Harnessing embryogenic cell suspension culture for Agrobacterium-mediated transformation of microvine

Harnessing embryogenic cell suspension culture for Agrobacterium-mediated transformation of microvine | 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 Harnessing embryogenic cell suspension culture for Agrobacterium-mediated transformation of microvine Shivani Sharma, Toni Mohr, Joshua T. Polito, Roger Thilmony This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7661205/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Microvine 04C023V0004 (V4) is a model Vitis vinifera genotype that carries a heterozygous Gibberellin insensitive 1 ( Vvigail ) mutation making the plants compact in stature and constantly fruiting. While these traits make V4 desirable for research, genetic engineering is challenging because of long regeneration times and modest transformation efficiency levels. Results To improve microvine V4 transformation, we established a method utilizing embryogenic cell suspension (ECS) cultures and a novel protocol for Agrobacterium- mediated transformation. Friable translucent or cream-colored globular somatic embryos from microvine embryogenic calli were used to initiate suspension cultures. ECS cultures require a modest amount of time and effort to maintain and produce abundant rapidly growing explant material within several months. A protocol was also developed that utilized these embryogenic suspension cells for Agrobacterium -mediated transformation. The ECS cells were heat shocked at 45°C for 5 minutes and then combined with a co-cultivation medium containing 400 mM acetosyringone, Agrobacterium tumefaciens AGL1 (OD 600 0.2) carrying a binary vector with the microvine Ubiquitin 7 ( VviUbi7 ) promoter controlling mCherry expression allowing the use of red fluorescence as a visible marker. After co-cultivation, washing the ECS cells with cefotaxime (400 mg/L) medium successfully inhibited bacterial growth. Development of healthy, actively growing transgenic microvine plants was achieved with the addition of gibberellic acid (GA 3 ) (10 mg/L) to the shooting medium. Eighteen independent transgenic plants were characterized using droplet digital PCR (ddPCR) demonstrating that eight (44%) had one or two copies of the introduced transgene. This method produced approximately 30 transgenic plants per 100 mg of ECS culture within five months from the start of Agrobacterium co-cultivation. Conclusion Use of microvine V4 ECS cultures and a modified transformation protocol can efficiently generate transgenic plants advancing grapevine biotechnology research. In the future, this protocol can potentially be adapted for other grapevine genotypes. microvine 04C023V0004 grapevine Agrobacterium tumefaciens embryogenic cell suspension transformation droplet digital PCR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Grapevine is typically genetically engineered using somatic embryos or embryogenic calli derived from reproductive tissues. However, its large size, long juvenile period, and biannual flowering impedes research progress. The Pinot Meunier cultivar of grapevine carries the Vvigail ( gibberellic acid-insensitive 1 ) gene mutation in the epidermal cell L1 layer of its apical meristem. VviGAI1 is involved in gibberellin signaling and this single nucleotide mutation converts leucine to a histidine in the DELLA domain of the protein [ 1 – 3 ], resulting in a tomentose (densely covered with trichomes) phenotype. Plants somatically derived from Pinot Meunier that are heterozygous for the VviGAI1/Vvigai1 mutation are called “microvine” and have a dwarf and hairy phenotype along with the conversion of tendrils into inflorescences, producing constant but physiologically normal flowering and fruiting. Microvine therefore serves as a model system for grape research and functional genomic studies. The original microvine mutant was not amenable to Agrobacterium -mediated transformation, so to overcome this limitation, crosses were performed and the hybrid progeny tested for embryogenic callus development and transgenic plant regeneration. The microvine genotype 04C023V0004 (V4), resulting from a cross between the L1 mutant and the black-berried variety Grenache , showed improved transformation efficiency [ 4 ] and has been used to create transgenic plants in previous studies [ 4 , 5 ]. However, despite the advantages of microvine as an amenable model, its transformation efficiency is still relatively low, transgenic shoot regeneration and development is inefficient and slow, and establishment of the regenerated plants in soil is challenging. The prolonged period in tissue culture can also increase the chance of recovering plants with undesirable somaclonal variation. Previously, embryogenic cell suspension (ECS) cultures were used to significantly improve plant regeneration and transformation efficiency in multiple crop species [ 6 – 9 ]. ECS culture has a good cell division rate, is more easily subcultured than callus on semi-solid media, and has a high plant regeneration rate [ 10 , 11 ]. ECS has been shown to enhance Agrobacterium -mediated transformation due to higher explant tissue uniformity, accessibility to infection, and improved selection efficiency for transformed tissues [ 10 ]. Despite these advantages, a higher susceptibility to bacterial and fungal contamination, shorter subculture intervals, and early senescence are challenges with the use of ECS cultures. In grapevine, cell suspension cultures were previously shown to improve regeneration in Thompson Seedless, Chardonnay, Richter 110, Cabernet Sauvignon, Chancellor, and Mencίa [ 11 – 16 ]. Plant regeneration from somatic embryos of grapevine interspecific hybrids was also previously reported from liquid cultures [ 17 ]. Chardonnay suspension culture has also been used for transformation in gene editing studies [ 18 , 19 ]. Liquid medium derived somatic embryos can develop a distinct embryonic structure known as the suspensor (a cellular connection between the embryo and surrounding tissue that transfers nutrients and growth regulators for embryo development) [ 20 , 21 ] and smaller cotyledons than counterpart proembryogenic masses grown on solid media. These structures correlated with more advanced shoot apical meristem development, little dormancy during germination relative to solid media grown somatic embryos and produce plants more efficiently [ 20 , 21 ]. The objective of this study was to establish microvine V4 suspension culture from embryogenic calli and to rapidly and efficiently generate transgenic plants using Agrobacterium -mediated transformation. The new protocol successfully created high-viability suspension cultures, ample embryogenic explant material for transformation and produced numerous healthy transgenic microvine plants. Methods Callus induction and proliferation Microvine V4 plants (provided by Dr. Paul Boss and Dr. Ian Dry, CSIRO, via Dr. Laurent Deluc) were grown in a USDA greenhouse in Albany, California. Inflorescences were collected as an explant source just after the immature flowers had begun to separate from each other. The flowers were washed under running water, divided into small clusters, and were surface sterilized with 100 mL of fresh 20% bleach solution containing a drop of Tween-20 in a conical flask for 1 minute. The flowers were washed three times with sterile water to remove residual bleach, then drained and either used directly for dissection or stored at 4ºC for up to three days before dissection. Flowers were dissected as previously described [ 22 ] and cultured on callus initiation medium (PIV) [ 23 ]. The culture plates were incubated in the dark at 28ºC and the resulting callus was transferred onto callus proliferation and maintenance medium (CIM) after 3 weeks for further callus proliferation. Embryogenic callus was transferred every 3 weeks onto new CIM media and incubated at 28ºC in the dark until use. ECS culture establishment Immature somatic embryos from ~ 4 month-old embryogenic calli were used to initiate a suspension culture. The friable, translucent or cream-colored globular somatic embryos (~ 50 mg fresh weight) were transferred into a 50 mL Erlenmeyer flask containing 7 mL of suspension medium (Supplementary file 1) and incubated at 28ºC with 90 rotations per minute (rpm) in the dark on a rotary shaker. ECS cultures were refreshed once a week by replacing 6 mL of old medium with new suspension medium. As the suspension cells proliferate, small cell clusters were transferred to a sterile large flask (120/250 mL) maintaining a suspension cells/medium volume ratio of 5–10% (approximately 15 mL suspension medium in 120 mL flask, measured using settled cell volume of the suspension cells in a 50 mL Falcon tube) and the residual bigger cell clusters in initial 50mL flask were maintained as mentioned above. Subculturing large flasks of ECS cultures was performed every 10 days. After transferring to a large flask, the suspension culture can be used directly for transformation or grown for future use. Bigger cell clusters within large flasks were filtered out using a 3.3-inch sieve to synchronize the cultures’ developmental stage every 1–2 months (Figure S2 ). However, initial small flask cultures were not filtered, but rather just transferred and maintained in a 50 mL flask for continuous proliferation. Suspension cells along with the medium were placed on a glass slide, covered with a coverslip, and observed directly under an Olympus microscope BX51 (Olympus Corporation, Tokyo, Japan) to assess their status and produce the images shown in Fig. 2 . Regeneration and plant development Suspension cells (small cell clusters) along with a minimal amount of suspension medium was transferred with a pipette onto embryo development and maturation (EDM) medium (Supplementary file 1) for regeneration. Plates were incubated at 28ºC in the dark in a growth chamber (Percival Scientific, Iowa, US). After 30–50 days on EDM, small cell clusters developed into matured somatic embryos and at the torpedo/cotyledon stage were transferred to suspension shooting medium (SSM) (Supplementary file 1) for growth and maintained at 28ºC under a 16-hour light/8-hour dark cycle (40–70 µmol m − 2 s − 1 ). When plantlets regenerated, they were transferred onto SSM-I (Supplementary file 1) under the same light and temperature conditions for further development. Once plants had grown to 4–5 cm in height, they were transferred to soil in pots, covered with polythene bags or a plastic dome to retain humidity for acclimatization, and maintained at 28ºC under a 16-hour light (40–70 µmol m − 2 s − 1 ) /8-hour dark cycle in a growth chamber (Conviron, North Dakota, US). Construct design The VviUbi7 promoter (Accession number: PV929291), including it’s 5ʹ intron (2,151 bp) [ 24 ], was amplified from microvine V4 genomic DNA and cloned into a Level 0 Golden Gate acceptor backbone from the MoClo Golden Gate library [ 25 ]. PCR amplification was performed using Q5 high fidelity DNA Polymerase (New England Biolabs, MA, US) with sequence specific primers (Supplementary file 2: Table S1 ) and microvine V4 genomic DNA under the following cycling conditions: 98ºC for 30 seconds, 35 cycles of 98ºC for 10 seconds, 67ºC for 10 seconds, 72ºC for 1.10 minutes, and a final extension of 72ºC for 2 minutes. The Level 0 vector carrying the VviUbi7 promoter was confirmed with Sanger DNA sequencing and cloned into a Level 2 acceptor vector driving the expression of the red fluorescence gene mCherry with a CaMV35S terminator and a kanamycin resistance plant selection cassette. A diagram of the vector construct T-DNA is shown in Fig. 4 A. Agrobacterium -mediated transformation Suspension cells were transformed using the Agrobacterium tumefaciens strain AGL1 containing the VviUbi7-mCherry binary vector plasmid. Agrobacterium culture was prepared as previously described [ 22 ] and resuspended in co-cultivation medium (Supplementary file 1) with 400 µM acetosyringone at an OD 600 = 0.2. Five hundred milligrams of suspension cells were collected into a 50 mL falcon tube and the original medium replaced with fresh media. Cells were then heat shocked at 45ºC for 5 minutes just prior to Agrobacterium co-cultivation. Ten milliliters of the Agrobacterium cocultivation medium were added to 500 mg suspension cells and mixed gently at room temperature for 10 minutes on a rotatory shaker at 50 rpm. After shaking, suspension cells were allowed to settle to the bottom of the tube. Five milliliters of medium were removed, and the remaining solution and cells were transferred to filter paper (a stack of 2–3 sterile filter paper disks) placed on a sterile petri plate using a serological pipette. Once the cells appeared air-dried in the laminar flow hood, the filter paper with cells were transferred to CIM plates and co-cultured at 22ºC in dark for 2 days. After co-cultivation, suspension cells were transferred to a falcon tube containing 10 mL suspension medium with 400 mg/L cefotaxime using a cell scraper (Alkali Scientific, Florida, US) and mixed on a rotatory shaker (50 rpm) for 15–30 minutes at room temperature. The cells were washed 3–4 times with suspension medium to remove the Agrobacterium and then transferred onto stacked filter papers in a sterile petri dish to air dry (carefully without over drying the cells) in the laminar flow hood. The dried cells were then transferred with a cell scraper to semi-solid EDM medium containing cefotaxime (250 mg/L) and divided into small groups of cells to track individual transformation events. Cultures were incubated in the dark at 28ºC for 20 days and observed under a microscope for red fluorescence to confirm transgene expression. The transformed cells were subcultured onto EDM media containing 250 mg/L cefotaxime and plant selection antibiotic (50 mg/L kanamycin) once a month until they develop mature somatic embryos. Mature somatic embryos exhibiting red florescence were then transferred to SSM containing 250 mg/L cefotaxime and GA 3 (5 mg/L) and maintained at 28ºC in light (16h/8h light/dark at 40–70 µmol m − 2 s − 1 light intensity) in a growth chamber. Regenerated plantlets were transferred onto SSM-I with GA 3 (10 mg/L) for further development and later transferred to soil for acclimatization under the same conditions before being moved to the greenhouse. Molecular analysis Genomic DNA was extracted using a modified Puregene kit protocol (Qiagen, Redwood City, CA, US). Leaf samples (~ 0.5 g) were snap frozen in liquid nitrogen in 1.5 mL centrifuge tubes and ground into a fine powder using a plastic mortar. 400 µL of cell lysis solution and 25 µL of β-mercaptoethanol were added to the ground sample, vortexed, and incubated at 65ºC for 1 hour, with additional mixing at 30 and 60 minutes. 