Vitrification-cryopreservation of Shoot Tips Excised from Dormant Bulbs in Fritillaria przewalskii Maxim: Assessing Histological lnjuries and Evaluating Genetic Fidelity in Cryo-Derived Plants

Vitrification-cryopreservation of Shoot Tips Excised from Dormant Bulbs in Fritillaria przewalskii Maxim: Assessing Histological lnjuries and Evaluating Genetic Fidelity in Cryo-Derived Plants | 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 Vitrification-cryopreservation of Shoot Tips Excised from Dormant Bulbs in Fritillaria przewalskii Maxim: Assessing Histological lnjuries and Evaluating Genetic Fidelity in Cryo-Derived Plants Yuming He, Huan Sun, Hui Fan, Chunyu He, Qingyi Guo, yanhong zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8370894/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 Fritillaria przewalskii Maxim. an essential medicinal plant in the Fritillaria genus of the Liliaceae family, is widely used in traditional medicine. This study focused on developing a cryopreservation protocol for F. przewalskii , an endangered and endemic species in China. We aimed to determine if ex vitro bulbs from field-grown F. przewalskii plants could serve a source of shoot tips for cryopreservation. Key factors influencing cryopreservation outcomes were studied using a vitrification-based approach. Prior to cryopreservation, cold hardening and sucrose preculture conditions were optimized. The best results were achieved by hardening bulbs at 4℃ for 3–4 months,followed by surface disinfection and isolation of 2–5 mm Shoot tips. The tips were then then precultured on a medium with 0.5M sucrose for 3 days. Cryopreservation steps included a 20-minute loading treatment, 60-minute PVS2 dehydration at room temperature, and storage in liquid nitrogen more than one hour. After thawing, this protocol resulted in 93% shoot survival rate, with tips regenerating into small bulbs within 5 weeks without intermediary callus formation. Histological analysis showed that while cooling and rewarming caused severe bud damage, apical meristem cells are the primary survival sites. Although loading and PVS2 treatment induced cell plasmolysis, it was not lethal. Optimal PVS2 treatment enhanced cellular dehydration resistance, promoting cell viability at ultralow temperature. RAPD analysis of regenepercentaged plants showed no genetic variation, confirming strong genetic stability. The direct use of ex vitro shoot tips provides a straightforward and effective cryopreservation method for F. przewalskii , combining convenience, efficiency, and genetic stability. Genetic stability In vitro conservation Paraffin section Regenerated plant Shoot tips Vitrification cryopreservation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Statement on Prior Submission An earlier version of this manuscript was submitted to Current Plant Biology , where it underwent peer review and was accepted (He et al. 2024). However, due to specific institutional requirements mandating that published work be indexed in particular academic databases (including the Chinese Academy of Sciences database), and as Current Plant Biology was not indexed accordingly at that time, the authors made the decision to withdraw the accepted manuscript prior to its formal publication. This withdrawal was based solely on administrative compliance with institutional policies and was in no way related to the scientific content, quality, or integrity of the research. The present manuscript, submitted to Plant Cell, Tissue and Organ Culture (PCTOC), represents a revised version of that work. The core scientific data, results, and conclusions remain unchanged. The withdrawn article is formally cited in the reference list of this manuscript for full transparency. 1. Introduction Fritillaria przewalskii Maxim. is a perennial herbaceous species in the Liliaceae family and is a primary source of the traditional Chinese medicine "Chuan bei" (Chinese Pharmacopoeia Commission 2020 ). The dried underground bulbs of this plant have medical properties, including the ability to clear heat and moisture from the lungs, reduce phlegm, and treat carbuncles (Chinese Pharmacopoeia Commission 2020 ). F . przewalskii is distributed across the western provinces of China, including southern Gansu, western Sichuan, eastern and southern Qinghai. It typically inhabits alpine meadows or grasslands at elevations between 2800 and 4400 meters (Chang et al. 2010 ). This species prefers cool, shady and moist environments, which restricts its natural distribution (Chen et al. 2020 ). Due to its therapeutic properties, F. przewalskii Is in high demand. However,tis natural supply can not meet market needs because of its slow growth, long life cycle, and low yield in wild conditions (Carasso et al. 2011 ). Currently, most F. przewalskii in the medicinal market is sourced from wild populations, and over-harvesting has led to a sharp decline in its resourses, resulting in its classification as an endangered species (Zhang et al. 2010 ). Therefore, preserving and cultivating this medicinal plant is essential for sustainable use. Cryopreservation, which involves cooling cells or entire tissues to ultralow temperatures using liquid nitrogen (-196℃), halts all cellular division and metabolic processes, theoretically enabling indefinite storage under these conditions (Benelli et al. 2009 ; Matsumoto et al. 2001 ). Vitrification-based cryopreservation has become the predominant method for long-term storage of germplasm. This approach involves dehydrating plant cells in a highly concentrated vitrification protective solution, followed by rapid immersion in liquid nitrogen, which induces a vitrified state in the cell without allowing ice crystals to form (Chen et al. 2020 ). Consequently, vitrification-based cryopreservation is regarded as a simple, cost-effective, and safe method for the long-term conservation of plant germplasm resources (Choudhary et al. 2014 ; Gupta and Mir 2019 ). Plant materials used for cryopreservation include pollen, seeds, shoot tips, calluses, and suspension cells. Many of these plant materials present challenges for cryopreservation due to their high water content, elevated metabolic levels, and stress sensitivity (Roque-Borda et al. 2021 ). Compared to other parts of the plant, the apical meristem demonstrates a stronger meristematic capacity, and the shoot tip retains significant meristematic ability even after cryopreservation (Barraco et al. 2011 ). Consequently, shoot tips are among the preferred materials for cryopreservation, offering several advantages such as ease of handling, minimal space requirements, safety ,stability, and low storage costs (Pennycooke and Towill 2000 ; Uchendu et al. 2013 ). However, most current cryopreservation practices employ a slow-freezing technique that relies on freeze-induced dehydration. This approach is primarily used for cryopreserving undifferentiated cultures and the apices of cold-tolerant species, and it requires expensive controlled freezers, limiting its widespread application (Al-Jubori and Mohammed 2024 ). The vitrification-based cryopreservation technique used in this study, treats cells with a high concentration of plant vitrification solution. During osmotic dehydration, the cryoprotectant enters the cell, altering the thermodynamic properties of the intracellular liquid. Rapid cooling enables intracellular and extracellular vitrification, thereby preserving cell structure and regeneration capacity, which significantly expands the range of species that can be cryopreserved (Lin 2021). In this research, we utilized ex vitro sources of plant material for cryopreservation. A major advantage of this approach is that it eliminates the need to establish an in vitro culture, multiply it, and subsequently excise the shoot tip. Notably, shoot tips were excised directly from ex vitro material, saving considerable time and reducing costs. Research indicates that shoot tips from ex vitro source have been successfully cryopreserved in prior studies. For instance, Bettoni et al. used shoot tips derived from Vitis plants grown in growth chambers as a cost-effective alternative for grape cryopreservation, avoiding the costs of establishing an in vitro preservation system before cryopreservation (Bettoni et al. 2019 ). In addition, shoot tips obtained directly from ex vitro plants have been used for other species, such as citrus (Volk et al. 2017 ). To our knowledge, no previous studies have investigated the vitrification cryopreservation of shoot tips excised from bulbs of F . przewalskii . Therefore, the objective of this work is to establish a vitrification-based ceypreservation system for the shoot tips of F. przewalskii . To this end, we examined the key factors influencing cryopreservation outcomes and preformed histological analyses to assess cell survival during cryopreservation. 2. Material and Methods 2.1 Source of explants Bulbs of Fritillaria przewalskii Maxim. were harvested from Zhang County, Gansu Province (E 103° 57' 38'' to 104° 45' 04 '' N 34° 25' 43'' to 34° 57' 42''), in July 2022 (fall). It is recommended to harvest when the aboveground portion of the plant has not yet withered. 2.2 Cold hardening pretreatment The bulbs of F. przewalskii were stored at 4°C in dark for two, three, and four months prior to experiment. Following the cld hardening period, the bulbs were disinfected, and shoot tips were dissected for cryopreservation. 2.3 Explants disinfection The bulbs were gently cleaned under running water with a soft brush, following by peeling off the outer scales. The bulbs were then disinfected on a sterile workbench by immersing in 75% ethanol for 30 seconds, then in 0.1% mercuric chloride for one minute, and finally rinsed three times with sterile water (He et al, 2023 ). 2.4 Sucrose preculture Sterile shoot tips (from bulbs after three months of cold hardening) were inoculated on pre-culture media and cultured in dark at 25℃. The preculture media contained sucrose at three concentrations: 0.3 M, 0.5 M, and 0.7 M (1/2MS medium with 0.3–0.7% agar, pH 5.8-6.0). Preculture durations were set at 1 day and 3 days, with 30 shoot tips per treatment. Shoot tips without preculture served as the control group (CK), directly used for vitrification cryopreservation. The cryopreservation steps included a 20-minute loading, 60-minute PVS2 dehydration, storage in liquid nitrogen, and inoculation post-thawing. 2.5 Vitrification cryopreservation of shoot tips 2.5.1 Plant Vitrification Solution 2 (PVS2) treatment time After two months of cold hardening, bulbs were disinfected, and sterile stoot tips were dissected under aseptic conditions. The shoot tips were immediately placed in pre-autoclaved cryovials, followed by the addition of 2 mL loading solution (2 M glycerol, 0.4 M sucrose in MS liquid medium, pH5.8) (Sakai et al. 1990 ) at room temperature for 20 minutes. They were then treated with 2 mL PVS2 (30% w/v glycerol, 15% w/v ethylene glycol, and 15% w/v dimethyl sulfoxide in MS liquid medium with 0.4 M sucrose, pH5.8) (Sakai et al. 1990 ) at room temperature for 30, 60, 90, and 120 minutes. The loading solution and the PVS2 haved been previously autoclaved and filter sterilized, respectively. Five shoot tips were placed in each cryovial, and six cryovials were useed in every PVS2 treatment durations. The cryovials were subsequently placed in liquid nitrogen for storage and later thawed as described in section 2.5.3 . Finally, the shoot tips were inoculated onto the medium as described in 2.6. 