003c/strong\u003e Seedling after decapitation of the primary shoot but with cotyledons intact. \u003cstrong\u003ee\u003c/strong\u003e Cotyledons separated by longitudinal excision.\u003c/p\u003e","description":"","filename":"Figure24.png","url":"https://assets-eu.researchsquare.com/files/rs-7237557/v1/2dcdf9b2fb80059ef56cfdba.png"},{"id":89507560,"identity":"4681e902-fed4-4599-a7b8-f843f19aded7","added_by":"auto","created_at":"2025-08-20 17:35:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3418104,"visible":true,"origin":"","legend":"\u003cp\u003eS\u003csub\u003e1 \u003c/sub\u003e- Shoot regeneration from the CN with cotyledon explants of Han FNH. \u003cstrong\u003ea\u003c/strong\u003e CN with cotyledon explant pairs on control medium (left) and close-up of an explant showing axillary shoot growth between the cotyledon and excised primary shoot at the epicotyl (the region above the cotyledon) after 14 d (right). \u003cstrong\u003eb\u003c/strong\u003e CN with cotyledon explant pairs on SRM (left) and close-up of a regenerating explant with the cotyledon, excised primary shoot (white arrow), multiple regenerating shoots (red arrows) at the base of the axillary shoot and callus at the proximal end of the explant after 14 d\u0026nbsp; (right). c Regeneration efficiency on control medium compared to SRM after 14 d. The graph shows overall regeneration (light grey) and \u003cem\u003ede novo \u003c/em\u003eregeneration (dark grey) percentages. Data represent mean ± SD from three independent biological replicates (n=48). Asterisks (**) indicate a significant difference in overall regeneration between control and SRM treatments (Student's T-test, p = 0.01).\u003c/p\u003e","description":"","filename":"Figure33.png","url":"https://assets-eu.researchsquare.com/files/rs-7237557/v1/c0c1b6fc088a715954ebb2a4.png"},{"id":89506679,"identity":"f2997f0b-5a18-4bd8-bfe4-70f427abb83c","added_by":"auto","created_at":"2025-08-20 17:19:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4748364,"visible":true,"origin":"","legend":"\u003cp\u003eS\u003csub\u003e1 \u003c/sub\u003e- Histological observations of the \u003cem\u003ede novo \u003c/em\u003eshoot regeneration from the CN with cotyledon explants of Han FNH. \u003cstrong\u003ea\u003c/strong\u003e Image of CN with cotyledon explant after 7 d on SRM, showing axillary shoot development and the initiation of \u003cem\u003ede novo\u003c/em\u003e shoots (red arrows). Note the vasculature and surrounding cortex of the excised primary shoot. \u003cstrong\u003eb\u003c/strong\u003e Longitudinal cryosection of the same explant shown in (\u003cstrong\u003ea\u003c/strong\u003e), through the CN region, showing the developing axillary shoot is connected to pre-existing vasculature and \u003cem\u003ede novo\u003c/em\u003e shoots (red arrows) originating from the cortex, which appear to lack vascular connections.\u003c/p\u003e","description":"","filename":"Figure43.png","url":"https://assets-eu.researchsquare.com/files/rs-7237557/v1/42c16d5ac7dd6e59ccb02553.png"},{"id":89507379,"identity":"d7d08009-13cb-4863-b65c-7a7e2304ffe4","added_by":"auto","created_at":"2025-08-20 17:27:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2815420,"visible":true,"origin":"","legend":"\u003cp\u003eS\u003csub\u003e2 \u003c/sub\u003e- Shoot proliferation from the CN with cotyledon explants of Han FNH. \u003cstrong\u003ea\u003c/strong\u003e Multiple shoot clumps from the SRM-exposed explant pairs with cotyledon, callus and axillary shoot excised at 0 d (left) and shoot proliferation after 14 d on SPM (right). \u003cstrong\u003eb\u003c/strong\u003e A shoot clump derived from control medium after 14 d on SPM showing limited shoots mainly from the nodal region of the excised primary shoot or regrowth of axillary shoot (insert). \u003cstrong\u003ec\u003c/strong\u003e A SRM-exposed shoot clump after 14 d on SPM showing multiple \u003cem\u003ede novo\u003c/em\u003e shoots. \u003cstrong\u003ed\u003c/strong\u003e Number of shoots per responding explant after three subcultures on SPM in SRM-exposed explants compared to the control. Data are presented as a box and whisker plot showing individual data points from three independent biological replicates (n=48). Red, blue and yellow diamonds indicate the three replicates. The horizontal line in SRM represents the mean number of shoots per responding explant rather than the median. Asterisk (*) indicates a significant difference between control and SRM (Student’s T-test, \u003cem\u003ep \u003c/em\u003e= 0.02).\u003c/p\u003e","description":"","filename":"Figure53.png","url":"https://assets-eu.researchsquare.com/files/rs-7237557/v1/cd5d05a4578939d8287f7241.png"},{"id":89507383,"identity":"3daa7ade-15fc-43d2-894c-e26b2f83b289","added_by":"auto","created_at":"2025-08-20 17:27:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5369198,"visible":true,"origin":"","legend":"\u003cp\u003eS\u003csub\u003e3 \u003c/sub\u003eand S\u003csub\u003e4 \u003c/sub\u003e- Shoot elongation, rooting and acclimatisation of \u003cem\u003ein vitro\u003c/em\u003e regenerated Han FNH shoots. \u003cstrong\u003ea\u003c/strong\u003e Slightly elongated shoots (~2 cm) separated from shoot clumps after each subculture stage, transferred to SPM containing IAA to promote further elongation. \u003cstrong\u003eb, c\u003c/strong\u003e Fully elongated shoots with simultaneous root initiation after 4 weeks on SPM, showing healthy shoot and root growth. \u003cstrong\u003ed\u003c/strong\u003e Individual elongated shoot with well-developed roots, ready for deflasking. \u003cstrong\u003ee\u003c/strong\u003e Deflasked plants transferred into 500 mL pots filled with coco perlite medium grown under 18:6 h photoperiod conditions inside a plastic humidity chamber to prevent desiccation (insert). \u003cstrong\u003ef, g\u003c/strong\u003e Acclimatised plants after 14 d on cocopeat medium, showing healthy foliage growth. \u003cstrong\u003eh\u003c/strong\u003e A female plant showing sexual functionality after 8 weeks of growth in 8 L pots with coco perlite medium (insert shows unfertilised female inflorescence).\u003c/p\u003e","description":"","filename":"Figure63.png","url":"https://assets-eu.researchsquare.com/files/rs-7237557/v1/ce972ea4f3c67bcc26d32508.png"},{"id":89506673,"identity":"0090c4b7-26e9-4580-9b0e-a4912bfaf63b","added_by":"auto","created_at":"2025-08-20 17:19:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":9651753,"visible":true,"origin":"","legend":"\u003cp\u003eShoot regeneration and proliferation from cotyledonary node explants of several medicinal species. \u003cstrong\u003ea\u003c/strong\u003e Shoot proliferation in \u003cem\u003eArtemisia absinthium \u003c/em\u003e(common wormwood) after 4 weeks on SPM. \u003cstrong\u003eb\u003c/strong\u003e \u003cem\u003eDe novo \u003c/em\u003eshoot formation (red arrows) in \u003cem\u003eBorago officinalis \u003c/em\u003e(star flower) after 2 weeks on SRM. \u003cstrong\u003ec\u003c/strong\u003e Shoot proliferation in \u003cem\u003eArtemisia vulgaris \u003c/em\u003e(common mugwort) after 4 weeks on SPM. \u003cstrong\u003ed\u003c/strong\u003e Shoot proliferation (red arrows) in \u003cem\u003eAloe ferox \u003c/em\u003e(bitter aloe, a monocot)\u003cem\u003e \u003c/em\u003eafter 4 weeks on SPM in an altered pattern from an unknown origin rather than the CN region. \u003cstrong\u003ee\u003c/strong\u003e \u003cem\u003eDe novo \u003c/em\u003eshoot formation (red arrows) in \u003cem\u003eDelonix regia \u003c/em\u003e(royal poinciana) after 2 weeks on SRM, followed by proliferation after 4 weeks on SPM.\u003c/p\u003e","description":"","filename":"Figure71.png","url":"https://assets-eu.researchsquare.com/files/rs-7237557/v1/866357cb84672a01b79950a6.png"},{"id":95564726,"identity":"a3d8ba9a-28c3-4e47-9790-f0a5aa8c1c05","added_by":"auto","created_at":"2025-11-10 16:10:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":36535094,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7237557/v1/433df676-73cc-4405-a1b1-ad10ff9990ab.pdf"},{"id":89507389,"identity":"d45f17e3-f5f8-4f93-bfbf-ec60ed4f25f7","added_by":"auto","created_at":"2025-08-20 17:27:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16104609,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1\u003cbr\u003e\n \u003c/strong\u003eFile format: Microsoft Word (.docx)\u003cbr\u003e\nTitle of data: Supplementary Figures 1-8\u003cbr\u003e\nDescription: This file contains Supplementary Figures 1 to 8, which provide additional visual data supporting the main findings. Each figure is labelled and accompanied by a figure legend.\u003c/p\u003e","description":"","filename":"AdditionalFile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7237557/v1/12d70c3fc05defc7b35535d0.docx"},{"id":89507561,"identity":"88ed01a8-dd81-4b86-852c-1ea32916329a","added_by":"auto","created_at":"2025-08-20 17:35:19","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":34580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2\u003cbr\u003e\n \u003c/strong\u003eFile format: Microsoft Word (.docx)\u003cbr\u003e\nTitle of data: Supplementary Tables 1-9\u003cbr\u003e\nDescription: This file contains Supplementary Tables 1 to 9, which provide additional statistical data supporting the main findings. Each table has a title and legend.\u003c/p\u003e","description":"","filename":"AdditionalFile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7237557/v1/c74ed9bffeb6ea075f82524d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genotype-independent de novo regeneration protocol in Cannabis sativa L. through direct organogenesis from cotyledonary nodes","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant biotechnology has significantly advanced over the past few decades, driven by the ability to manipulate plant systems through tissue culture and genetic modification techniques. The success of genetic engineering in most plant species fundamentally relies on efficient tissue culture, as most stable transformation protocols require regeneration, a process involving the formation of new shoots and/or roots through \u003cem\u003ede novo\u003c/em\u003e organogenesis (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) from, in this case, transformed tissues, organs, unorganised calli or even single cells. Tissue culture serves as a foundational tool for various other biotechnological techniques, including micropropagation for rapid plant multiplication (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), double haploid production for breeding programs (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), protoplast fusion for somatic hybridisation (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), and gene editing for plant improvement (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), which enables applications ranging from germplasm conservation (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) to large-scale commercial plant production. These methods offer immense potential for enhancing plant traits, improving yields, and introducing novel characteristics. However, the effectiveness of tissue culture-based techniques is often constrained by the inherent recalcitrance in certain species. Recalcitrance includes regeneration recalcitrance, referring to the failure to regenerate tissues such as embryos or shoots through standard protocols, and genetic transformation recalcitrance, defined as the inability of a plant to incorporate foreign DNA into its genome (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). This poses significant challenges in developing efficient, reliable, and reproducible regeneration protocols, ultimately impacting the consistency of plant biotechnology outcomes.\u003c/p\u003e\u003cp\u003eAmong medicinal plants, \u003cem\u003eCannabis sativa\u003c/em\u003e L. (commonly referred to as cannabis) is a multi-purpose crop with both medicinal and industrial applications, renowned for its therapeutic compounds, high-quality stem fibre, nutrient-rich seeds, and its potential in phytoremediation (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). This crop is classified into industrial hemp and medicinal cannabis (MC), with legal distinction in many countries based on the concentration of Δ\u003csup\u003e9\u003c/sup\u003e-tetrahydrocannabinol (THC). Typically, genotypes with \u0026lt;\u0026thinsp;1.0% THC are designated as hemp, while those exceeding this threshold are classified as MC (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Despite its economic significance and diverse utility, regeneration and genetic transformation of cannabis pose substantial challenges due to its recalcitrant nature (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). While cannabis can be readily propagated through conventional methods such as stem cuttings (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) or \u003cem\u003ein vitro\u003c/em\u003e micropropagation through shoot tips (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), nodal segments (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) and inflorescences (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), these clonal approaches do not enable genetic manipulation studies necessary to rapidly improve traits such as disease resistance, yield, or cannabinoid profile. Therefore, developing efficient regeneration protocols is critical for advancing biotechnological research in this important crop.\u003c/p\u003e\u003cp\u003ePlant regeneration through organogenesis, which facilitates the recovery of whole plants from a single cell or tissue, is an essential prerequisite for genetic transformation and genome editing (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Despite significant advances in cannabis micropropagation and shoot proliferation, reliable \u003cem\u003ede novo\u003c/em\u003e plant regeneration protocols remain limited (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Previous studies have demonstrated indirect (with an intermediate callus phase) or direct (from preexisting meristems) regeneration from explants such as leaves (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and petioles (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), epicotyls (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), cotyledons (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), and hypocotyls (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), typically using combinations of cytokinins and auxins to induce shoot organogenesis. However, these methods have shown high genotype dependency, which limits the applicability of transgenic studies to other cannabis germplasm (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). For example, Zhang et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) screened 100 hemp cultivars and found regeneration rates ranging from 0-7.09%, with significant variation between genotypes, where most were generally on the low end of regeneration efficiency.\u003c/p\u003e\u003cp\u003eProtocols leveraging \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation have achieved transgenic hairy root cultures (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) (i.e., autonomous root systems devoid of other plant organs), yet the regeneration of fully developed transgenic plants remains inconsistent. Recent efforts to overcome these limitations have included exploring different explant types (e.g. embryonic/meristematic tissues), plant growth regulators (e.g. synthetic cytokinins), novel approaches such as the introduction of morphogenic genes (e.g., GRF/GIF chimeras), nanoparticle delivery systems and integration of cutting-edge techniques such as CRISPR-Cas9 for targeted gene editing (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). While progress has been made with some transformation protocols showing success within specific research settings (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), the development of standardised protocols that work consistently across diverse cannabis germplasm remains an ongoing challenge for the field. Given the rising global demand for cannabis in various applications, there is an urgent need to develop robust and scalable regeneration methods to unlock its full potential.