5 µL of RNase A (10 mg/mL) solution was added, mixed gently, and incubated at 37ºC for 30 minutes. The sample was placed on ice for 1 minute to cool, and 133 µL of protein precipitation buffer was added. The mixture was vortexed for 20 seconds and put on ice for 20 minutes. The samples were then centrifuged for 3 minutes at 13,000 x g, and the supernatant was transferred into a new 1.5 mL microcentrifuge tube. Equal amounts of chloroform: isoamyl alcohol were added, inverted 50 times, and centrifuged for 5 minutes at 13,000 x g. The supernatant was transferred into a new 1.5 mL microcentrifuge tube, an equal amount of 100% isopropanol was added and mixed by inverting 50 the tube times. The samples were then incubated on ice for 5 minutes and then centrifuged for 1 minute at 13,000 x g. The supernatant was discarded, and the DNA pellet was washed with 300 µL of 70% ethanol, mixed by inverting, and centrifuged at 13,000 x g for 1 minute. The supernatant was discarded, and the genomic DNA pellet was air dried and then dissolved in 30 µL of sterile ultra-pure water. Genomic DNA was quantified with a DeNovix microvolume spectrophotometer (DeNovix, Davis, CA) and its integrity evaluated by separation on a 1% agarose gel prior to use. Droplet digital PCR (ddPCR) was used to detect the copy number in the T 0 transgenic events. The grapevine chalcone synthase (VviCHI) gene [ 26 , 27 ] was used as a single copy reference gene. Primers and probes (Supplementary file 2: Table S1 ) were designed for the reference gene and as used previously for nptII [ 28 ] following the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, US). A FAM™-labeled probe was used to detect the reference gene amplicon and a HEX™-labeled probe for the nptII amplicon detection. Genomic DNA (200 ng) of transgenic and control plants was digested with EcoRI-HF restriction enzyme (New England Biolabs, MA, US) prior to ddPCR. Each 20 µL reaction contained 10 µL of 2x ddPCR supermix for probes (no dUTP) (Bio-Rad), 0.5µl of each primer (22.5 µM), 0.25 µl of reference and target probe (25µM), 3µl of digested DNA (60–100 ng), and 5.5µl water for a total reaction volume of 20 µl. Droplets were generated in the QX200 droplet generator for each sample and PCR was carried out at: 95ºC for 10 minutes, 40 cycles (94 ºC for 30s, 60 ºC for 1 minute), 98 ºC for 10 minutes, 12 ºC hold. After PCR, the droplets were read in the Bio-Rad QX200 droplet reader, and the data was analyzed using QuantaSoft software. Results Development of microvine ECS The development of callus and suspension cultures using stamen explants are illustrated in Fig. 1 . An early stage microvine inflorescence is shown in Fig. 1 A. Immature flowers that are the most suitable for initiating embryogenic callus are at the stage when they begin to separate from each other within the floral cluster (Fig. 1 B). At this stage, the anthers are pale yellow and appear translucent (Fig. 1 C). We tested stamens, filaments, anthers, pistils, and whole flowers with the calyptra and without the calyptra as tissue types for callus induction and found that intact flowers without the calyptra produced the greatest number of embryogenic calli, but stamens and dissected filaments also generate embryogenic calli. Within 20 days of dissection, anthers turn brown, and the filament tips attached to the ovary swell (Fig. 1 D). Induced callus starts to proliferate within two rounds of subculturing on CIM and was further maintained by transferring to new CIM medium every 2–4 weeks. Embryogenic callus generation efficiency in microvine V4 was 2–6% (number of embryogenic calli/total number of stamens * 100) from healthy, greenhouse-grown plants. After approximately four months of subculturing on CIM, friable, translucent or cream-colored globular somatic embryos (Fig. 1 E, 1 F) were selected to initiate ECS culture in 50 mL flasks (Fig. 1 G). Selection of globular somatic embryos is the most critical step in establishing an ECS culture. Compact callus pieces fail to develop ECS and eventually turn brown within 2–3 weeks in liquid medium (Figure S1 A-C). Approximately 50 mg fresh weight of somatic embryos were used to initiate the cell suspension culture in 7 mL of suspension medium in a 50 mL Erlenmeyer flask. During subculturings, removal of brown cell clusters (if any) helps to maintain a healthy ECS culture. Embryogenic cultures typically proliferate within 2 months (Fig. 1 H). After proliferation, the small cell clusters from culture were transferred into larger flasks (120/250mL), leaving behind the bigger cell clusters in the original flask for further proliferation, maintaining cells and suspension medium ratio of 5–10% as shown in Figure S2 . When bigger cell clusters appear in the larger flask suspension cultures, they were strained through a sieve and removed. This process aids in maintaining a culture with uniform smaller sized cell clusters within the ECS culture (Figure S2 ). Microscopic observations of ECS cultures show many small clusters of cells (Fig. 2 A). A healthy embryogenic suspension cell culture that can efficiently generate shoots will typically contain small spherical cell clusters where the cells are filled with dense cytoplasm (starch granules) as shown in Fig. 2 A-C. Non-embryogenic cultures contain cells and cell clusters with irregular shapes, a large vacuole and no/less starch granules (Fig. 2 D). These non-embryogenic cells and cell clusters were frequently observed in older ECS cultures and became more abundant as they aged. When these non-embryogenic cells predominate in a culture, it is no longer useful for plant regeneration. Plant regeneration from microvine ECS cultures Suspension cells (along with a minimal amount of the suspension medium) were transferred onto EDM plates to examine their shoot regeneration capacity (Figure S3 A). The suspension cells developed into mature somatic embryos on EDM after approximately 30–50 days of subculturing on the semi-solid medium (Figure S3 B), although the exact timeframe can vary between independently derived ECS cultures. Mature globular and torpedo staged somatic embryos were observed under the microscope (Figure S3 C) for cultures with the capacity to regenerate shoots. These explants turned green and grew into small shoots with roots on SSM after 15–30 days (Figure S3 D). These shoots then developed into plantlets after 1–2 months of subculturing on SSM-I containing gibberellin hormone (Figure S4). Subsequently, healthy 4–5 cm tall plantlets were transferred to soil for acclimatization and further growth (Figure S3 F). Agrobacterium -mediated transformation of microvine ECS To evaluate the efficiency of transformation in microvine ECS cultures, we used an Agrobacterium -mediated transformation approach. A flow-chart of the procedure is shown in Fig. 3 . Schematic diagram of the transformation construct is shown in Fig. 4 A. Co-cultivation of suspension cells with Agrobacterium carrying the binary vector construct is shown in Fig. 4 B. After co-cultivation, the plant cells were washed and transferred as small groups of cell clusters to semi-solid EDM containing kanamycin and cefotaxime (Fig. 4 C). Transformed cell foci exhibited red florescence four days post co-cultivation (Fig. 4 D). The transformed cells matured into the torpedo and cotyledon staged somatic embryos on EDM selection medium within two-three months (Fig. 4 E). These mature somatic embryos exhibited bright red fluorescence confirming the stable integration and expression of the mCherry transgene (Fig. 4 F). The transformed embryos were then transferred to SSM medium and developed roots and shoots (Fig. 4 G), but some of these shoots exhibited slow and/or abnormal growth (Figure S5). To improve shoot growth and development, we supplemented the SSM medium with four different concentrations of GA 3 : 5 mg/L, 8 mg/L, 10 mg/L and 15 mg/L. Microvine shoots developed into healthy plantlets on SSM-I containing GA 3 after an additional 60 days (Fig. 4 H). We observed a greater number of normal healthy shoots with the addition of GA 3 , and the highest percentage of normal shoots were observed with 10mg/L GA 3 (Figure S4, Supplementary file 2: Table S2 ). Plants grown with 15mg/L GA 3 showed some curling of shoot tips. After two months, well-established rooted plants were transferred to soil and exhibited normal growth (Fig. 4 I). This approach generated approximately 150 red fluorescent transgenic plants from 500 mg fresh weight of starting ECS culture. Eighteen randomly selected plants were grown in soil for further characterization. The overall timeline for the generation of transgenic microvine V4 plants from ECS cultures is illustrated in Fig. 5 . Molecular confirmation of transgenic plants Eighteen independent putative transgenic plants were analyzed with ddPCR to confirm T-DNA integration and measure nptII copy number. The transgene quantification was calculated using the endogenous single-copy grapevine gene VviChi as a reference [ 26 , 27 ]. Among the tested plants, four carried a single-copy integration, five had two copies of nptII , and nine plants contained three or more transgene copies (Fig. 6 , Table S3 ). These results demonstrated that the use of ECS cultures with Agrobacterium transformation generates stably transformed microvine V4 plants carrying 1 or 2 copies of an introduced transformation construct half of the time. Discussion A major bottleneck in grapevine biotechnology is the rapid regeneration of transgenic plants carrying constructs of interest. Microvine is a desirable grapevine research model system because of several favorable attributes, but it is somewhat recalcitrant for shoot regeneration and grows relatively slowly in culture in part due to its gibberellin insensitivity [ 2 – 4 ]. In this study, we report the development of ECS cultures from microvine V4 embryogenic calli and a streamlined protocol for Agrobacterium -mediated transformation. The first step for producing ECS is the establishment of embryogenic calli and the selection of suitable explant starting material. In previous studies, stamens, pistils and whole flowers produced embryogenic calli on PIV medium in grapevine [ 29 – 31 ]. In the current study, microvine filament tissues induced callus from stamens as well as flowers without calyptra. We observed more embryogenic callus production with whole flowers without calyptra as compared to stamens only as observed for other grapevine cultivars [ 29 – 31 ]. This may be due to the presence of endogenous hormone levels within these reproductive structures that contribute to more efficient callus induction [ 32 – 34 ]. The second most important consideration for establishing ECS is the selection of good quality starting material from the embryogenic calli. Inoculation of whole embryogenic callus even with good somatic embryos may result in mixed culture and can turn brown later. The selection of globular somatic embryos for ECS initiation, significantly influences the quality and uniformity of the resulting suspension culture [ 11 ]. An earlier study also reported that somatic embryos from Chardonnay callus produced a rapidly growing suspension culture [ 11 ]. However, specificity of starting material used for establishing Chardonnay ECS is not mentioned in other studies [ 15 , 18 , 35 ]. Some articles reported use of embryogenic cells, embryo masses and pro-embryonic masses in Chardonnay, Cabernet Sauvignon, rootstocks and Mencίa grapevine varieties [ 14 , 16 , 19 ]. Our study demonstrated that immature, friable, and translucent/cream colored somatic embryos at the globular stage exhibit the highest embryogenic potential in liquid culture, consistent with previous studies in other crop species [ 7 , 36 ]. For maintaining ECS, the ratio of suspension cells to suspension medium also plays an important role, as an excessive number of cells in the culture can lead to browning and cell death as observed previously in grapevine and palm [ 11 , 36 ]. Frequently subdividing cells into new flasks reduces cell browning as reported earlier in Chardonnay and Thompson seedless [ 11 ]. Cell debris sticking to the inner wall of the flask may also contribute to the browning of the cells and the removal of these dead cells by transferring the culture to a new flask is necessary to maintain healthy suspension cultures. Using the reported protocol, we were able to maintain high quality microvine ECS cultures for more than one year before they lost their capacity to efficiently regenerate plants. Suspension cells have been shown to be a good source material for protoplast isolation [ 37 , 38 ], so this may be a useful approach to produce microvine protoplasts as well. Previously, grapevine cell suspension culture has been used for biolistic and Agrobacterium- mediated transformation but has been a relatively inefficient approach for producing plants [ 15 , 18 , 19 , 39 , 40 ]. In contrast, one report produced 240 plants/10 ml settled cell volume with Chardonnay suspension culture and another generated 37 positive plants with Chancellor cell suspension using a biolistic approach [ 35 , 41 ]. Another study of Chardonnay Agrobacterium -mediated transformation that used liquid selection medium to generate resistant cell masses, reported the generation of four transgenic plants [ 18 ]. However, microvine transformation has been only reported using calli explants with Agrobacterium- mediated transformation and no reports describe the development of microvine cell suspension cultures. One of those previous studies reported the recovery of nine microvine transgenic plants [ 42 ] and other studies have reported the time for the production of microvine transgenic plants was 6–10 months [ 3 , 5 ]. Conversely, ECS have been reported to be highly responsive to Agrobacterium infection and amenable to efficient shoot regeneration in crops like banana, citrus, switchgrass, and medicinal plants [ 6 , 7 , 43 , 44 ]. The reported protocol generated approximately 150 transgenic microvine plants in about five months from the start of Agrobacterium cocultivation and all eighteen of candidate transgenic plants that were selected for molecular characterized were red fluorescent and transgene positive. For better recovery of transgenic plants, we heat shocked suspension cells prior to Agrobacterium infection as reported previously to improve transformation efficiency [ 45 – 48 ] and discontinued the use of antibiotic selection at the shooting stage as a negative impact of kanamycin selection on shoot growth and regeneration was reported previously [ 49 – 51 ]. Heat shock promotes the survival of transformed cells due to the prevention of programmed cell death [ 47 ] and induces the production of heat