2.5.2 Loading treatment For one treatment group, shoot tips were exposed to 2 mL of loading solution at room temperature for 20 minutes, followed by 2 mL PVS2 dehydrated for 60 minutes, and then exposed to liquid nitrogen for more than one hour. As a control to evaluate the effect of the loading solution, another group was directly dehydration in 2 mL PVS2 without prior loading, with all other conditions kept consistent. 2.5.3 Unloading process Cryovial were removed from liquid nitrogen and rewarmed in a 38℃ water bath for 2 minutes. The shoot tips were then divided into two groups: one group was immersed in an unloading solution (1.2 M sucrose in MS liquid medium, pH5.8) at room temperature for 20 minutes before inoculation while the other group was directly inoculated onto the medium without the unloading process. The unloading solution was filter-sterilized in advance. Thirty shoot tips were included in each group. 2.5.4 Shoot tips size The shoot tips were categorized into three size groups: large (greater than 4–5 mm), medium (2–4 mm), and small (less than 2 mm). Vitrification cryopreservation was performed, and thirty shoot tips from each size group were selected. The protocol included treating theshoot tips with 2 mL of loading solution at room temperature for 20 minutes, followed by 2 mL of PVS2 for 60 minutes at room temperature, and then exposured to liquid nitrogen for more than one hour. Cryovials were thawed in a 38℃ water bath for 2 minutes, and shoot tips were directly inoculated onto the recovery medium without unloading. 2.6 Recovery growth after cryopreservation The shoot tips were inoculated onto 1/2 MS medium supplemented with 0.5 mg·L − 1 6-BA (6-Benzylaminopurine), 0.1 mg·L − 1 NAA (1-naphthlcetic acid), 3% sucrose, and 0.7% agar, pH 5.8–6.0, for recovery. During the first week, explants were kept in the dark, then transferred to 12-hour photoperiod with 1500–2000 lx light intensity at 25℃ (He et al, 2023 ). Survival rate was recorded 7 days post-culture and expressed as the percentage of the shoot tips turning green. The regeneration rate was defined as the percentage of shoot tips developing into bulblets 5 weeks post-culture. 2.7 Histological analysis of shoot tips during cryopreservation Shoot tips were collected at various stages for histological analysis: Control (CK+): Untreated shoot tips. Negative control (CK-): Shoot tips plunged directly into liquid nitrogen for more than 60 minutes, followed by rewarming. Loading (LS): Shoot tips were immersed in 2 mL cryovials at room temperature for 20 minutes with a loading solution. PVS2 (P 60 ): After loading shoot tips were treated with PVS2 for 60 minutes at room temperature. Liquid nitrogen (LN 60 ): Shoot tips after dehydration in cryovials were rapidly plunged into liquid nitrogen (LN) and held for 60 minutes. Rewarming: Shoot tips after LN freezing rewarmed in 38℃ water bath for 2 minutes. Recovery (R 60 ): Shoot tips cultured in the dark for one week on 1/2MS medium supplemented with 0.5 mg·L − 1 6-BA ,0.1 mg·L − 1 NAA, 3% sucrose, 0.7% agar, pH 5.8–6.0. Shoot tips were fixed in FAA (ethanol:formalin:acetic acid = 18:1:1) solution for 24 hours, dehydrated through an ethanol series (70%, 85%, 95%, and 100% ethanol) and embedded in paraffin. For light microscopy studies, 5 µm sections were prepared using a Leica RM2125 microtome and stained with 0.01% toluidine blue for microscopy (Li 2009 ). Untreated shoot tips served as a positive control, while those that were freshly excised, directly immersed in LN served as a negative control. Each treatment contained at least five shoot tips. 2.8 Genetic stability evaluation in regenerated plants 2.8.1 Morphological markers Fresh regenerated bulbs were selected after five subcultures, and morphological traits of the regenerated bulbs and the original bulbs were observed, including bulb color and leaf shape and color of plantlets. 2.8.2 Genomic DNA isolation and RAPD markers Genetic fidelity was evaluated by random amplified polymorphic DNA (RAPD). Five shoot tips were randomly selected from 30 replicates under the same treatment, and the genetic fidelity of the ultra-low temperature regenerated bulblets of these shoot tips was evaluated after five subcultures. DNA was extracted by modified CTAB method (Coelho et al. 2014 ). Four regenerated bulblets from the same shoot tip were randomly selected, and 0.02 g fresh bulblets were weighed in each part. A total of 20 samples were collected from 5 shoot tips. DNA was purified and quantified using ultraviolet spectrophotometry. Each sample was diluted to 50 ng/µL in deionized water and stored at -20℃ until use. Suitable primers were selected from 20 screened primers for genetic stability assessment. PCR was conducted in a reaction mixture containing 12.5 µL 2×Taq PCR Master MixⅡ(with Dye), 1 µL of 10 µmol/L primer and 2 µL of 50 ng/µL DNA template in a final volume of 25 µL. PCR amplification was performed in a thermal cycler under the following reaction conditions: initial denaturation step for 4 minutes at 94℃, 38 cycles at 94℃ for 30 seconds, annealing at 40℃ for 60 seconds, and extension at 72℃ for 2 minutes followed by the final extension step at 72℃ for 8 minutes (Jiang et al. 2012 ). The PCR products were separated by electrophoresis in 1.5% agarose gel containing GoldView Ⅰ and the image was photographed under a gel image system (Gel Doc XRTM). The molecular mass ladder DNA Marker DL2000 was used for estimating the size of the amplified products. 2.9 Statistical Analysis Each cryopreservation treatments included 30 explants, with each cryovial containing 5 shoots as one replicate. The data were compared by Duncan multiple-range test, and comparisons between the mean values ere evaluated by the least significant different test at P < 0.05. For genetic stability assessments, RAPD profiles were manually scored for the presence or absence of bands. Bands with low visual intensity that could not be readily distinguished were not scored. 3 Results 3.1 Factors affecting vitrification-based cryopreservation of shoot tips 3.1.1 Cold hardening time of bulbs As the duration of cold acclimation increased, the survival and regeneration rates of shoot tips improved markably (Fig. 1 ). Specifically, after two months of acclimation at 4℃, the survival and regeneration rates of excised shoot tips were observed to be 47% and 37%, respectively. When the acclimation period was extended to three months, these rates significantly rose to 77% and 60%, respectively. Extending the acclimation period to four monthsprovided a further increase, although the difference compared to three months was not statistically significant. Therefore, maintaining bulbs of F. przewalskii at 4°C for three to four months before cryopreservation optimizes recovery outcomes. 3.1.2 Sucrose preculture Comparisons were mad among vary sucrose concentrations and preculture durations for their effects on the shoot tip survival and regeneration rates post-cryopreservation. When shoot tips were precultured for one day, increasing sucrose concentration led to a slight decline in survival and regeneration rates. However, extending the preculture period to three days yielded improved survival and regeneration rates with 0.3 M and 0.5 M sucrose, while 0.7 M sucrose resulted in a slight decrease. (Fig. 2 ). Notably, The treatment with 0.5 M sucrose for three days achieved the highest survival and regeneration rates of 93%, following exposure to the loading solution for 20 minutes and PVS2 for 60 minutes at room temperature before immersion in liquid nitrogen. Therefore, a sucrose concentration of 0.5 M for three days was identified as the optimal preculture condition. 3.1.3 The treatment of PVS2 dehydration The duration of PVS2 dehydration influenced regeneration, although it had minimal effect on survival rate (Fig. 3 ). While shoot tips survival did not significantly fluctuate across PVS2 treatment times ranging from 30 to 120 minutes,the regeneration rate was notably affected, showing a peak at 60 minutes, which survival and regeneration rate were 47% and 43% respectively. Shoot tips treated with PVS2 for 60 minutes exhibited the highest regeneration rate, suggesting that this duration provides an optimal balance for cellular dehydration and viability under ultralow temperatures. 3.1.4 Loading and unloading Pre-treatment with loading solution did not significantly impact shoot tip survival; however, it did contribute to a 30% increase in regeneration rate (Fig. 4 ). Without loading, the regeneration rate was only 37% when the shoot tips were directly treated with PVS2 for 60 minutes. In contrast, pre-treatment with the a loading solution for 20 minutes, followed by 60 minutes of PVS2 dehydration, increased the regeneration rate to 77%. Though the unloading step did not lead to a statistically significant difference, the treatment group that underwent unloading exhibited slightly higher survival and regeneration rates (83% and 77%, respectively) compared to those without unloading. 3.1.5 Shoot tips size The length of the shoot tips significantly affected the post-cryopreservation recovery, with survival and regeneration rates improving as shoot tip size increased (Fig. 5 ). Shoot tips shorter than 2 mm displayed the lowest survival and regeneration rates following the standard cryopeservation procedure. In contrast, shoot tips measuring between 2–4 mm achieved the highest survival and regeneration rates, at 77% and 70%, respectively, under 20 minutes of loading and 60 minutes of PVS2 dehydration at room temperature. For shoot tip exceeding 4 mm, both survival and regeneration rates decreased, indicating that 2–4 mm is the optimal size for successful cryopreservation. 3.2 Recovery culture Before cryopreservation, the shoot tips exhibited a slender, conical shape with a pale yellow hue (Fig. 6 a), Post-cryopreservation, the shoot tips turned white (Fig. 6 b). Within the initial 7-day culture period, some buds showed signs of viability by turning green (white arrow in Fig. 6 c), while those that did not change color or turned brown were considered non-viable (black arrow in Fig. 6 c). After a 5-week culture period, viable shoot tips developed into 4–5 small, white, and spherical bulblets on the surface (Fig. 6 d),which could propagate further through subcultures (Fig. 6 e). Transferred to rooting media, these bulblets developed into complete plantlets (Fig. 6 f). 