\u003c/p\u003e\u003cp\u003eIn this study, we sought to develop a high-efficiency shoot regeneration protocol through direct organogenesis using various seed explants, including cotyledons, hypocotyls, and particularly the cotyledonary node (CN) across a range of cannabis lines. By systematically optimising key stages (S\u003csub\u003e0\u003c/sub\u003e-S\u003csub\u003e4\u003c/sub\u003e) of the regeneration process and with careful attention to both explant type and the optimal duration of plant growth hormone treatments, we have established a reliable and scalable \u003cem\u003ede novo\u003c/em\u003e regeneration protocol for cannabis. Additionally, we assessed the effectiveness of our protocol in a selection of other medicinal species with limited or no published methods, demonstrating its potential broader applicability beyond cannabis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003ePlant material\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIndustrial hemp seeds of cultivars Han FNH, Han FNQ, Bama, Puma, Yuma, Han Cold, Han NE and Si-1 were sourced from The Hemp Corporation Pty Ltd (Vacy, NSW, Australia) while CRS-1, CFX-2, Katani, Futura-75, and Ferimon-12 were procured from Midlands Seed Pty Ltd (Richmond, Tasmania, Australia). Seeds from all cultivars were stored at 4\u0026deg;C in sealed containers with silica bead desiccant until use. Fresh seeds of Han FNQ were produced in-house. Briefly, Han FNQ seedlings were initially cultivated following the method described by Welling et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). At reproductive maturity, pollen was collected from dehiscing anthers of male plants and dusted onto the stigmas of flowers within each inflorescence of female plants. Successful pollination was confirmed by stigma senescence and bract swelling. Seed maturity was determined by yellowing leaves and bract drying. Once mature, the seeds were harvested manually and any underdeveloped seeds were discarded.\u003c/p\u003e\u003cp\u003eSeeds from three MC lines-MC-1 (7.5% total THC, 8.6% total CBD), MC-2 (4.2% total THC, 8.1% total CBD), and MC-3 (2.6% total THC, 4.5% total CBD) were supplied by Cann Group Ltd (Port Melbourne, Victoria, Australia). These MC seeds were ~\u0026thinsp;8 yrs old and originated from a controlled glasshouse environment. Each line was derived from a feminised population pollinated by a single pollen donor.\u003c/p\u003e\u003cp\u003eSeeds of eight medicinal species, \u003cem\u003eAloe ferox\u003c/em\u003e, \u003cem\u003eArtemisia absinthium\u003c/em\u003e, \u003cem\u003eAr. annua, Ar. vulgaris\u003c/em\u003e, \u003cem\u003eBorago officinalis\u003c/em\u003e, \u003cem\u003eCaesalpinia pulcherrima, Delonix regia\u003c/em\u003e and \u003cem\u003eEchinacea purpurea\u003c/em\u003e, were purchased from The Seed Vine (Tenterfield, NSW, Australia).\u003c/p\u003e\u003cp\u003e\u003cb\u003eS\u003c/b\u003e\u003csub\u003e\u003cb\u003e0\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e- Seed sterilisation and germination\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBatches of 40 seeds were initially surface-sterilised by soaking in 45 mL 1% (v/v) H₂O₂ (hydrogen peroxide, 7.5%, Milestone Chemicals Australia Pty Ltd, Heidelberg West, VIC) in a sterile 50 mL tube and incubated in the dark at 24\u0026deg;C for 24 h to initiate germination. The solution was then replaced with fresh 1% (v/v) H₂O₂, and seeds were incubated for an additional 24 h under identical conditions and germination (seeds displaying radicle emergence) scored at 48 h. After 48 h, germinated seeds were dissected to remove both the outer pericarp (hull) and seed coat. The dissected embryos underwent secondary sterilisation in 45 mL 1% (v/v) H₂O₂ with shaking at 150 rpm, 24\u0026deg;C for 1 h. Sterilised embryos were transferred (12 per plate) to 90 mm diameter plastic Petri dishes containing germination medium (GM; 0.5x Murashige and Skoog (MS) salts and vitamins (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) (M519, PhytoTech Labs, Kansas, USA), 1.5% (w/v) commercial-grade sucrose, 0.65% (w/v) agar (A296, PhytoTech Labs), pH 5.7) and grown under 16/8 h L/D photoperiod (140\u0026ndash;170 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e PAR) conditions at 24\u0026deg;C for 5 d. Each embryo was then scored for both endophyte contamination and responsiveness, i.e. whether it grew beyond radicle emergence.\u003c/p\u003e\u003cp\u003e\u003cb\u003eS\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e- Explant preparation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter 5 d on GM, various explants were prepared from 7 d-old seedlings and assessed for regeneration potential. Using sterile forceps and a scalpel (blade no. 11), cotyledons were excised from the seedling by making cuts at the distal end to remove the cotyledon tip and at the proximal end where the cotyledon attaches to the primary shoot, to create wound sites at both ends. For hypocotyls, cuts were made at both the proximal end below the CN and the distal end above the radicle. For the CN with cotyledon explant, the hypocotyl and radicle were excised just below the CN, the region where the two cotyledons converge, followed by removal of the primary shoot. The cotyledons were then separated through a complete vertical cut. The prepared explant pairs were subsequently used for regeneration. For comparative analysis, additional CN-based explants were also prepared: half CN with half-cotyledon (CN with cotyledon explant halved longitudinally), whole CN (intact CN without cotyledons) and CN with hypocotyl (CN with top portion of hypocotyl).\u003c/p\u003e\u003cp\u003e\u003cb\u003eS\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e- Shoot regeneration\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate regeneration potential, a maximum of 10 cotyledons (abaxial side down) or hypocotyls (oriented horizontally) were placed per 90 mm Petri dish containing shoot regeneration medium (SRM; 1x MS, 0.4 mg/L thidiazuron (TDZ, T888, PhytoTech Labs), 0.2 mg/L α-naphthaleneacetic acid (NAA, N600, PhytoTech Labs), 3% (w/v) commercial-grade sucrose, 0.8% (w/v) agar, pH 5.7). Similarly, up to four CN with cotyledon explant pairs (8 explants in total) were placed per dish with the abaxial side down and the proximal end embedded in the medium. Control plates containing the same medium without growth hormones were used for comparison. All plates were maintained in a Conviron GEN1000 growth incubator (Controlled Environments Ltd, Winnipeg, Canada) at 24\u0026deg;C, 16/8 h L/D photoperiod with white light from broad-spectrum 12W T5 LEDs (140\u0026ndash;170 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e PAR). After 7 d, the primary shoot (identified as the central emerging shoot) from each explant was re-excised to promote multiple shoot initiation, and explants returned to control or SRM plates for an additional 7 d. After 14 d, the overall regeneration efficiency was calculated as the percentage of explants forming one or more shoots. This included both axillary shoot growth (observed on both control and SRM plates) and \u003cem\u003ede novo\u003c/em\u003e shoot formation (exclusively observed on SRM). \u003cem\u003eDe novo\u003c/em\u003e regeneration efficiency was calculated as the percentage of explants exhibiting \u003cem\u003ede novo\u003c/em\u003e shoot organogenesis on SRM after 14 d.\u003c/p\u003e\u003cp\u003eTo determine minimum SRM exposure time for \u003cem\u003ede novo\u003c/em\u003e shoot induction, CN with cotyledon explants were subjected to varying SRM durations (1, 2, 4, 6, 8, 10, 12, and 14 d) including a control (no SRM exposure, direct transfer to proliferation medium) and 5 min vacuum infiltration in liquid SRM (to enhance medium penetration into explant tissues). After treatment, explants were transferred to proliferation medium for the remainder of the 14 d period to standardise total culture duration across all treatments and then evaluated for \u003cem\u003ede novo\u003c/em\u003e regeneration.\u003c/p\u003e\u003cp\u003e\u003cb\u003eShoot proliferation (S\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e), elongation and rooting (S\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMultiple shoot clumps were excised from regenerating CN with cotyledon explants after 14 d on SRM or control medium and transferred to shoot proliferation medium (SPM; 1x MS, 0.175 mg/L indole-3-acetic acid (IAA, I885, PhytoTech Labs), 1% (w/v) commercial-grade sucrose, 0.8% (w/v) agar, pH 5.7). During the excision process, the axillary shoot, cotyledon and callus tissue were removed. Plates were maintained under the same growth conditions as the shoot regeneration phase. After 14 d on SPM, elongated shoots (~\u0026thinsp;2 cm in length) were separated from shoot clumps and transferred to fresh SPM in 500 mL screw-cap polycarbonate culture vessels (84 x 102 mm, Thermo Fisher Scientific NZ Ltd, Auckland, New Zealand) and incubated under the same conditions for 3\u0026ndash;4 weeks to enable further elongation and rooting. The remaining shoot clumps were divided and subcultured at 14 d intervals for three cycles on fresh SPM plates. At each subculture, elongated shoots were counted and transferred to culture vessels. For MC lines, while the shoot proliferation phase utilised standard SPM plates, a modified SPM containing 0.5 mg/L indole-3-butyric acid (IBA, I538, PhytoTech Labs) instead of IAA was used specifically for rooting the elongated shoots to allow a direct comparison with rooting in vegetative nodal cuttings treated with Clonex rooting hormone gel (Yates, Australia), which contains IBA as the active ingredient.\u003c/p\u003e\u003cp\u003e\u003cb\u003eS\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e- Acclimatisation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eElongated plantlets with developed roots were gently removed from SPM culture vessels and thoroughly rinsed with reverse osmosis (RO) water to eliminate any residual medium. They were then transplanted into 500 mL plastic pots filled with coco perlite (70:30) medium (Epping Hydroponics, Epping, VIC, Australia) saturated with RO water and supplemented with half-strength CANNA Classic Vega A and Vega B nutrient solutions (4 mL of each Vega/L of RO water) (CANNA Australasia, Subiaco, WA, Australia). The pots were placed inside a plastic humidity chamber where the plantlets acclimatised for 2 d under 100% relative humidity at 24\u0026deg;C, 18/6 h L/D photoperiod with white fluorescent lights (140\u0026ndash;160 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e PAR) in a controlled environment room. After 2 d, plantlets were removed from the humidity chamber and grown for another 14 d before being transferred to 1.5 L pots for two weeks and then 8 L pots containing coco perlite medium to support further growth and development till seed harvest.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicroscopy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStereomicroscopy images were captured using a Leica M80 stereo microscope equipped with a Leica DMS4500 digital microscope camera. Image acquisition and processing were performed with the Leica Application Suite (version 4.12.0), using the Image Builder application. Photographs of cultures and plants were captured on a Canon 90D camera fitted with a 24\u0026ndash;105 mm f/4-7.1 IS Canon lens. To examine internal tissue structure, CN with cotyledon explants exposed to SRM for 7 d were embedded in Super Cryoembedding Medium (SCEM, Section-Lab, Japan). Transverse sections of 30 \u0026micro;m were then taken with a Leica CM3050 S cryostat and visualised using an Olympus BX63 compound microscope. Images were captured with an Olympus DP60 camera. Schematic diagrams were hand-drawn on an iPad using the Procreate app (version 5.3.15).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll experiments followed a randomised design. Data were analysed using Microsoft Excel (version 2410). Graphs were prepared using Microsoft Excel and BioRender 2024.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe availability of an efficient plant regeneration protocol is a prerequisite for large-scale \u003cem\u003ein vitro\u003c/em\u003e propagation and/or genetic manipulation of any plant. This study aimed to develop a reproducible, genotype-independent regeneration protocol for cannabis for both applications. To ensure systematic protocol optimisation, the process was divided into distinct ordered stages: S\u003csub\u003e0\u003c/sub\u003e, seed sterilisation and germination; S\u003csub\u003e1\u003c/sub\u003e, explant excision and shoot induction; S\u003csub\u003e2\u003c/sub\u003e, shoot proliferation; S\u003csub\u003e3\u003c/sub\u003e, elongation and rooting; and S\u003csub\u003e4\u003c/sub\u003e, acclimatisation (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe optimisation of each stage focused on refining key parameters that critically influence regeneration efficiency, building on previous findings by Chaohua et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), Gal\u0026aacute;n-\u0026Aacute;vila et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) and Ahsan et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In S\u003csub\u003e0\u003c/sub\u003e, we modified an established H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e sterilisation protocol to significantly enhance seed germination rates and reduce endophyte contamination, thereby increasing the availability of viable explants (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). S\u003csub\u003e1\u003c/sub\u003e involved a systematic evaluation of embryonic explants, including cotyledons (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) and hypocotyls (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), which had been previously explored for regeneration, in addition to the cotyledonary node (CN). Rather than extensively testing different media formulations and hormonal combinations, we specifically optimised exposure time to a previously validated shoot regeneration medium (SRM) containing TDZ and NAA (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). For S\u003csub\u003e2\u003c/sub\u003e, we used a shoot proliferation medium (SPM) containing IAA in conjunction with subculturing intervals to maximise shoot production. In S\u003csub\u003e3\u003c/sub\u003e, we used IAA (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) and IBA (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) for shoot elongation and root induction, aligning with published protocols. Finally, in S\u003csub\u003e4\u003c/sub\u003e, the acclimatisation process was optimised by creating an optimal \u003cem\u003eex vitro\u003c/em\u003e environment to enhance survival rates and facilitate the successful establishment of regenerated cannabis plants. The following sections provide a detailed account of the optimisation process of each stage.