shock proteins that promotes DNA transfer during Agrobacterium -mediated transformation [ 52 – 54 ]. The use of the mCherry red fluorescent reporter enabled the reliable recovery of nonchimeric transgenic microvine plants. Conclusions In the present study, a method for producing rapidly growing microvine ECS cultures is described. These ECS cultures were demonstrated to be a viable source material for Agrobacterium -mediated transformation producing abundant fluorescent transgenic shoots and healthy transgenic plants (150 transgenic plants/500 mg ECS) within approximately 5–7 months. An advantage of using microvine ECS cultures is that they grow rapidly and can provide an ample source material for transformation and other experiments while requiring a modest amount of time and effort to maintain compared to embryogenic callus. The protocol presented can be used to efficiently engineer novel traits, or to deploy CRISPR genome editing reagents to better understand gene function in microvine. In the future, this approach could potentially be adapted for other grapevine genotypes enabling more efficient biotechnology research in this important crop. Abbreviations V4 microvine genotype 04C023V0004 ECS embryogenic cell suspension GAI 1 Gibberellic acid insensitive 1 VviUbi7 Microvine Ubiquitin 7 GA 3 Gibberellic acid PCR Polymerase Chain Reaction ddPCR droplet digital PCR PIV Callus initiation medium CIM Callus proliferation medium EDM Embryo development and maturation medium SSM Suspension shooting medium CaMV Cauliflower mosaic virus CRISPR Clustered Regularly Interspaced Short Palindromic Repeats Declarations Ethics and consent to participate declarations Not applicable Consent for publication Not applicable Availability of data and materials The microvine VviUbi7 promoter sequence used in the study is available from GenBank (accession number PV929291). Competing interests The authors declare no competing interests. Funding This work was supported by a United States Department of Agriculture-Agricultural Research Service (USDA-ARS) project 2030-21220-003-000-D and an appointment to the ARS Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the United States Department of Energy (DOE) and the USDA. ORISE is managed by Oak Ridge Associated Universities under DOE contract number DE-SC0014664. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. Author contributions RT and S designed the study. S, TM and JTP performed experiments. S wrote the manuscript and designed the figures. All authors contributed to editing and revising the manuscript. RT provided supervisory guidance and funding support. Acknowledgement We would also like to thank James Horstman for his support in developing and maintaining embryogenic calli and for skilled greenhouse management. References Boss PK, Thomas MR. Association of dwarfism and floral induction with a grape ‘green revolution’ mutation. Nat 2002, 416(6883):847–50. Pellegrino A, Romieu C, Rienth M, Torregrosa L. The microvine: a versatile plant model to boost grapevine studies in physiology and genetics. In: Adv grape wine Biotechnol IntechOpen; 2019. Torregrosa LJ-M, Rienth M, Romieu C, Pellegrino A. The microvine, a model for studies in grapevine physiology and genetics. OENO One. 2019;53(3):373–91. Chaib J, Torregrosa L, Mackenzie D, Corena P, Bouquet A, Thomas MR. The grape microvine - a model system for rapid forward and reverse genetics of grapevines. Plant J 2010, 62(6):1083–92. Gouthu S, Deluc LG. Use of the microvine and plant gene switch system for functional studies of genes involved in the control of ripening initiation in Vitis vinifera . Acta Hortic 2019(1248):187–94. Ondzighi-Assoume CA, Willis JD, Ouma WK, Allen SM, King Z, Parrott WA, Liu W, Burris JN, Lenaghan SC, Stewart CN Jr.. Embryogenic cell suspensions for high-capacity genetic transformation and regeneration of switchgrass ( Panicum virgatum L). Biotechnol Biofuels. 2019;12:290. Shivani TS. Enhanced Agrobacterium -mediated transformation efficiency of banana cultivar Grand Naine by reducing oxidative stress. Sci Hortic. 2019;246:675–85. Tripathi JN, Oduor RO, Tripathi L. high-throughput regeneration and transformation platform for production of genetically modified banana. Front Plant Sci. 2015;6:1025. Woo HA, Ku SS, Jie EY, Kim H, Kim HS, Cho HS, Jeong WJ, Park SU, Min SR, Kim SW. Efficient plant regeneration from embryogenic cell suspension cultures of Euonymus alatus . Sci Rep. 2021;11(1):15120. Finer JJ. Plant regeneration via embryogenic suspension cultures. In: Plant Cell Cult 1995: 99–126. Jayasankar S, Gray DJ, Litz RE. High-efficiency somatic embryogenesis and plant regeneration from suspension cultures of grapevine. Plant Cell Rep. 1999;18:533–7. Forgács I, Suller B, Zok A, Pedryc A, Oláh R, Deák T, Bisztray GD, Szegedi E. Establishment of grapevine embryogenic liquid culture and induced somatic embryogenesis. Acta Hortic 2017(1157):113–8. Jiao W, Ya-li Z, Rong-rong H, Lei Z, Chao-xia W, Xia X, Jiang L. Optimization of transformation efficiency of suspension cultured Vitis vinifera cv. Chardonnay embryogenic cells. J Integr Agric. 2012;11(3):387–96. Amar AB, Cobanov P, Boonrod K, Krczal G, Bouzid S, Ghorbel A, Reustle GM. Efficient procedure for grapevine embryogenic suspension establishment and plant regeneration: role of conditioned medium for cell proliferation. Plant Cell Rep. 2007;26:1439–47. Torregrosa L, verries C, Tesniere C. Grapevine ( Vitis vinifera L.) promoter analysis by biolistic-mediated transient transformation of cell suspensions. Vitis. 2002;41(1):27–32. Acanda Y, Martínez Ó, González MV, Prado MJ, Rey M. Highly efficient in vitro tetraploid plant production via colchicine treatment using embryogenic suspension cultures in grapevine ( Vitis vinifera cv. Mencía). Plant Cell Tissue Organ Cult. 2015;123(3):547–55. Zlenko V, Kotikov IV, Troshin LP. Plant regeneration from somatic embryos of interspecific hybrids of grapevine formed in liquid medium. J Hortic Sci Biotechnol. 2005;80(4):461–5. Ren C, Liu X, Zhang Z, Wang Y, Duan W, Li S, Liang Z. CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay ( Vitis vinifera L). Sci Rep. 2016;6:32289. Villette J, Lecourieux F, Bastiancig E, Heloir M-C, Poinssot B. New improvements in grapevine genome editing: high efficiency biallelic homozygous knock-out from regenerated plantlets by using an optimized zCas9i. Plant Methods. 2024;20:45. Jayasankar S, Bondada BR, Li Z, Gray DJ. Comparative anatomy and morphology of Vitis vinifera (Vitaceae) somatic embryos from solid- and liquid-culture-derived proembryogenic masses. Am J Bot. 2003;90(7):973–9. Kawashima T, Goldberg RB. The suspensor: not just suspending the embryo. Trends Plant Sci. 2010;15(1):23–30. Torregrosa L, Vialet S, Adiveze A, Iocco-Corena P, Thomas MR. Grapevine ( Vitis vinifera L). Methods Mol Biol. 2015;1224:177–94. Franks T, He D, Thomas M. Regeneration of transgenic Vitis vinifera L. Sultana plants: genotypic and phenotypic analysis. Mol Breeding. 1998;4:321–33. Li ZT, Kim KH, Jasinski JR, Creech MR, Gray DJ. Large-scale characterization of promoters from grapevine ( Vitis spp.) using quantitative anthocyanin and GUS assay systems. Plant Sci. 2012;196:132–42. Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLoS ONE. 2008;3(11):e3647. Harris NN, Luczo JM, Robinson SP, Walker AR. Transcriptional regulation of the three grapevine chalcone synthase genes and their role in flavonoid synthesis in Shiraz. Aust J Grape Wine Res. 2013;19(2):221–9. Costa LD, Vinciguerra D, Giacomelli L, Salvagnin U, Piazza S, Spinella K, Malnoy M, Moser C, Marchesi U. Integrated approach for the molecular characterization of edited plants obtained via Agrobacterium tumefaciens –mediated gene transfer. Eur Food Res Technol. 2022;248:289–99. Thilmony R, Dasgupta K, Shao M, Harris D, Hartman J, Harden LA, Chan R, Thomson JG. Tissue-specific expression of Ruby in Mexican lime ( C. aurantifolia ) confers anthocyanin accumulation in fruit. Front Plant Sci 2022, 13. Capriotti L, Limera C, Mezzetti B, Ricci A, Sabbadini S. From induction to embryo proliferation: improved somatic embryogenesis protocol in grapevine for Italian cultivars and hybrid Vitis rootstocks. Plant Cell, Tissue and Organ Cult 2022, 151(2):221–33. Gambino G, Ruffa P, Vallania R, Gribaudo I. Somatic embryogenesis from whole flowers, anthers and ovaries of grapevine ( Vitis spp.). Plant Cell, Tissue and Organ Cult 2007, 90(1):79–83. Dhekney SA, Li ZT, Compton ME, Gray DJ. HortScience : Optimizing initiation and maintenance of Vitis embryogenic cultures. 2009, 44:1400–6. Jiménez VM. Regulation of in vitro somatic embryogenesis with emphasis on the role of endogenous hormones. Braz J Plant Physiol. 2001;13(2):196–223. Jiménez VM, Bangerth F. Relationship between endogenous hormone levels of grapevine callus cultures and their morphogenetic behaviour. Vitis 2000, 39(4):151–157. Mostafa HHA, Wang H, Song J, Li X. Effects of genotypes and explants on garlic callus production and endogenous hormones. Sci Rep. 2020;10(1):4867. Vidal JR, Kikkert JR, Wallace PG, Reisch BI. High-efficiency biolistic co-transformation and regeneration of 'Chardonnay' ( Vitis vinifera L.) containing npt-II and antimicrobial peptide genes. Plant Cell Rep. 2003;22(4):252–60. Abohatem M, Zouine J, Hadrami IE. Low concentrations of BAP and high rate of subcultures improve the establishment and multiplication of somatic embryos in date palm suspension cultures by limiting oxidative browning associated with high levels of total phenols and peroxidase activities. Sci Hortic. 2011;130:344–8. Li S-F, Ye T-W, Xu X, Yuan D-Y, Xiao S-X. Callus induction, suspension culture and protoplast isolation in Camellia oleifera . Sci Hortic 2021, 286(110193). Masani MYA, Noll G, Parveez GKA, Sambanthamurthi R, Prüfer D. Regeneration of viable oil palm plants from protoplasts by optimizing media components, growth regulators and cultivation procedures. Plant Sci. 2013;210:118–27. Mandolino LM. Genetic transformation and regeneration of transgenic plants in grapevine ( Vitis rupestris S). Theor Appl Genet. 1994;88:621–8. Hebert D, Kikkert JR, Smith FD, Reisch BI. Optimization of biolistic transformation of embryogenic grape cell suspensions. Plant Cell Rep. 1993;12:585–9. Kikkert JR, Hebert-Soule D, Wallace PG, Striem MJ, Reisch, Bl. Transgenic plantlets of 'Chancellor' grapevine ( Vitis sp.) from biolistic transformation of embryogenic cell suspensions. Plant Cell Rep. 1996;15:311–6. Dalla Costa L, Emanuelli F, Trenti M, Moreno-Sanz P, Lorenzi S, Coller E, Moser S, Slaghenaufi D, Cestaro A, Larcher R, Gribaudo I, Costantini L, Malnoy M. Grando MS:Induction of terpene biosynthesis in berries of microvine transformed with VvDXS1 alleles. Front Plant Sci. 2018;8:2244. Babich O, Sukhikh S, Pungin A, Ivanova S, Asyakina L, Prosekov A. Modern trends in the In Vitro production and use of callus, suspension cells androot cultures of medicinal plants. Molecules 2020, 25(24):5805. Moniruzzaman M, Zhong Y, Huang Z, Yan H, Yuanda L, Jiang B, Zhong G. Citrus cell suspension culture establishment, maintenance, efficient transformation and regeneration to complete transgenic plant. Plants. 2021;10(4):664. Gurel S, Gurel E, Kaur R, Wong J, Meng L, Tan HQ, Lemaux PG. Efficient, reproducible Agrobacterium -mediated transformation of sorghum using heat treatment of immature embryos. Plant Cell Rep. 2009;28(3):429–44. Hiei Y, Ishida Y, Kasaoka K, Komari T. Improved frequency of transformation in rice and maize by treatment of immature embryos with centrifugation and heat prior to infection with Agrobacterium tumefacien s. Plant Cell Tissue Organ Cult. 2006;87:233–43. Khanna H, Becker D, Kleidon J, Dale J. Centrifugation Assisted Agrobacterium tumefaciens -mediated Transformation (CAAT) of embryogenic cell suspensions of banana ( Musa spp. Cavendish AAA and Lady finger AAB). Mol Breed. 2004;14:239–52. Patel M, Dewey RE, Qu R. Enhancing Agrobacterium tumefaciens -mediated transformation efficiency of perennial ryegrass and rice using heat and high maltose treatments during bacterial infection. Plant Cell Tissue Organ Cult. 2013;114(1):19–29. Bon RS, Noia FD, Segura A, Vidal JR. Toxic effect of antibiotics in grapevine ( Vitis vinifera 'Albariño') for embryo emergence and transgenic plant regeneration from embryogenic cell suspension. Vitis. 2014;53(2):89–94. Li ZT, Dhekney S, Dutt M, Aman M, Tattersall J, Kelley KT, Gray DJ. Optimizing Agrobacterium -mediated transformation of grapevine. Vitro Cell Dev Biol Plant. 2006;42(3):220–7. Torregrosa L, Lopez G, Bouquet A. Antibiotic sensitivity of grapevine: A comparison between the effect of hygromycin and kanamycin on shoot development of transgenic 110 richter rootstock ( Vitis Berlandieri x Vitis rupestris ). S Afr J Enol Vitic 2016, 21(1). Park SY, Yin X, Duan K, Gelvin SB, Zhang ZJ. Heat shock protein 90.1 plays a role in Agrobacterium -mediated plant transformation. Mol Plant. 2014;7(12):1793–6. Hwang HH, Liu YT, Huang SC, Tung CY, Huang FC, Tsai YL, Cheng TF, Lai EM. Overexpression of the HspL promotes Agrobacterium tumefaciens virulence in Arabidopsis under heat shock conditions. Phytopathology. 2015;105(2):160–8. Tsai YL, Wang MH, Gao C, Klusener S, Baron C, Narberhaus F, Lai EM. Small heat-shock protein HspL is induced by VirB protein(s) and promotes VirB/D4-mediated DNA transfer in Agrobacterium tumefaciens . Microbiology. 2009;155:3270–80. Additional Declarations No competing interests reported. Supplementary Files FiguresS1S5.pdf TablesS1S3.xlsx Additionalfile1Mediarecipes.