3.3 Histological analysis of cryopreserved shoot tips Microscopic Examination of Cellular Integrity Control shoot tip showed distinct staining with a well-defined nucleus and nucleolus, indicating intact cellular structure (Fig. 7b1, b2). The apical meristem cells and leaf primordium cells are tiny, virtually spherical with dense cytoplasm and a high nuclear-to-cytoplasm ratio. Nucleus and nucleolus were clearly visible (Fig. 7b1). The bud axis cells are approximately rectangular, the cells are large, the nucleus is located in the center of the cell, and there are one or more nucleoli in the nucleus (Fig. 7b2). In the negative control (untreated shoot tips directly plunged into liquid nitrogen), cells appeared lightly stained with disrupted plasma membranes, dissolved nuclei, and absent nucleoli, suggesting extensive cryo-injury (Fig. 7c1, c2). Plasmolysis Observations Treatment with loading solution caused minor plasmolysis without severe cellular damage (Fig. 7d1, d2). Subsequent exposure to PVS2 intensified plasmolysis, particularly in the lower half of the shoot tips (Fig. 7e1, e2). During the freezing and rewarming steps, serious damage was observed, including ruptured cell walls, cytoplasmic degeneration, and nucleolar disappearance in most cells, except in the apical meristem, which largely remained viable (Fig. 7f1, f2). Upon recovery in culture, visible cells exhibited a restored structure with clear nuclei and visible nucleoli in certain cells, suggesting the apical meristem as a key survival site during cryopreservation (Fig. 7g1, g2). Histological Implications The histological analysis underscores the effectiveness of PVS2 in inducing protective dehydration, though excessive plasmolysis occurs in some regions. The resilience of the apical meristem cells aligns with the survival outcomes, emphasizing their crucial role in shoot tip viability after cryopreservation. 3.4 Genetic stability of cryo-derived regenerants 3.4.1 Morphological analysis Morphological Consistency Bulblets regenerated from cryopreserved shoot tips consistently exhibited a dense texture, white coloration, and 2–4 scales, with the characteristic lanceolate, green leaves of F. przewalskii. uniform morphological traits across regenerants indicate that cryopreservation did not induce phenotypic deviations, supporting the preservation of genotype integrity. 3.4.2 RAPD analysis for genetic fidelity Using three RAPD primers, a total of 400 monomorphic bands were generated from 20 regenerat samples, with no variations (absence or gain of loci) detected across samples (Fig. 8 ). This genetic consistency confirms the absence of somaclonal variation, validating the genetic stability of the cryopreserved germplasm. 4 Discussions Cryopreservation Challenges and Significance The preservation of endemic and endangered poses unique challenges due to their ecological specificity, genetic diversity, and often limited accessibility. In the case of Fritillaria przewalskii, an endangered medicinal plant, developing an effective cryopreservation protocol is critical for its long-term conservation and sustainable utilization. This study presents a comprehensive vitrification-based cryopreservation protocol for F. przewalskii, providing insights into factors such as shoot tips size, cold acclimation, and PVS2 dehydration time, which are essential for optimizing cryopreservation outcomes. These findings the broader body of knowledge on plant germplasm preservation, offering practical guidelines that may be adapted for other endangered species with similar conservation needs. (Charoensub et al. 2003 ; Chen et al. 2011 ; Lambardi et al. 2000 ). Effect of Explant Size on Cryopreservation In general, small material can be seriously damaged during the dehydration process, making regeneration difficult. Conversely, if the material is too large, the cryoprotectant solution may struggle to infiltrate the interior cells, resulting in inadequate protection during the subsequent freezing step and a low regeneration rate. It has been found that the optimal shoot-tip size of cryopreservation of sweet potato [ Ipomoea batatas (L.) Lam. ] using the vitrification method is 0.5-1.0 mm long (Pennycooke and Towill 2000 ). For garlic, 1.5×3.0 mm shoot apices were precultured at 10°C for 3 days on medium containing 0.1 M sucrose, followed by dehydrated in PVS3 solution for 150 min at 23°C prior to freezing, resulting in a survival percentage as high as 85% and a regeneration rate exceeding 73% (Baek et al. 2003 ). In our study, the 2–4 mm long shoot tips were exposed in the loading solution for 20 minutes and then to PVS2 for 60 minutes at room temperature. We achieved a high survival percentage of 77% and a regeneration rate of 70%. Furthermore, the appropriate PVS2 treatment time varies depending on the size of the material. Even with the same treatment time, regeneration of plant materials can differ at various temperatures. Therefore, it is essential to formulate the most suitable cryopreservation method according to the specific plant materials. Optimizing Sucrose Preculture and Cold Acclimation Pretreating materials before vitrification effectively reduces tissue water content and enhance cold resistance, allowing the materials to maintain high viability after cryopreservation (Salaj et al. 2010 ; Hong and Yin 2012 ; Sershen et al. 2012). Sucrose preculture and cold acclimation are the two most commonly used methods for improving plant recovery in cryopreservation. Numerous studies, such as those involving Vitis (Volk et al. 2018 ), kiwifruit (Mathew et al. 2018 ), potato (Kaczmarczyk et al. 2008 ), Pyrus cordata (Chang and Reed 2001 ), and Prunus cerasus (Barraco et al. 2012 ), have reported favorable results using high concentration of sucrose for preculture. In this study, both the survival percentage and regeneration rate of shoot tips from F. przewalskii reached a maximum of 93% when the tips were precultured on 0.5 M sucrose for 3 days, followed by 20 minutes in the loading solution and 60 minutes in PVS2 at room temperature before exposure to liquid nitrogen. This indicated that a high concentration of sucrose in the medium can induce cell dehydration by increasing osmotic pressure, thereby reducing the free water content in cells and enhancing cold resistance (Bettoni et al. 2021 ; Chen et al. 2014 ; Kulus et al. 2018 ). Many studies have shown that cold acclimation can activate cold resistance mechanism in plants and improve the survival of shoot tips during cryopreservation, particularly for low-temperature sensitive species (Channuntapipat et al. 2000 ; Engelmann 2011 ; Harding et al. 2009 ). For example, with cold hardening for 6 to 14 weeks, the regrowth percentage of Malus domestica Borkh. shoot tips peaked at 88% during the 6th and 8th weeks, and the plants remained capable of regenerating even at 14 weeks (Bilavčík et al. 2012 ). The survival percentage of blueberry shoot tips was highest (91%) when treated at low temperature for 2 to 4 weeks (Wang et al. 2017 ). In the present study, both the survival and regeneration rates of F. przewalskii shoot tips after cryopreservation increased with the duration of cold acclimation. Dehydration with PVS2 and the Importance of Dehydration Duration Plant Vitrification Solution (PVS) prevents intracellular water from forming ice crystals or limits the time available for ice crystals to grow, resulting in cells entering a state of artificial complete vitrification (Volk and Walters 2006 ; Halmagyi et al. 2017 ). Currently, the most commonly used vitrification solution is PVS2, and the optimal dehydration time of PVS2 is varies among different plant species (Bilavčík 2012; Matsumoto et al. 2015 ; Sant et al. 2006 ). If the PVS2 treatment time is insufficient, the cell dehydration may be not be complete, leading to the formation of ice crystals during freeziing in liquid nitrogen, which can seriously injure the cells. Conversely, if the treatment time is too long, the material can be damaged by PVS2, resulting in a significant reduction in survival percentage and regeneration rate (Matsumoto et al. 2015 ; Wang et al. 2014 ). Therefore, determining the appropriate dehydration time in PVS2 is crucial for minimizing cell damage and enhancing cell tolerance to freezing. Unloading is a standard step in the cryopreservation process. The unloading solution treatment is implemented to prevent the secondary toxicity of the PVS2 solution on shoot tips, necessitating thorough washing of the tips. However, in our study, unloading had little effect on the survival and regeneration of the shoot tips after cryopreservation. Research has shown that unloading significantly influences cryopreservation outcomes, with factors such as unloading time, unloading conditions, and sucrose concentration in the unloading solution playing critical roles. The optimal unloading conditions vary for each plant species and are closely related to specific interspecies differences (Rajasekharan and Prakashkumar 2010 ; Sajini et al. 2011 ). Therefore, there was no significant difference in the survival of F. przewalskii after cryopreservation, regardless of whether unloading solution treatment was applied. Histological Analysis of Shoot Tip Damage During Cryopreservation The alternation and damage of shoot tips during cryopreservation can be observed through histological sections. After dehydration with the loading solution, the cells in each part of the shoot tips lost water and exhibited slight plasmolysis. The treatment with PVS2 subjected the cells to higher osmotic pressure, resulting in severe dehydration and further plasmolysis (Popov et al. 2004 ). Cryopreserved cells also experienced mechanical damage, characterized by severe plasmolysis, cell membrane rupture, nuclear fragmentation, and nucleolus disappearance. After direct cryopreservation of shoot tips, most cells sustained irreversible damage, except for the apical meristem, which maintained structural integrity. The occurrence of cell damage is likely attributed to the extreme decline in water content, resulting in the inability of protoplasts to contract and concentrate cell fluid (Ganino et al. 2012 ). Additionally, freezing damage that results in cell death is caused by intracellular water crystallization during the freezing or rewarming processes. Consequently, the freezing and rewarming process inflicted significant damage to the buds, primarily manifested as the breakage of the cell wall in the bud axis, blurring of the nuclear boundary, ablation of the cytoplasm, and disappearance of the nucleolus. Although the shoot apical meristem exhibited slight plasmolysis, it remained the least damaged part, maintaining overall cell integrity. This region is crucial for the survival of shoot tips. Similar phenomena have also been observed in the cryopreservation of other species, including apple (Feng et al. 2013 ), Rubus (Chang and Reed 1999 ), Dendrobium (Antony et al. 2011 ; Ching et al. 2012 ; Poobathy et al. 2013 ), and Brassidium (Mubbarakh et al. 2014 ). Genetic Fidelity of Cryo-Derived Plants The genetic fidelity of cryo-derived plants is essential for ensuring the effectiveness of techniques applied to plant germplasm conservation. The RAPD analysis confirmed the genetic stability of regenerants, indicating that the vitrification-based protocol preserved the genetic fidelity of F. przewalskii . This is essential for germplasm conservation, as it ensures that preserved plants retain their original genetic composition, a prerequisite for any effective conservation strategy. Similarly, genetic stability has been confirmed in shoot tips or dormant buds following cryopreservation in Dioscorea rotundata Poir (Mandal et al. 2008 ), apple (Liu et al. 2008 ), sugarcane (Kaya and Souza 2017 ), Dendranthema grandiflora Tzvelev (Martín and González-Benito 2005 ), and both grape and kiwi (Zhai et al. 2003 ). The genetic fidelity of regenerated plants supports the established vitrification-based cryopreservation protocol as a reliable technique for the long-term storage of F. przewalskii germplasm. Conclusion Conclusion and Future Directions In this paper, we present, for the first time, an effective vitrification-based cryopreservation protocol for F. przewalskii shoot tips, achieving high