\u003c/p\u003e\u003cp\u003e\u003cb\u003eS\u003c/b\u003e\u003csub\u003e\u003cb\u003e0\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e- Effect of H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eon seed germination and endophyte contamination\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePlant tissue sterilisation is an essential first step to obtain aseptic, healthy explants as donor material for shoot regeneration. Standardising a sterilisation protocol is especially important when starting with wild collections or field-grown seed populations arising from open pollination, as is the case with hemp. Such seeds often exhibit phenotypic variability, reflecting their genetic diversity as a result of outcrossing. Also, exposure to external environmental conditions can lead to a range of biological contaminants that accumulate on the external surface of the seed as well as the presence of non-pathogenic microbes (endophytes) within the seed. This necessitates the optimisation of sterilisation protocols to address microbial contamination without compromising seed viability. In cannabis, seed-associated endophytes are known to vary between genotypes, cultivation environment (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) and post-harvest storage conditions (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). While endophytes can benefit plant growth in field conditions, they severely hinder tissue culture by introducing persistent contamination (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur early efforts focused on hemp cultivars due to larger seed volumes being readily available. Published protocols have successfully used ethanol and sodium hypochlorite for seed sterilisation (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). However, when applied to field-grown seeds of hemp cultivars Han FNH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b) and Han FNQ, these methods resulted in poor germination and persistent bacterial, fungal and endophyte contamination. The radicle was observed to emerge from the rigid outer pericarp (hull) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), but often this layer remained attached to the embryo together with the membranous seed coat (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), raising the possibility that microbes may persist in parts of the embryo surface less accessible to these sterilisation agents. In an attempt to improve germination rate and reduce contamination, we explored alternative approaches and drawing from previous studies (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), adopted a 1% (v/v) H₂O₂ treatment with modifications to enhance the efficacy of the sterilisation solution.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTreatment of hemp seeds with fresh 1% (v/v) H₂O₂ twice for 24 h followed by an additional 1 h wash with fresh 1% (v/v) H₂O₂ after manually removing both the pericarp and seed coat (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) significantly improved germination rates from ~\u0026thinsp;20% when seeds are treated with ethanol and sodium hypochlorite to an average of 78.2% in Han FNH and 63.3% in Han FNQ (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, this modified seed sterilisation regime was found to completely eliminate surface-derived bacterial and fungal contamination.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of 1% (v/v) H₂O₂ sterilisation treatment on seed germination and endophyte contamination in hemp cultivars Han FNH and Han FNQ\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eHemp cultivar\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAfter 48 h in 1% (v/v) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003eAfter 5 d on GM\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGermination rate (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEndophyte-free seeds (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEndophyte-contaminated seeds (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eUnresponsive seeds (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHan FNH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e78.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e60.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHan FNQ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e63.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e29.6\u0026thinsp;\u0026plusmn;\u0026thinsp;11.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e28.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHan FNQ\u003c/p\u003e\u003cp\u003e(fresh seeds)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e99.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e95.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eValues show mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from three independent replicates (Han FNH-230, 160, 75; Han FNQ-116, 100, 200; Han FNQ fresh seeds-50, 50, 240). Data presented as a percentage of total seeds used\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFollowing sterilisation, seeds were placed on germination medium (GM) (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) for 5 d (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef) to determine which were endophyte-free and capable of further growth, thus suitable for explant excision for regeneration purposes. After 5 d on GM, an average of 60.4% of total Han FNH and 29.6% of Han FNQ seeds were found to be endophyte-free (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), while 6.3% and 28.2%, respectively, showed endophyte contamination (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Unresponsive seeds where the embryo had germinated but exhibited minimal growth (i.e., had poor viability) accounted for 10.8% of total seeds in Han FNH and 5.4% in Han FNQ on average (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). Therefore, the proportion of seeds identified as responsive and endophyte-free on GM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej) differed considerably between the two hemp cultivars.\u003c/p\u003e\u003cp\u003eThe standardised 1% (v/v) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e sterilisation protocol was applied to an additional five hemp cultivars and three MC lines to determine if this variability in endophyte-free seed amongst cannabis genotypes is typical, as this impacts the total number of seeds required per experiment to achieve successful regeneration. Among the additional hemp cultivars, germination rates again varied widely, ranging from 33.8% in Futura-75 to 81.3% in CFX-2 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The proportion of total seeds free from endophyte contamination followed a similar trend, with Futura-75 displaying the lowest at 20.6%, while CFX-2 showed the highest at 63.5% after 5 d on GM (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Seeds from the MC lines, produced in an indoor facility, exhibited notably higher germination rates of 86\u0026ndash;96% after 1% (v/v) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These lines also demonstrated a high proportion of seeds free from endophyte contamination, ranging between 68\u0026ndash;76%, indicating that the sterilisation process can effectively eliminate endophyte contaminants. It was noted that a relatively high proportion of MC seeds were unresponsive, between 14\u0026ndash;28% (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), indicating a loss in seed viability that was likely due to a prolonged storage period.\u003c/p\u003e\u003cp\u003eThe hemp results suggested that poor seed germination and high endophyte contamination may be associated traits. To determine whether improving germination could assist in reducing both endophyte contamination and seed unresponsiveness, we generated fresh seeds of Han FNQ under controlled indoor conditions and evaluated them using our 1% (v/v) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e sterilisation protocol. Fresh seeds exhibited significant improvements compared to the original seed batch (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The germination rate increased to 99.9%, seeds free from endophyte contamination rose to 95.7%, and unresponsive seeds decreased to 1.5% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These data suggest that undertaking a growth cycle to generate fresh seed is advantageous to boost responsive explant numbers and thereby enhance regeneration efficiency in the long term.\u003c/p\u003e\u003cp\u003e\u003cb\u003eS\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e- Effect of different explant types and SRM exposure time on direct regeneration\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eComparative evaluation of embryo-derived explants\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe shoot induction step (S\u003csub\u003e1\u003c/sub\u003e) typically involves exposing explants to a specific regime of plant growth hormones that either activate pre-existing (sometimes dormant) meristematic cells or stimulate \u003cem\u003ede novo\u003c/em\u003e shoot organogenesis in responsive tissue. To systematically evaluate the regeneration potential of seed explants, we first replicated the published protocols with cotyledons (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) and hypocotyls (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) excised from 7 d-old seedlings subjected to SRM containing TDZ and NAA, the hormone combination optimised in previous cannabis regeneration studies (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the hemp cultivars previously tested, we selected CRS-1, CFX-2, Katani, Han FNH and Han FNQ due to the availability of more endophyte-free seeds compared to other cultivars (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Despite meticulously replicating the published methods, including SRM composition and exposure time of 2\u0026ndash;5 weeks, cotyledons and hypocotyls repeatedly failed to regenerate shoots at the previously observed rates across all five hemp cultivars tested. Instead, they predominantly formed callus at their cut surfaces, particularly at their proximal end (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). While previous studies have reported regeneration rates of 4\u0026ndash;55% from cotyledons (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) and ~\u0026thinsp;50% from hypocotyls (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), our rates were dramatically lower. Only two of 2,358 cotyledon explants from CRS-1 (0.1%), two of 100 from Han FNH (2%) and a single cotyledon explant from 60 Han FNQ (1.7%) regenerated shoots, all at the proximal end of the cotyledon (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). No regeneration was observed with hypocotyl explants of any of the five hemp cultivars.\u003c/p\u003e\u003cp\u003eThe consistency of negative results across multiple hemp cultivars supports the notion that non-meristematic seed explants are unable to undergo direct regeneration on SRM (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Closer examination of the regenerating cotyledon explants suggested that these rare cases may have resulted from inadvertent retention of a portion of the CN, the region at the point of insertion of the cotyledon into the embryonic axis (near the primary shoot) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and b). Hence, we hypothesised that successful direct regeneration in cannabis may require an explant with inherent meristematic activity. We therefore explored the CN, which retains such characteristics, as a potentially more effective explant alternative.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnatomically, in most epigeal species in which cotyledons emerge above-ground during seedling growth, the CN is a critical junction (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Formed during early embryogenesis, this region houses both dormant axillary meristems at each cotyledon base and undifferentiated cells with significant regenerative potential that can be activated by exogenous hormones (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In contrast, hypogeal species where cotyledons remain underground during seedling growth often lack clearly defined or functionally competent axillary nodes at this junction (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). This anatomical distinction between germination types further supports the use of CN explants in cannabis, an epigeal species, for efficient direct shoot regeneration.\u003c/p\u003e\u003cp\u003eEleven hemp cultivars were used to investigate the regenerative potential of CN explants and evaluate possible genotype dependency. The CN was excised along with the attached cotyledon from 7 d-old seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-e) following protocols developed in other epigeal species (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Using the same SRM, regrowth of the primary shoot was observed in most explants after 7 d and was removed to reduce apical dominance (i.e., the more vigorous growth of the primary shoot due to endogenous auxin accumulation that suppresses axillary bud growth). After 4\u0026ndash;5 weeks, shoot regeneration was observed at the proximal end of the cotyledon in all 11 cultivars, typically in the form of multiple shoots with one dominant central shoot surrounded by numerous smaller shoots (Fig. S3a). The overall regeneration efficiency ranged from 3.2% in Yuma to 22.2% in Han FNQ (Table S3). This indicates that the CN with cotyledon explant is far superior to cotyledon or hypocotyl in producing shoot regeneration in a seemingly genotype-independent manner. However, while shoot induction was successful, prolonged exposure to TDZ led to extensive callus formation (Fig. S3a).\u003c/p\u003e\u003cp\u003e\u003cem\u003eOptimising exposure time to SRM to enhance shoot regeneration\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo mitigate TDZ-induced limitations on shoot regeneration, we investigated whether shortening the SRM exposure time from 5 to 3 weeks could improve regeneration outcomes. We selected four hemp cultivars, CFX-2 and Katani, which showed moderate regeneration efficiency, along with Futura-75 and Ferimon-12, previously reported to regenerate successfully (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). With the shortened exposure time, a reduction in callus proliferation was observed, though still persistent, together with a substantial improvement in the overall regeneration efficiency, increasing to 67.5%-90% across cultivars (Table S4).