1 Droplet vitrification: A lifeline for long-term conservation of threatened species Garcinia 1 indica 2 Vartika Srivastavaa, Bart Panisb, Anuradha Agrawalc* 3 a Division of Germplasm Conservation, Indian Council of Agricultural Research (ICAR)-National 4 Bureau of Plant Genetic Resources (NBPGR), New Delhi, 110012, India 5 b Alliance of Bioversity International and CIAT, KU Leuven, 3001, Belgium 6 c National Agricultural Higher Education Project (NAHEP), ICAR, Krishi Anusandhan Bhawan-7 II, Pusa Campus, New Delhi, 110012, India 8 9 *Corresponding author email: Anuradha.Agrawal@icar.gov.in 10 11 ORCID ID 12 Vartika Srivastava: 0000-0002-3625-0728 13 Bart Panis: 0000-0001-6717-947X 14 Anuradha Agrawal: 0000-0003-1499-8572 15 16 17 18 mailto:Anuradha.Agrawal@icar.gov.in 2 Abstract 19 Cryopreservation is a promising technique for the ex situ long-term conservation of plant 20 biodiversity particularly of species that are not amenable to seed bank conservation. Garcinia 21 indica (Thouars) Choisy is a species endemic to the biodiversity hotspot of the Western Ghats of 22 India. Conservation of this species is difficult as it produces recalcitrant seeds, and, therefore, it 23 can only be conserved in the field genebanks or in their natural habitats. This high-value fruit tree 24 species is listed as vulnerable due to the rapid loss of the natural population, and, therefore, the 25 conservation of its genetic diversity is imperative. This study described the first successful 26 cryopreservation protocol for G. indica through a modified droplet-vitrification technique using in 27 vitro derived shoots conserved in the In Vitro Genebank of the Indian Council of Agricultural 28 Research - National Bureau of Plant Genetic Resources (ICAR- NBPGR), New Delhi. Among 29 vitrification (V) and droplet-vitrification (DV) techniques, only the DV technique yielded explant 30 regeneration after cryopreservation. Apical shoots (2 mm long) collected from 24-wk-old explants 31 (IC638183), without any pre-culture, showed maximum regeneration (51.76%) on Murashige and 32 Skoog (MS) medium supplemented with 2.22 µM 6-benzylaminopurine (BAP). The regenerated 33 shoots were hardened in a mist chamber successfully (85% survival), which is essential for the 34 future restoration of the species in the field. With genotypic-dependent variation, this protocol was 35 applicable to the other three accessions (IC638184, IC638185, and IC638186) with an average of 36 43.7% regeneration after cryopreservation, which can be implemented for the effective 37 conservation of G. indica germplasm. 38 Keywords Cryopreservation, Garcinia indica, Threatened plant species, Ex situ conservation 39 40 3 Introduction 41 The irreversible loss of species by extinction due to human activity is addressed in SDG 15 (Life 42 on Land), a part of the United Nations' Sustainable Development Goals, which calls for the 43 protection, restoration, and sustainable utilization of terrestrial ecosystems to prevent biodiversity 44 loss. The overall extinction risk indicated in the Red List Index has increased in the last three 45 decades with each successive decade witnessing a faster deterioration. These trends are inhibiting 46 the goal of halting biodiversity loss and preventing the extinction of endangered species. Science-47 led solutions, including the adoption of different conservation strategies, are necessary to address 48 this issue. These strategies can be related to in situ or ex situ maintenance of plant populations. 49 However, in situ conservation in coastal areas is exposed to multiple climate-related hazards, 50 including tropical cyclones, sea level rise and flooding, marine heatwaves, sea-ice loss, and 51 permafrost thaw (https://sdgs.un.org/publications/publication-making-peace-nature-scientific-52 blueprint-tackle-climate-biodiversity-and). Therefore, to supplement the in situ conservation, ex 53 situ conservation of at least 75% of threatened plant species preferably in the country of origin, is 54 considered as an additional measure under Target 8 of the Global Strategy for Plant Conservation 55 (https://www.cbd.int/gspc/targets.shtml). Wyse and Dickie (2017) estimated that 7.5% to 19.6% 56 of the world’s seed-plant species (mean value 8%) bear desiccation-sensitive seeds. Importantly, 57 18.5% of the seed-plant flora in tropical and subtropical moist broadleaf species possessed the 58 trait, due to which their conservation in traditional seed genebank is difficult. The conservation of 59 threatened plant species with recalcitrant seeds is challenging, necessitating the use of alternative 60 conservation techniques for their timely and safe conservation (Wyse et al. 2018). 61 The Indian subcontinent is known for its biodiversity-rich areas contributing to 11.9% of 62 the world’s flora, including 33% endemic species in its four biodiversity hotspots, such as the 63 https://sdgs.un.org/publications/publication-making-peace-nature-scientific-blueprint-tackle-climate-biodiversity-and https://sdgs.un.org/publications/publication-making-peace-nature-scientific-blueprint-tackle-climate-biodiversity-and 4 Western Ghats, harboring nearly 1,600 endemic species (Arora 1991). Known for its humid 64 tropical climate, the Western Ghats is home to a wide variety of Garcinia spp. The genus Garcinia, 65 encompassing about 250 species, is the largest within the family Clusiaceae of which the habitat 66 is restricted to the South-East Asian region (Mabberley 2008). Garcinia species have recently 67 received a lot of attention from the scientific and industrial sectors all over the world, and as a 68 result, several novel structures, bioactive compounds, and potential applications have been 69 reported. This potentially valuable group of trees is the focal point for various industrial sectors, 70 such as pharmaceutical, nutraceutical, paint, and food additives (Hemshekhar et al. 2011; 71 Magadula and Mbwambo 2014). Secondary metabolites, like xanthones, benzophenones, and 72 bioflavonoids, in major quantities and flavonoids, biphenyls, acyl phloroglucinols, depsidones, 73 and triterpenoids in minor quantities are found in Garcinia species (Arvind et al. 2016; Akoro et 74 al. 2018). Nearly 43 species and five varieties of this genus are known to occur in India; 37 species 75 and four varieties are found in the wild, and only six species and one variety are reported to be 76 cultivated (Baruah et al. 2021). A rich diversity of this species is found in the Western Ghats of 77 India with nine species and two varieties of which seven species and two varieties are endemic 78 (Shameer et al. 2016). 