survival and regeneration rates with minimal genetic variation. A great advantage of this method is the ability to collect frozen material directly from the bulbs of F. przewalskii. This protocol is efficient, low-cost, and provides a reliable method for long-term conservation. Future research should explore the application of this protocol to other rare and endangered species, assessing potential modifications to optimize cryopreservation outcomes. Additionally, further investigation into the molecular mechanisms underlying cryotolerance in F. przewalskii could provide valuable insights, enhancing our understanding of plant responses to extreme dehydration and freezing conditions. Implications for Cryopreservation in Plant Conservation : This protocol offers a practical and replicable method for the cryopreservation of endangered plant germplasm. By contributing to the preservation of F. przewalskii , a valuable medicinal resource, this research supports the broader goals of biodiversity conservation and sustainable resource utilization, offering a model that may encourage the implementation of similar protocols for other medicinal and endemic plant species. Declarations Author contribution Yuming He conducted experiments, analyzed data, and wrote the manuscript. Huan Sun wrote, and revised the manuscript. Hui Fan analyzed data. Chunyu He, Qingyi Guo and Yanhong Zhang conceived and designed research. Yanhong Zhang conceived and designed research and revised the manuscript. Acknowledgements This work was supported by the National Natural Science Foundation of China (81960683); the Science and Technology Project of Gansu Province (No. 21JR1RA263); the Gansu Provincial University Industry Support Project (2020C-09); Gansu University of Traditional Chinese Medicine 2023 Postgraduate Innovation and Entrepreneurship Fund. Conflict of interest We declare that this work was done by the authors mentioned in this article and that all responsibility for claims relating to the content of this article will be borne by the authors. Data availability statement The data that support the findings of this study are available on request from the corresponding author, upon reasonable request. Ethics Declaration The plant material used in this study was commercially cultivated medicinal herb, purchased from licensed local herb farmers. As the samples were obtained from cultivated (non-wild) sources and did not involve endangered or protected species, no specific permit for wild collection was required. The procurement and use of the material comply with relevant national/institutional guidelines for botanical research. References He YM, Sun H, Fan H, He CY, Guo QY, Zhang YH (2024) Vitrification-cryopreservation of shoot tips excised from dormant bulbs in Fritillaria przewalskii maxim: Assessing histological injuries and evaluating genetic fidelity in cryo-derived plants. https://doi.org/10.1016/j.cpb.2024.100415 . 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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-8370894","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":568442597,"identity":"1a33f760-ff43-4790-bdc3-c4960fb1c1dd","order_by":0,"name":"Yuming He","email":"","orcid":"","institution":"Gansu University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuming","middleName":"","lastName":"He","suffix":""},{"id":568442598,"identity":"a1487863-2a4a-4f11-b402-a8f894131663","order_by":1,"name":"Huan Sun","email":"","orcid":"","institution":"Gansu University of Chinese 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12:58:03","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":140223,"visible":true,"origin":"","legend":"","description":"","filename":"PCTOD25009411structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8370894/v1/ccb1f269e74ec3b51cab9f7e.xml"},{"id":99511430,"identity":"ba2ee933-ebac-4969-aff0-d02610748fdf","added_by":"auto","created_at":"2026-01-05 09:36:17","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":156226,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8370894/v1/104c2a737d22489aa8c396ac.html"},{"id":99790576,"identity":"d6beee43-24c2-42d7-93ec-60f99586f7e4","added_by":"auto","created_at":"2026-01-08 12:58:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":162411,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCold hardening pretreatment\u003c/strong\u003e \u003cstrong\u003eon survival and regeneration rates of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFritillaria przewalskii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Maxim shoot tips excised from bulbsafter vitrification cryopreservation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferent letters are significantly different from each other at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, using Duncan’s Multiple Range Test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8370894/v1/030887442ada361f07ea2d24.png"},{"id":99791096,"identity":"47daa5bd-9cad-45d2-89a3-04727562cae8","added_by":"auto","created_at":"2026-01-08 12:59:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":236196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of preculture sucrose concentration and incubation time on survival and regeneration rates xxxxfrom bulbs after xx cryopreservation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferent letters are significantly different from each other at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, using Duncan’s Multiple Range Test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8370894/v1/7e8300337435ea5938660879.png"},{"id":99511409,"identity":"02604aa2-cb76-4b32-967e-d0d345918a21","added_by":"auto","created_at":"2026-01-05 09:36:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":208735,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of PVS2 dehydration time on survival and regeneration rates of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFritillaria przewalskii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Maxim shoot tips excised from bulbs after Vitrification\u003c/strong\u003e \u003cstrong\u003ecryopreservation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferent letters are significantly different from each other at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, using Duncan’s Multiple Range Test.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8370894/v1/0274713d7a46cae6bca71a8c.png"},{"id":99790925,"identity":"5e8b0bf7-3462-49d4-9483-92a0785f4daf","added_by":"auto","created_at":"2026-01-08 12:58:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":180833,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of loading and unloading on survival rate and regeneration rate of shoot tips after cryopreservation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferent letters are significantly different from each other at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, using Duncan’s Multiple Range Test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8370894/v1/d0a2b73134a3e1647284927e.png"},{"id":99511410,"identity":"350ac5df-fcc6-421d-936d-f76b354b63cf","added_by":"auto","created_at":"2026-01-05 09:36:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":138754,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of size (length) of shoot tips on Survival and regeneration rates from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFritillaria przewalskii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Maxim bulbs after vitrification cryopreservation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferent letters are significantly different from each other at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, using Duncan’s Multiple Range Test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8370894/v1/ce117b55a9be0c6b35ff4aea.png"},{"id":99790759,"identity":"2e5ed84f-de3e-4830-a15b-496495d4e1c1","added_by":"auto","created_at":"2026-01-08 12:58:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1869542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe anatomical structure of bulbs of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFritillaria przewalskii \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand the recovery process of shoot tips excised from bulbs after vitrification cryopreservation. \u003c/strong\u003ea Bulb morphology (1, Large scale petal; 2, Small scale petal; 3, Small scale leaf; 4, Shoot tips). b Shoot tipsafter cryopreservation. \u003cstrong\u003ec \u003c/strong\u003eRecovery of shoot tips cultured for 7 days (white arrows indicate viable shoot tips, black arrows indicate dead \u0026nbsp;shoot tips). \u003cstrong\u003ed \u003c/strong\u003eShoot tipsproduce small bulblets.\u003cstrong\u003e e \u003c/strong\u003eSubculture propagation of small bulblets. \u003cstrong\u003ef \u003c/strong\u003eRegenerated seedlings from bulblets.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8370894/v1/1c3be78bbef4263490d1431d.png"},{"id":99511418,"identity":"c204b241-e4aa-48b2-b515-b0c3a18ad372","added_by":"auto","created_at":"2026-01-05 09:36:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4032391,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological changes of shoot tips excised from bulbs of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. przewalskii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e following vitrification cryopreservation. Tissues slices were stained with toluidine blue. \u003c/strong\u003ea1 and a2 represent the complete picture of shoot tips at 4x; b1-g2 represents the CK+, CK-, LS, P\u003csub\u003e60\u003c/sub\u003e, LN\u003csub\u003e60\u003c/sub\u003e, and R\u003csub\u003e60\u003c/sub\u003e leaf primordia and bud axis cells at 40x, respectively; abbreviation \u003cstrong\u003ecu,\u003c/strong\u003e cell nucleus; \u003cstrong\u003enu, \u003c/strong\u003enucleolus; \u003cstrong\u003epl,\u003c/strong\u003e plasmolysis; \u003cstrong\u003ebc,\u003c/strong\u003e broken cells; and \u003cstrong\u003eca,\u003c/strong\u003e cell cavity.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8370894/v1/cfa0b44d78ee918ef2240518.png"},{"id":99511433,"identity":"f9123689-0380-4aa3-8882-5f65a78ba8e1","added_by":"auto","created_at":"2026-01-05 09:36:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1361580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe RAPD genotypic profiles for 20 regenerated plants from of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eprzewalskii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e after vitrification cryopreservation. \u003c/strong\u003eImage a and b is the amplification profiles from primer s303, c and d from primer s308, and e and f from primer s28.