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7661205","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":518973520,"identity":"126600e9-d378-49f2-b97b-63fa156292fd","order_by":0,"name":"Shivani Sharma","email":"","orcid":"","institution":"USDA-Agricultural Research Service","correspondingAuthor":false,"prefix":"","firstName":"Shivani","middleName":"","lastName":"Sharma","suffix":""},{"id":518973521,"identity":"6cbb8845-8363-4ff0-92cd-3b7460440086","order_by":1,"name":"Toni Mohr","email":"","orcid":"","institution":"USDA-Agricultural Research Service","correspondingAuthor":false,"prefix":"","firstName":"Toni","middleName":"","lastName":"Mohr","suffix":""},{"id":518973522,"identity":"410c6e39-95d0-4fc8-8ba6-34201a8e6d33","order_by":2,"name":"Joshua T. Polito","email":"","orcid":"","institution":"USDA-Agricultural Research Service","correspondingAuthor":false,"prefix":"","firstName":"Joshua","middleName":"T.","lastName":"Polito","suffix":""},{"id":518973523,"identity":"b04bd04c-20f1-4e1a-a4b5-7d1836b27f9e","order_by":3,"name":"Roger Thilmony","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIiWNgGAWjYBAC+wYw9T+BjR3MsAHiBPxaDA6AKYkENmYwI40ELQwQLYeJ0HK8/eLjCgaJPD5m7sTHhW3nE+e3Jz9g/FJxGKcW+54zxYZnGCSK2Zh5NxvPbLuduOHMMwNmmTO4tdhJ5KRJNjBIJLYx826T5gVpkUgwYJZsS8OpxVgiJ/0nkpZzifNnpH/Aq8VwRvoxRiQtBxIbbuQYMH5ss8Ht/TNnmCUbDMBaNhvznEs23nDmTcFhhjN4tBxvf/ixoUICGFC9Gx/zlNnJzm9P3/jwR4UETi0MDDwGQI1QNiMbhD7Mg0cDAwP7AyTOH6jWH3i1jIJRMApGwQgDANiXVp5oIYDaAAAAAElFTkSuQmCC","orcid":"","institution":"USDA-Agricultural Research Service","correspondingAuthor":true,"prefix":"","firstName":"Roger","middleName":"","lastName":"Thilmony","suffix":""}],"badges":[],"createdAt":"2025-09-19 21:23:14","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7661205/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7661205/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":92238546,"identity":"0fbf9112-7962-43f9-9a3f-7c2b81e48c1f","added_by":"auto","created_at":"2025-09-26 08:02:50","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8522259,"visible":true,"origin":"","legend":"","description":"","filename":"MicrovineembryogeniccellsuspensionShivanietal..docx","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/775cf5c305a7b506513bfa1e.docx"},{"id":92238325,"identity":"012f87db-c885-44e2-b22d-9041a8953838","added_by":"auto","created_at":"2025-09-26 07:54:49","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6699,"visible":true,"origin":"","legend":"","description":"","filename":"1ed61b7676464cccbc181cdd016d67de.json","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/c88465d1ee2dcfa4a1847fc6.json"},{"id":92238542,"identity":"16033430-bd65-4be0-8513-6a6dea7f6cc9","added_by":"auto","created_at":"2025-09-26 08:02:49","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":34135,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1Mediarecipes.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/0870e5dc116c054f7ccab553.xlsx"},{"id":92238336,"identity":"177c8f34-5659-44b3-8c79-26fa6eb8f53f","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2244570,"visible":true,"origin":"","legend":"","description":"","filename":"FiguresS1S5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/d0431f3a59e50bcb3d227361.pdf"},{"id":92238351,"identity":"dbfa8f6f-87c0-4da4-a159-896b07d4075e","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17761,"visible":true,"origin":"","legend":"","description":"","filename":"TablesS1S3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/fe9b579e5b22f6df577774a4.xlsx"},{"id":92238343,"identity":"b3bf83dd-3642-4ebc-b01c-7f9b37c24e94","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"xml","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":135874,"visible":true,"origin":"","legend":"","description":"","filename":"1ed61b7676464cccbc181cdd016d67de1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/3967c6e2091e7e7118bb23b8.xml"},{"id":92238337,"identity":"626fa1ce-2dec-4cae-b13f-e21610e46992","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1189430,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/024614f230177f751142f773.jpeg"},{"id":92238330,"identity":"4474d5f8-8f7f-4745-9e95-b2efbed03fd9","added_by":"auto","created_at":"2025-09-26 07:54:49","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2114,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/bff13173d7700d458e1f9db8.jpeg"},{"id":92238335,"identity":"b88444bc-15d0-4aa3-9d1d-9bf2e01146e1","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2114,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/181963eaa098277eec7b2ab7.jpeg"},{"id":92238346,"identity":"ee0706af-83e7-42bf-9ed7-bb59a0fd1ef4","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2114,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/8fb705550ae4f67d8889332e.jpeg"},{"id":92238333,"identity":"a5c8220b-978d-485e-999d-eb81e936f9d6","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2114,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/2a0a3b88c95870d03def6c0a.jpeg"},{"id":92238359,"identity":"0d614810-1866-4b64-9a1d-a102dc04c486","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1316529,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage14.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/06cb77bcb289e74b14a35ba4.jpeg"},{"id":92238347,"identity":"e775b311-f85c-4b38-b1f2-3b01da260117","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"jpeg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":596271,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage15.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/e79319a2283cbe202f9cc1b0.jpeg"},{"id":92238348,"identity":"fdd130ac-2eb0-427a-be0e-f71597566161","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"jpeg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":179173,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage16.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/fde5dd30c673653378c7e643.jpeg"},{"id":92238367,"identity":"6ba64c77-0311-43b5-b236-2328b4ef1491","added_by":"auto","created_at":"2025-09-26 07:54:51","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3233124,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/6536cfa48628b6e03afa472e.png"},{"id":92238352,"identity":"b8842723-c250-439a-8457-b9bf1504c744","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"jpeg","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":16507,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/ef56cf84b75c8ee1c0c940ff.jpeg"},{"id":92238549,"identity":"b49d6875-8c09-44fe-bc7b-47179dec9a01","added_by":"auto","created_at":"2025-09-26 08:02:50","extension":"jpeg","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2114,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/b0173d6edaffdd118022edd1.jpeg"},{"id":92238552,"identity":"87b26200-c716-467d-8d45-f411bb809d82","added_by":"auto","created_at":"2025-09-26 08:02:50","extension":"jpeg","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2114,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/db9829b78956b6f1801cee9f.jpeg"},{"id":92238344,"identity":"ed947d14-8bd6-425e-9992-13408d21b78a","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"jpeg","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2114,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/5156c799c219b52aa44ac541.jpeg"},{"id":92238339,"identity":"57aadb9b-5f94-4a10-949a-eb67d1fd1c05","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"jpeg","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2114,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/96109cf92ca5b1b49038f992.jpeg"},{"id":92239253,"identity":"0ac97854-41a8-4084-a181-db72f80aa3d0","added_by":"auto","created_at":"2025-09-26 08:10:50","extension":"jpeg","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2114,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/2a66d569c1d116f4f86d2cc3.jpeg"},{"id":92239254,"identity":"2e066e43-0b17-4931-b679-bcc5d40ddb95","added_by":"auto","created_at":"2025-09-26 08:10:50","extension":"jpeg","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2114,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/8fab4b0faa89e247c0672b0f.jpeg"},{"id":92238358,"identity":"cf2e3607-8118-4943-80e3-f0e91c5ee969","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":413281,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/091c96c891fc19dcf0f94118.png"},{"id":92238365,"identity":"99b88d75-12d4-4110-9a69-a04862c6e7c3","added_by":"auto","created_at":"2025-09-26 07:54:51","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1132,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/5171ce3821c2f7af834ca0b1.png"},{"id":92238371,"identity":"a8cf8101-adfa-4eb2-b924-d75f92b38979","added_by":"auto","created_at":"2025-09-26 07:54:51","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1132,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/d17fbba0d94ab9fce6b3fb96.png"},{"id":92238341,"identity":"7049def1-58da-47cf-bf2b-4a33292817af","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1132,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/1bba9a885524a05ee78240da.png"},{"id":92238554,"identity":"af9386f8-9f45-4483-94f0-16857ed4652c","added_by":"auto","created_at":"2025-09-26 08:02:51","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1132,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/1fdaaac6d8e76e5961cc488f.png"},{"id":92238550,"identity":"12fe79c1-1493-400c-b3c5-b759d69ccea3","added_by":"auto","created_at":"2025-09-26 08:02:50","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":371046,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/01d2b14726d06873487c04a5.png"},{"id":92238551,"identity":"d7dceb65-ffe1-462a-8988-3b0c93f3fabf","added_by":"auto","created_at":"2025-09-26 08:02:50","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":174250,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/49bc84bbe70fe93b4fe3aeca.png"},{"id":92238338,"identity":"72f86d10-1021-44bf-b26c-ef38d3d5852f","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":81525,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage16.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/c330ad765680872b8e87e251.png"},{"id":92238364,"identity":"838d2c47-2ffd-4465-98fa-6c19993b1444","added_by":"auto","created_at":"2025-09-26 07:54:51","extension":"png","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":293548,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/844f0cb9ccf3ae48b25d6427.png"},{"id":92238357,"identity":"93a91a50-744d-4baa-b4af-93da6b745274","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3933,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/245ff9f651b955e0d04d40fd.png"},{"id":92238555,"identity":"8e9ed1b2-cab9-4690-b2ab-826acc51a17c","added_by":"auto","created_at":"2025-09-26 08:02:51","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1132,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/cf7ee21e5bddb21bce3ff74c.png"},{"id":92238355,"identity":"d99ee9a2-6e34-440f-9f81-a028f0da5fe0","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"png","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1132,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/28a3504b586242de22a562fd.png"},{"id":92238368,"identity":"b38de54f-f8a1-49c8-b558-d37b06cb5e63","added_by":"auto","created_at":"2025-09-26 07:54:51","extension":"png","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1132,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/c567173c4617780c8bd781c3.png"},{"id":92238372,"identity":"a9e69c97-0671-44ba-90f8-bbd89be8fee6","added_by":"auto","created_at":"2025-09-26 07:54:52","extension":"png","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1132,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/6c085cdcba39bd9dd27c792a.png"},{"id":92238363,"identity":"444342ef-d435-4ce0-b977-8b0420335844","added_by":"auto","created_at":"2025-09-26 07:54:51","extension":"png","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1132,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/f11ba5f6fce68db0db1a664b.png"},{"id":92238545,"identity":"1a376cfd-258c-4f99-9438-fa2deaafe607","added_by":"auto","created_at":"2025-09-26 08:02:50","extension":"png","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1132,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/b44d33f216563edfa2e2743a.png"},{"id":92238548,"identity":"1154ae18-447e-4ec0-8bb3-11bcd8ae058a","added_by":"auto","created_at":"2025-09-26 08:02:50","extension":"xml","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133852,"visible":true,"origin":"","legend":"","description":"","filename":"1ed61b7676464cccbc181cdd016d67de1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/dcdd04ca87b558a465ac236e.xml"},{"id":92238366,"identity":"f2b192b8-5602-48a9-a62b-ec0770c275b0","added_by":"auto","created_at":"2025-09-26 07:54:51","extension":"html","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":147002,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/360f7ff2b35a24e72ef3426f.html"},{"id":92238329,"identity":"26cc9275-c00d-4fe7-a9c9-3f634ec42cfc","added_by":"auto","created_at":"2025-09-26 07:54:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":680509,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicrovine Callus and ECS development. A\u003c/strong\u003e Flowering microvine plant. \u003cstrong\u003eB\u003c/strong\u003e Immature inflorescence used for dissection (with flowers that have just began to separate from each other). \u003cstrong\u003eC\u003c/strong\u003e Slightly transparent and yellow stage of stamen used for callus induction. \u003cstrong\u003eD\u003c/strong\u003e Reproductive floral tissues (after removing calyptra) on PIV medium producing callus. \u003cstrong\u003eE\u003c/strong\u003e Friable, transparent somatic embryos emerging from embryogenic callus after ~4 months on CIM. Desirable friable somatic embryos marked with red arrows. \u003cstrong\u003eF\u003c/strong\u003eCream/translucent globular somatic embryos (~50 mg fresh weight) used for initiating ECS. \u003cstrong\u003eG\u003c/strong\u003e Inoculation of somatic embryos in suspension medium for ECS culture initiation. \u003cstrong\u003eH \u003c/strong\u003eProliferation of suspension cells after two months with continuous shaking under dark conditions at 28˚C (view from the bottom of the flask).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/0e033b72b067959290b91d25.png"},{"id":92238324,"identity":"c959886e-acef-46ac-955e-356be2a6dfd0","added_by":"auto","created_at":"2025-09-26 07:54:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":986338,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroscopic observation of ECS. A-C\u003c/strong\u003e are of the same ECS cell line culture. \u003cstrong\u003eA\u003c/strong\u003e Large cell cluster of embryogenic cells (E with arrow) with dense cytoplasm (filled with starch granules). \u003cstrong\u003eB \u0026amp; C\u003c/strong\u003e Different sized embryogenic cell clusters showing a compact and generally spherical shape. \u003cstrong\u003eD\u003c/strong\u003e Non-embryogenic cells (NE with arrow) with irregular shapes and large vacuoles without starch granules from an old cell suspension culture that cannot efficiently regenerate shoots.