\u003c/p\u003e\u003cp\u003eNext, we proceeded to test whether reducing SRM exposure to 14 d could further enhance shoot regeneration while mitigating persistent callus formation. Han FNH was selected for this experiment due to its relatively high proportion of endophyte-free seeds (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To better assess the effects of TDZ and NAA on shoot regeneration, we included a control medium (MS without hormones) to enable a detailed comparison of regeneration efficiency and shoot development on SRM. Unlike previous experiments where explants were evaluated individually, CN explants with attached cotyledons were tracked in pairs to capture the full regenerative potential of the CN from individual seeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eAs expected, regrowth of the primary shoot was observed within 4\u0026ndash;7 d of exposing the explants to either medium. After 7 d, it was excised at the epicotyl region to reduce apical dominance. On both media, this excision led to the elongation of the axillary shoot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b), the dominant central shoot identified in earlier experiments. This response mirrors the natural hormonal regulation in seedlings where removal of the primary shoot disrupts basipetal auxin flow from the shoot apex while allowing cytokinin synthesis, shifting the hormonal balance to favour axillary shoot outgrowth (Fig. S4a) (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). In contrast, on SRM, elongation of the axillary shoot was accompanied by additional shoot regeneration at the base of the excised primary shoot and on both sides of the axillary shoot in a symmetrical pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). We have termed this direct \u003cem\u003ede novo\u003c/em\u003e regeneration. Consequently, the overall regeneration efficiency varied significantly between the two treatments. On control medium, regeneration reached 24.3% after 14 d, consisting of axillary shoot elongation only (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). However, SRM significantly enhanced regeneration due to the additional \u003cem\u003ede novo\u003c/em\u003e shoot formation, increasing overall efficiency to 77.8% over the same period (Student T-test, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), with \u003cem\u003ede novo\u003c/em\u003e regeneration accounting for 70.1% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Axillary shoot elongation occurred to a similar extent in both treatments, but \u003cem\u003ede novo\u003c/em\u003e shoot initiation was exclusive to SRM-treated explants following removal of the primary shoot (compare Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and b). Axillary shoot elongation may be supported by endogenous cytokinin, potentially present in the cotyledons, along with nutritional cues such as sugars (Fig. S4b). In contrast, explants cultured on SRM were exposed to exogenous cytokinin (TDZ), which may act in conjunction with endogenous signals to facilitate \u003cem\u003ede novo\u003c/em\u003e shoot formation (Fig. S4b). Furthermore, minimal callus formation was observed at the proximal end of explants compared to those maintained for 3 weeks on SRM, suggesting the reduction in TDZ and NAA exposure was beneficial.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnother noteworthy observation was that the paired explants exhibited distinct responses under different media conditions. On control medium, an average of 8.3% of seeds produced shoots from both explants, whereas this increased substantially to 54.2% on SRM (Table S5). However, the percentage of seeds with at least one regenerating explant was similar between control and SRM (~\u0026thinsp;32%), suggesting that SRM primarily promoted regeneration in both explants rather than affecting the likelihood of at least one explant regenerating (Table S5). Additionally, the proportion of seeds where neither explant regenerated was significantly reduced on SRM, decreasing from 59.7% in the control to 13.9% (Table S5). These results highlight the strong stimulatory effect of TDZ and NAA in SRM on direct shoot regeneration. Importantly, the ability to obtain two regenerative explants from a single seed demonstrates that our excision method preserves the zone(s) critical for shoot regeneration.\u003c/p\u003e\u003cp\u003eTo determine whether 14 d of SRM exposure was optimal across multiple cannabis genotypes, we tested its efficacy in the Han FNQ hemp cultivar and three MC lines. In Han FNQ, SRM exposure for 14 d resulted in a \u003cem\u003ede novo\u003c/em\u003e regeneration efficiency of 78% (Table S6) with minimal callus growth, similar to the observations made in Han FNH. The protocol also successfully induced regeneration in all three tested MC lines, with \u003cem\u003ede novo\u003c/em\u003e shoot formation comparable to that observed in hemp cultivars (Fig. S5a). Notably, MC lines exhibited even higher \u003cem\u003ede novo\u003c/em\u003e regeneration efficiencies (84-91.7%) than the hemp cultivars tested, highlighting the robustness of the 14 d SRM treatment across genotypes (Table S6).\u003c/p\u003e\u003cp\u003e\u003cem\u003eEvaluating variants of CN explants to define regeneration competence\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWith the aim of maximising regeneration potential, we also evaluated how different excisions of the CN region affect shoot regeneration. Using Han FNH due to its strong regeneration response (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), we excised and compared the performance of three other CN-based explant types from 7 d-old seedlings relative to CN with cotyledon by culturing them on SRM for 14 d (Fig. S6). The three CN variants, half CN with half-cotyledon (CN with cotyledon explant halved longitudinally, 4 per seed), whole CN (intact CN without cotyledons, 1 per seed), and CN with hypocotyl (whole CN with the top portion of hypocotyl, 1 per seed), allowed us to evaluate if there is a trade-off between explant quantity and regeneration efficiency.\u003c/p\u003e\u003cp\u003eCN with cotyledon explants exhibited efficient \u003cem\u003ede novo\u003c/em\u003e shoot formation (57.5%, Table S7) with minimal callus growth, consistent with our previous trials (Fig. S6a-c). Half-CN with half-cotyledon explants were hypothesised to increase shoot regeneration due to the separation of the axillary meristematic zone by longitudinal bisection of the CN and cotyledon, thereby potentially enhancing axillary meristem activation (Fig. S6d and e). Instead, these explants exhibited a greatly reduced \u003cem\u003ede novo\u003c/em\u003e regeneration efficiency of 23.8% (Table S7). The high degree of wounding led to excessive callusing at the cut edges of the cotyledons but mainly at the axillary node region, probably compromising the structural integrity of the CN and impairing \u003cem\u003ede novo\u003c/em\u003e shoot formation (Fig. S6f). Axillary shoot regeneration was reduced to \u0026lt;\u0026thinsp;2%, likely due to the damage caused to the axillary node from the vertical bisection (Table S7).\u003c/p\u003e\u003cp\u003eWhole CN explants without attached cotyledons retained intact CN structure and avoided damage to the axillary nodes (Fig. S6g and h), enabling assessment of cotyledon necessity for efficient \u003cem\u003ede novo\u003c/em\u003e regeneration. Axillary shoot growth was observed in 24 of 40 explants (60%) (Table S7), approximately twice the amount of the CN with cotyledon explants, as expected, due to two axillary meristem regions being present rather than one (Fig. S4b). However, the \u003cem\u003ede novo\u003c/em\u003e regeneration was severely compromised, often delayed and accompanied by substantial callus proliferation at the base of the explant (Fig. S6i). Moreover, since only one explant could be obtained per seed and regeneration was both slower and less prolific, whole CN explants exhibited the lowest \u003cem\u003ede novo\u003c/em\u003e regeneration efficiency at 12.5% (Table S7).\u003c/p\u003e\u003cp\u003eWhen CN with hypocotyl explants were tested, regeneration was exclusively observed at the CN region, while the remaining hypocotyl sections developed callus (Fig. S6j-l). Despite differences in efficiency, all CN-based explants exhibited some level of regeneration, indicating that the CN plays an indispensable role in direct shoot organogenesis. Among them, CN with cotyledon emerged as the most efficient and reliable explant, demonstrating superior regeneration capacity.\u003c/p\u003e\u003cp\u003e\u003cem\u003eDetermining the onset and cellular origin of regeneration from CN explant\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo further examine the timing and progression of \u003cem\u003ede novo\u003c/em\u003e shoot formation, we conducted a time-course analysis over 14 d on SRM using CN with cotyledon explants from Han FNH and Han FNQ cultivars. Morphological changes of representative explants were documented daily on both control medium (Han FNH) and SRM (both cultivars). As previously observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), regrowth of the primary shoot and axillary shoot development occurred within 4\u0026ndash;7 d across all conditions (Fig. S7). However, only axillary shoot elongation was noted in control explants throughout the 14 d period (Fig. S7a\u0026ndash;e). For SRM-treated explants, by day 10 in Han FNH (Fig. S7f-j) and as early as day 7 in Han FNQ (Fig. S7k-o), multiple \u003cem\u003ede novo\u003c/em\u003e shoots emerged around the axillary shoot and at the base of the excised primary shoot (Fig. S7h and k). These findings suggested that 7\u0026ndash;10 d SRM exposure represents a suitable timeframe to capture early organogenic events.\u003c/p\u003e\u003cp\u003eIn a separate experiment using Han FNH CN with cotyledon explants, regeneration was observed at 7 d on SRM, similar to Han FNQ in the earlier time-course study (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). These explants were then used for histological analysis. Microscopic observations revealed distinct origins of the two types of shoot regeneration in these explants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Transverse sections showed that the axillary shoot emerged from the vascular ring of the primary shoot, confirming its origin from pre-existing meristematic tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Concurrently, multiple \u003cem\u003ede novo\u003c/em\u003e shoots differentiated from peripheral cortical cells at the junction between the base of the excised primary shoot and the cotyledon attachment site. These \u003cem\u003ede novo\u003c/em\u003e shoots developed independently of the vasculature and without intervening callus formation, indicating true \u003cem\u003ede novo\u003c/em\u003e organogenesis induced by exogenous TDZ and NAA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven the emergence of \u003cem\u003ede novo\u003c/em\u003e shoots by 7\u0026ndash;10 d, we investigated whether the standard 14 d SRM exposure could be shortened further without compromising efficient regeneration. Brief exposures (\u0026lt;\u0026thinsp;2 d) failed to induce \u003cem\u003ede novo\u003c/em\u003e shoots in Han FNH CN with cotyledon explants, but exhibited only axillary shoot elongation, similar to the control (Table S8), suggesting these were insufficient to activate \u003cem\u003ede novo\u003c/em\u003e shoot formation. However, regeneration efficiency steadily increased with longer exposures, reaching 6.7% at 2 d, 30% at 6 d, and peaking at 50% at 10 d before slightly declining to 43% at 12 d and 14 d (Table S8). These findings indicate that a minimum of 2 d is required to trigger \u003cem\u003ede novo\u003c/em\u003e regeneration, while exposures at 10\u0026ndash;14 d are optimal for maximising shoot induction.\u003c/p\u003e\u003cp\u003e\u003cb\u003eS\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e- Shoot proliferation in response to IAA\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFollowing shoot induction on SRM, the developing multiple shoots must undergo further proliferation to allow the separation of individual shoots. Without timely separation and regular subculturing, apical dominance results in the elongation of only one or two shoots while suppressing the growth of adjacent \u003cem\u003ede novo\u003c/em\u003e regenerated shoots (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), limiting overall shoot yield per explant and increasing resource competition. Shoot proliferation is achieved on an auxin-only medium (SPM) with reduced sucrose concentration (1% w/v) relative to SRM, which gradually supports the transition to autotrophy, a critical step for subsequent rooting and acclimatisation. Our preliminary work in MC compared different auxins such as IAA and IBA in SPM, revealing that IBA-supplemented medium caused excessive callusing at the primary shoot excision site. Given the small size of the shoot clumps and the negative impact of callusing we observed on shoot proliferation, IAA was selected as the preferred auxin for this process.\u003c/p\u003e\u003cp\u003eWe initially assessed shoot proliferation from explants maintained on SRM for long durations. Five weeks of SRM exposure followed by 4\u0026ndash;5 weeks of proliferation on the same medium resulted in restricted proliferation (\u0026le;\u0026thinsp;4 shoots per explant) across cultivars (Table S3), accompanied by excessive callus formation and shoot vitrification (translucent, brittle, and water-soaked tissues) (Fig. S3b). Reducing SRM exposure to 3 weeks mitigated, but did not eliminate, TDZ-induced callusing. However, when explants were subsequently transferred to TDZ-free SPM, shoot proliferation improved substantially, with yields increasing to 3-7.2 shoots per explant depending on cultivar (Table S4). Although callusing persisted, vitrification was notably absent (Fig. S3c).\u003c/p\u003e\u003cp\u003eTo further optimise proliferation efficiency, Han FNH was assessed for shoot proliferation response following a shortened 14 d SRM exposure. Shoot clumps formed after 14 d on either control media or SRM were excised from residual tissues (cotyledons, callus, and axillary shoot) and transferred to SPM where they began to proliferate within 14 d (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Clumps from the control medium exhibited limited shoot proliferation either from the nodal region of the excised primary shoot or from the regrowth of axillary shoot (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). In contrast, clumps from SRM displayed active proliferation on SPM, with multiple shoots emerging from most of the explant pairs with less callus growth at the base of the shoot clump compared to 3 weeks on SRM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and Fig. S3c). Over the course of three fortnightly subcultures on SPM, control-derived clumps showed a marked decline in proliferation, producing fewer shoots in the first and second subcultures and none by the third (Fig. S8a). However, SRM-derived clumps exhibited significantly enhanced and sustained shoot proliferation across all three subcultures, with the highest shoot numbers observed in the third round (Fig. S8a). The mean number of shoots per responding explant (determined by averaging the total number of shoots observed across all subcultures and the remainder on the original explant) increased from 1.4 shoots per explant (1.7 per seed) exposed to control medium to 7 (11.4 per seed) in the SRM-exposed explants, with the highest observed response being 28 shoots from a single explant (50 shoots from a single seed) (Student\u0026rsquo;s T-test, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further assess the reproducibility of shoot proliferation, the protocol was extended to the Han FNQ cultivar and three MC lines. Similar trends were observed in Han FNQ, which produced an average of 7.1 shoots per explant (11.6 per seed), closely aligning with the results obtained for Han FNH (Table S6). Likewise, all three MC lines demonstrated similarly consistent shoot proliferation, with shoot numbers ranging between 6-7.6 shoots per responding explant (~\u0026thinsp;13 per seed) (Fig. S5b-e and Table S6).