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8370894/v1/4ac51b0d756603e31152f443.png"},{"id":102294908,"identity":"454ec01c-46ca-4921-bd28-b9e48af21589","added_by":"auto","created_at":"2026-02-10 10:04:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12408590,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8370894/v1/ea0d5460-45a1-4fc6-8ac1-ac5a81331ff2.pdf"}],"financialInterests":"","formattedTitle":"Vitrification-cryopreservation of Shoot Tips Excised from Dormant Bulbs in Fritillaria przewalskii Maxim: Assessing Histological lnjuries and Evaluating Genetic Fidelity in Cryo-Derived Plants","fulltext":[{"header":"Statement on Prior Submission","content":"\u003cp\u003eAn earlier version of this manuscript was submitted to \u003cem\u003eCurrent Plant Biology\u003c/em\u003e, where it underwent peer review and was accepted (He et al. 2024). However, due to specific institutional requirements mandating that published work be indexed in particular academic databases (including the Chinese Academy of Sciences database), and as \u003cem\u003eCurrent Plant Biology\u003c/em\u003e was not indexed accordingly at that time, the authors made the decision to withdraw the accepted manuscript prior to its formal publication. This withdrawal was based solely on administrative compliance with institutional policies and was in no way related to the scientific content, quality, or integrity of the research. The present manuscript, submitted to Plant Cell, Tissue and Organ Culture (PCTOC), represents a revised version of that work. The core scientific data, results, and conclusions remain unchanged. The withdrawn article is formally cited in the reference list of this manuscript for full transparency.\u003c/p\u003e\n"},{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eFritillaria przewalskii\u003c/em\u003e Maxim. is a perennial herbaceous species in the \u003cem\u003eLiliaceae\u003c/em\u003e family and is a primary source of the traditional Chinese medicine \"Chuan bei\" (Chinese Pharmacopoeia Commission \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The dried underground bulbs of this plant have medical properties, including the ability to clear heat and moisture from the lungs, reduce phlegm, and treat carbuncles (Chinese Pharmacopoeia Commission \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eF\u003c/em\u003e. \u003cem\u003eprzewalskii\u003c/em\u003e is distributed across the western provinces of China, including southern Gansu, western Sichuan, eastern and southern Qinghai. It typically inhabits alpine meadows or grasslands at elevations between 2800 and 4400 meters (Chang et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This species prefers cool, shady and moist environments, which restricts its natural distribution (Chen et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Due to its therapeutic properties, \u003cem\u003eF. przewalskii\u003c/em\u003e Is in high demand. However,tis natural supply can not meet market needs because of its slow growth, long life cycle, and low yield in wild conditions (Carasso et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Currently, most \u003cem\u003eF. przewalskii\u003c/em\u003e in the medicinal market is sourced from wild populations, and over-harvesting has led to a sharp decline in its resourses, resulting in its classification as an endangered species (Zhang et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Therefore, preserving and cultivating this medicinal plant is essential for sustainable use.\u003c/p\u003e \u003cp\u003eCryopreservation, which involves cooling cells or entire tissues to ultralow temperatures using liquid nitrogen (-196℃), halts all cellular division and metabolic processes, theoretically enabling indefinite storage under these conditions (Benelli et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Matsumoto et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Vitrification-based cryopreservation has become the predominant method for long-term storage of germplasm. This approach involves dehydrating plant cells in a highly concentrated vitrification protective solution, followed by rapid immersion in liquid nitrogen, which induces a vitrified state in the cell without allowing ice crystals to form (Chen et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Consequently, vitrification-based cryopreservation is regarded as a simple, cost-effective, and safe method for the long-term conservation of plant germplasm resources (Choudhary et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gupta and Mir \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlant materials used for cryopreservation include pollen, seeds, shoot tips, calluses, and suspension cells. Many of these plant materials present challenges for cryopreservation due to their high water content, elevated metabolic levels, and stress sensitivity (Roque-Borda et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Compared to other parts of the plant, the apical meristem demonstrates a stronger meristematic capacity, and the shoot tip retains significant meristematic ability even after cryopreservation (Barraco et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Consequently, shoot tips are among the preferred materials for cryopreservation, offering several advantages such as ease of handling, minimal space requirements, safety ,stability, and low storage costs (Pennycooke and Towill \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Uchendu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, most current cryopreservation practices employ a slow-freezing technique that relies on freeze-induced dehydration. This approach is primarily used for cryopreserving undifferentiated cultures and the apices of cold-tolerant species, and it requires expensive controlled freezers, limiting its widespread application (Al-Jubori and Mohammed \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The vitrification-based cryopreservation technique used in this study, treats cells with a high concentration of plant vitrification solution. During osmotic dehydration, the cryoprotectant enters the cell, altering the thermodynamic properties of the intracellular liquid. Rapid cooling enables intracellular and extracellular vitrification, thereby preserving cell structure and regeneration capacity, which significantly expands the range of species that can be cryopreserved (Lin 2021).\u003c/p\u003e \u003cp\u003eIn this research, we utilized ex vitro sources of plant material for cryopreservation. A major advantage of this approach is that it eliminates the need to establish an in vitro culture, multiply it, and subsequently excise the shoot tip. Notably, shoot tips were excised directly from ex vitro material, saving considerable time and reducing costs. Research indicates that shoot tips from ex vitro source have been successfully cryopreserved in prior studies. For instance, Bettoni et al. used shoot tips derived from \u003cem\u003eVitis\u003c/em\u003e plants grown in growth chambers as a cost-effective alternative for grape cryopreservation, avoiding the costs of establishing an in vitro preservation system before cryopreservation (Bettoni et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition, shoot tips obtained directly from ex vitro plants have been used for other species, such as citrus (Volk et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo our knowledge, no previous studies have investigated the vitrification cryopreservation of shoot tips excised from bulbs of \u003cem\u003eF\u003c/em\u003e. \u003cem\u003eprzewalskii\u003c/em\u003e. Therefore, the objective of this work is to establish a vitrification-based ceypreservation system for the shoot tips of \u003cem\u003eF. przewalskii\u003c/em\u003e. To this end, we examined the key factors influencing cryopreservation outcomes and preformed histological analyses to assess cell survival during cryopreservation.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Source of explants\u003c/h2\u003e \u003cp\u003eBulbs of \u003cem\u003eFritillaria przewalskii\u003c/em\u003e Maxim. were harvested from Zhang County, Gansu Province (E 103\u0026deg; 57' 38'' to 104\u0026deg; 45' 04 '' N 34\u0026deg; 25' 43'' to 34\u0026deg; 57' 42''), in July 2022 (fall). It is recommended to harvest when the aboveground portion of the plant has not yet withered.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cold hardening pretreatment\u003c/h2\u003e \u003cp\u003eThe bulbs of \u003cem\u003eF. przewalskii\u003c/em\u003e were stored at 4\u0026deg;C in dark for two, three, and four months prior to experiment. Following the cld hardening period, the bulbs were disinfected, and shoot tips were dissected for cryopreservation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Explants disinfection\u003c/h2\u003e \u003cp\u003eThe bulbs were gently cleaned under running water with a soft brush, following by peeling off the outer scales. The bulbs were then disinfected on a sterile workbench by immersing in 75% ethanol for 30 seconds, then in 0.1% mercuric chloride for one minute, and finally rinsed three times with sterile water (He et al, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Sucrose preculture\u003c/h2\u003e \u003cp\u003eSterile shoot tips (from bulbs after three months of cold hardening) were inoculated on pre-culture media and cultured in dark at 25℃. The preculture media contained sucrose at three concentrations: 0.3 M, 0.5 M, and 0.7 M (1/2MS medium with 0.3\u0026ndash;0.7% agar, pH 5.8-6.0). Preculture durations were set at 1 day and 3 days, with 30 shoot tips per treatment. Shoot tips without preculture served as the control group (CK), directly used for vitrification cryopreservation. The cryopreservation steps included a 20-minute loading, 60-minute PVS2 dehydration, storage in liquid nitrogen, and inoculation post-thawing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Vitrification cryopreservation of shoot tips\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Plant Vitrification Solution 2 (PVS2) treatment time\u003c/h2\u003e \u003cp\u003eAfter two months of cold hardening, bulbs were disinfected, and sterile stoot tips were dissected under aseptic conditions. The shoot tips were immediately placed in pre-autoclaved cryovials, followed by the addition of 2 mL loading solution (2 M glycerol, 0.4 M sucrose in MS liquid medium, pH5.8) (Sakai et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) at room temperature for 20 minutes. They were then treated with 2 mL PVS2 (30% w/v glycerol, 15% w/v ethylene glycol, and 15% w/v dimethyl sulfoxide in MS liquid medium with 0.4 M sucrose, pH5.8) (Sakai et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) at room temperature for 30, 60, 90, and 120 minutes. The loading solution and the PVS2 haved been previously autoclaved and filter sterilized, respectively. Five shoot tips were placed in each cryovial, and six cryovials were useed in every PVS2 treatment durations. The cryovials were subsequently placed in liquid nitrogen for storage and later thawed as described in section \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003e2.5.3\u003c/span\u003e. Finally, the shoot tips were inoculated onto the medium as described in 2.6.