79 Garcinia indica (Thouars) Choisy, commonly known as ‘Kokum’, Kokum butter tree, 80 Goa-butter tree, or wild mangosteen, is an important tree species indigenous to the Indian 81 subcontinent. Even though it is considered an underutilized fruit due to unorganized and scattered 82 plantations, G. indica is listed as one of the 32 prioritized species by the National Medicinal Plant 83 Board (NMPB) of India (Ravi et al. 2022). Its distribution extends from the northern parts of 84 central Sahyadri to the South Konkan coast of the Western Ghats of India (Watt 1890; Baliga et 85 al. 2011). Natural populations are recorded in Kerala (Badi Baduka, Thaliparamba), Maharashtra 86 5 (Thungar Hill, North Kanara), Karnataka (Tinai Ghat), and in some parts of Assam (Karbi 87 Anglong) (Shameer et al. 2016). The seed is a rich source of edible fat known as 'kokum butter', 88 which has a high economic value in the food and pharmaceutical industries. The dried rinds of the 89 fruits are used to prepare a refreshing drink in summer and are used as an alternative to tamarind 90 in various Indian food preparations. Garcinol is an important benzophenone of nutraceutical 91 importance found in G. indica that can be used as an anti-cancer agent (Padhye et al. 2012). 92 Bioactive compounds, for example guttiferones, another class of benzophenones, are of great 93 interest in pharmaceutical research, particularly due to their anti-HIV, trypanocidal, and cytotoxic 94 activities (Acuna et al. 2009). The fruit rind is high in hydroxycitric acid (HCA), a biologically 95 active plant metabolite used as an anti-obesity drug that inhibits the conversion of carbohydrates 96 into fats by inhibiting the ATP citrate lyase enzyme (Heymsfield et al. 1998). Apart from its health 97 and pharmaceutical applications, G. indica seed oil with its high fatty acid methyl ester content 98 has the potential to serve as biofuel and improve fuel efficiency when combined with other fuels 99 (Hosamani et al. 2009). This versatile tree species carries the potential to address the global 100 demand for clean energy-based biofuels, extending its significance well beyond India. 101 Nonetheless, the diminishing population trend of G. indica has led to its classification as 102 ‘Vulnerable’ on the IUCN Red List (Ved et al. 2015). A recent analysis underscores the severity 103 of the situation, particularly in the North Western Ghats, where G. indica distribution has reached 104 critical levels. The analysis done using a prediction map of habitat suitability that used MaxEnt 105 modeling indicated that by 2050 a substantial portion of the current distribution area of G. indica 106 is expected to convert to a low-potential habitat, and this trend is predicted to gradually increase 107 by 2070 (Pramanik et al. 2018). This implies that the habitat conditions for G. indica are forecasted 108 to deteriorate significantly over time, potentially impacting its population and survival. This 109 6 emphasizes the pressing need for proactive measures to safeguard the genetic diversity of this 110 important species as in situ conservation in its natural habitat appears increasingly unsafe in the 111 near future. 112 For ex situ conservation in field genebanks (FGBs), the diverse accessions of G. indica are 113 collected through exploration missions in diversity-rich areas of Western Ghats by ICAR-NBPGR, 114 New Delhi in collaboration with other ICAR research institutes and Universities (Fig. 1). FGB 115 conservation of G. indica is only limited to a few sites with over 60% of accessions maintained in 116 Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli, (Maharashtra). Meanwhile, only 15% 117 of collections are conserved in the ICAR-NBPGR, Regional Station, Thrissur, (Kerala), and the 118 remaining accessions, over 25%, are conserved in FGBs across only six institutes (Table 1). 119 Furthermore, with these sites in coastal regions, concentrating most accessions in one location is 120 suboptimal, increasing the risk of germplasm loss due to various stresses. Thus, it's crucial to 121 explore alternative measures using cryopreservation to conserve the genetic diversity of this 122 species. 123 Due to the recalcitrant nature of the seeds of G. indica that lose viability completely with 124 slight desiccation, they are not amenable to genebank conservation (Malik et al. 2005a). Hence in 125 vitro-derived shoots are preferred for medium- and long-term conservation of its germplasm. In 126 vitro plantlets can be generated by adventitious bud differentiation from mature apomictic seeds 127 of G. indica (Malik et al. 2005b). Protocols for mass multiplication and medium-term conservation 128 of G. indica have recently been standardized using in vitro-derived shoots obtained through the 129 direct organogenesis of mature seeds (Srivastava et al. 2022). For the first time, four G. indica 130 accessions and one G. gummi-gutta accession were successfully conserved as safety backup 131 collections in the In Vitro Gene Bank (IVGB) of ICAR- NBPGR, New Delhi. These accessions 132 7 have been maintained in the in vitro facility since 2020 under slow growth conditions with 133 subculture every 20 to 24 mo. However, the germplasm could be conserved under such conditions 134 only for a limited period as this predisposes explants to covert endophytic bacteria which become 135 visible after repeated subculture over prolonged periods (Srivastava et al. 2021). Hence, the 136 present study aimed to develop an efficient cryopreservation protocol for safe and long-term 137 conservation of G. indica germplasm using in vitro multiplied shoots. 