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/d3816b63724c4adcb23cce84.png"},{"id":92239252,"identity":"7c79428d-54be-4e09-a4b6-ead53c3196e1","added_by":"auto","created_at":"2025-09-26 08:10:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":30291,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAgrobacterium-\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emediated transformation protocol of ECS\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/2b2beb0148e03983c4c21cd0.png"},{"id":92238340,"identity":"065fe62a-a282-48f1-ac28-f2c0da04e8f2","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":821665,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAgrobacterium-\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emediated transformation of ECS cultures and the recovery of microvine transgenic plants. A\u003c/strong\u003e Schematic diagram of the T-DNA construct used for ECS \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation. The \u003cem\u003enopaline synthase \u003c/em\u003epromoter (NOSp) and terminator (NOSt) controls expression of \u003cem\u003enptII\u003c/em\u003e (to confer plant resistance to kanamycin) and the microvine \u003cem\u003eubiquitin 7\u003c/em\u003e promoter (\u003cem\u003eVviUbi7\u003c/em\u003ep) with 5ʹ intron (dashed arrow) and CaMV35S terminator (35St) controls \u003cem\u003emCherry\u003c/em\u003e reporter gene expression, \u003cem\u003eAgrobacterium\u003c/em\u003e T-DNA (Transfer-DNA) left border (LB) and right border (RB). \u003cstrong\u003eB\u003c/strong\u003e Suspension cells after infection with \u003cem\u003eAgrobacterium\u003c/em\u003e. \u003cstrong\u003eC\u003c/strong\u003e The suspension cells on EDM and divided into groups after washing with cefotaxime. \u003cstrong\u003eD\u003c/strong\u003e Transformed cells showing red fluorescence (3 second exposure) four days after co-cultivation. \u003cstrong\u003eE\u003c/strong\u003e Somatic embryo development on EDM with cefotaxime (250 mg/L) and kanamycin (50 mg/L) three months after co-cultivation. \u003cstrong\u003eF\u003c/strong\u003e Transformed suspension cells exhibiting red florescence (1 second exposure) three months after co-cultivation. \u003cstrong\u003eG\u003c/strong\u003e Regenerating shoots and roots on SSM medium (without selection antibiotic) 20 days after subculturing. \u003cstrong\u003eH\u003c/strong\u003e Regenerated plants growing on SSM-I medium containing GA3\u0026nbsp;(10 mg/L) 60 days after subculturing. \u003cstrong\u003eI\u003c/strong\u003e Transgenic microvine plants growing in soil.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/be069329a4f8bf9bdaf8f054.png"},{"id":92238332,"identity":"7711c35f-8c85-4f8e-a8b6-2f983ab88149","added_by":"auto","created_at":"2025-09-26 07:54:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":281018,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTimeline to develop plants from ECS after \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAgrobacterium\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-mediated transformation\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/63b019f50fc6ff596b437b3a.png"},{"id":92238331,"identity":"9246534d-73a9-4a90-bef8-7b460fd02309","added_by":"auto","created_at":"2025-09-26 07:54:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53525,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCopy number of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enptII\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in eighteen independent microvine transgenic plants derived from ECS.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/338a8c03df3952696cd0bf7a.png"},{"id":100549395,"identity":"439bc7b6-b705-485c-96ac-b989f9213755","added_by":"auto","created_at":"2026-01-19 08:23:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3985594,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/2b5531c8-1b46-42b3-9098-692beebe456b.pdf"},{"id":92238544,"identity":"35fa6d2a-6929-4cb8-8846-f4100c9ca0a1","added_by":"auto","created_at":"2025-09-26 08:02:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2244570,"visible":true,"origin":"","legend":"","description":"","filename":"FiguresS1S5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/e84b3a30e1c618613fbf9b39.pdf"},{"id":92238334,"identity":"b326e6a3-ac38-48dd-bccd-71f97da4a028","added_by":"auto","created_at":"2025-09-26 07:54:50","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17761,"visible":true,"origin":"","legend":"","description":"","filename":"TablesS1S3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/0f73b3daa508511c66f5554b.xlsx"},{"id":92238328,"identity":"ecd900b2-bd32-4c69-b4b4-8c400e685f93","added_by":"auto","created_at":"2025-09-26 07:54:49","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":34135,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1Mediarecipes.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7661205/v1/2bde25f826e7f5b12ea066f3.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Harnessing embryogenic cell suspension culture for Agrobacterium-mediated transformation of microvine","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGrapevine is typically genetically engineered using somatic embryos or embryogenic calli derived from reproductive tissues. However, its large size, long juvenile period, and biannual flowering impedes research progress. The Pinot Meunier cultivar of grapevine carries the \u003cem\u003eVvigail\u003c/em\u003e (\u003cem\u003egibberellic acid-insensitive 1\u003c/em\u003e) gene mutation in the epidermal cell L1 layer of its apical meristem. \u003cem\u003eVviGAI1\u003c/em\u003e is involved in gibberellin signaling and this single nucleotide mutation converts leucine to a histidine in the DELLA domain of the protein [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], resulting in a tomentose (densely covered with trichomes) phenotype. Plants somatically derived from Pinot Meunier that are heterozygous for the \u003cem\u003eVviGAI1/Vvigai1\u003c/em\u003e mutation are called \u0026ldquo;microvine\u0026rdquo; and have a dwarf and hairy phenotype along with the conversion of tendrils into inflorescences, producing constant but physiologically normal flowering and fruiting. Microvine therefore serves as a model system for grape research and functional genomic studies. The original microvine mutant was not amenable to \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation, so to overcome this limitation, crosses were performed and the hybrid progeny tested for embryogenic callus development and transgenic plant regeneration. The microvine genotype 04C023V0004 (V4), resulting from a cross between the L1 mutant and the black-berried variety \u003cem\u003eGrenache\u003c/em\u003e, showed improved transformation efficiency [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and has been used to create transgenic plants in previous studies [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, despite the advantages of microvine as an amenable model, its transformation efficiency is still relatively low, transgenic shoot regeneration and development is inefficient and slow, and establishment of the regenerated plants in soil is challenging. The prolonged period in tissue culture can also increase the chance of recovering plants with undesirable somaclonal variation.\u003c/p\u003e\u003cp\u003ePreviously, embryogenic cell suspension (ECS) cultures were used to significantly improve plant regeneration and transformation efficiency in multiple crop species [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. ECS culture has a good cell division rate, is more easily subcultured than callus on semi-solid media, and has a high plant regeneration rate [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. ECS has been shown to enhance \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation due to higher explant tissue uniformity, accessibility to infection, and improved selection efficiency for transformed tissues [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Despite these advantages, a higher susceptibility to bacterial and fungal contamination, shorter subculture intervals, and early senescence are challenges with the use of ECS cultures. In grapevine, cell suspension cultures were previously shown to improve regeneration in Thompson Seedless, Chardonnay, Richter 110, Cabernet Sauvignon, Chancellor, and Mencίa [\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Plant regeneration from somatic embryos of grapevine interspecific hybrids was also previously reported from liquid cultures [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Chardonnay suspension culture has also been used for transformation in gene editing studies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Liquid medium derived somatic embryos can develop a distinct embryonic structure known as the suspensor (a cellular connection between the embryo and surrounding tissue that transfers nutrients and growth regulators for embryo development) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and smaller cotyledons than counterpart proembryogenic masses grown on solid media. These structures correlated with more advanced shoot apical meristem development, little dormancy during germination relative to solid media grown somatic embryos and produce plants more efficiently [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe objective of this study was to establish microvine V4 suspension culture from embryogenic calli and to rapidly and efficiently generate transgenic plants using \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation. The new protocol successfully created high-viability suspension cultures, ample embryogenic explant material for transformation and produced numerous healthy transgenic microvine plants.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCallus induction and proliferation\u003c/h2\u003e\u003cp\u003eMicrovine V4 plants (provided by Dr. Paul Boss and Dr. Ian Dry, CSIRO, via Dr. Laurent Deluc) were grown in a USDA greenhouse in Albany, California. Inflorescences were collected as an explant source just after the immature flowers had begun to separate from each other. The flowers were washed under running water, divided into small clusters, and were surface sterilized with 100 mL of fresh 20% bleach solution containing a drop of Tween-20 in a conical flask for 1 minute. The flowers were washed three times with sterile water to remove residual bleach, then drained and either used directly for dissection or stored at 4\u0026ordm;C for up to three days before dissection. Flowers were dissected as previously described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and cultured on callus initiation medium (PIV) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The culture plates were incubated in the dark at 28\u0026ordm;C and the resulting callus was transferred onto callus proliferation and maintenance medium (CIM) after 3 weeks for further callus proliferation. Embryogenic callus was transferred every 3 weeks onto new CIM media and incubated at 28\u0026ordm;C in the dark until use.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eECS culture establishment\u003c/h3\u003e\n\u003cp\u003eImmature somatic embryos from ~\u0026thinsp;4 month-old embryogenic calli were used to initiate a suspension culture. The friable, translucent or cream-colored globular somatic embryos (~\u0026thinsp;50 mg fresh weight) were transferred into a 50 mL Erlenmeyer flask containing 7 mL of suspension medium (Supplementary file 1) and incubated at 28\u0026ordm;C with 90 rotations per minute (rpm) in the dark on a rotary shaker. ECS cultures were refreshed once a week by replacing 6 mL of old medium with new suspension medium. As the suspension cells proliferate, small cell clusters were transferred to a sterile large flask (120/250 mL) maintaining a suspension cells/medium volume ratio of 5\u0026ndash;10% (approximately 15 mL suspension medium in 120 mL flask, measured using settled cell volume of the suspension cells in a 50 mL Falcon tube) and the residual bigger cell clusters in initial 50mL flask were maintained as mentioned above. Subculturing large flasks of ECS cultures was performed every 10 days. After transferring to a large flask, the suspension culture can be used directly for transformation or grown for future use. Bigger cell clusters within large flasks were filtered out using a 3.3-inch sieve to synchronize the cultures\u0026rsquo; developmental stage every 1\u0026ndash;2 months (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). However, initial small flask cultures were not filtered, but rather just transferred and maintained in a 50 mL flask for continuous proliferation. Suspension cells along with the medium were placed on a glass slide, covered with a coverslip, and observed directly under an Olympus microscope BX51 (Olympus Corporation, Tokyo, Japan) to assess their status and produce the images shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eRegeneration and plant development\u003c/h3\u003e\n\u003cp\u003eSuspension cells (small cell clusters) along with a minimal amount of suspension medium was transferred with a pipette onto embryo development and maturation (EDM) medium (Supplementary file 1) for regeneration. Plates were incubated at 28\u0026ordm;C in the dark in a growth chamber (Percival Scientific, Iowa, US). After 30\u0026ndash;50 days on EDM, small cell clusters developed into matured somatic embryos and at the torpedo/cotyledon stage were transferred to suspension shooting medium (SSM) (Supplementary file 1) for growth and maintained at 28\u0026ordm;C under a 16-hour light/8-hour dark cycle (40\u0026ndash;70 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). When plantlets regenerated, they were transferred onto SSM-I (Supplementary file 1) under the same light and temperature conditions for further development. Once plants had grown to 4\u0026ndash;5 cm in height, they were transferred to soil in pots, covered with polythene bags or a plastic dome to retain humidity for acclimatization, and maintained at 28\u0026ordm;C under a 16-hour light (40\u0026ndash;70 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) /8-hour dark cycle in a growth chamber (Conviron, North Dakota, US).