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Loading treatment\u003c/h2\u003e \u003cp\u003eFor one treatment group, shoot tips were exposed to 2 mL of loading solution at room temperature for 20 minutes, followed by 2 mL PVS2 dehydrated for 60 minutes, and then exposed to liquid nitrogen for more than one hour. As a control to evaluate the effect of the loading solution, another group was directly dehydration in 2 mL PVS2 without prior loading, with all other conditions kept consistent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3 Unloading process\u003c/h2\u003e \u003cp\u003eCryovial were removed from liquid nitrogen and rewarmed in a 38℃ water bath for 2 minutes. The shoot tips were then divided into two groups: one group was immersed in an unloading solution (1.2 M sucrose in MS liquid medium, pH5.8) at room temperature for 20 minutes before inoculation while the other group was directly inoculated onto the medium without the unloading process. The unloading solution was filter-sterilized in advance. Thirty shoot tips were included in each group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4 Shoot tips size\u003c/h2\u003e \u003cp\u003eThe shoot tips were categorized into three size groups: large (greater than 4\u0026ndash;5 mm), medium (2\u0026ndash;4 mm), and small (less than 2 mm). Vitrification cryopreservation was performed, and thirty shoot tips from each size group were selected. The protocol included treating theshoot tips with 2 mL of loading solution at room temperature for 20 minutes, followed by 2 mL of PVS2 for 60 minutes at room temperature, and then exposured to liquid nitrogen for more than one hour. Cryovials were thawed in a 38℃ water bath for 2 minutes, and shoot tips were directly inoculated onto the recovery medium without unloading.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Recovery growth after cryopreservation\u003c/h2\u003e \u003cp\u003eThe shoot tips were inoculated onto 1/2 MS medium supplemented with 0.5 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 6-BA (6-Benzylaminopurine), 0.1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA (1-naphthlcetic acid), 3% sucrose, and 0.7% agar, pH 5.8\u0026ndash;6.0, for recovery. During the first week, explants were kept in the dark, then transferred to 12-hour photoperiod with 1500\u0026ndash;2000 lx light intensity at 25℃ (He et al, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSurvival rate was recorded 7 days post-culture and expressed as the percentage of the shoot tips turning green. The regeneration rate was defined as the percentage of shoot tips developing into bulblets 5 weeks post-culture.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Histological analysis of shoot tips during cryopreservation\u003c/h2\u003e \u003cp\u003eShoot tips were collected at various stages for histological analysis:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eControl (CK+): Untreated shoot tips.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eNegative control (CK-): Shoot tips plunged directly into liquid nitrogen for more than 60 minutes, followed by rewarming.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eLoading (LS): Shoot tips were immersed in 2 mL cryovials at room temperature for 20 minutes with a loading solution.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ePVS2 (P\u003csub\u003e60\u003c/sub\u003e): After loading shoot tips were treated with PVS2 for 60 minutes at room temperature.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eLiquid nitrogen (LN\u003csub\u003e60\u003c/sub\u003e): Shoot tips after dehydration in cryovials were rapidly plunged into liquid nitrogen (LN) and held for 60 minutes.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eRewarming: Shoot tips after LN freezing rewarmed in 38℃ water bath for 2 minutes.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eRecovery (R\u003csub\u003e60\u003c/sub\u003e): Shoot tips cultured in the dark for one week on 1/2MS medium supplemented with 0.5 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 6-BA ,0.1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA, 3% sucrose, 0.7% agar, pH 5.8\u0026ndash;6.0.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eShoot tips were fixed in FAA (ethanol:formalin:acetic acid\u0026thinsp;=\u0026thinsp;18:1:1) solution for 24 hours, dehydrated through an ethanol series (70%, 85%, 95%, and 100% ethanol) and embedded in paraffin. For light microscopy studies, 5 \u0026micro;m sections were prepared using a Leica RM2125 microtome and stained with 0.01% toluidine blue for microscopy (Li \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Untreated shoot tips served as a positive control, while those that were freshly excised, directly immersed in LN served as a negative control. Each treatment contained at least five shoot tips.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Genetic stability evaluation in regenerated plants\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.8.1 Morphological markers\u003c/h2\u003e \u003cp\u003eFresh regenerated bulbs were selected after five subcultures, and morphological traits of the regenerated bulbs and the original bulbs were observed, including bulb color and leaf shape and color of plantlets.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.8.2 Genomic DNA isolation and RAPD markers\u003c/h2\u003e \u003cp\u003eGenetic fidelity was evaluated by random amplified polymorphic DNA (RAPD). Five shoot tips were randomly selected from 30 replicates under the same treatment, and the genetic fidelity of the ultra-low temperature regenerated bulblets of these shoot tips was evaluated after five subcultures. DNA was extracted by modified CTAB method (Coelho et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Four regenerated bulblets from the same shoot tip were randomly selected, and 0.02 g fresh bulblets were weighed in each part. A total of 20 samples were collected from 5 shoot tips. DNA was purified and quantified using ultraviolet spectrophotometry. Each sample was diluted to 50 ng/\u0026micro;L in deionized water and stored at -20℃ until use. Suitable primers were selected from 20 screened primers for genetic stability assessment. PCR was conducted in a reaction mixture containing 12.5 \u0026micro;L 2\u0026times;Taq PCR Master MixⅡ(with Dye), 1 \u0026micro;L of 10 \u0026micro;mol/L primer and 2 \u0026micro;L of 50 ng/\u0026micro;L DNA template in a final volume of 25 \u0026micro;L. PCR amplification was performed in a thermal cycler under the following reaction conditions: initial denaturation step for 4 minutes at 94℃, 38 cycles at 94℃ for 30 seconds, annealing at 40℃ for 60 seconds, and extension at 72℃ for 2 minutes followed by the final extension step at 72℃ for 8 minutes (Jiang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The PCR products were separated by electrophoresis in 1.5% agarose gel containing GoldView Ⅰ and the image was photographed under a gel image system (Gel Doc XRTM). The molecular mass ladder DNA Marker DL2000 was used for estimating the size of the amplified products.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical Analysis\u003c/h2\u003e \u003cp\u003eEach cryopreservation treatments included 30 explants, with each cryovial containing 5 shoots as one replicate. The data were compared by Duncan multiple-range test, and comparisons between the mean values ere evaluated by the least significant different test at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. For genetic stability assessments, RAPD profiles were manually scored for the presence or absence of bands. Bands with low visual intensity that could not be readily distinguished were not scored.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Factors affecting vitrification-based cryopreservation of shoot tips\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Cold hardening time of bulbs\u003c/h2\u003e \u003cp\u003eAs the duration of cold acclimation increased, the survival and regeneration rates of shoot tips improved markably (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Specifically, after two months of acclimation at 4℃, the survival and regeneration rates of excised shoot tips were observed to be 47% and 37%, respectively. When the acclimation period was extended to three months, these rates significantly rose to 77% and 60%, respectively. Extending the acclimation period to four monthsprovided a further increase, although the difference compared to three months was not statistically significant. Therefore, maintaining bulbs of \u003cem\u003eF. przewalskii\u003c/em\u003e at 4\u0026deg;C for three to four months before cryopreservation optimizes recovery outcomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Sucrose preculture\u003c/h2\u003e \u003cp\u003eComparisons were mad among vary sucrose concentrations and preculture durations for their effects on the shoot tip survival and regeneration rates post-cryopreservation. When shoot tips were precultured for one day, increasing sucrose concentration led to a slight decline in survival and regeneration rates. However, extending the preculture period to three days yielded improved survival and regeneration rates with 0.3 M and 0.5 M sucrose, while 0.7 M sucrose resulted in a slight decrease. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Notably, The treatment with 0.5 M sucrose for three days achieved the highest survival and regeneration rates of 93%, following exposure to the loading solution for 20 minutes and PVS2 for 60 minutes at room temperature before immersion in liquid nitrogen. Therefore, a sucrose concentration of 0.5 M for three days was identified as the optimal preculture condition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3 The treatment of PVS2 dehydration\u003c/h2\u003e \u003cp\u003eThe duration of PVS2 dehydration influenced regeneration, although it had minimal effect on survival rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). While shoot tips survival did not significantly fluctuate across PVS2 treatment times ranging from 30 to 120 minutes,the regeneration rate was notably affected, showing a peak at 60 minutes, which survival and regeneration rate were 47% and 43% respectively. Shoot tips treated with PVS2 for 60 minutes exhibited the highest regeneration rate, suggesting that this duration provides an optimal balance for cellular dehydration and viability under ultralow temperatures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.1.4 Loading and unloading\u003c/h2\u003e \u003cp\u003ePre-treatment with loading solution did not significantly impact shoot tip survival; however, it did contribute to a 30% increase in regeneration rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Without loading, the regeneration rate was only 37% when the shoot tips were directly treated with PVS2 for 60 minutes. In contrast, pre-treatment with the a loading solution for 20 minutes, followed by 60 minutes of PVS2 dehydration, increased the regeneration rate to 77%. Though the unloading step did not lead to a statistically significant difference, the treatment group that underwent unloading exhibited slightly higher survival and regeneration rates (83% and 77%, respectively) compared to those without unloading.