138 Materials and methods 139 Plant material 140 G. indica accessions IC638183, IC638184, IC638185, and IC638186, collected from Goa and 141 Maharashtra regions of the Western Ghats of India and maintained in the IVGB at ICAR-NBPGR, 142 New Delhi, were used as mother cultures (Srivastava et al. 2022). For the development of the 143 cryopreservation protocol, accession IC638183 was used initially for all experiments. The 144 standardized protocol was tested on the three other accessions (IC638184, IC638185, and 145 IC638186) for the validation of results. All accessions of G. indica were maintained in the culture 146 room of the IVGB at a temperature of 25 ± 2 °C under a 16 hr light/8 hr dark photoperiod with a 147 light intensity of 40 µmol m-2s-1 maintained by cool white, fluorescent tubes (Philips, Mumbai, 148 India) herein referred to as standard culture room conditions (SCC). 149 Micropropagation and isolation of explant 150 To obtain the explants for cryopreservation experiments, shoots (8 to 10 cm long with 6 to 8 151 internodes) were multiplied by subculturing on shoot multiplication medium consisting of 152 Murashige and Skoog (MS; Murashige and Skoog 1962; Hi-Media®, Mumbai, India) medium 153 supplemented with 17.76 µM 6-benzylaminopurine (BAP) and 0.53 µM 1-naphthaleneacetic acid 154 8 (NAA) (Sigma-Aldrich® St. Louis, MO) in glass test tubes (15 × 10 cm) (Borosil, New Delhi, 155 India) with polypropylene caps (Srivastava et al. 2022). Shoots were subcultured every 6 to 8 wk 156 to proliferate until enough apical and axillary shoots were available for cryopreservation 157 experiments. Since G. indica is a woody perennial and grows slowly under in vitro conditions, 158 explants for cryopreservation experiments were collected from 24-wk-old plantlets. Under a stereo 159 zoom microscope (Olympus, Gurugram, India), apical shoots (AS) were isolated by removing the 160 lateral leaves till the apical meristem was visible; and, finally, a sharp horizontal cut was made just 161 below the meristem to isolate the meristematic dome covered with two leaf primordia. The axillary 162 shoots (AxS) were excised by removing the lateral leaves by making vertical cuts upside down, 163 followed by severing the axillary meristem from the main stem, and, finally, the basal portion was 164 trimmed by making a sharp horizontal cut (Fig. 2). Two sizes of explant, including 1 mm and 2 165 mm, were tested for their regeneration potential. The explants were cultured on three different 166 media for testing their regeneration capacity – (i) MS basal medium, (ii) MS containing 2.22 µM 167 BAP, and (iii) MS containing 4.44 µM BAP – and were observed after 15 d of culture. 168 Standardizing Cryopreservation Protocol 169 Effect of pre-culture 170 Two sets of experiments were conducted: one with “no pre-culture method” and another “with 171 pre-culture method”. For the “no pre-culture method”, the excised meristems were kept on MS 172 medium [30.0 g L-1 sucrose (Hi-Media®); 7.0 g L-1 agar (Hi-Media®); pH set to 5.85] over a piece 173 of sterile filter paper until all the meristems were isolated. These meristems were subjected to 174 cryopreservation on the same day of isolation. For the “with pre-culture method”, the isolated 175 meristems were first transferred on a sterile filter paper placed on MS medium in a Petri plate. As 176 9 soon as all the meristems were excised, the filter paper was transferred to a Petri plate containing 177 MS medium with 0.3 M sucrose (102.7 g L-1 sucrose; 7.0 g L-1 agar; pH set to 5.85) and was kept 178 in the dark for 24 hr and 48 hr before cryopreservation. 179 Effect of the cryopreservation technique 180 Two cryopreservation techniques were tested with fresh as well as pre-cultured explants namely 181 Vitrification (V) and Droplet Vitrification (DV) for effective long-term conservation of G. indica 182 as presented in the experimental schema (Fig. 3). For vitrification, the explants were submerged 183 in a loading solution (LS) consisting of MS salts, 0.4 M sucrose, and 2.0 M glycerol (Sigma-184 Aldrich) at room temperature (RT; 25 ± 2˚C) for 20 min in a 50 mL gamma-sterilized plastic vial 185 (TPP, Switzerland). This was followed by removing the LS using a sterile pipette and dispensing 186 chilled plant vitrification solution 2 [PVS2; comprising 30.0% (w/v) glycerol, 15.0% (w/v) 187 ethylene glycol (Sigma-Aldrich), 15.0% (w/v) dimethyl sulfoxide (DMSO) (Sigma-Aldrich), and 188 0.4 M sucrose in MS] in the vials. The vials were kept in an icebox for 10, 20, 30, 40, and 60 min, 189 and the solution was swirled intermittently. Before completion of the PVS2 duration, the explants 190 were shifted to 1.8 mL cryovials (Greiner, Germany) containing fresh PVS2 (chilled) and then 191 directly plunged into liquid nitrogen (LN) and stored in an LN tank for 1 hr. For the droplet 192 vitrification (DV), explants were exposed to LS for 20 min at RT in a 50 mL gamma-sterilized 193 plastic vial (TPP, Switzerland). After this, the loading solution was removed from the vial using a 194 sterile pipette leaving behind the explants. Chilled PVS2 was dispensed in the vial and kept in an 195 ice box after swirling the solution slowly and treatment was given for various durations, for 196 example 10, 20, 30, 40, and 60 min. Five min before the completion of exposure time to PVS2, 197 the explants were taken out using a sterile pipette and placed over a pre-sterilized aluminum foil 198 strip (20 × 5 mm) kept in a Petri plate over an ice pack (to maintain 0°C temperature of PVS2) 199 10 with a drop of PVS2 (5.0 µL). Upon completion of the exposure time, the aluminum foil strip was 200 rapidly plunged in LN. For uniform freezing of the material, the foil was held in LN for a few s 201 until the bubble formation stopped. The aluminum foils containing the explants were transferred 202 to cryovials submerged in LN one by one. The caps of the vials were closed, and cryostorage was 203 done in the LN tank for at least 1 hr. 204 Thawing and recovery after cryopreservation 205 For thawing, the cryo-box containing the frozen cryovials was taken out from LN tank, placed in 206 a Styrofoam box containing LN, and held completely submerged before thawing. The explants 207 conserved using the V technique were thawed by rewarming