\u003c/p\u003e\n\u003ch3\u003eConstruct design\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eVviUbi7\u003c/em\u003e promoter (Accession number: PV929291), including it\u0026rsquo;s 5ʹ intron (2,151 bp) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], was amplified from microvine V4 genomic DNA and cloned into a Level 0 Golden Gate acceptor backbone from the MoClo Golden Gate library [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. PCR amplification was performed using Q5 high fidelity DNA Polymerase (New England Biolabs, MA, US) with sequence specific primers (Supplementary file 2: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and microvine V4 genomic DNA under the following cycling conditions: 98\u0026ordm;C for 30 seconds, 35 cycles of 98\u0026ordm;C for 10 seconds, 67\u0026ordm;C for 10 seconds, 72\u0026ordm;C for 1.10 minutes, and a final extension of 72\u0026ordm;C for 2 minutes. The Level 0 vector carrying the \u003cem\u003eVviUbi7\u003c/em\u003e promoter was confirmed with Sanger DNA sequencing and cloned into a Level 2 acceptor vector driving the expression of the red fluorescence gene \u003cem\u003emCherry\u003c/em\u003e with a CaMV35S terminator and a kanamycin resistance plant selection cassette. A diagram of the vector construct T-DNA is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAgrobacterium\u003c/b\u003e\u003cb\u003e-mediated transformation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSuspension cells were transformed using the \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain AGL1 containing the VviUbi7-mCherry binary vector plasmid. \u003cem\u003eAgrobacterium\u003c/em\u003e culture was prepared as previously described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and resuspended in co-cultivation medium (Supplementary file 1) with 400 \u0026micro;M acetosyringone at an OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.2. Five hundred milligrams of suspension cells were collected into a 50 mL falcon tube and the original medium replaced with fresh media. Cells were then heat shocked at 45\u0026ordm;C for 5 minutes just prior to \u003cem\u003eAgrobacterium\u003c/em\u003e co-cultivation. Ten milliliters of the \u003cem\u003eAgrobacterium\u003c/em\u003e cocultivation medium were added to 500 mg suspension cells and mixed gently at room temperature for 10 minutes on a rotatory shaker at 50 rpm. After shaking, suspension cells were allowed to settle to the bottom of the tube. Five milliliters of medium were removed, and the remaining solution and cells were transferred to filter paper (a stack of 2\u0026ndash;3 sterile filter paper disks) placed on a sterile petri plate using a serological pipette. Once the cells appeared air-dried in the laminar flow hood, the filter paper with cells were transferred to CIM plates and co-cultured at 22\u0026ordm;C in dark for 2 days. After co-cultivation, suspension cells were transferred to a falcon tube containing 10 mL suspension medium with 400 mg/L cefotaxime using a cell scraper (Alkali Scientific, Florida, US) and mixed on a rotatory shaker (50 rpm) for 15\u0026ndash;30 minutes at room temperature. The cells were washed 3\u0026ndash;4 times with suspension medium to remove the \u003cem\u003eAgrobacterium\u003c/em\u003e and then transferred onto stacked filter papers in a sterile petri dish to air dry (carefully without over drying the cells) in the laminar flow hood.\u003c/p\u003e\u003cp\u003eThe dried cells were then transferred with a cell scraper to semi-solid EDM medium containing cefotaxime (250 mg/L) and divided into small groups of cells to track individual transformation events. Cultures were incubated in the dark at 28\u0026ordm;C for 20 days and observed under a microscope for red fluorescence to confirm transgene expression. The transformed cells were subcultured onto EDM media containing 250 mg/L cefotaxime and plant selection antibiotic (50 mg/L kanamycin) once a month until they develop mature somatic embryos. Mature somatic embryos exhibiting red florescence were then transferred to SSM containing 250 mg/L cefotaxime and GA\u003csub\u003e3\u003c/sub\u003e (5 mg/L) and maintained at 28\u0026ordm;C in light (16h/8h light/dark at 40\u0026ndash;70 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e light intensity) in a growth chamber. Regenerated plantlets were transferred onto SSM-I with GA\u003csub\u003e3\u003c/sub\u003e (10 mg/L) for further development and later transferred to soil for acclimatization under the same conditions before being moved to the greenhouse.\u003c/p\u003e\n\u003ch3\u003eMolecular analysis\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted using a modified Puregene kit protocol (Qiagen, Redwood City, CA, US). Leaf samples (~\u0026thinsp;0.5 g) were snap frozen in liquid nitrogen in 1.5 mL centrifuge tubes and ground into a fine powder using a plastic mortar. 400 \u0026micro;L of cell lysis solution and 25 \u0026micro;L of β-mercaptoethanol were added to the ground sample, vortexed, and incubated at 65\u0026ordm;C for 1 hour, with additional mixing at 30 and 60 minutes. 5 \u0026micro;L of RNase A (10 mg/mL) solution was added, mixed gently, and incubated at 37\u0026ordm;C for 30 minutes. The sample was placed on ice for 1 minute to cool, and 133 \u0026micro;L of protein precipitation buffer was added. The mixture was vortexed for 20 seconds and put on ice for 20 minutes. The samples were then centrifuged for 3 minutes at 13,000 x g, and the supernatant was transferred into a new 1.5 mL microcentrifuge tube. Equal amounts of chloroform: isoamyl alcohol were added, inverted 50 times, and centrifuged for 5 minutes at 13,000 x g. The supernatant was transferred into a new 1.5 mL microcentrifuge tube, an equal amount of 100% isopropanol was added and mixed by inverting 50 the tube times. The samples were then incubated on ice for 5 minutes and then centrifuged for 1 minute at 13,000 x g. The supernatant was discarded, and the DNA pellet was washed with 300 \u0026micro;L of 70% ethanol, mixed by inverting, and centrifuged at 13,000 x g for 1 minute. The supernatant was discarded, and the genomic DNA pellet was air dried and then dissolved in 30 \u0026micro;L of sterile ultra-pure water. Genomic DNA was quantified with a DeNovix microvolume spectrophotometer (DeNovix, Davis, CA) and its integrity evaluated by separation on a 1% agarose gel prior to use.\u003c/p\u003e\u003cp\u003eDroplet digital PCR (ddPCR) was used to detect the copy number in the T\u003csub\u003e0\u003c/sub\u003e transgenic events. The grapevine \u003cem\u003echalcone synthase (VviCHI)\u003c/em\u003e gene [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] was used as a single copy reference gene. Primers and probes (Supplementary file 2: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were designed for the reference gene and as used previously for \u003cem\u003enptII\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] following the manufacturer\u0026rsquo;s instructions (Bio-Rad Laboratories, Hercules, CA, US). A FAM\u0026trade;-labeled probe was used to detect the reference gene amplicon and a HEX\u0026trade;-labeled probe for the \u003cem\u003enptII\u003c/em\u003e amplicon detection. Genomic DNA (200 ng) of transgenic and control plants was digested with EcoRI-HF restriction enzyme (New England Biolabs, MA, US) prior to ddPCR. Each 20 \u0026micro;L reaction contained 10 \u0026micro;L of 2x ddPCR supermix for probes (no dUTP) (Bio-Rad), 0.5\u0026micro;l of each primer (22.5 \u0026micro;M), 0.25 \u0026micro;l of reference and target probe (25\u0026micro;M), 3\u0026micro;l of digested DNA (60\u0026ndash;100 ng), and 5.5\u0026micro;l water for a total reaction volume of 20 \u0026micro;l. Droplets were generated in the QX200 droplet generator for each sample and PCR was carried out at: 95\u0026ordm;C for 10 minutes, 40 cycles (94 \u0026ordm;C for 30s, 60 \u0026ordm;C for 1 minute), 98 \u0026ordm;C for 10 minutes, 12 \u0026ordm;C hold. After PCR, the droplets were read in the Bio-Rad QX200 droplet reader, and the data was analyzed using QuantaSoft software.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eDevelopment of microvine ECS\u003c/h2\u003e\u003cp\u003eThe development of callus and suspension cultures using stamen explants are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. An early stage microvine inflorescence is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. Immature flowers that are the most suitable for initiating embryogenic callus are at the stage when they begin to separate from each other within the floral cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). At this stage, the anthers are pale yellow and appear translucent (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). We tested stamens, filaments, anthers, pistils, and whole flowers with the calyptra and without the calyptra as tissue types for callus induction and found that intact flowers without the calyptra produced the greatest number of embryogenic calli, but stamens and dissected filaments also generate embryogenic calli. Within 20 days of dissection, anthers turn brown, and the filament tips attached to the ovary swell (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Induced callus starts to proliferate within two rounds of subculturing on CIM and was further maintained by transferring to new CIM medium every 2\u0026ndash;4 weeks. Embryogenic callus generation efficiency in microvine V4 was 2\u0026ndash;6% (number of embryogenic calli/total number of stamens\u003csup\u003e*\u003c/sup\u003e100) from healthy, greenhouse-grown plants.\u003c/p\u003e\u003cp\u003eAfter approximately four months of subculturing on CIM, friable, translucent or cream-colored globular somatic embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) were selected to initiate ECS culture in 50 mL flasks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Selection of globular somatic embryos is the most critical step in establishing an ECS culture. Compact callus pieces fail to develop ECS and eventually turn brown within 2\u0026ndash;3 weeks in liquid medium (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e A-C). Approximately 50 mg fresh weight of somatic embryos were used to initiate the cell suspension culture in 7 mL of suspension medium in a 50 mL Erlenmeyer flask. During subculturings, removal of brown cell clusters (if any) helps to maintain a healthy ECS culture. Embryogenic cultures typically proliferate within 2 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). After proliferation, the small cell clusters from culture were transferred into larger flasks (120/250mL), leaving behind the bigger cell clusters in the original flask for further proliferation, maintaining cells and suspension medium ratio of 5\u0026ndash;10% as shown in Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. When bigger cell clusters appear in the larger flask suspension cultures, they were strained through a sieve and removed. This process aids in maintaining a culture with uniform smaller sized cell clusters within the ECS culture (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMicroscopic observations of ECS cultures show many small clusters of cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). A healthy embryogenic suspension cell culture that can efficiently generate shoots will typically contain small spherical cell clusters where the cells are filled with dense cytoplasm (starch granules) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C. Non-embryogenic cultures contain cells and cell clusters with irregular shapes, a large vacuole and no/less starch granules (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These non-embryogenic cells and cell clusters were frequently observed in older ECS cultures and became more abundant as they aged. When these non-embryogenic cells predominate in a culture, it is no longer useful for plant regeneration.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePlant regeneration from microvine ECS cultures\u003c/h3\u003e\n\u003cp\u003eSuspension cells (along with a minimal amount of the suspension medium) were transferred onto EDM plates to examine their shoot regeneration capacity (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). The suspension cells developed into mature somatic embryos on EDM after approximately 30\u0026ndash;50 days of subculturing on the semi-solid medium (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB), although the exact timeframe can vary between independently derived ECS cultures. Mature globular and torpedo staged somatic embryos were observed under the microscope (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC) for cultures with the capacity to regenerate shoots. These explants turned green and grew into small shoots with roots on SSM after 15\u0026ndash;30 days (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD). These shoots then developed into plantlets after 1\u0026ndash;2 months of subculturing on SSM-I containing gibberellin hormone (Figure S4). Subsequently, healthy 4\u0026ndash;5 cm tall plantlets were transferred to soil for acclimatization and further growth (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAgrobacterium\u003c/b\u003e\u003cb\u003e-mediated transformation of microvine ECS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the efficiency of transformation in microvine ECS cultures, we used an \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation approach. A flow-chart of the procedure is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Schematic diagram of the transformation construct is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. Co-cultivation of suspension cells with \u003cem\u003eAgrobacterium\u003c/em\u003e carrying the binary vector construct is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. After co-cultivation, the plant cells were washed and transferred as small groups of cell clusters to semi-solid EDM containing kanamycin and cefotaxime (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Transformed cell foci exhibited red florescence four days post co-cultivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The transformed cells matured into the torpedo and cotyledon staged somatic embryos on EDM selection medium within two-three months (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These mature somatic embryos exhibited bright red fluorescence confirming the stable integration and expression of the \u003cem\u003emCherry\u003c/em\u003e transgene (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). The transformed embryos were then transferred to SSM medium and developed roots and shoots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), but some of these shoots exhibited slow and/or abnormal growth (Figure S5). To improve shoot growth and development, we supplemented the SSM medium with four different concentrations of GA\u003csub\u003e3\u003c/sub\u003e: 5 mg/L, 8 mg/L, 10 mg/L and 15 mg/L. Microvine shoots developed into healthy plantlets on SSM-I containing GA\u003csub\u003e3\u003c/sub\u003e after an additional 60 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). We observed a greater number of normal healthy shoots with the addition of GA\u003csub\u003e3\u003c/sub\u003e, and the highest percentage of normal shoots were observed with 10mg/L GA\u003csub\u003e3\u003c/sub\u003e (Figure S4, Supplementary file 2: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Plants grown with 15mg/L GA\u003csub\u003e3\u003c/sub\u003e showed some curling of shoot tips. After two months, well-established rooted plants were transferred to soil and exhibited normal growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). This approach generated approximately 150 red fluorescent transgenic plants from 500 mg fresh weight of starting ECS culture. Eighteen randomly selected plants were grown in soil for further characterization. The overall timeline for the generation of transgenic microvine V4 plants from ECS cultures is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMolecular confirmation of transgenic plants\u003c/h2\u003e\u003cp\u003eEighteen independent putative transgenic plants were analyzed with ddPCR to confirm T-DNA integration and measure \u003cem\u003enptII\u003c/em\u003e copy number. The transgene quantification was calculated using the endogenous single-copy grapevine gene \u003cem\u003eVviChi\u003c/em\u003e as a reference [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Among the tested plants, four carried a single-copy integration, five had two copies of \u003cem\u003enptII\u003c/em\u003e, and nine plants contained three or more transgene copies (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). These results demonstrated that the use of ECS cultures with \u003cem\u003eAgrobacterium\u003c/em\u003e transformation generates stably transformed microvine V4 plants carrying 1 or 2 copies of an introduced transformation construct half of the time.