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.1.5 Shoot tips size\u003c/h2\u003e \u003cp\u003eThe length of the shoot tips significantly affected the post-cryopreservation recovery, with survival and regeneration rates improving as shoot tip size increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Shoot tips shorter than 2 mm displayed the lowest survival and regeneration rates following the standard cryopeservation procedure. In contrast, shoot tips measuring between 2\u0026ndash;4 mm achieved the highest survival and regeneration rates, at 77% and 70%, respectively, under 20 minutes of loading and 60 minutes of PVS2 dehydration at room temperature. For shoot tip exceeding 4 mm, both survival and regeneration rates decreased, indicating that 2\u0026ndash;4 mm is the optimal size for successful cryopreservation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Recovery culture\u003c/h2\u003e \u003cp\u003eBefore cryopreservation, the shoot tips exhibited a slender, conical shape with a pale yellow hue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), Post-cryopreservation, the shoot tips turned white (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Within the initial 7-day culture period, some buds showed signs of viability by turning green (white arrow in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), while those that did not change color or turned brown were considered non-viable (black arrow in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). After a 5-week culture period, viable shoot tips developed into 4\u0026ndash;5 small, white, and spherical bulblets on the surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed),which could propagate further through subcultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Transferred to rooting media, these bulblets developed into complete plantlets (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Histological analysis of cryopreserved shoot tips\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eMicroscopic Examination of Cellular Integrity\u003c/strong\u003e \u003cp\u003eControl shoot tip showed distinct staining with a well-defined nucleus and nucleolus, indicating intact cellular structure (Fig.\u0026nbsp;7b1, b2). The apical meristem cells and leaf primordium cells are tiny, virtually spherical with dense cytoplasm and a high nuclear-to-cytoplasm ratio. Nucleus and nucleolus were clearly visible (Fig.\u0026nbsp;7b1). The bud axis cells are approximately rectangular, the cells are large, the nucleus is located in the center of the cell, and there are one or more nucleoli in the nucleus (Fig.\u0026nbsp;7b2). In the negative control (untreated shoot tips directly plunged into liquid nitrogen), cells appeared lightly stained with disrupted plasma membranes, dissolved nuclei, and absent nucleoli, suggesting extensive cryo-injury (Fig.\u0026nbsp;7c1, c2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePlasmolysis Observations\u003c/strong\u003e \u003cp\u003eTreatment with loading solution caused minor plasmolysis without severe cellular damage (Fig.\u0026nbsp;7d1, d2). Subsequent exposure to PVS2 intensified plasmolysis, particularly in the lower half of the shoot tips (Fig.\u0026nbsp;7e1, e2). During the freezing and rewarming steps, serious damage was observed, including ruptured cell walls, cytoplasmic degeneration, and nucleolar disappearance in most cells, except in the apical meristem, which largely remained viable (Fig.\u0026nbsp;7f1, f2). Upon recovery in culture, visible cells exhibited a restored structure with clear nuclei and visible nucleoli in certain cells, suggesting the apical meristem as a key survival site during cryopreservation (Fig.\u0026nbsp;7g1, g2).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHistological Implications\u003c/strong\u003e \u003cp\u003eThe histological analysis underscores the effectiveness of PVS2 in inducing protective dehydration, though excessive plasmolysis occurs in some regions. The resilience of the apical meristem cells aligns with the survival outcomes, emphasizing their crucial role in shoot tip viability after cryopreservation.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Genetic stability of cryo-derived regenerants\u003c/h2\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Morphological analysis\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eMorphological Consistency\u003c/strong\u003e \u003cp\u003eBulblets regenerated from cryopreserved shoot tips consistently exhibited a dense texture, white coloration, and 2\u0026ndash;4 scales, with the characteristic lanceolate, green leaves of \u003cem\u003eF.\u003c/em\u003e przewalskii. uniform morphological traits across regenerants indicate that cryopreservation did not induce phenotypic deviations, supporting the preservation of genotype integrity.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2 RAPD analysis for genetic fidelity\u003c/h2\u003e \u003cp\u003eUsing three RAPD primers, a total of 400 monomorphic bands were generated from 20 regenerat samples, with no variations (absence or gain of loci) detected across samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This genetic consistency confirms the absence of somaclonal variation, validating the genetic stability of the cryopreserved germplasm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4 Discussions","content":"\u003cp\u003e \u003cstrong\u003eCryopreservation Challenges and Significance\u003c/strong\u003e \u003cp\u003eThe preservation of endemic and endangered poses unique challenges due to their ecological specificity, genetic diversity, and often limited accessibility. In the case of Fritillaria przewalskii, an endangered medicinal plant, developing an effective cryopreservation protocol is critical for its long-term conservation and sustainable utilization. This study presents a comprehensive vitrification-based cryopreservation protocol for F. przewalskii, providing insights into factors such as shoot tips size, cold acclimation, and PVS2 dehydration time, which are essential for optimizing cryopreservation outcomes. These findings the broader body of knowledge on plant germplasm preservation, offering practical guidelines that may be adapted for other endangered species with similar conservation needs. (Charoensub et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Lambardi et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEffect of Explant Size on Cryopreservation\u003c/strong\u003e \u003cp\u003eIn general, small material can be seriously damaged during the dehydration process, making regeneration difficult. Conversely, if the material is too large, the cryoprotectant solution may struggle to infiltrate the interior cells, resulting in inadequate protection during the subsequent freezing step and a low regeneration rate. It has been found that the optimal shoot-tip size of cryopreservation of sweet potato [ \u003cem\u003eIpomoea batatas\u003c/em\u003e (L.) Lam. ] using the vitrification method is 0.5-1.0 mm long (Pennycooke and Towill \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). For garlic, 1.5\u0026times;3.0 mm shoot apices were precultured at 10\u0026deg;C for 3 days on medium containing 0.1 M sucrose, followed by dehydrated in PVS3 solution for 150 min at 23\u0026deg;C prior to freezing, resulting in a survival percentage as high as 85% and a regeneration rate exceeding 73% (Baek et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In our study, the 2\u0026ndash;4 mm long shoot tips were exposed in the loading solution for 20 minutes and then to PVS2 for 60 minutes at room temperature. We achieved a high survival percentage of 77% and a regeneration rate of 70%. Furthermore, the appropriate PVS2 treatment time varies depending on the size of the material. Even with the same treatment time, regeneration of plant materials can differ at various temperatures. Therefore, it is essential to formulate the most suitable cryopreservation method according to the specific plant materials.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eOptimizing Sucrose Preculture and Cold Acclimation\u003c/strong\u003e \u003cp\u003ePretreating materials before vitrification effectively reduces tissue water content and enhance cold resistance, allowing the materials to maintain high viability after cryopreservation (Salaj et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Hong and Yin \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sershen et al. 2012). Sucrose preculture and cold acclimation are the two most commonly used methods for improving plant recovery in cryopreservation. Numerous studies, such as those involving \u003cem\u003eVitis\u003c/em\u003e (Volk et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), kiwifruit (Mathew et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), potato (Kaczmarczyk et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), \u003cem\u003ePyrus\u003c/em\u003e cordata (Chang and Reed \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and \u003cem\u003ePrunus cerasus\u003c/em\u003e (Barraco et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), have reported favorable results using high concentration of sucrose for preculture. In this study, both the survival percentage and regeneration rate of shoot tips from \u003cem\u003eF. przewalskii\u003c/em\u003e reached a maximum of 93% when the tips were precultured on 0.5 M sucrose for 3 days, followed by 20 minutes in the loading solution and 60 minutes in PVS2 at room temperature before exposure to liquid nitrogen. This indicated that a high concentration of sucrose in the medium can induce cell dehydration by increasing osmotic pressure, thereby reducing the free water content in cells and enhancing cold resistance (Bettoni et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kulus et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Many studies have shown that cold acclimation can activate cold resistance mechanism in plants and improve the survival of shoot tips during cryopreservation, particularly for low-temperature sensitive species (Channuntapipat et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Engelmann \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Harding et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). For example, with cold hardening for 6 to 14 weeks, the regrowth percentage of \u003cem\u003eMalus domestica\u003c/em\u003e Borkh. shoot tips peaked at 88% during the 6th and 8th weeks, and the plants remained capable of regenerating even at 14 weeks (Bilavč\u0026iacute;k et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The survival percentage of blueberry shoot tips was highest (91%) when treated at low temperature for 2 to 4 weeks (Wang et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In the present study, both the survival and regeneration rates of \u003cem\u003eF. przewalskii\u003c/em\u003e shoot tips after cryopreservation increased with the duration of cold acclimation.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDehydration with PVS2 and the Importance of Dehydration Duration\u003c/strong\u003e \u003cp\u003ePlant Vitrification Solution (PVS) prevents intracellular water from forming ice crystals or limits the time available for ice crystals to grow, resulting in cells entering a state of artificial complete vitrification (Volk and Walters \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Halmagyi et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Currently, the most commonly used vitrification solution is PVS2, and the optimal dehydration time of PVS2 is varies among different plant species (Bilavč\u0026iacute;k 2012; Matsumoto et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sant et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). If the PVS2 treatment time is insufficient, the cell dehydration may be not be complete, leading to the formation of ice crystals during freeziing in liquid nitrogen, which can seriously injure the cells. Conversely, if the treatment time is too long, the material can be damaged by PVS2, resulting in a significant reduction in survival percentage and regeneration rate (Matsumoto et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, determining the appropriate dehydration time in PVS2 is crucial for minimizing cell damage and enhancing cell tolerance to freezing. Unloading is a standard step in the cryopreservation process. The unloading solution treatment is implemented to prevent the secondary toxicity of the PVS2 solution on shoot tips, necessitating thorough washing of the tips. However, in our study, unloading had little effect on the survival and regeneration of the shoot tips after cryopreservation. Research has shown that unloading significantly influences cryopreservation outcomes, with factors such as unloading time, unloading conditions, and sucrose concentration in the unloading solution playing critical roles. The optimal unloading conditions vary for each plant species and are closely related to specific interspecies differences (Rajasekharan and Prakashkumar \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Sajini et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Therefore, there was no significant difference in the survival of \u003cem\u003eF. przewalskii\u003c/em\u003e after cryopreservation, regardless of whether unloading solution treatment was applied.