the cryovials in a water bath (38 ± 2 208 ˚C) for 3 min followed by exposure to the recovery solution (RS; comprising MS with 1.2 M 209 sucrose; pH 5.8) at room temperature (RT) for 20 min. For the thawing of the explants 210 cryopreserved using DV, the caps of stored cryovials were removed using sterile forceps while 211 held in LN. Aluminum foil strips containing frozen explants were taken out from LN and directly 212 placed in a vial (50 mL) containing 30 mL RS for 20 min at RT. After thawing explants were 213 placed over a piece of filter paper on the standardized recovery medium (MS containing 2.22 µM 214 BAP with 3.0% sucrose). and were kept in the dark overnight under SCC in both V and DV 215 procedures. After 17 hr (overnight) of incubation, all explants were transferred to fresh 216 standardized recovery media without filter paper using sterile forceps and were kept in dark 217 conditions for five days by wrapping the Petri plates with aluminum foil. After five d, the 218 aluminum foils were removed and shifted to diffused light conditions for the next two d before 219 being transferred to full illumination. As the growth of the shoots in G. indica is slow, the survival 220 of explants (for example, number of explants turning green and showing signs of growth) after 221 cryopreservation was recorded 4 wk after thawing. The explants that regenerated and formed a 222 11 complete shoot after 2 mo of thawing were recorded in terms of percent regenerated explants. The 223 plantlets were cultured in rooting media containing MS with 5.37 µM NAA for 2 mo before being 224 transferred to field conditions. 225 Ex vitro hardening of control and cryopreserved plantlets 226 For hardening, about 10 to 15 cm long plants (2 to 3 mo old) with at least 3 or 4 roots were washed 227 under running tap water to remove the adhering nutrient medium. Plantlets were transferred to the 228 plastic pots containing autoclaved potting mixture. The plantlets were irrigated on alternate days 229 with half-strength MS media for 2 wk. Once planted, the non-cryopreserved control (-LN) and 230 cryopreserved (+LN) plantlets were covered with transparent perforated polybags and 231 subsequently exposed to ex vitro conditions of a mist chamber under 25 ± 2˚C temperature and 232 85% relative humidity (RH) for the initial 2 wk. Sixty plantlets, each from control and 233 cryopreserved treatment, were transferred to ex vitro conditions, and final survival was recorded 234 after 2 mo. 235 Applicability of the standardized cryopreservation protocol to other accessions of G. indica 236 The standardized droplet vitrification protocol for long-term ex situ conservation of G. indica was 237 further tested in three different accessions (IC638184, IC638185, and IC638186). For this, the 238 multiplied cultures were used to isolate AS explants from 24-wk-old plantlets under SCC. Freshly 239 isolated explants placed over a piece of filter paper on MS medium were suspended in LS for 20 240 min at RT followed by dehydration in chilled PVS2 for 40 min using DV. The cryopreserved 241 shoots were thawed with RS for 20 min at RT, and regeneration of the shoots was recorded after 242 2 mo of culture. 243 Statistical analysis 244 12 Experiments were performed in a complete randomized design (CRD). Each treatment consisted 245 of three replications containing a minimum of 20 explants. Data was recorded at different intervals 246 and presented as mean ± standard error (SE) for percent shoot survival and percent shoot 247 regenerated. Percent data was transformed using arcsine transformation before analysis. Statistical 248 analysis was performed with the SPSS program. Data were analyzed using analysis of variance 249 (ANOVA), and the mean values were compared for a significant difference using Duncan’s 250 multiple range test (DMRT) at P ≤ 0.05. For the representation of areas explored for G. indica on 251 the Indian map, the PGR informatics tool 252 (http://pgrinformatics.nbpgr.ernet.in/pgrmap/SpeciesDetails.aspx) was used. The schematic 253 representation of shoot tip isolation and standardizing the cryopreservation protocol was created 254 with BioRender.com. 255 Results 256 Effect of media, explant size, and position on the shoot regeneration 257 Media composition and explant size were found to have a significant effect on the regeneration 258 capacity of the explants. Media containing MS and 2.22 µM BAP gave the highest regeneration of 259 shoots as compared to other media across the explant types and size with maximum regeneration 260 (77.71%) in 2 mm long apical shoots (Fig. 4). The regeneration of shoots ranged from a minimum 261 of 46.92% (1 mm axillary bud) to a maximum of 59% (2 mm apical bud) in MS media without 262 growth regulators; however, in MS supplemented with 4.44 µM BAP, the regeneration of shoots 263 were recorded to be as low as 30.78% (1 mm axillary bud) to a maximum of 59% (2 mm apical 264 bud), indicating both of these media were not suitable for regeneration of the smaller explants of 265 axillary and apical origin. Overall, as compared to axillary shoots, apical shoots of 2 mm length 266 http://pgrinformatics.nbpgr.ernet.in/pgrmap/SpeciesDetails.aspx 13 were observed to be faster-growing and more responsive towards regeneration in all the treatments 267 with the maximum being on MS containing 2.22 µM BAP. In the subsequent experiments, apical 268 shoots (2 mm long) were used for the development of cryo-protocol due to their higher 269 morphogenetic potential. 270 Effect of PVS2 dehydration on explants 271 The dehydration tolerance of apical shoots (2 mm long) subjected to successive exposure durations 272 of PVS2 at 0°C, using either V and DV techniques, showed a persistent decline in survival of the 273 explants. Notably, when comparing the two techniques, the DV method outperformed the V 274 method in terms of both the survival and regrowth of the desiccated explants. Within the DV 275 approach, the survival rates of explants ranged from 90% to 55.77% whereas, in the V technique, 276 these rates spanned from 73.4% to 37.26% with the highest recorded survival at 10 min followed 277 by subsequent decline as PVS2 exposure time increased (Fig. 5). 