\u003c/p\u003e\u003cp\u003e\u003cb\u003eS\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e- Shoot elongation and rooting in response to IAA and IBA\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFollowing shoot proliferation, the shoots continuously removed from shoot clumps during each subculture require elongation and root formation to develop into individual plants. While the proliferation stage maximises shoot production, these detached shoots need further growth before \u003cem\u003eex vitro\u003c/em\u003e establishment to complete the regeneration process. Their transfer from Petri dishes to larger culture vessels provides increased space, allowing them to elongate rapidly and develop sufficiently to support independent growth. Different auxins, particularly IAA, are widely used for both shoot elongation and rooting in cannabis regeneration protocols. Building on our findings from the proliferation stage (S\u003csub\u003e2\u003c/sub\u003e), we maintained IAA in SPM as the primary auxin for elongation and rooting, creating a streamlined protocol that minimises media changes while effectively supporting both processes.\u003c/p\u003e\u003cp\u003eThe shoots of the Han FNH cultivar derived from SRM and measuring\u0026thinsp;~\u0026thinsp;2 cm in length were transferred to SPM for elongation and rooting (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Within four weeks, these shoots successfully elongated and simultaneously developed root systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and c). The rooting efficiency of this cultivar was evaluated across the three successive subcultures on SPM. A progressive increase in mean rooting efficiency was observed, from 18.1% in subculture 1 to 29% in subculture 2 and 34.7% in subculture 3 (Fig. S8b). Interestingly, \u003cem\u003ein vitro\u003c/em\u003e flowering was also observed in this cultivar, with no flowering occurring during subculture 1, followed by 34.2% during subculture 2, and 12.4% during subculture 3 (Fig. S8c). This precocious flowering phenomenon, while potentially reducing the number of shoots, indicates the capacity of regenerated plantlets to complete their life cycle and may offer insights into the cultivar's response to \u003cem\u003ein vitro\u003c/em\u003e conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven the relatively low rooting efficiency observed with IAA in Han FNH and other hemp cultivars (Han FNQ) used in this study, we explored IBA as an alternative auxin for root induction in MC. IBA has been widely documented for its effectiveness in cannabis regeneration protocols (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). It has significant commercial relevance in the cannabis industry, particularly for MC production, where it serves as the active ingredient in Clonex, a commonly used rooting agent for vegetative nodal cuttings (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). When evaluated on SPM substituted with IBA instead of IAA, the shoots of MC lines exhibited substantially higher rooting efficiencies compared to IAA-based rooting in hemp. MC-1, MC-2, and MC-3 achieved 66.7%, 81%, and 75% rooting success, respectively, with no signs of reproductive growth (Fig. S8d). The marked difference in flowering response between Han FNH and the MC lines suggests that \u003cem\u003ein vitro\u003c/em\u003e flowering may be genotype-dependent, though comprehensive comparisons across additional cultivars are necessary to confirm this observation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eS\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e- Acclimatisation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe final step in a successful regeneration protocol is the acclimatisation of regenerated plantlets to \u003cem\u003eex vitro\u003c/em\u003e conditions. Well-developed shoots with established roots, as obtained in the previous step (S\u003csub\u003e3\u003c/sub\u003e), are crucial for effective deflasking and successful transition to non-sterile environments. Given the robust rooting achieved in our protocol, we tested the acclimatisation potential, a relatively straightforward step, of regenerated plantlets to confirm they could complete their life cycle.\u003c/p\u003e\u003cp\u003eAmong the 173 rooted shoots of Han FNH, 15 were randomly selected and transferred from the culture vessels containing SPM supplemented with IAA to coco perlite medium for acclimatisation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed and e). The plantlets acclimatised exceptionally well inside a humidity chamber for 2 d, with a 100% survival rate observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Following this initial phase, the plantlets were removed and grown for an additional 14 d under standard conditions. Throughout this period, the plantlets demonstrated normal vegetative growth, comparable to nodal cuttings, the conventional method for commercial propagation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef and g). Over time, they exhibited significant vegetative and reproductive growth, with both male and female plants observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh), the latter producing viable seeds, confirming that plants regenerated via tissue culture are capable of completing their growth and reproduction cycles.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDe novo\u003c/b\u003e \u003cb\u003eshoot regeneration in other medicinal species\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHaving established an efficient protocol for \u003cem\u003ede novo\u003c/em\u003e shoot regeneration from the CN with cotyledon explants of cannabis, we sought to investigate whether this approach could be applied to other medicinally important plant species that have traditionally been considered recalcitrant to tissue culture methods. We selected eight angiosperm medicinal species representing diverse growth habits, taxonomic classifications, morphological characteristics and germination patterns to assess the broader applicability of our protocol. This selection allowed us to evaluate whether the regenerative capacity of the CN region observed in cannabis was a species-specific phenomenon or potentially a more universal characteristic that could be exploited for regeneration across various medicinal plants.\u003c/p\u003e\u003cp\u003eA limited number of seeds (\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) of the woody and herbaceous eudicot and monocot medicinal species (Table S9) were subjected to the identical regeneration procedure outlined in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. \u003cem\u003eDe novo\u003c/em\u003e shoot regeneration was observed in five out of eight species: \u003cem\u003eArtemisia absinthium\u003c/em\u003e and \u003cem\u003eA. vulgaris\u003c/em\u003e (wormwoods), \u003cem\u003eBorago officinalis\u003c/em\u003e (borage), \u003cem\u003eAloe ferox\u003c/em\u003e (bitter aloe) and \u003cem\u003eDelonix regia\u003c/em\u003e (royal poinciana) after exposure to at least 14 d on SRM (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Notably, four of these species (\u003cem\u003eArtemisia\u003c/em\u003e spp., \u003cem\u003eB. officinalis\u003c/em\u003e, and \u003cem\u003eD. regia\u003c/em\u003e) are epigeal, with regeneration originating from the CN region, similar to cannabis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-c, e). Interestingly, \u003cem\u003eA. ferox\u003c/em\u003e, a hypogeal species, also regenerated shoots but with an altered pattern and unclear origin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). This suggests that the CN is a worthy explant to trial for \u003cem\u003ede novo\u003c/em\u003e shoot regeneration, particularly in epigeal species.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study aimed to establish an efficient and reproducible shoot regeneration protocol in cannabis. Our findings provide critical insights into the spatial origin and mechanisms of direct shoot organogenesis, revealing the CN region as the principal site of \u003cem\u003ede novo\u003c/em\u003e shoot regeneration in the young seedling, an aspect largely overlooked or ambiguously addressed in previous cannabis protocols (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe CN region is the site of dual regeneration pathways in cannabis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe identified two distinct regeneration pathways from CN explants: 1) direct shoot initiation from pre-existing meristematic tissue (axillary buds) with vascular connectivity to the primary shoot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and 2) \u003cem\u003ede novo\u003c/em\u003e organogenesis from peripheral cortical cells, independent of vasculature, induced by TDZ and NAA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Histological analysis confirmed that \u003cem\u003ede novo\u003c/em\u003e shoots occurred without intervening callus and pre-existing meristem, as observed in other epigeal species (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThese findings challenge earlier reports attributing regeneration to callus-mediated pathways without considering underlying meristems in the cotyledon axils (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). TDZ and NAA alone were insufficient to induce shoot regeneration in explants lacking meristems (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), instead promoting only callus formation in cotyledon and hypocotyl explants. This limitation aligns with previous studies in hemp, which attributed the lack of organogenic potential in these tissues to the absence of meristems (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Moreover, similar \u003cem\u003ede novo\u003c/em\u003e regeneration systems have enabled successful transformation in other recalcitrant species (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e), underscoring the value of targeting accessible organogenic cells for both regeneration and transformation purposes.\u003c/p\u003e\u003cp\u003eThe low shoot regeneration (\u0026lt;\u0026thinsp;2 shoots per explant) in previous hypocotyl-based cannabis protocols is likely due to the retention of axillary meristems rather than true \u003cem\u003ede novo\u003c/em\u003e organogenesis, exemplified by the typical initiation pattern of two shoots per explant (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). This is consistent with our control treatment, where CN with cotyledon explants produced few shoots (avg. 1.4 per explant, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), primarily from axillary nodes. Another study (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) also reported improved regeneration when the CN region was retained in hypocotyl explants, reinforcing the importance of meristem inclusion, though without distinguishing between axillary and \u003cem\u003ede novo\u003c/em\u003e origins. While axillary meristems are inherently regeneration-competent and have been used in cannabis regeneration (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), our identification of \u003cem\u003ede novo\u003c/em\u003e organogenesis provides a complementary pathway that expands regeneration potential.\u003c/p\u003e\u003cp\u003eAmong the CN-based explants tested, cotyledon presence proved critical for efficient \u003cem\u003ede novo\u003c/em\u003e regeneration, with CN with cotyledon explants demonstrating the highest regeneration potential (Table S7). This likely reflects the role of cotyledon-derived morphogenic signals, nutritional support, and hormone homeostasis, as reported in legumes such as \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e (green bean) (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), \u003cem\u003eCajanus cajan\u003c/em\u003e (pigeonpea) (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e), \u003cem\u003eVigna radiata\u003c/em\u003e (mungbean) (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) and \u003cem\u003eV. mungo\u003c/em\u003e (blackgram) (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Detection of endogenous cytokinins in cannabis cotyledons supports this contributory role when retained alongside the CN region (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Additionally, the partial or complete removal of the cotyledons, as in half-CN and half-cotyledon and whole CN explants, respectively, may have disrupted the cotyledon-derived hormonal and nutritional signals, further limiting regenerative competence (Table S7).\u003c/p\u003e\u003cp\u003eStructural integrity of the node region also proved to be critical, exhibiting differential responses to TDZ and NAA. Significant damage to axillary nodes, as seen in half-CN and half-cotyledon explants, substantially reduced axillary shoot formation and increased callusing, thereby decreasing \u003cem\u003ede novo\u003c/em\u003e regeneration (Fig. S6d-f and Table S7). This is consistent with previous reports in \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e) and \u003cem\u003eGlycine max\u003c/em\u003e (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e), emphasising the importance of intact axillary buds for successful regeneration. In contrast, while structurally intact, whole CN explants exhibited low and delayed regeneration, likely due to the apical dominance exerted by the two axillary shoots and profuse callusing, along with cotyledon absence (Fig. S6g-i and Table S7). Similar suppression of lateral shoot development due to apical dominance has been reported in cannabis tissue culture (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). However, the relative contribution of each of these factors remains unclear and warrants further investigation to refine explant selection for maximal regeneration.\u003c/p\u003e\u003cp\u003eUnderstanding the precise cellular origin of shoot regeneration is critical for advancing cannabis transformation. Our histological evidence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) shows that \u003cem\u003ede novo\u003c/em\u003e shoots originate from outer cortical cells, making them ideal targets for transformation due to their greater accessibility compared to deeper cell layers (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). This may enhance DNA delivery and reduce chimerism, a limitation in previous hypocotyl-based transformations (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) where regeneration likely arose from deeper, more mature meristematic tissues that cannot be uniformly transformed. Additionally, bypassing the callus phase reduces the risk of somaclonal variation, enhancing the genetic stability of regenerated lines (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Clarifying the distinct regeneration pathways, therefore, provides a mechanistic foundation for improving transformation efficiency in cannabis, an area that continues to present technical challenges despite recent advances (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eOptimising exogenous hormone exposure time is essential for efficient\u003c/b\u003e \u003cb\u003ede novo\u003c/b\u003e \u003cb\u003eregeneration\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSuccessful shoot organogenesis depends on optimal auxin:cytokinin balance in conjunction with the explant\u0026rsquo;s endogenous hormonal status (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). In our study, the strategic combination of the synthetic cytokinin TDZ and auxin NAA proved essential for inducing \u003cem\u003ede novo\u003c/em\u003e regeneration from CN explants, while hormone-free medium failed to elicit the same. This combination has consistently outperformed other tested cytokinin-auxin combinations in cannabis (BAP, zeatin, kinetin, and meta-topolin coupled with NAA and IBA) in promoting shoot induction (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). TDZ's high morphogenic potential stimulates shoot initiation and early developmental processes (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e), while NAA supports cell differentiation and elongation (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, our results highlight a critical nuance: the duration of hormone exposure significantly affects regeneration outcomes. While short-term TDZ\u0026thinsp;+\u0026thinsp;NAA treatments (2\u0026ndash;6 d) were effective, 7\u0026ndash;14 d provided optimal conditions for maximum shoot induction with minimal callusing (Fig. S7). Prolonged exposure (3\u0026ndash;5 weeks) led to excessive callusing, vitrification and shoot inhibition, effects primarily attributed to TDZ (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e), with a marked decline in regenerative potential (Fig. S3). This sensitivity is consistent with reports in legumes, where short-term exposure (typically 7 d) induced maximal regeneration while avoiding cytokinin dominance effects (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). Previous cannabis protocols using higher TDZ concentrations or extended exposure often achieved regeneration at the cost of increased callusing (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), reduced shoot proliferation and lengthy protocols (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), further supporting our optimised approach.