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHistological Analysis of Shoot Tip Damage During Cryopreservation\u003c/strong\u003e \u003cp\u003eThe alternation and damage of shoot tips during cryopreservation can be observed through histological sections. After dehydration with the loading solution, the cells in each part of the shoot tips lost water and exhibited slight plasmolysis. The treatment with PVS2 subjected the cells to higher osmotic pressure, resulting in severe dehydration and further plasmolysis (Popov et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Cryopreserved cells also experienced mechanical damage, characterized by severe plasmolysis, cell membrane rupture, nuclear fragmentation, and nucleolus disappearance. After direct cryopreservation of shoot tips, most cells sustained irreversible damage, except for the apical meristem, which maintained structural integrity. The occurrence of cell damage is likely attributed to the extreme decline in water content, resulting in the inability of protoplasts to contract and concentrate cell fluid (Ganino et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Additionally, freezing damage that results in cell death is caused by intracellular water crystallization during the freezing or rewarming processes. Consequently, the freezing and rewarming process inflicted significant damage to the buds, primarily manifested as the breakage of the cell wall in the bud axis, blurring of the nuclear boundary, ablation of the cytoplasm, and disappearance of the nucleolus. Although the shoot apical meristem exhibited slight plasmolysis, it remained the least damaged part, maintaining overall cell integrity. This region is crucial for the survival of shoot tips. Similar phenomena have also been observed in the cryopreservation of other species, including apple (Feng et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), \u003cem\u003eRubus\u003c/em\u003e (Chang and Reed \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), \u003cem\u003eDendrobium\u003c/em\u003e (Antony et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ching et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Poobathy et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and \u003cem\u003eBrassidium\u003c/em\u003e (Mubbarakh et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eGenetic Fidelity of Cryo-Derived Plants\u003c/strong\u003e \u003cp\u003eThe genetic fidelity of cryo-derived plants is essential for ensuring the effectiveness of techniques applied to plant germplasm conservation. The RAPD analysis confirmed the genetic stability of regenerants, indicating that the vitrification-based protocol preserved the genetic fidelity of \u003cem\u003eF. przewalskii\u003c/em\u003e. This is essential for germplasm conservation, as it ensures that preserved plants retain their original genetic composition, a prerequisite for any effective conservation strategy. Similarly, genetic stability has been confirmed in shoot tips or dormant buds following cryopreservation in \u003cem\u003eDioscorea rotundata\u003c/em\u003e Poir (Mandal et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), apple (Liu et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), sugarcane (Kaya and Souza \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), \u003cem\u003eDendranthema grandiflora\u003c/em\u003e Tzvelev (Mart\u0026iacute;n and Gonz\u0026aacute;lez-Benito \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and both grape and kiwi (Zhai et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The genetic fidelity of regenerated plants supports the established vitrification-based cryopreservation protocol as a reliable technique for the long-term storage of \u003cem\u003eF. przewalskii\u003c/em\u003e germplasm.\u003c/p\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cstrong\u003eConclusion and Future Directions\u003c/strong\u003e \u003cp\u003eIn this paper, we present, for the first time, an effective vitrification-based cryopreservation protocol for F. przewalskii shoot tips, achieving high survival and regeneration rates with minimal genetic variation. A great advantage of this method is the ability to collect frozen material directly from the bulbs of \u003cem\u003eF. przewalskii.\u003c/em\u003e This protocol is efficient, low-cost, and provides a reliable method for long-term conservation. Future research should explore the application of this protocol to other rare and endangered species, assessing potential modifications to optimize cryopreservation outcomes. Additionally, further investigation into the molecular mechanisms underlying cryotolerance in \u003cem\u003eF. przewalskii\u003c/em\u003e could provide valuable insights, enhancing our understanding of plant responses to extreme dehydration and freezing conditions.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImplications for Cryopreservation in Plant Conservation\u003c/b\u003e: This protocol offers a practical and replicable method for the cryopreservation of endangered plant germplasm. By contributing to the preservation of \u003cem\u003eF. przewalskii\u003c/em\u003e, a valuable medicinal resource, this research supports the broader goals of biodiversity conservation and sustainable resource utilization, offering a model that may encourage the implementation of similar protocols for other medicinal and endemic plant species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuming He conducted experiments, analyzed data, and wrote the manuscript. Huan Sun wrote, and revised the manuscript. Hui Fan analyzed data. Chunyu He, Qingyi Guo and Yanhong Zhang conceived and designed research. Yanhong Zhang conceived and designed research and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (81960683); the Science and Technology Project of Gansu Province (No. 21JR1RA263); the Gansu Provincial University Industry Support Project (2020C-09); Gansu University of Traditional Chinese Medicine 2023 Postgraduate Innovation and Entrepreneurship Fund.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare that this work was done by the authors mentioned in this article and that all responsibility for claims relating to the content of this article will be borne by the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available on request from the corresponding author, upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plant material used in this study was commercially cultivated medicinal herb, purchased from licensed local herb farmers. As the samples were obtained from cultivated (non-wild) sources and did not involve endangered or protected species, no specific permit for wild collection was required. The procurement and use of the material comply with relevant national/institutional guidelines for botanical research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHe YM, Sun H, Fan H, He CY, Guo QY, Zhang YH (2024) Vitrification-cryopreservation of shoot tips excised from dormant bulbs in Fritillaria przewalskii maxim: Assessing histological injuries and evaluating genetic fidelity in cryo-derived plants. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cpb.2024.100415\u003c/span\u003e\u003cspan address=\"10.1016/j.cpb.2024.100415\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 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Biochem Syst Ecol 38:236\u0026ndash;242\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"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":"Genetic stability, In vitro conservation, Paraffin section, Regenerated plant, Shoot tips, Vitrification cryopreservation","lastPublishedDoi":"10.21203/rs.3.rs-8370894/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8370894/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eFritillaria przewalskii\u003c/em\u003e Maxim. an essential medicinal plant in the \u003cem\u003eFritillaria\u003c/em\u003e genus of the Liliaceae family, is widely used in traditional medicine. This study focused on developing a cryopreservation protocol for \u003cem\u003eF. przewalskii\u003c/em\u003e, an endangered and endemic species in China. We aimed to determine if ex vitro bulbs from field-grown \u003cem\u003eF. przewalskii\u003c/em\u003e plants could serve a source of shoot tips for cryopreservation. Key factors influencing cryopreservation outcomes were studied using a vitrification-based approach. Prior to cryopreservation, cold hardening and sucrose preculture conditions were optimized. The best results were achieved by hardening bulbs at 4℃ for 3\u0026ndash;4 months,followed by surface disinfection and isolation of 2\u0026ndash;5 mm Shoot tips. The tips were then then precultured on a medium with 0.5M sucrose for 3 days. Cryopreservation steps included a 20-minute loading treatment, 60-minute PVS2 dehydration at room temperature, and storage in liquid nitrogen more than one hour. After thawing, this protocol resulted in 93% shoot survival rate, with tips regenerating into small bulbs within 5 weeks without intermediary callus formation. Histological analysis showed that while cooling and rewarming caused severe bud damage, apical meristem cells are the primary survival sites. Although loading and PVS2 treatment induced cell plasmolysis, it was not lethal. Optimal PVS2 treatment enhanced cellular dehydration resistance, promoting cell viability at ultralow temperature. RAPD analysis of regenepercentaged plants showed no genetic variation, confirming strong genetic stability. The direct use of ex vitro shoot tips provides a straightforward and effective cryopreservation method for \u003cem\u003eF. przewalskii\u003c/em\u003e, combining convenience, efficiency, and genetic stability.\u003c/p\u003e","manuscriptTitle":"Vitrification-cryopreservation of Shoot Tips Excised from Dormant Bulbs in Fritillaria przewalskii Maxim: Assessing Histological lnjuries and Evaluating Genetic Fidelity in Cryo-Derived Plants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-05 09:36:12","doi":"10.21203/rs.3.rs-8370894/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":"bc4cbdd7-3f76-4ace-82a3-57bef2009465","owner":[],"postedDate":"January 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-23T19:03:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-05 09:36:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8370894","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8370894","identity":"rs-8370894","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}