278 Furthermore, prolonged exposure to PVS2 had a detrimental influence on the explant 279 regeneration in both techniques. Specifically, in the V technique, the regeneration percentage 280 declined significantly at each level of PVS2 desiccation, ranging from 64.69% to 21.33%. In the 281 case of DV, maximum regeneration of explants was recorded after 10 min of PVS2 exposure 282 (77.71%), which reduced with increasing PVS2 time (Fig. 6). The regeneration of apical shoots 283 was at par at 30 and 40 min PVS2 exposure (63.55% and 60.2%, respectively) and reduced 284 significantly (44.03%) in explants subjected to 60 min PVS2 exposure. 285 Effect of Pre-culture and Cryopreservation Technique 286 The explants (2 mm long apical shoots), if pre-cultured, showed very low survival (<15%) of 287 control (-LN), and did not exhibit any survival after cryopreservation, irrespective of technique (V 288 14 or DV) employed (data not presented). When the V technique was applied without preculturing, 289 there was a very low survival (16.59%) at 40 min PVS2 exposure. However, the surviving tissues 290 failed to regenerate and turned black. 291 In contrast, explants without pre-culture and cryopreserved using DV technique displayed 292 varying degrees of success (Fig. 6). Initially, shoot survival increased gradually from 31% to 293 64.69% when apical shoot tips were exposed for 10 to 40 min PVS2 treatment followed by LN 294 exposure and culture on the standardized recovery media (MS containing 2.22 µM BAP with 3% 295 sucrose). However, the survival of cryopreserved explants declined significantly when PVS2 296 exposure was extended to 60 min, resulting in only 43.09% survival. After exposure to LN, the 297 highest shoot regeneration (51.76%) was recorded when the explants were exposed to PVS2 for 298 40 min (Fig. 7). 299 Ex vitro hardening and survival of plantlets 300 After the thawing process, the regenerated shoots of accession IC0638183 were cultured in a 301 rooting medium. Subsequently, the plantlets, each possessing 4 to 6 roots, were transferred to a 302 mist chamber for hardening. The results demonstrated that non-cryopreserved G. indica plantlets 303 achieved a 90% regrowth. In comparison, cryo-retrieved plantlets exhibited an 85% regrowth in 304 the mist chamber, closely resembling the control. Moreover, these cryo-retrieved plantlets 305 displayed normal growth comparable to that of the non-treated control plants, as depicted in Figure 306 8. These findings suggest that the cryopreservation process did not adversely affect the survival 307 and growth of G. indica plantlets, underscoring the efficacy of the cryopreservation method 308 employed in this study. 309 Applicability of the standardized cryopreservation protocol to other accessions of G. indica 310 15 In this investigation, it was noted that all accessions of G. indica exhibited favorable outcomes 311 following cryopreservation utilizing the standardized DV protocol (Fig. 9). The survival rates of 2 312 mm-long apical shoots (AS) varied among different accessions, ranging from 48.86% to 60%. 313 Similarly, the rates of shoot regeneration also exhibited variability, spanning from 40% to 48%. 314 Accession IC0638186 demonstrated the highest rates of both shoot survival (60%) and 315 regeneration (48%) followed by accessions IC0638184 and IC0638185, which showed statistically 316 comparable results for survival (53.76% and 48.86%, respectively) and regeneration (42.12% and 317 40.16%, respectively). These findings implied that the cryopreservation method applied in this 318 study effectively preserved the apical shoots of diverse G. indica accessions with certain 319 accessions displaying notably high levels of success in terms of both survival and regeneration 320 Discussion 321 The present study indicated that in G. indica, a highly recalcitrant species, long-term conservation 322 of the shoot tips using a modified droplet vitrification method (Fig. 10) was successful in contrast 323 to the vitrification method that did not give any post-thaw regeneration. Droplet vitrification can 324 be considered a generic cryopreservation method due to its simplicity, time efficiency, and high 325 rates of success. Applied to diverse crops, including banana (Panis et al. 2005), magnolia (Folgado 326 and Panis 2019), Arabian pea (Gisbert et al. 2015), apple (Condello et al. 2011), and Himalayan 327 gentian (Sharma et al. 2021), this method is often adjusted to the different plant species and tissues, 328 for example in wild Indian Musa spp. (Agrawal et al. 2014) and sweet potato (Wilms et al. 2020). 329 Panis et al. (2005) devised a step-by-step methodology for determining optimal parameters for 330 each species. In the present study, for the first time in G. indica, the focus was on some of the 331 important steps, including explant size, position, regeneration media, PVS2 dehydration, pre-332 culture, and regeneration. 333 16 One of the most important factors in cryopreservation success was the selection and 334 isolation of the suitable explant (size and position of the explant) so that excised apices remain in 335 a physiological state that is suitable for the acquisition of osmotic tolerance leading to vigorous 336 growth recovery. In G. indica, apical shoots responded better to regeneration than axillary shoots 337 within 15 days of culture. In order to regenerate, different plant species had varying degrees of 338 success when using different meristems. While apical meristems yielded better results in dianthus 339 (Dereuddre et al. 1988), Stevia rebaudiana (Benelli et al. 2021), and dahlia (Gowthami et al. 340 2023), axillary meristems were found to be the best explants in chrysanthemum (Lee et al. 2011) 341 and sweet potato (Wilms et al. 2020). While experiments comparing two types of meristems in 342 Garcinia species are lacking, the present work clearly demonstrated that apical meristems have a 343 higher potential to regenerate and form a normal shoot compared to axillary ones. A larger explant 344 (2 mm long) was found to respond better to all media tested while smaller explants (1 mm long) 345 lost the capability to regenerate. In comparison to MS basal media, regeneration of shoots in MS 346 media with 2.22 µM BAP was found to be optimal; a higher concentration was not beneficial for 347 rapid plantlet regeneration. 