Encapsulation-dehydration Technique Research Articles

Cryopreservation of Arum palaestinum plant callus as a strategy for mitigating extinction risks

Arum palaestinum is a wild perennial plant commonly known as "Al-Loof" in Jordan. Due to overharvesting, climate change, and increasing demand, its natural populations are threatened with extinction. Cryopreservation, an effective method for conserving plant material at ultra-low temperatures, is explored for A. palaestinum calli. We investigated the applicability of encapsulation-vitrification (using different plant vitrification solutions (PVS) and incubation times), encapsulation-dehydration (using sucrose or sorbitol at different concentrations and dehydration times), and the v-cryoplate (using different pre-culture times and temperatures) techniques. In the encapsulation-vitrification experiment, a notable 82.4 % regrowth rate was achieved by desiccating calli in plant vitrification solution 2 (PVS2) for 10 minutes at 25 °C. The encapsulation-dehydration technique resulted in an 82.6 % regrowth rate by incubating calli for one day in low sucrose levels (0.1 M sucrose) following one hour of air dehydration, where the moisture content of the beads was 30 %. The moisture content of the beads decreased from 81 % before chemical and air dehydration to 71 % after 0 hours of air dehydration combined with chemical dehydration using 0.1 M sucrose or sorbitol. It further dropped to 30–34 % after one day of chemical dehydration with 0.1 M sucrose and 1 hour of air dehydration. The v-cryoplate technique successfully conserved calli, showing impressive survival and regrowth percentages (96.8 %) when the callus was pre-cultured with 0.3 M sucrose for three days at 5 °C. Temperature during pre-culture significantly influenced regrowth percentages in the v-cryoplate technique. The study establishes promising cryopreservation protocols for A. palaestinum calli, offering a means to conserve germplasm and contribute to environmental and biodiversity protection by reintroducing endangered plants to their native habitats.

Integrated ex situ conservation strategies for endangered New Zealand Myrtaceae species

ABSTRACT The current threat of myrtle rust (Austropuccinia psidii) to New Zealand Myrtaceae, including a number of indigenous and socio-economically important species, requires that ex situ conservation is used to complement in situ populations. New Zealand’s Myrtaceae have received little attention in terms of ex situ conservation. In this study, we assessed the integrated ex situ conservation strategies for selected New Zealand Myrtaceae. We particularly investigated seed banking options by assessing seed desiccation tolerance, in vitro culture, pollen cryopreservation and zygotic embryo cryopreservation of the recalcitrant Syzygium maire. A desiccation trial was conducted on six Myrtaceae species: Lophomyrtus bullata, L. obcordata, Metrosideros diffusa, M. umbellata, M. bartlettii and Syzygium maire. S. maire seeds and embryos showed extreme sensitivity to desiccation confirming its recalcitrant behaviour. The seeds of the other species were desiccation tolerant i.e. orthodox. Zygotic embryos of S. maire were successfully cryopreserved using an encapsulation-dehydration technique. Pollen cryopreservation was successful for M. excelsa following desiccation to about 5% moisture content, rapid freezing and rapid thawing. For M. bartlettii, one of the most endangered and a nationally critical Myrtaceae species in New Zealand, we tested the efficacy of hand pollination in producing viable seeds. Our assessment confirmed that one of the M. bartlettii trees at Otari is self-incompatible, and successful hand pollination using pollen obtained from different genotypes growing in the gardens at the University of Auckland resulted in seed production with c. 20% germination. Tissue culture protocols were successfully developed for selected Myrtaceae. In addition, photoautotrophic micropropagation techniques were developed for the first time for L. scoparium. This paper highlights the importance of holistic conservation strategies to ensure future access to New Zealand’s unique Myrtaceae germplasm as a key component of long-term management response to the threat posed by A. psidii.

Callus induction, plant regeneration, and cryopreservation ofDendrobium findlayanumbased on an encapsulation-dehydration technique

Callus induction, plant regeneration, and cryopreservation of<i>Dendrobium findlayanum</i>based on an encapsulation-dehydration technique

Cryopreservation ofArundina graminifolia(D. Don) Hochr. seeds using D cryo-plate method

In this study, cryopreservation of Arundina graminifolia seeds by D cryo-plate and encapsulation dehydration technique were investigated. The results showed that the D cryo-plate method gave the highest regrowth of 82%, followed by encapsulation-dehydration (74%). The D cryo-plate protocol is as follows: pour the alginate solution containing 2% (w/v) sodium alginate in calcium-free ½ MS basal medium with 0.4 M sucrose in the wells of the cryo-plate. For encapsulation-dehydration the protocol is the same as D cryo-plate without using cryo-plate. Place 20 to 50 seeds from 3-month-old selfing capsules in each well. Pour calcium chloride solution containing 0.1 M calcium chloride in ½ MS basal medium with 0.4 M sucrose. Expose to 0.4 M sucrose + 2 M glycerol (loading solution) for 30 min and then dehydrate under a laminar air-flow cabinet for 3 h. Put each cryo-plate in a 2-mL cryotube and plunge into liquid nitrogen for 1 d. Warm in 1.2 M sucrose solution (unloading solution) for 15 min then remove the solution and culture cryopreserved beads on ½ MS agar medium.