\u003c/p\u003e\u003cp\u003eNotably, \u003cem\u003ede novo\u003c/em\u003e regeneration was observed in 6.7% of explants in 2 d of SRM treatment (Table S8), and histological studies confirmed that by 7 d, most peripheral cells were already committed to the organogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). This indicates hormonal signalling required for \u003cem\u003ede novo\u003c/em\u003e regeneration primes cortical cells within the first 1\u0026ndash;2 d of exposure, suggesting optimal timing for infection with \u003cem\u003eAgrobacterium\u003c/em\u003e or other DNA delivery methods is before or very early in this window, while cells remain uncommitted and accessible for transformation. To our knowledge, no previous cannabis regeneration study has reported such an early onset of \u003cem\u003ede novo\u003c/em\u003e organogenesis or considered its relevance for transformation efficiency.\u003c/p\u003e\u003cp\u003eCytokinin profiling studies in hemp (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) showed hypocotyl-derived callus, even when cultured on cytokinin-rich medium (BAP\u0026thinsp;+\u0026thinsp;Kinetin), failed to accumulate active cytokinins, indicating a limited endogenous-exogenous hormonal interaction and response capacity. This underscores the importance of both appropriate hormonal treatment and biological competence of the explant, in our case, the CN's unique combination of meristematic structure and hormone responsiveness that enables \u003cem\u003ede novo\u003c/em\u003e regeneration.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMultiple subculturing of\u003c/b\u003e \u003cb\u003ede novo\u003c/b\u003e \u003cb\u003eshoots enables scalable regeneration\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur CN-based approach uniquely enables large-scale shoot production from a single seed through continuous proliferation achieved by periodic subculturing. Up to three subcultures were performed, with shoot numbers peaking in the third culturing; although a slight decline was observed at the fourth (reminder shoots on the original explant), further proliferation remained possible (Fig. S8a). This scalable regeneration system achieved a maximum of 50 shoots per seed in the Han FNH cultivar, with performance being genotype-dependent across the tested varieties. The protocol significantly reduces the cost per shoot while generating numerous genetically identical plants, making it valuable for both breeding programs and commercial propagation. The protocol\u0026rsquo;s efficiency is further enhanced by the timely transfer of regenerated shoots from cytokinin-rich to cytokinin-free medium, which is critical to prevent the inhibitory effects of prolonged cytokinin (TDZ) exposure on elongation and rooting, a limitation commonly reported in similar protocols (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Moreover, in transformation workflows, iterative subculturing aids in the recovery and selection of uniformly transformed tissues while progressively eliminating chimeric shoots (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e), making our protocol a robust platform for downstream applications.\u003c/p\u003e\u003cp\u003eOur rooting experiments revealed genotype-specific challenges requiring further optimisation. While both IAA and IBA induced roots, efficiency with IAA remained suboptimal in hemp (Fig. S8b), likely attributed to the relatively low concentration used (0.175 mg/L) compared to the higher IBA concentration (0.5 mg/L) employed in MC lines, where rooting responses were more favourable (Fig. S8d) and consistent with previous protocols (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). However, IBA\u0026rsquo;s tendency to promote callus formation highlights the need for tailored rooting strategies across different cannabis genotypes (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe occurrence of \u003cem\u003ein vitro\u003c/em\u003e flowering during the rooting stage, despite strict long-day photoperiod conditions, may reflect genotype-specific responses and light intensity stress. This aligns with previous findings that certain cannabis genotypes can initiate flowering \u003cem\u003ein vitro\u003c/em\u003e even under extended photoperiods, demonstrating the species' unique capacity for photoperiod-insensitive floral induction (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). While reproductive development may redirect metabolic energy from root development, it presents opportunities for accelerating breeding cycles (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e), studying the regulation of secondary metabolites (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e) and production of floral tissue for regeneration (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Such an approach could facilitate multiple crosses within a limited space and time, offering a scalable tool for both research and commercial breeding programs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCN explants enable efficient genotype-independent regeneration\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCompared to existing protocols, our CN-based regeneration system delivers significantly higher efficiency and consistent shoot proliferation across diverse cannabis genotypes. Cotyledon-based methods have reported highly variable efficiencies (4\u0026ndash;55%) and modest shoots per explant (1.3-8) (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), while hypocotyl-based approaches typically achieved\u0026thinsp;~\u0026thinsp;50% efficiency with only 1.7 shoots per explant (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Furthermore, recent protocols using CN with hypocotyl, though reporting higher efficiencies, demonstrated substantial variability (26\u0026ndash;87% efficiency; 2.6\u0026ndash;8.6 shoots per explant) across cultivars (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCritically, these earlier studies failed to distinguish shoots arising from pre-existing meristems from true \u003cem\u003ede novo\u003c/em\u003e regeneration, likely inflating their reported efficiencies. In contrast, our protocol applied stringent criteria to exclude primary and axillary shoots, quantifying only those initiated \u003cem\u003ede novo\u003c/em\u003e. Using this conservative approach, we consistently achieved 70\u0026ndash;90% efficiency with an average of 7 \u003cem\u003ede novo\u003c/em\u003e shoots per responding explant (~\u0026thinsp;11.4 shoots per seed), representing approximately a 2-fold improvement over cotyledon-based and 5-fold over hypocotyl-based methods under comparable culture conditions. Moreover, by using paired CN with cotyledon explants, our protocol effectively doubled explant yield per seed, enhancing scalability for high-throughput applications.\u003c/p\u003e\u003cp\u003eThis positions the CN as a universally responsive explant type that circumvents the genotype dependency barriers that have long constrained cannabis biotechnology, with implications extending beyond cannabis (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). Successful regeneration in five out of eight medicinal plant species spanning multiple families and growth forms demonstrates the broad applicability of our protocol across taxonomically diverse angiosperms (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and Table S9). Notably, four of the responsive species (\u003cem\u003eArtemisia\u003c/em\u003e spp., \u003cem\u003eB. officinalis\u003c/em\u003e and \u003cem\u003eD. regia\u003c/em\u003e) exhibit epigeal germination similar to cannabis, supporting previous observations in \u003cem\u003eVigna\u003c/em\u003e species where regeneration from CN was consistently observed in epigeal but not in hypogeal types, unless modified by genomic factors such as allotetraploidy (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). This observation suggests that epigeal species, characterised by photosynthetically active cotyledons and the presence of axillary buds, exhibit greater regenerative competence than hypogeal species, where cotyledons remain underground and axillary buds are often less developed or absent (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). These physiological and anatomical differences likely influence the availability and transport of morphogenic signals, nutrients, and hormones, thereby affecting regeneration potential.\u003c/p\u003e\u003cp\u003eInterestingly, \u003cem\u003eAloe ferox\u003c/em\u003e, a hypogeal monocot, also exhibited \u003cem\u003ede novo\u003c/em\u003e shoot formation, although its precise anatomical origin remains unclear. This suggests that CN-based regeneration may extend to at least some hypogeal taxa, potentially due to variation in nodal architecture or hormone responsiveness, but further analysis is needed to clarify this. While our observations are derived from a limited number of species, they support the hypothesis that the CN region harbours a conserved, regeneration-competent meristematic architecture that can be reliably activated across epigeal species, similar to legumes (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e) and possibly beyond, offering a high-efficiency, transformation-compatible platform for genetic improvement in cannabis and other recalcitrant species.\u003c/p\u003e\u003cp\u003eHowever, while the regeneration phase itself demonstrated genotype independence, upstream challenges remain. Seed germination and endophyte contamination varied significantly across cannabis cultivars (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), influenced by genetic background, storage conditions, and seed age (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). Freshly harvested seeds achieved near-complete germination and minimal contamination, underscoring their advantages (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). However, producing fresh seeds under controlled conditions is often impractical, particularly for perennial species or in low-resource settings and is disadvantageous when the genetic eliteness of a line is to be retained. Although the 1% (v/v) H₂O₂ protocol significantly improved explant availability across genotypes, these findings highlight that the full potential of this genotype-independent regeneration system is contingent upon the availability of healthy explants, particularly when working with long-stored or field-grown material common in cannabis and other medicinal species.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we developed a high-efficiency regeneration protocol for cannabis by optimising five stages (S\u003csub\u003e0\u003c/sub\u003e-S\u003csub\u003e4\u003c/sub\u003e) of tissue culture and identifying CN with attached cotyledon as the optimal explant for \u003cem\u003ede novo\u003c/em\u003e shoot regeneration. Optimising hormone exposure time significantly enhanced regeneration efficiency, while the protocol's genotype-independent nature enabled reliable performance across diverse hemp cultivars and high-THC MC lines. This approach addresses long-standing recalcitrance barriers in cannabis tissue culture and establishes a foundation for large-scale propagation of genetically identical shoots, genetic transformation, and germplasm conservation. Its successful extension to other medicinal plants and its prior validation in legumes suggests broader applicability to a range of recalcitrant species. Overall, this work offers a valuable platform for advanced biotechnological applications that could enhance sustainable cultivation practices, improve desirable traits, and unlock the therapeutic and industrial potential of cannabis and other medicinal plants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge the funding provided by the Australian Research Council and the Australian Department of Education Regional Research Collaboration Program. We would like to thank La Trobe University and the University of Melbourne for their support on this project and Cann Group Ltd for providing access to their facilities and plant materials. Further thanks are extended to A/Prof Berin Boughton for training and use of the cryostat and Prof Phil Brewer for invaluable discussions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePLB, MOB, SCF and MSD: conceptualisation and experiment design; PLB, MOB and SCF: performed the experiments; PLB: writing the original draft, figure preparation and data analysis; MOB and MSD: supervision and manuscript revision; PLB, MOB, and MSD: review and editing. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was funded by\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethe Australian Research Council to the Industrial Transformation Research Hub for Medicinal Agriculture (ARC MedAg Hub, IH180100006) and the Australian Department of Education Regional Research Collaboration Program \u0026ndash; Next Generation Protected Cropping in a Regional Manufacturing Facility.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAltpeter F, Springer NM, Bartley LE, Blechl AE, Brutnell TP, Citovsky V, et al. Advancing crop transformation in the era of genome editing. Plant Cell. 2016;28(7):1510\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/tpc.16.00196\u003c/span\u003e\u003cspan address=\"10.1105/tpc.16.00196\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoraes RM, Cerdeira AL, Louren\u0026ccedil;o MV. Using micropropagation to develop medicinal plants into crops. Molecules. 2021;26(6):1752. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules26061752\u003c/span\u003e\u003cspan address=\"10.3390/molecules26061752\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDwivedi SL, Britt AB, Tripathi L, Sharma S, Upadhyaya HD, Ortiz R, Haploids. Constraints and opportunities in plant breeding. Biotechnol Adv. 2015;33(6):812\u0026ndash;29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biotechadv.2015.07.001\u003c/span\u003e\u003cspan address=\"10.1016/j.biotechadv.2015.07.