348 The two-step dehydration of the explants in which they were treated with the LS at 25°C 349 for 20 min. followed by PVS2 exposure at 0°C yielded good results in most of the tropical species, 350 including banana (Panis et al. 2005), cassava (Wilms et al 2020), and Garcinia hombroniana 351 (Sulong et al. 2018). Hence, 20 min. treatment with LS was used before PVS2 exposure to the 352 shoot tips. The survival of the shoot tips was reduced drastically as the PVS2 exposure duration 353 increased from 10 to 60 min. both in V and DV techniques. However, explants dehydrated using 354 DV showed higher survival and regeneration as compared to the V technique. 355 17 Between the two vitrification methods during PVS2 dehydration, a large difference in the 356 regeneration of non-cryopreserved (–LN) explants after the same incubation times may be 357 attributed to various reasons. One factor may be the desiccation-sensitive nature of Garcinia spp. 358 in general. The species that are more desiccation-sensitive required very precisely controlled 359 desiccation and cooling conditions (Dussert et al. 1997; 1998). The highly concentrated 360 vitrification solution may cause injury to the tissue due to chemical toxicity or excessive osmotic 361 stress (Sakai et al. 2008). Sometimes, minor modifications may lead to improvement in the 362 recovery rates for some species (Abdelnour et al. 1992; Normah and Vengadasalam 1992). Hence, 363 it was important to modify the solution or dehydration procedures for desiccation-sensitive species. 364 Another factor contributing to better results in DV may stem from a slight procedural difference. 365 In DV, the explants were transferred onto an aluminum foil strip placed over an icepack whereas 366 in the V technique, they were suspended in the PVS2 within a vial kept in an ice box. Consequently, 367 the temperature of PVS2 in the vial tended to be marginally higher and less uniform compared to 368 DV. In DV, the application of cryoprotectant solution directly onto the explant over aluminum foil 369 in smaller volumes ensured a more consistent temperature and dehydration rate. 370 As temperature plays a very important role in the penetration of PVS2 in the tissues, it 371 appeared that when desiccation-sensitive tissues were placed directly over chilled aluminum foil, 372 the penetration of PVS2 was reduced due to the increased viscosity of the solution. Therefore, the 373 damage of the explants was less in the DV as compared to the V. This was more evident at 30 and 374 40 min of incubation time of PVS2 where regeneration of control explants (–LN) in the DV method 375 showed double the rates as compared to the V method (Fig. 5 & 6). This indicated the strong role 376 of terminal temperature variation in reduced PVS2 penetration and thereby higher regeneration as 377 compared to the vitrification method. In line with the present study’s findings, stepwise 378 18 dehydration using a slower exposure to the full concentration of PVS2 was suggested as an 379 alternative way to reduce the toxicity of the PVS2 during vitrification in G. hombroniana where 380 the shoot tips, when dehydrated with 50% PVS2 for 15 min and thereafter by 100 % PVS2 for 10 381 min, showed an increase in survival from 25% to 41.67% (Sulong et al. 2018). 382 Due to the non-uniform desiccation and cooling of the explants, there was no shoot 383 regeneration after cryopreservation following the V technique while the DV technique showed 384 good shoot survival and regeneration, indicating the effectiveness of the technique. To increase 385 the osmotolerance of the explants, pre-culturing in high sucrose-containing media was suggested 386 for many plant species in order to increase the survival of cryopreserved shoots. In G. 387 hombroniana, 48 h pre-culture on MS media with 0.3 M sucrose resulted in significantly higher 388 survival (75%) compared to 24 h pre-culture (50%) after cryopreservation (Sulong et al. 2018). 389 The shoot tips of G. mangostana showed increased survival (13.7%) when pre-cultured on MS 390 with 0.6 M sucrose for 2 days (Ibrahim and Normah 2013). However, pre-culture with sugar or 391 sorbitol did not result in significant increases in growth recovery after vitrification in apical 392 meristems from a variety of species. Wilms et al. (2020) also reported that pre-cultured explants 393 of sweet potatoes showed increased survival as compared to non-precultured ones, but 394 regeneration rates were lower. They argued that preculturing of the shoots led to the survival of 395 non-meristematic cells, which eventually formed callus and outgrew the surviving meristem after 396 thawing (Wilms et al. 2020). In the present study’s experiments, it was found that pre-cultured 397 shoots of G. indica on MS media with high sucrose (0.3 M) for 24 hr and 48 h had no beneficial 398 effect on survival after cryopreservation in both V and DV techniques. According to Ishikawa et 399 al. (2000), this could be due to an excessive accumulation of sucrose in the cytoplasm, which can 400 cause toxic effects. In line with the present study’s findings, Yap et al. (2011) reported that 401 19 increasing pre-culture duration on 0.3 M sucrose medium from 0 to 3 days in G. cowa shoot tips 402 increased explant tolerance to PVS2 from 5.6% (no pre-culture) to 49.2% (3-day pre-culture); 403 however, no survival was observed after cryopreservation. They also reported the steady 404 accumulation of an electron-dense substance in meristematic cells with increasing exposure 405 durations to 0.3 M sucrose pre-culture by using transmission electron microscopy. 406 In the present study’s experiments, no success in terms of regeneration was achieved in the 407 vitrification technique after cryopreservation. The DV technique gave a higher survival as well as 408 regeneration of the non-precultured apical shoots at 40 min. of chilled PVS2 exposure followed 409 by cryopreservation. This may be due to the effect of PVS2 dehydration and loss of free water 410 molecules from the cellular masses making the tissue more tolerant to the cryogenic temperature. 