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eA major bottleneck in grapevine biotechnology is the rapid regeneration of transgenic plants carrying constructs of interest. Microvine is a desirable grapevine research model system because of several favorable attributes, but it is somewhat recalcitrant for shoot regeneration and grows relatively slowly in culture in part due to its gibberellin insensitivity [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In this study, we report the development of ECS cultures from microvine V4 embryogenic calli and a streamlined protocol for \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation.\u003c/p\u003e\u003cp\u003eThe first step for producing ECS is the establishment of embryogenic calli and the selection of suitable explant starting material. In previous studies, stamens, pistils and whole flowers produced embryogenic calli on PIV medium in grapevine [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In the current study, microvine filament tissues induced callus from stamens as well as flowers without calyptra. We observed more embryogenic callus production with whole flowers without calyptra as compared to stamens only as observed for other grapevine cultivars [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This may be due to the presence of endogenous hormone levels within these reproductive structures that contribute to more efficient callus induction [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The second most important consideration for establishing ECS is the selection of good quality starting material from the embryogenic calli. Inoculation of whole embryogenic callus even with good somatic embryos may result in mixed culture and can turn brown later. The selection of globular somatic embryos for ECS initiation, significantly influences the quality and uniformity of the resulting suspension culture [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. An earlier study also reported that somatic embryos from Chardonnay callus produced a rapidly growing suspension culture [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, specificity of starting material used for establishing Chardonnay ECS is not mentioned in other studies [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Some articles reported use of embryogenic cells, embryo masses and pro-embryonic masses in Chardonnay, Cabernet Sauvignon, rootstocks and Mencίa grapevine varieties [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our study demonstrated that immature, friable, and translucent/cream colored somatic embryos at the globular stage exhibit the highest embryogenic potential in liquid culture, consistent with previous studies in other crop species [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. For maintaining ECS, the ratio of suspension cells to suspension medium also plays an important role, as an excessive number of cells in the culture can lead to browning and cell death as observed previously in grapevine and palm [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Frequently subdividing cells into new flasks reduces cell browning as reported earlier in Chardonnay and Thompson seedless [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Cell debris sticking to the inner wall of the flask may also contribute to the browning of the cells and the removal of these dead cells by transferring the culture to a new flask is necessary to maintain healthy suspension cultures. Using the reported protocol, we were able to maintain high quality microvine ECS cultures for more than one year before they lost their capacity to efficiently regenerate plants. Suspension cells have been shown to be a good source material for protoplast isolation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], so this may be a useful approach to produce microvine protoplasts as well.\u003c/p\u003e\u003cp\u003ePreviously, grapevine cell suspension culture has been used for biolistic and \u003cem\u003eAgrobacterium-\u003c/em\u003emediated transformation but has been a relatively inefficient approach for producing plants [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In contrast, one report produced 240 plants/10 ml settled cell volume with Chardonnay suspension culture and another generated 37 positive plants with Chancellor cell suspension using a biolistic approach [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Another study of Chardonnay \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation that used liquid selection medium to generate resistant cell masses, reported the generation of four transgenic plants [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, microvine transformation has been only reported using calli explants with \u003cem\u003eAgrobacterium-\u003c/em\u003emediated transformation and no reports describe the development of microvine cell suspension cultures. One of those previous studies reported the recovery of nine microvine transgenic plants [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and other studies have reported the time for the production of microvine transgenic plants was 6\u0026ndash;10 months [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Conversely, ECS have been reported to be highly responsive to \u003cem\u003eAgrobacterium\u003c/em\u003e infection and amenable to efficient shoot regeneration in crops like banana, citrus, switchgrass, and medicinal plants [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The reported protocol generated approximately 150 transgenic microvine plants in about five months from the start of \u003cem\u003eAgrobacterium\u003c/em\u003e cocultivation and all eighteen of candidate transgenic plants that were selected for molecular characterized were red fluorescent and transgene positive. For better recovery of transgenic plants, we heat shocked suspension cells prior to \u003cem\u003eAgrobacterium\u003c/em\u003e infection as reported previously to improve transformation efficiency [\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and discontinued the use of antibiotic selection at the shooting stage as a negative impact of kanamycin selection on shoot growth and regeneration was reported previously [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Heat shock promotes the survival of transformed cells due to the prevention of programmed cell death [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and induces the production of heat shock proteins that promotes DNA transfer during \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation [\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The use of the mCherry red fluorescent reporter enabled the reliable recovery of nonchimeric transgenic microvine plants.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn the present study, a method for producing rapidly growing microvine ECS cultures is described. These ECS cultures were demonstrated to be a viable source material for \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation producing abundant fluorescent transgenic shoots and healthy transgenic plants (150 transgenic plants/500 mg ECS) within approximately 5\u0026ndash;7 months. An advantage of using microvine ECS cultures is that they grow rapidly and can provide an ample source material for transformation and other experiments while requiring a modest amount of time and effort to maintain compared to embryogenic callus. The protocol presented can be used to efficiently engineer novel traits, or to deploy CRISPR genome editing reagents to better understand gene function in microvine. In the future, this approach could potentially be adapted for other grapevine genotypes enabling more efficient biotechnology research in this important crop.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eV4\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;microvine genotype 04C023V0004\u003c/p\u003e\n\u003cp\u003eECS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;embryogenic cell suspension\u003c/p\u003e\n\u003cp\u003eGAI 1\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/em\u003eGibberellic acid insensitive 1\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eVviUbi7\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/em\u003eMicrovine \u003cem\u003eUbiquitin 7\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGA\u003csub\u003e3\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/sub\u003eGibberellic acid\u003c/p\u003e\n\u003cp\u003ePCR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003eddPCR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;droplet digital PCR\u003c/p\u003e\n\u003cp\u003ePIV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Callus initiation medium\u003c/p\u003e\n\u003cp\u003eCIM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Callus proliferation medium\u003c/p\u003e\n\u003cp\u003eEDM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Embryo development and maturation medium\u003c/p\u003e\n\u003cp\u003eSSM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Suspension shooting medium\u003c/p\u003e\n\u003cp\u003eCaMV \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cauliflower mosaic virus\u003c/p\u003e\n\u003cp\u003eCRISPR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Clustered Regularly Interspaced Short Palindromic Repeats\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics and consent to participate declarations\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\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe microvine \u003cem\u003eVviUbi7\u003c/em\u003e promoter sequence used in the study is available from GenBank (accession number\u0026nbsp;PV929291).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a United States Department of Agriculture-Agricultural Research Service (USDA-ARS) project 2030-21220-003-000-D and an appointment to the ARS Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the United States Department of Energy (DOE) and the USDA. ORISE is managed by Oak Ridge Associated Universities under DOE contract number DE-SC0014664. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT and S designed the study. S, TM and JTP performed experiments. S wrote the manuscript and designed the figures. All authors contributed to editing and revising the manuscript. RT provided supervisory guidance and funding support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would also like to thank James Horstman for his support in developing and maintaining embryogenic calli and for skilled greenhouse management.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBoss PK, Thomas MR. Association of dwarfism and floral induction with a grape \u0026lsquo;green revolution\u0026rsquo; mutation. Nat 2002, 416(6883):847\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePellegrino A, Romieu C, Rienth M, Torregrosa L. The microvine: a versatile plant model to boost grapevine studies in physiology and genetics. In: Adv grape wine Biotechnol IntechOpen; 2019.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTorregrosa LJ-M, Rienth M, Romieu C, Pellegrino A. The microvine, a model for studies in grapevine physiology and genetics. OENO One. 2019;53(3):373\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChaib J, Torregrosa L, Mackenzie D, Corena P, Bouquet A, Thomas MR. The grape microvine - a model system for rapid forward and reverse genetics of grapevines. Plant J 2010, 62(6):1083\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGouthu S, Deluc LG. Use of the microvine and plant gene switch system for functional studies of genes involved in the control of ripening initiation in \u003cem\u003eVitis vinifera\u003c/em\u003e. Acta Hortic 2019(1248):187\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOndzighi-Assoume CA, Willis JD, Ouma WK, Allen SM, King Z, Parrott WA, Liu W, Burris JN, Lenaghan SC, Stewart CN Jr.. Embryogenic cell suspensions for high-capacity genetic transformation and regeneration of switchgrass (\u003cem\u003ePanicum virgatum\u003c/em\u003e L). Biotechnol Biofuels. 2019;12:290.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShivani TS. Enhanced \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation efficiency of banana cultivar Grand Naine by reducing oxidative stress. Sci Hortic. 2019;246:675\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTripathi JN, Oduor RO, Tripathi L. high-throughput regeneration and transformation platform for production of genetically modified banana. Front Plant Sci. 2015;6:1025.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWoo HA, Ku SS, Jie EY, Kim H, Kim HS, Cho HS, Jeong WJ, Park SU, Min SR, Kim SW. Efficient plant regeneration from embryogenic cell suspension cultures of \u003cem\u003eEuonymus alatus\u003c/em\u003e. Sci Rep. 2021;11(1):15120.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFiner JJ. Plant regeneration via embryogenic suspension cultures. In: Plant Cell Cult 1995: 99\u0026ndash;126.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJayasankar S, Gray DJ, Litz RE. High-efficiency somatic embryogenesis and plant regeneration from suspension cultures of grapevine. Plant Cell Rep. 1999;18:533\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eForg\u0026aacute;cs I, Suller B, Zok A, Pedryc A, Ol\u0026aacute;h R, De\u0026aacute;k T, Bisztray GD, Szegedi E. Establishment of grapevine embryogenic liquid culture and induced somatic embryogenesis. Acta Hortic 2017(1157):113\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJiao W, Ya-li Z, Rong-rong H, Lei Z, Chao-xia W, Xia X, Jiang L. Optimization of transformation efficiency of suspension cultured \u003cem\u003eVitis vinifera\u003c/em\u003e cv. Chardonnay embryogenic cells. J Integr Agric. 