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDutt M, Mahmoud LM, Chamusco K, Stanton D, Chase CD, Nielsen E, et al. Utilization of somatic fusion techniques for the development of HLB tolerant breeding resources employing the Australian finger lime (\u003cem\u003eCitrus australasica\u003c/em\u003e). PLoS ONE. 2021;16(8):e0255842. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0255842\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0255842\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMohammadi MA, Wai MH, Rizwan HM, Qarluq AQ, Xu M, Wang L, et al. Advances in micropropagation, somatic embryogenesis, somatic hybridizations, genetic transformation and cryopreservation for Passiflora improvement. Plant Methods. 2023;19:50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13007-023-01030-0\u003c/span\u003e\u003cspan address=\"10.1186/s13007-023-01030-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRichins M, Montes C, Merkle S. Conservation of green and white ash germplasm using the cryopreservation of embryogenic cultures. Plants. 2024;13(3):352. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants13030352\u003c/span\u003e\u003cspan address=\"10.3390/plants13030352\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBennur PL, O'Brien M, Fernando SC, Doblin MS. Improving transformation and regeneration efficiency in medicinal plants: Insights from other recalcitrant species. J Exp Bot. 2024;76(1):52\u0026ndash;75. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jxb/erae189\u003c/span\u003e\u003cspan address=\"10.1093/jxb/erae189\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMonthony AS, Page SR, Hesami M, Jones AMP. The past, present and future of \u003cem\u003eCannabis sativa\u003c/em\u003e tissue culture. Plants. 2021;10(1):185. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants10010185\u003c/span\u003e\u003cspan address=\"10.3390/plants10010185\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCaplan D, Stemeroff J, Dixon MA, Zheng Y. Vegetative propagation of cannabis by stem cuttings: effects of leaf number, cutting position, rooting hormone, and leaf tip removal. Can J Plant Sci. 2018;98(5):1126\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1139/cjps-2018-0038\u003c/span\u003e\u003cspan address=\"10.1139/cjps-2018-0038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChandra S, Lata H, ElSohly MA, Walker LA, Potter D. Cannabis cultivation: Methodological issues for obtaining medical-grade product. Epilepsy Behav. 2017;70:302\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.yebeh.2016.11.029\u003c/span\u003e\u003cspan address=\"10.1016/j.yebeh.2016.11.029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStephen C, Zayas VA, Galic A, Bridgen MP. Micropropagation of hemp (\u003cem\u003eCannabis sativa\u003c/em\u003e L). HortScience. 2023;58(3):307\u0026ndash;16. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21273/hortsci16969-22\u003c/span\u003e\u003cspan address=\"10.21273/hortsci16969-22\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWr\u0026oacute;bel T, Dreger M, Wielgus K, Słomski R. Modified nodal cuttings and shoot tips protocol for rapid regeneration of \u003cem\u003eCannabis sativa\u003c/em\u003e L. J Nat Fibers. 2020;19(2):536\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/15440478.2020.1748160\u003c/span\u003e\u003cspan address=\"10.1080/15440478.2020.1748160\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLata H, Chandra S, Khan I, ElSohly MA. Thidiazuron-induced high-frequency direct shoot organogenesis of \u003cem\u003eCannabis sativa\u003c/em\u003e L. Vitro Cell Dev Biol Plant. 2009;45:12\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11627-008-9167-5\u003c/span\u003e\u003cspan address=\"10.1007/s11627-008-9167-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLata H, Chandra S, Khan IA, ElSohly MA. Propagation through alginate encapsulation of axillary buds of \u003cem\u003eCannabis sativa\u003c/em\u003e L. - an important medicinal plant. Physiol Mol Biol Plants. 2009;15:79\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12298-009-0008-8\u003c/span\u003e\u003cspan address=\"10.1007/s12298-009-0008-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLata H, Chandra S, Techen N, Khan IA, ElSohly MA. \u003cem\u003eIn vitro\u003c/em\u003e mass propagation of \u003cem\u003eCannabis sativa\u003c/em\u003e L.: A protocol refinement using novel aromatic cytokinin meta-topolin and the assessment of eco-physiological, biochemical and genetic fidelity of micropropagated plants. J Appl Res Med Aromat Plants. 2016;3(1):18\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jarmap.2015.12.001\u003c/span\u003e\u003cspan address=\"10.1016/j.jarmap.2015.12.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePiunno KF, Golenia G, Boudko EA, Downey C, Jones AMP. Regeneration of shoots from immature and mature inflorescences of \u003cem\u003eCannabis sativa\u003c/em\u003e. Can J Plant Sci. 2019;99(4):556\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1139/cjps-2018-0308\u003c/span\u003e\u003cspan address=\"10.1139/cjps-2018-0308\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMonthony AS, Bagheri S, Zheng Y, Jones AMP. Flower power: floral reversion as a viable alternative to nodal micropropagation in \u003cem\u003eCannabis sativa\u003c/em\u003e. Vitro Cell Dev Biol Plant. 2021;57:1018\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11627-021-10181-5\u003c/span\u003e\u003cspan address=\"10.1007/s11627-021-10181-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHesami M, Baiton A, Alizadeh M, Pepe M, Torkamaneh D, Jones AMP. Advances and perspectives in tissue culture and genetic engineering of cannabis. Int J Mol Sci. 2021;22(11):5671. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms22115671\u003c/span\u003e\u003cspan address=\"10.3390/ijms22115671\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMonthony AS, Kyne ST, Grainger CM, Jones AMP. Recalcitrance of \u003cem\u003eCannabis sativa\u003c/em\u003e to \u003cem\u003ede novo\u003c/em\u003e regeneration; a multi-genotype replication study. PLoS ONE. 2021;16(8):e0235525. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0235525\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0235525\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdhikary D, Kulkarni M, El-Mezawy A, Mobini S, Elhiti M, Gjuric R, et al. Medical cannabis and industrial hemp tissue culture: Present status and future potential. Front Plant Sci. 2021;12:627240. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2021.627240\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2021.627240\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLata H, Chandra S, Khan IA, Elsohly MA. High frequency plant regeneration from leaf derived callus of high ∆\u003csup\u003e9\u003c/sup\u003e-tetrahydrocannabinol yielding \u003cem\u003eCannabis sativa\u003c/em\u003e L. Planta Med. 2010;76(14):1629\u0026ndash;33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1055/s-0030-1249773\u003c/span\u003e\u003cspan address=\"10.1055/s-0030-1249773\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhsan SM, Kwon DB, Injamum-Ul-Hoque M, Rahman MM, Yeam I, Choi HW. De novo regeneration of \u003cem\u003eCannabis sativa\u003c/em\u003e cv. Cheungsam and evaluation of secondary metabolites of its callus. Horticulturae. 2024;10(12):1331. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/horticulturae10121331\u003c/span\u003e\u003cspan address=\"10.3390/horticulturae10121331\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSlusarkiewicz-Jarzina A, Ponitka A, Kaczmarek Z. Influence of cultivar, explant source and plant growth regulator on callus induction and plant regeneration of \u003cem\u003eCannabis sativa\u003c/em\u003e L. Acta Biol Crac Ser Bot. 2005;47(2):145\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://abcbot.pl/pdf/47_2/145-151.pdf\u003c/span\u003e\u003cspan address=\"https://abcbot.pl/pdf/47_2/145-151.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMovahedi M, Ghasemi-Omran V, Torabi S. The effect of different concentrations of TDZ and BA on \u003cem\u003ein vitro\u003c/em\u003e regeneration of Iranian cannabis (\u003cem\u003eCannabis sativa\u003c/em\u003e) using cotyledon and epicotyl explants. J Plant Mol Breed. 2015;3(2):20\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.22058/jpmb.2015.15371\u003c/span\u003e\u003cspan address=\"10.22058/jpmb.2015.15371\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChaohua C, Gonggu Z, Lining Z, Chunsheng G, Qing T, Jianhua C, et al. A rapid shoot regeneration protocol from the cotyledons of hemp (\u003cem\u003eCannabis sativa\u003c/em\u003e L). Ind Crops Prod. 2016;83:61\u0026ndash;5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.indcrop.2015.12.035\u003c/span\u003e\u003cspan address=\"10.1016/j.indcrop.2015.12.035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWielgus K, Luwanska A, Lassocinski W, Kaczmarek Z. Estimation of \u003cem\u003eCannabis sativa\u003c/em\u003e L. tissue culture conditions essential for callus induction and plant regeneration. J Nat Fibers. 2008;5(3):199\u0026ndash;207. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/15440470801976045\u003c/span\u003e\u003cspan address=\"10.1080/15440470801976045\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGal\u0026aacute;n-\u0026Aacute;vila A, Garc\u0026iacute;a-Fortea E, Prohens J, Herraiz FJ. Development of a direct \u003cem\u003ein vitro\u003c/em\u003e plant regeneration protocol from \u003cem\u003eCannabis sativa\u003c/em\u003e L. seedling explants: Developmental morphology of shoot regeneration and ploidy level of regenerated plants. Front Plant Sci. 2020;11:645. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2020.00645\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2020.00645\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSorokin A, Kovalchuk I. Development of efficient and scalable regeneration tissue culture method for \u003cem\u003eCannabis sativa\u003c/em\u003e. Plant Sci. 2025;350:112296. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.plantsci.2024.112296\u003c/span\u003e\u003cspan address=\"10.1016/j.plantsci.2024.112296\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeeney M, Punja ZK. Tissue culture and \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation of hemp (\u003cem\u003eCannabis sativa\u003c/em\u003e L). Vitro Cell Dev Biol Plant. 2003;39:578\u0026ndash;85. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1079/IVP2003454\u003c/span\u003e\u003cspan address=\"10.1079/IVP2003454\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGal\u0026aacute;n-\u0026Aacute;vila A, Gramazio P, Ron M, Prohens J, Herraiz FJ. A novel and rapid method for Agrobacterium-mediated production of stably transformed \u003cem\u003eCannabis sativa\u003c/em\u003e L. plants. Ind Crops Prod. 2021;170:113691. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.indcrop.2021.113691\u003c/span\u003e\u003cspan address=\"10.1016/j.indcrop.2021.113691\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang X, Xu G, Cheng C, Lei L, Sun J, Xu Y, et al. Establishment of an \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated genetic transformation and CRISPR/Cas9-mediated targeted mutagenesis in Hemp (\u003cem\u003eCannabis Sativa\u003c/em\u003e L). Plant Biotechnol J. 2021;19(10):1979\u0026ndash;87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/pbi.13611\u003c/span\u003e\u003cspan address=\"10.1111/pbi.13611\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWahby I, Caba JM, Ligero F. \u003cem\u003eAgrobacterium\u003c/em\u003e infection of hemp (\u003cem\u003eCannabis sativa\u003c/em\u003e L.): establishment of hairy root cultures. J Plant Interact. 2013;8(4):312\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/17429145.2012.746399\u003c/span\u003e\u003cspan address=\"10.1080/17429145.2012.746399\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhmed S, Gao X, Jahan MA, Adams M, Wu N, Kovinich N. Nanoparticle-based genetic transformation of \u003cem\u003eCannabis sativa\u003c/em\u003e. J Biotechnol. 2021;326:48\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jbiotec.2020.12.014\u003c/span\u003e\u003cspan address=\"10.1016/j.jbiotec.2020.12.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu G, Liu Y, Yu S, Kong D, Tang K, Dai Z, et al. \u003cem\u003eCsMIKC1\u003c/em\u003e regulates inflorescence development and grain production in \u003cem\u003eCannabis sativa\u003c/em\u003e plants. Hortic Res. 2024;11(8):uhae161. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/hr/uhae161\u003c/span\u003e\u003cspan address=\"10.1093/hr/uhae161\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWelling MT, Deseo MA, Bacic A, Doblin MS. Untargeted metabolomic analyses reveal chemical complexity of dioecious cannabis flowers. Aust J Chem. 2021;74(6):463\u0026ndash;79. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1071/CH21033\u003c/span\u003e\u003cspan address=\"10.1071/CH21033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMurashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962;15(3):473\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1399-3054.1962.tb08052.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1399-3054.1962.tb08052.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhsan SM, Shin JH, Choi HW. Availability of hydrogen peroxide solutions as a germination liquid medium for contamination-free \u003cem\u003ein vitro\u003c/em\u003e seedling development of \u003cem\u003eCannabis sativa\u003c/em\u003e. Hortic Sci Technol. 2022;40(6):605\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7235/HORT.20220055\u003c/span\u003e\u003cspan address=\"10.7235/HORT.20220055\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLobato C, de Freitas JM, Habich D, K\u0026ouml;gl I, Berg G, Cernava T. Wild again: recovery of a beneficial Cannabis seed endophyte from low domestication genotypes. Microbiome. 2024;12:239. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s40168-024-01951-5\u003c/span\u003e\u003cspan address=\"10.1186/s40168-024-01951-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBuirs L, Punja ZK. Endophytes in \u003cem\u003eCannabis sativa\u003c/em\u003e: Identifying and characterizing microbes with beneficial and detrimental effects on plant health. Plants. 2025;14(8):1247. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants14081247\u003c/span\u003e\u003cspan address=\"10.3390/plants14081247\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQuambusch M, Winkelmann T. Bacterial endophytes in plant tissue culture: Mode of action, detection, and control. In: Loyola-Vargas VM, Ochoa-Alejo N, editors. Plant cell culture protocols. 4th ed. New York: Springer; 2018. pp. 69\u0026ndash;88. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-1-4939-8594-4_4\u003c/span\u003e\u003cspan address=\"10.1007/978-1-4939-8594-4_4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSorokin A, Yadav NS, Gaudet D, Kovalchuk I. Development and standardization of rapid and efficient seed germination protocol for \u003cem\u003eCannabis sativa\u003c/em\u003e. Bio Protoc. 2021;11(1):e3875. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21769/BioProtoc.3875\u003c/span\u003e\u003cspan address=\"10.21769/BioProtoc.3875\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSm\u0026yacute;kalov\u0026aacute; I, Vrbov\u0026aacute; M, Cvečkov\u0026aacute; M, Plačkov\u0026aacute; L, Žukauskaitė A, Zatloukal M, et al. The effects of novel synthetic cytokinin derivatives and endogenous cytokinins on the \u003cem\u003ein vitro\u003c/em\u003e growth responses of hemp (\u003cem\u003eCannabis sativa\u003c/em\u003e L.) explants. Plant Cell Tissue Organ Cult. 2019;139:381\u0026ndash;94. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-019-01693-5\u003c/span\u003e\u003cspan address=\"10.1007/s11240-019-01693-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAvenido RA, Hattori K. Differences in shoot regeneration response from cotyledonary node explants in Asiatic \u003cem\u003eVigna\u003c/em\u003e species support genomic grouping within subgenus \u003cem\u003eCeratotropis\u003c/em\u003e (Piper) Verdc. Plant Cell Tissue Organ Cult. 1999;58:99\u0026ndash;110. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1023/A:1006325327624\u003c/span\u003e\u003cspan address=\"10.1023/A:1006325327624\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu H, Guo Y, Qiu L, Ran Y. Progress in soybean genetic transformation over the last decade. Front Plant Sci. 2022;13:900318. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2022.900318\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2022.900318\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAvenido R, Motoda J, Hattori K. Direct shoot regeneration from cotyledonary nodes as a marker for genomic groupings within the Asiatic \u003cem\u003eVigna\u003c/em\u003e (subgenus \u003cem\u003eCeratotropis\u003c/em\u003e {Piper} Verdc.) species. Plant Growth Regul. 2001;35:59\u0026ndash;67. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1023/A:1013874902530\u003c/span\u003e\u003cspan address=\"10.1023/A:1013874902530\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHinchee MAW, Connor-Ward DV, Newell CA, McDonnell RE, Sato SJ, Gasser CS, et al. Production of transgenic soybean plants using \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated DNA transfer. Nat Biotechnol. 1988;6:915\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nbt0888-915\u003c/span\u003e\u003cspan address=\"10.1038/nbt0888-915\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFranklin CI, Trieu TN, Gonzales RA, Dixon RA. Plant regeneration from seedling explants of green bean (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e L) via organogenesis. Plant Cell Tissue Organ Cult. 1991;24:199\u0026ndash;206. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF00033477\u003c/span\u003e\u003cspan address=\"10.1007/BF00033477\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKebrom TH. A growing stem inhibits bud outgrowth \u0026ndash; The overlooked theory of apical dominance. Front Plant Sci. 2017;8:1874. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2017.01874\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2017.01874\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKurtz LE, Borbas LN, Brand MH, Lubell-Brand JD. Ex vitro rooting of \u003cem\u003eCannabis sativa\u003c/em\u003e microcuttings and their performance compared to retip and stem cuttings. HortScience. 2022;57(12):1576\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21273/HORTSCI16890-22\u003c/span\u003e\u003cspan address=\"10.21273/HORTSCI16890-22\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShiva prakash N, Pental D, Bhalla-Sarin N. Regeneration of pigeonpea (\u003cem\u003eCajanus cajan\u003c/em\u003e) from cotyledonary node via multiple shoot formation. Plant Cell Rep. 1994;13:623\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF00232933\u003c/span\u003e\u003cspan address=\"10.1007/BF00232933\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJackson JA, Hobbs SLA. Rapid multiple shoot production from cotyledonary node explants of pea (\u003cem\u003ePisum sativum\u003c/em\u003e L). Vitro Cell Dev Biol. 1990;26:835\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF02623626\u003c/span\u003e\u003cspan address=\"10.1007/BF02623626\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHsieh Y-F, Jain M, Wang J, Gallo M. Direct organogenesis from cotyledonary node explants suitable for \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation in peanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e L). Plant Cell Tissue Organ Cult. 2017;128:161\u0026ndash;75. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-016-1095-1\u003c/span\u003e\u003cspan address=\"10.1007/s11240-016-1095-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePatial V, Krishna R, Arya G, Singh VK, Agarwal M, Goel S, et al. Development of an efficient, genotype independent plant regeneration and transformation protocol using cotyledonary nodes in safflower (\u003cem\u003eCarthamus tinctorius\u003c/em\u003e L). J Plant Biochem Biotechnol. 2016;25(4):421\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13562-016-0354-x\u003c/span\u003e\u003cspan address=\"10.1007/s13562-016-0354-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaz MM, Martinez JC, Kalvig AB, Fonger TM, Wang K. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated soybean transformation. Plant Cell Rep. 2006;25:206\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00299-005-0048-7\u003c/span\u003e\u003cspan address=\"10.1007/s00299-005-0048-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGulati A, Jaiwal PK. Plant regeneration from cotyledonary node explants of mungbean (\u003cem\u003eVigna radiata\u003c/em\u003e (L.) Wilczek). Plant Cell Rep. 1994;13:523\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF00232949\u003c/span\u003e\u003cspan address=\"10.1007/BF00232949\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSen J, Guha-Mukherjee S. \u003cem\u003evitro\u003c/em\u003e induction of multiple shoots and plant regeneration in \u003cem\u003eVigna\u003c/em\u003e. Vitro Cell Dev Biol Plant. 1998;34:276\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF02822733\u003c/span\u003e\u003cspan address=\"10.1007/BF02822733\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShan Z, Raemakers K, Tzitzikas EN, Ma Z, Visser RGF. Development of a highly efficient, repetitive system of organogenesis in soybean (\u003cem\u003eGlycine max\u003c/em\u003e (L.) Merr). Plant Cell Rep. 2005;24:507\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00299-005-0971-7\u003c/span\u003e\u003cspan address=\"10.1007/s00299-005-0971-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShujat S, Robinson GI, Norouzkhani F, Kovalchuk I. Using advanced biotechnological techniques to improve cannabis cultivars. Biocatal Agric Biotechnol. 2024;60:103250. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bcab.2024.103250\u003c/span\u003e\u003cspan address=\"10.1016/j.bcab.2024.103250\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eErland LAE, Giebelhaus RT, Victor JMR, Murch SJ, Saxena PK. The morphoregulatory role of Thidiazuron: Metabolomics-guided hypothesis generation for mechanisms of activity. Biomol. 2020;10(9):1253. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/biom10091253\u003c/span\u003e\u003cspan address=\"10.3390/biom10091253\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCampanoni P, Nick P. Auxin-dependent cell division and cell elongation. 1-Naphthaleneacetic Acid and 2,4-Dichlorophenoxyacetic Acid activate different pathways. Plant Physiol. 2005;137(3):939\u0026ndash;48. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.104.053843\u003c/span\u003e\u003cspan address=\"10.1104/pp.104.053843\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDewir YH, Nurmansyah, Naidoo Y, Teixeira da Silva JA. Thidiazuron-induced abnormalities in plant tissue cultures. Plant Cell Rep. 2018;37:1451\u0026ndash;70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00299-018-2326-1\u003c/span\u003e\u003cspan address=\"10.1007/s00299-018-2326-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMalik KA, Saxena PK. Regeneration in \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e L.: High-frequency induction of direct shoot formation in intact seedlings by N(6)-benzylaminopurine and thidiazuron. Planta. 1992;186:384\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF00195319\u003c/span\u003e\u003cspan address=\"10.1007/BF00195319\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang H, Wei Y, Zhai Y, Ouyang K, Chen X, Bai L. High frequency regeneration of plants via callus-mediated organogenesis from cotyledon and hypocotyl cultures in a multipurpose tropical tree (\u003cem\u003eNeolamarkia Cadamba\u003c/em\u003e). Sci Rep. 2020;10:4558. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-020-61612-z\u003c/span\u003e\u003cspan address=\"10.1038/s41598-020-61612-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMathews H, Dewey V, Wagoner W, Bestwick RK. Molecular and cellular evidence of chimaeric tissues in primary transgenics and elimination of chimaerism through improved selection protocols. Transgenic Res. 1998;7:123\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1023/A:1008872425917\u003c/span\u003e\u003cspan address=\"10.1023/A:1008872425917\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoher M, Jones M, Zheng Y. Photoperiodic response of in vitro \u003cem\u003eCannabis sativa\u003c/em\u003e plants. HortScience. 2021;56(1):108\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21273/HORTSCI15452-20\u003c/span\u003e\u003cspan address=\"10.21273/HORTSCI15452-20\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLavie O, Buxdorf K, Eshed Williams L. Optimizing cannabis cultivation: an efficient in vitro system for flowering induction. Plant Methods. 2024;20:141. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13007-024-01265-5\u003c/span\u003e\u003cspan address=\"10.1186/s13007-024-01265-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaren NA, Duan H, Da K, Yencho GC, Ranney TG, Liu W. Genotype-independent plant transformation. Hortic Res. 2022;9:uhac047. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/hr/uhac047\u003c/span\u003e\u003cspan address=\"10.1093/hr/uhac047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAvenido RA, Hattori K. Benzyladenine-preconditioning in germinating mungbean seedlings stimulates axillary buds in cotyledonary nodes resulting in multiple shoot regeneration. Breed Sci. 2001;51(2):137\u0026ndash;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1270/jsbbs.51.137\u003c/span\u003e\u003cspan address=\"10.1270/jsbbs.51.137\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eScott M, Rani M, Samsatly J, Charron JB, Jabaji S. Endophytes of industrial hemp (\u003cem\u003eCannabis sativa\u003c/em\u003e L.) cultivars: identification of culturable bacteria and fungi in leaves, petioles, and seeds. Can J Microbiol. 2018;64(10):664\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1139/cjm-2018-0108\u003c/span\u003e\u003cspan address=\"10.1139/cjm-2018-0108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCollado R, Veit\u0026iacute;a N, Berm\u0026uacute;dez-Caraballoso I, Garc\u0026iacute;a LR, Torres D, Romero C, et al. Efficient \u003cem\u003ein vitro\u003c/em\u003e plant regeneration via indirect organogenesis for different common bean cultivars. Sci Hortic. 2013;153:109\u0026ndash;16. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scienta.2013.02.007\u003c/span\u003e\u003cspan address=\"10.1016/j.scienta.2013.02.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cannabis, recalcitrance, de novo regeneration, genotype-independent, cotyledonary node, TDZ","lastPublishedDoi":"10.21203/rs.3.rs-7237557/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7237557/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEfficient regeneration protocols are essential for large-scale propagation and genetic manipulation of recalcitrant medicinal species such as \u003cem\u003eCannabis sativa\u003c/em\u003e. Existing direct and indirect regeneration methods are highly genotype and explant-dependent, limiting broader applicability. Here, we report a five-stage (S\u003csub\u003e0\u003c/sub\u003e-S\u003csub\u003e4\u003c/sub\u003e) optimised protocol that is reproducible and achieves high-efficiency direct \u003cem\u003ede novo\u003c/em\u003e regeneration using cotyledonary node explants from both hemp and medicinal cannabis genotypes. A 1% (v/v) H₂O₂-based sterilisation method significantly improved seed germination and reduced endophyte contamination. Among embryo-derived explants, the cotyledonary node attached to the cotyledon showed superior regeneration efficiency through two distinct pathways: axillary shoot initiation and \u003cem\u003ede novo\u003c/em\u003e regeneration, the latter achieving\u0026thinsp;~\u0026thinsp;70\u0026ndash;90% efficiency in six hemp cultivars and three medicinal cannabis lines on TDZ and NAA containing shoot regeneration medium. Histological analysis confirmed true \u003cem\u003ede novo\u003c/em\u003e shoot formation from peripheral cortical cells, independent of pre-existing meristems or callus. \u003cem\u003eDe novo\u003c/em\u003e shoots were initiated within 2 d of shoot regeneration medium treatment, indicating rapid cellular commitment to organogenesis, with optimal regeneration between 7\u0026ndash;14 d. Prolonged exposure proved detrimental, causing excessive callusing and vitrification. Repeated subculturing during proliferation stage enabled scalable shoot multiplication, yielding an average of 7 shoots per responding explant (~\u0026thinsp;11.4 shoots per seed), outperforming previously published cotyledon-based (~\u0026thinsp;2-fold) and hypocotyl-based (~\u0026thinsp;5-fold) methods under comparable conditions. Regenerated plantlets developed healthy roots (with IAA or IBA) and acclimatised readily, exhibiting normal vegetative and reproductive growth. The protocol\u0026rsquo;s reproducibility across diverse cannabis genotypes and its applicability to other medicinal angiosperm species in this study highlights its value for both research and commercial applications.\u003c/p\u003e","manuscriptTitle":"Genotype-independent de novo regeneration protocol in Cannabis sativa L. through direct organogenesis from cotyledonary nodes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-20 17:19:14","doi":"10.21203/rs.3.rs-7237557/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-15T06:37:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-07T06:49:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-28T15:40:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"225648601427250299639043061919900116214","date":"2025-08-17T03:23:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158054106213058874637826290146369035273","date":"2025-08-15T13:53:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-13T00:50:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-29T07:34:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-29T07:31:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Methods","date":"2025-07-28T23:23:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3ca5a092-fea1-42b9-8d74-daab41b16251","owner":[],"postedDate":"August 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T16:08:58+00:00","versionOfRecord":{"articleIdentity":"rs-7237557","link":"https://doi.org/10.1186/s13007-025-01468-4","journal":{"identity":"plant-methods","isVorOnly":false,"title":"Plant Methods"},"publishedOn":"2025-11-08 15:57:49","publishedOnDateReadable":"November 8th, 2025"},"versionCreatedAt":"2025-08-20 17:19:14","video":"","vorDoi":"10.1186/s13007-025-01468-4","vorDoiUrl":"https://doi.org/10.1186/s13007-025-01468-4","workflowStages":[]},"version":"v1","identity":"rs-7237557","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7237557","identity":"rs-7237557","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}