411 Droplet vitrification has been used in many crops to achieve higher regeneration after 412 cryopreservation; however, only vitrification has been tested for Garcinia species conservation to 413 date. To the best of the present authors’ knowledge, no report on the long-term conservation of G. 414 indica is available. In general, conserving Garcinia species long-term through cryopreservation 415 using the vitrification technique proved to be quite challenging as the success rates fluctuated 416 widely with shoot survival after cryopreservation ranging from 0% in G. cowa (Yap et al. 2011), 417 50% in G. mangostana (Ibrahim and Normah 2013), and 66.67% in G. hombroniana (Sulong et 418 al. 2018). Typically, the results were limited to survival only, and none of these studies discussed 419 successful regeneration or recovery of a complete plant for transplantation to field conditions. The 420 present experiments were not only limited to survival but also recorded regeneration till complete 421 plantlet formation and hardening. In addition to this, when tested on the other three accessions of 422 G. indica, the observations indicated the reproducibility and success of the protocol that was 423 developed. The variation between the accessions for survival and regeneration was observed, 424 20 indicating the genotype-dependent differences towards the success of the cryopreservation 425 protocol. 426 Conclusion 427 This study focused on the development of a cryopreservation protocol for in vitro meristems of G. 428 indica, a highly recalcitrant tropical fruit tree species. The experimental findings indicated that the 429 droplet vitrification technique was effective for long-term conservation of G. indica whereas the 430 vitrification method did not result in any success. Sucrose pre-culture adversely affected explant 431 regeneration in both the control and LN treatments, likely due to the cytotoxic effects stemming 432 from an excessive accumulation of sucrose within the cytoplasm. Consequently, there was no 433 observable recovery in the explants that underwent pre-culture following cryopreservation, 434 indicating a sensitivity of the shoot tips to this procedure. Apical shoots were observed to show 435 high regeneration after cryopreservation, resulting in tissue swelling followed by the emergence 436 of multiple shoots on the regeneration media containing MS with 2.22 µM BAP. However, this 437 type of de novo shoot organogenesis rather than the regeneration of the existing shoot tip may 438 induce somaclonal variation after cryopreservation, and, therefore, there is a need to ascertain the 439 genetic integrity of the regenerated plantlets after cryopreservation. The 2 mm long apical shoot 440 tip explants without any pre-culture were incubated in a loading solution at room temperature for 441 20 min. followed by exposure to chilled PVS2 for 40 min, and LN exposure using the DV 442 technique yielded maximum recovery after thawing in a recovery solution at room temperature for 443 20 min. The optimized DV protocol yielded impressive regeneration rates, surpassing 40% for all 444 four tested accessions coupled with the simplicity of the protocol (such as the absence of a pre-445 culture step and use of apical shoots for better success), establishing it as an acceptable and 446 dependable method for cryobanking of G. indica. This suggests that the protocol is well-suited for 447 21 preserving the genetic diversity and resources of G. indica, ensuring their conservation and 448 availability for future use and research. Cryopreservation of in vitro-derived shoots has the 449 potential for use in the conservation of the genetic resources of this highly recalcitrant endemic 450 tropical fruit species of Indian origin. 451 452 Acknowledgments 453 The authors greatly acknowledge the Director, ICAR-National Bureau of Plant Genetic Resources 454 (NBPGR), New Delhi, for supporting this study. Thanks to Dr J.K. Ranjan, Principal Scientist, 455 Indian Agricultural Research Institute (IARI), New Delhi, for helping with the statistical analysis. 456 The authors are thankful to Dr K. Pradheep, Principal Scientist and Officer-in-Charge, ICAR-457 NBPGR, Regional Station-Thrissur for his valuable input. The first author thanks the Belgian 458 Government for training on “Cryopreservation of tropical crop species” at the Laboratory of 459 Tropical Crop Improvement, KU Leuven, Belgium. 460 Author contribution 461 VS: Conceptualization, Methodology, Formal analysis, Investigation, Writing- original draft. AA: 462 Writing- review & editing, Supervision. BP: Writing- review & editing, Supervision. 463 Funding 464 This work was done as part of the intra-mural project “In vitro conservation of tropical fruit crops” 465 (project code: PGR/TCCUBUR-DEL-01.05) at ICAR-NBPGR that was funded by the Indian 466 Council of Agricultural Research (ICAR). 467 22 Declaration 468 Competing Interest The authors declare that they have no conflict of interest. 469 References 470 Abdelnour–Esquivel A, Villalobos V and Engelmann F (1992) Cryopreservation of zygotic 471 embryos of Coffea spp. CryoLett 13:297–302. 472 Acuna UM, Jancovski N, Kennelly EJ (2009) Polyisoprenylated benzophenones from Clusiaceae: 473 Potential drugs and lead compounds. Curr Top Med Chem 9:1560-1580 474 Agrawal A, Verma S, Sharma N, Vijay P, Meena DPS, Tyagi RK (2014) Cryoconservation of 475 some wild species of Musa L. 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Nat 609 Plants 4:848–850. https:// doi. org/10. 