2012;11(3):387\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAmar AB, Cobanov P, Boonrod K, Krczal G, Bouzid S, Ghorbel A, Reustle GM. Efficient procedure for grapevine embryogenic suspension establishment and plant regeneration: role of conditioned medium for cell proliferation. Plant Cell Rep. 2007;26:1439\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTorregrosa L, verries C, Tesniere C. Grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e L.) promoter analysis by biolistic-mediated transient transformation of cell suspensions. Vitis. 2002;41(1):27\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAcanda Y, Mart\u0026iacute;nez \u0026Oacute;, Gonz\u0026aacute;lez MV, Prado MJ, Rey M. Highly efficient in vitro tetraploid plant production via colchicine treatment using embryogenic suspension cultures in grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e cv. Menc\u0026iacute;a). Plant Cell Tissue Organ Cult. 2015;123(3):547\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZlenko V, Kotikov IV, Troshin LP. Plant regeneration from somatic embryos of interspecific hybrids of grapevine formed in liquid medium. J Hortic Sci Biotechnol. 2005;80(4):461\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRen C, Liu X, Zhang Z, Wang Y, Duan W, Li S, Liang Z. CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (\u003cem\u003eVitis vinifera\u003c/em\u003e L). Sci Rep. 2016;6:32289.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVillette J, Lecourieux F, Bastiancig E, Heloir M-C, Poinssot B. New improvements in grapevine genome editing: high efficiency biallelic homozygous knock-out from regenerated plantlets by using an optimized zCas9i. Plant Methods. 2024;20:45.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJayasankar S, Bondada BR, Li Z, Gray DJ. Comparative anatomy and morphology of \u003cem\u003eVitis vinifera\u003c/em\u003e (Vitaceae) somatic embryos from solid- and liquid-culture-derived proembryogenic masses. Am J Bot. 2003;90(7):973\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKawashima T, Goldberg RB. The suspensor: not just suspending the embryo. Trends Plant Sci. 2010;15(1):23\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTorregrosa L, Vialet S, Adiveze A, Iocco-Corena P, Thomas MR. Grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e L). Methods Mol Biol. 2015;1224:177\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFranks T, He D, Thomas M. Regeneration of transgenic \u003cem\u003eVitis vinifera\u003c/em\u003e L. Sultana plants: genotypic and phenotypic analysis. Mol Breeding. 1998;4:321\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi ZT, Kim KH, Jasinski JR, Creech MR, Gray DJ. Large-scale characterization of promoters from grapevine (\u003cem\u003eVitis\u003c/em\u003e spp.) using quantitative anthocyanin and GUS assay systems. Plant Sci. 2012;196:132\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEngler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLoS ONE. 2008;3(11):e3647.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHarris NN, Luczo JM, Robinson SP, Walker AR. Transcriptional regulation of the three grapevine \u003cem\u003echalcone synthase\u003c/em\u003e genes and their role in flavonoid synthesis in Shiraz. Aust J Grape Wine Res. 2013;19(2):221\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCosta LD, Vinciguerra D, Giacomelli L, Salvagnin U, Piazza S, Spinella K, Malnoy M, Moser C, Marchesi U. Integrated approach for the molecular characterization of edited plants obtained via \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e\u0026ndash;mediated gene transfer. Eur Food Res Technol. 2022;248:289\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThilmony R, Dasgupta K, Shao M, Harris D, Hartman J, Harden LA, Chan R, Thomson JG. Tissue-specific expression of \u003cem\u003eRuby\u003c/em\u003e in Mexican lime (\u003cem\u003eC. aurantifolia\u003c/em\u003e) confers anthocyanin accumulation in fruit. Front Plant Sci 2022, 13.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCapriotti L, Limera C, Mezzetti B, Ricci A, Sabbadini S. From induction to embryo proliferation: improved somatic embryogenesis protocol in grapevine for Italian cultivars and hybrid Vitis rootstocks. Plant Cell, Tissue and Organ Cult 2022, 151(2):221\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGambino G, Ruffa P, Vallania R, Gribaudo I. Somatic embryogenesis from whole flowers, anthers and ovaries of grapevine (\u003cem\u003eVitis\u003c/em\u003e spp.). Plant Cell, Tissue and Organ Cult 2007, 90(1):79\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDhekney SA, Li ZT, Compton ME, Gray DJ. HortScience : Optimizing initiation and maintenance of \u003cem\u003eVitis\u003c/em\u003e embryogenic cultures. 2009, 44:1400\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJim\u0026eacute;nez VM. Regulation of in vitro somatic embryogenesis with emphasis on the role of endogenous hormones. Braz J Plant Physiol. 2001;13(2):196\u0026ndash;223.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJim\u0026eacute;nez VM, Bangerth F. Relationship between endogenous hormone levels of grapevine callus cultures and their morphogenetic behaviour. Vitis 2000, 39(4):151\u0026ndash;157.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMostafa HHA, Wang H, Song J, Li X. Effects of genotypes and explants on garlic callus production and endogenous hormones. Sci Rep. 2020;10(1):4867.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVidal JR, Kikkert JR, Wallace PG, Reisch BI. High-efficiency biolistic co-transformation and regeneration of 'Chardonnay' (\u003cem\u003eVitis vinifera\u003c/em\u003e L.) containing npt-II and antimicrobial peptide genes. Plant Cell Rep. 2003;22(4):252\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbohatem M, Zouine J, Hadrami IE. Low concentrations of BAP and high rate of subcultures improve the establishment and multiplication of somatic embryos in date palm suspension cultures by limiting oxidative browning associated with high levels of total phenols and peroxidase activities. Sci Hortic. 2011;130:344\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi S-F, Ye T-W, Xu X, Yuan D-Y, Xiao S-X. Callus induction, suspension culture and protoplast isolation in \u003cem\u003eCamellia oleifera\u003c/em\u003e. Sci Hortic 2021, 286(110193).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMasani MYA, Noll G, Parveez GKA, Sambanthamurthi R, Pr\u0026uuml;fer D. Regeneration of viable oil palm plants from protoplasts by optimizing media components, growth regulators and cultivation procedures. Plant Sci. 2013;210:118\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMandolino LM. Genetic transformation and regeneration of transgenic plants in grapevine (\u003cem\u003eVitis rupestris\u003c/em\u003e S). Theor Appl Genet. 1994;88:621\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHebert D, Kikkert JR, Smith FD, Reisch BI. Optimization of biolistic transformation of embryogenic grape cell suspensions. Plant Cell Rep. 1993;12:585\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKikkert JR, Hebert-Soule D, Wallace PG, Striem MJ, Reisch, Bl. Transgenic plantlets of 'Chancellor' grapevine (\u003cem\u003eVitis\u003c/em\u003e sp.) from biolistic transformation of embryogenic cell suspensions. Plant Cell Rep. 1996;15:311\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDalla Costa L, Emanuelli F, Trenti M, Moreno-Sanz P, Lorenzi S, Coller E, Moser S, Slaghenaufi D, Cestaro A, Larcher R, Gribaudo I, Costantini L, Malnoy M. Grando MS:Induction of terpene biosynthesis in berries of microvine transformed with VvDXS1 alleles. Front Plant Sci. 2018;8:2244.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBabich O, Sukhikh S, Pungin A, Ivanova S, Asyakina L, Prosekov A. Modern trends in the In Vitro production and use of callus, suspension cells androot cultures of medicinal plants. Molecules 2020, 25(24):5805.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoniruzzaman M, Zhong Y, Huang Z, Yan H, Yuanda L, Jiang B, Zhong G. Citrus cell suspension culture establishment, maintenance, efficient transformation and regeneration to complete transgenic plant. Plants. 2021;10(4):664.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGurel S, Gurel E, Kaur R, Wong J, Meng L, Tan HQ, Lemaux PG. Efficient, reproducible \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation of sorghum using heat treatment of immature embryos. Plant Cell Rep. 2009;28(3):429\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHiei Y, Ishida Y, Kasaoka K, Komari T. Improved frequency of transformation in rice and maize by treatment of immature embryos with centrifugation and heat prior to infection with \u003cem\u003eAgrobacterium tumefacien\u003c/em\u003es. Plant Cell Tissue Organ Cult. 2006;87:233\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhanna H, Becker D, Kleidon J, Dale J. Centrifugation Assisted \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e-mediated Transformation (CAAT) of embryogenic cell suspensions of banana (\u003cem\u003eMusa\u003c/em\u003e spp. Cavendish AAA and Lady finger AAB). Mol Breed. 2004;14:239\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePatel M, Dewey RE, Qu R. Enhancing \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e-mediated transformation efficiency of perennial ryegrass and rice using heat and high maltose treatments during bacterial infection. Plant Cell Tissue Organ Cult. 2013;114(1):19\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBon RS, Noia FD, Segura A, Vidal JR. Toxic effect of antibiotics in grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e 'Albari\u0026ntilde;o') for embryo emergence and transgenic plant regeneration from embryogenic cell suspension. Vitis. 2014;53(2):89\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi ZT, Dhekney S, Dutt M, Aman M, Tattersall J, Kelley KT, Gray DJ. Optimizing \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation of grapevine. Vitro Cell Dev Biol Plant. 2006;42(3):220\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTorregrosa L, Lopez G, Bouquet A. Antibiotic sensitivity of grapevine: A comparison between the effect of hygromycin and kanamycin on shoot development of transgenic 110 richter rootstock (\u003cem\u003eVitis Berlandieri x Vitis rupestris\u003c/em\u003e). S Afr J Enol Vitic 2016, 21(1).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePark SY, Yin X, Duan K, Gelvin SB, Zhang ZJ. Heat shock protein 90.1 plays a role in \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated plant transformation. Mol Plant. 2014;7(12):1793\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHwang HH, Liu YT, Huang SC, Tung CY, Huang FC, Tsai YL, Cheng TF, Lai EM. Overexpression of the HspL promotes \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e virulence in \u003cem\u003eArabidopsis\u003c/em\u003e under heat shock conditions. Phytopathology. 2015;105(2):160\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTsai YL, Wang MH, Gao C, Klusener S, Baron C, Narberhaus F, Lai EM. Small heat-shock protein HspL is induced by VirB protein(s) and promotes VirB/D4-mediated DNA transfer in \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e. Microbiology. 2009;155:3270\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"microvine 04C023V0004, grapevine, Agrobacterium tumefaciens, embryogenic cell suspension, transformation, droplet digital PCR","lastPublishedDoi":"10.21203/rs.3.rs-7661205/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7661205/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eMicrovine 04C023V0004 (V4) is a model \u003cem\u003eVitis vinifera\u003c/em\u003e genotype that carries a heterozygous \u003cem\u003eGibberellin insensitive 1\u003c/em\u003e (\u003cem\u003eVvigail\u003c/em\u003e) mutation making the plants compact in stature and constantly fruiting. While these traits make V4 desirable for research, genetic engineering is challenging because of long regeneration times and modest transformation efficiency levels.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eTo improve microvine V4 transformation, we established a method utilizing embryogenic cell suspension (ECS) cultures and a novel protocol for \u003cem\u003eAgrobacterium-\u003c/em\u003emediated transformation. Friable translucent or cream-colored globular somatic embryos from microvine embryogenic calli were used to initiate suspension cultures. ECS cultures require a modest amount of time and effort to maintain and produce abundant rapidly growing explant material within several months. A protocol was also developed that utilized these embryogenic suspension cells for \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation. The ECS cells were heat shocked at 45\u0026deg;C for 5 minutes and then combined with a co-cultivation medium containing 400 mM acetosyringone, \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e AGL1 (OD\u003csub\u003e600\u003c/sub\u003e 0.2) carrying a binary vector with the microvine \u003cem\u003eUbiquitin 7\u003c/em\u003e (\u003cem\u003eVviUbi7\u003c/em\u003e) promoter controlling \u003cem\u003emCherry\u003c/em\u003e expression allowing the use of red fluorescence as a visible marker. After co-cultivation, washing the ECS cells with cefotaxime (400 mg/L) medium successfully inhibited bacterial growth. Development of healthy, actively growing transgenic microvine plants was achieved with the addition of gibberellic acid (GA\u003csub\u003e3\u003c/sub\u003e) (10 mg/L) to the shooting medium. Eighteen independent transgenic plants were characterized using droplet digital PCR (ddPCR) demonstrating that eight (44%) had one or two copies of the introduced transgene. This method produced approximately 30 transgenic plants per 100 mg of ECS culture within five months from the start of \u003cem\u003eAgrobacterium\u003c/em\u003e co-cultivation.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eUse of microvine V4 ECS cultures and a modified transformation protocol can efficiently generate transgenic plants advancing grapevine biotechnology research. In the future, this protocol can potentially be adapted for other grapevine genotypes.\u003c/p\u003e","manuscriptTitle":"Harnessing embryogenic cell suspension culture for Agrobacterium-mediated transformation of microvine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-26 07:54:44","doi":"10.21203/rs.3.rs-7661205/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2439576b-ee03-40ae-8c12-5ddf74c700d8","owner":[],"postedDate":"September 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-18T23:38:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-26 07:54:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7661205","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7661205","identity":"rs-7661205","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}