1038/ s41477- 018- 0298-3 610 https://doi.org/10.1038/s41598-020-70869-3 29 Yap LV, Normah NM, Clyde MM, Chi HF (2011) Cryopreservation of Garcinia cowa shoot tips 611 by vitrification: the effects of sucrose pre-culture and loading treatment on ultrastructural changes 612 in meristematic cells. CryoLett 32:188-196 613 614 30 615 616 Figure 1. Diversity-rich areas of Garcinia indica (Thouars) Choisy explored by ICAR-National Bureau of Plant 617 Genetic Resources (NBPGR). Source: (http://pgrinformatics.nbpgr.ernet.in/pgrmap/SpeciesDetails.aspx) 618 619 31 Table 1. Status of Garcinia indica (Thouars) Choisy and other species conserved in various field genebanks of India 620 Institute/State Agriculture University Number of accessions of Garcinia indica conserved Other Garcinia spp. conserved Reference Department of Horticulture, College of Agriculture, Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli, (Maharashtra) >300 - Haldankar et al. (2012) ICAR- National Bureau of Plant Genetic Resources (NBPGR)-Regional Station, Thrissur (Kerala) 76 Garcinia andamanica, G. cowa, G. dhanikariensis, G. dulcis, G. hombroniana, G. kydia, G. livingstonei, G. mangostana, G. nervosa, G. speciosa, G. xanthochymus Murthy et al. (2018) College of Forestry, Sirsi, University of Agricultural Sciences, Dharwad (Karnataka) 40 Garcinia gummi-gutta Patil et al. (2010) Indian Institute of Spices Research (IISR), Kozhikode, (Kerala) 32 Garcinia morella, G. talbotii, G. atroviridis G. macrophylla, G. acuminata, G. assamica, G. dulcis, G. lanceifolia, G. pedunculata, G. sibeswarii, G. xanthochymus, G. talbotii, G. conicarpa, G. pushpangadaniana, G. puat Praveena et al. (2022) ICAR- Central Horticulture Experimental Station (CHES), Indian Institute of Horticultural Research (IIHR), Chettalli (Karnataka) 30 Garcinia gummi-gutta, G. xanthochymus ICAR-IIHR (2022) 32 ICAR- Central Coastal Agricultural Research Institute (CCARI), Goa (Maharashtra) 19 Garcinia tinctoria, G. hombroniana, G. mangostana, G. cambogia ICAR-CCARI (2019, 2021); Priya et al. (2013) Jawaharlal Nehru Tropical Botanical Garden and Research Institute, Thiruvananthapuram (Kerala) 1 Garcinia spicata, G. gummi-gutta, G. imbertii, G. talbotti, G. wightii, G. xanthochymus Nazarudeen et al. (2022) ICAR- Central Island Agricultural Research Institute (CIARI), Port Blair (Andaman and Nicobar Islands) 1 (550 seedlings) Garcinia celebica, G. dhanikhariensis, G. kydia, G. xanthochymus, G. andamanica, G. cowa, G. gummi- gutta, G. mangostana ICAR-CIARI (2022) 621 622 33 623 Figure 2. Isolation of explants from in vitro derived plantlets of Garcinia indica (Thouars) Choisy ; (A) Pictorial 624 representation of two types of explants i.e., Apical shoot (AS) and Axillary shoot (AxS) used for cryopreservation, 625 and (B) Schematic representation of details of explant isolation before cryopreservation (Created with 626 BioRender.com). 627 628 34 629 630 Figure 3. Experimental schema followed for standardizing the cryopreservation protocol from in vitro derived 631 plantlets of Garcinia indica (Thouars) Choisy (IC0638183). V: Vitrification; DV: Droplet Vitrification; LS: Loading 632 Solution; PVS2: Plant Vitrification Solution 2; LN: Liquid Nitrogen, RS: Recovery Solution (Created with 633 BioRender.com). 634 35 635 Figure 4. Effect of explant size and position on shoot regeneration of Garcinia indica (Thouars) Choisy (IC0638183) 636 on three media types. Bars marked by the same letter for each parameter measured are not significantly different 637 according to Duncan’s test after arcsine transformation (P ≤ 0.05). The error bars represent standard error. 638 36 639 Figure 5. Effect of duration of PVS2 exposure on survival (%) and regeneration (%) in cryopreserved (+LN) and non-640 cryopreserved (-LN) apical shoots (2 mm) of Garcinia indica (Thouars) Choisy (IC0638183) using Vitrification (V). 641 Bars marked by the same letter for each parameter measured are not significantly different according to Duncan’s test 642 after arcsine transformation (P ≤ 0.05). The error bars represent standard error. 643 0 10 20 30 40 50 60 70 80 10 min 20 min 30 min 40 min 60 min P e rc e n t PVS2 Duration (-LN) Survival (-LN) Regeneration (+LN) Survival (+LN) Regeneration E a A B C D a b c d e 37 644 645 Figure 6. Effect of duration of PVS2 exposure on survival (%) and regeneration (%) in cryopreserved (+LN) and non-646 cryopreserved (-LN) apical shoots (2 mm) of Garcinia indica (Thouars) Choisy (IC0638183) using Droplet 647 Vitrification (DV). Bars marked by the same letter for each parameter measured are not significantly different 648 according to Duncan’s test after arcsine transformation (P ≤ 0.05). The error bars represent standard error. 649 0 10 20 30 40 50 60 70 80 90 100 10 min 20 min 30 min 40 min 60 min P e rc e n t (-LN) Survival (-LN) Regeneration (+LN) Survival (+LN) Regeneration A α a a B B B C ab b b cβ β β γ b bc c d PVS2 Duration 38 650 Figure 7. Stages of Garcinia indica (Thouars) Choisy (IC0638183) cryopreservation: A) Apical shoot-tip isolation 651 under the microscope; B) Shoot induction and survival after 40 min. PVS2 treatment after 40 d of inoculation; C) 652 Surviving ST at 15 d of inoculation after Droplet Vitrification D) Shoot regeneration at 30 d after Droplet Vitrification 653 E) Shoot proliferation at 60 d after Droplet Vitrification; F) Plantlet formation at 90 d after Droplet Vitrification. 654 39 655 656 Figure 8. Stages of hardening and survival of cryopreserved shoots of Garcinia indica (Thouars) Choisy (IC0638183). 657 (A) Multiplied shoots from cryo-retrieved shoot-tips; (B) In vitro rooting of the shoots; (C) Transferring the rooted 658 plantlets into the pro-trays for hardening; (D) Primary hardening with shoots covered in perforated transparent 659 polybags; (E) Control plant after 1 mo of hardening; (F) Cryo-retrieved plant after 1 mo of hardening in mist chamber. 660 40 661 Figure 9. Regeneration percentage obtained in cryopreserved (+LN) and non-cryopreserved (-LN) shoot tips by 662 applying the standardized Droplet Vitrification protocol to three different accessions of Garcinia indica (Thouars) 663 Choisy . Bars marked by the same letter for each parameter measured are not significantly different according to 664 Duncan’s test after arcsine transformation (P ≤ 0.05). The error bars represent standard error. (LN: Liquid nitrogen) 665 41 666 667 Figure 10. Flow diagram showing various steps in the standardized Droplet Vitrification protocol of Garcinia indica 668 (Thouars) Choisy. BAP: 6-Benzylaminopurine; LS: Loading Solution; PVS2: Plant Vitrification Solution 2; LN: 669 Liquid Nitrogen, RS: Recovery Solution, MS: Murashige and Skoog medium. 670 671