The Use of Encapsulation-Dehydration Technique for Short-Term Preservation of Endangered Sweet Potato Landraces ( Ipomoea batatas Lam) from Benin

Ex situ conservation of genetic resources remains a challenge for preservation of vegetatively propagated species such as sweet potato. The objective of this study was to establish the protocol based on the encapsulation-dehydration technique for short term preservation of endangered sweet potato landraces produced in Benin. Thus, the evaporative dehydration duration on silicagel was previously determinated on the empty beads which were made using alginate sodium (3%) and calcium chloride (1.32M). Then, the young shoots of two sweet potato landraces (Koidokpon and Dokoui carotte) growing in the screen house were cutted aseptically and desinfected with 10% sodium hypochlorite. The apices were excised on stereoscope and were encapsulated prior their dehydration on silicagel during 5 and 6h. The encapsulated apices were finally conserved in eppendorf tubes at 2°C in batches for 15 days and 90 days. The encapsulated apices were cultured in MS medium supplemented with 0.15 mg/l BAP, 0.2 mg/l NAA, 0.08 mg/l GA3 and 80 mg/l adenine sulfate. The survival and the regeneration rates were then evaluated. At our experimentation condition, the dehydration duration which allowed around 20% water content of the beads was 6h. For the encapsulated apices, the highest survival rates (59.26% and 37.04%) and the highest regeneration rate (37.04% and 11.11%) were recorded respectivelly with the landraces ''Koidokpon'' and ''Dokoui carotte'' when the apices were dehydrated by 6h and stored for 15 days. The regeneration rates decreased according to the stored duration. Significant difference was noted on the regeneration of apices for the landraces tested. This method can be used to preserve the endangered sweet potato landraces and other species during at least three months without subcultures. It also reduce the cost of conservation in terms of consumables and permit better genotype stability during the storage.

EFFECT OF VARIOUS PRECULTURE AND OSMOTIC DEHYDRATION CONDITIONS ON CRYOPRESERVATION EFFICIENCY AND MORPHOGENETIC RESPONSE OF CHRYSANTHEMUM SHOOT TIPS

The aim of this study was to evaluate the influence of sucrose concentration in the preculture medium, as well as the duration of osmotic dehydration on the efficiency of chrysanthemum ‘Richmond’, ‘Lady Orange’ and ‘Lady Salmon’ cryopreservation by encapsulation-dehydration technique. For all cultivars tested, the best regrowth of cryopreserved shot tips was recorded with 0.25 M sucrose concentration and 10&nbsp;µM ABA during a two-week preculture, followed by a 4- or 7-day osmotic dehydration. The survival rate ranged from 56.8–58.0% (the Lady group) to 63.6% (‘Richmond’). However, the ability to grow was smaller and reached 18.2–50.7%. It was found that higher sucrose concentration during the preculture slowed the growth of chrysanthemum shoot tips and led to an increased formation of multiple shoots (by activating axillary buds) or deformed adventitious shoots (incapable of further growth). The frequency of tissue hyperhydricity also increased, while the rhizogenesis efficiency decreased when higher sucrose concentration in the preculture medium was applied. The influence of osmotic dehydration duration on the explants morphogenetic response was cultivar-dependent

Morphogenetic response of shoot tips to cryopreservation by encapsulation-dehydration in a solid mutant and periclinal chimeras of Chrysanthemum\xa0\xd7\xa0grandiflorum /Ramat./Kitam.

Cryopreservation is widely applied to many economically important species excluding chimera plants which are problematic for long-term conservation. Their storage problems can be circumvented only by cryopreserving meristems. This study looked at the morphogenetic response of shoot tips of periclinal chimera chrysanthemum ‘Lady Orange’ and ‘Lady Salmon’, as well as the solid mutant ‘Richmond’, that were cryopreserved by encapsulation-dehydration technique. By applying 10 µM ABA in the preculture medium followed by 4-day-long dehydration treatment, the explant survival reached up to 67%. Besides the stimulation of typical single shoot recovery, cryopreservation led to direct or indirect multiple shoot formation, shoot malformation, as well as inhibited their spontaneous rooting. Microscopic analysis revealed three types of structural damages of shoot tips which can correspond with their morphogenetic response in recovery culture. No influence of cryostorage on the acclimatisation efficiency of the recovered chrysanthemums was observed.

Propagation, micropropagation and cryopreservation of Buxus hyrcana Pojark., an endangered ornamental shrub

Buxus hyrcana Pojark. is an important ornamental shrub, widely used for landscape and gardens. This ornamental shrub is a critically endangered species due to invasion of some pathogens. Rooting of this species is slow using propagation technique such as cutting. This study was carried out in three sections: 1) the effect of indole-3-butiyric acid (IBA) and ɑ-naphthalene acetic acid (NAA) on rooting of semi-hardwood cuttings in greenhouse condition; and 2) the effect of 6-benzylaminopurine (BAP), IBA and NAA on micropropagation in tissue culture condition and 3) cryopreservation of shoot tip by encapsulation-dehydration technique. For the first experiment, the basal region of the cutting was treated with various IBA and NAA solutions: 0 (free of IBA), 500, 1000, 2000 and 3000mgl−1. The 1000mgl−1 IBA along with 1000mgl−1 NAA induced the largest number of root (8.700/plantlet). For the second experiment, shoot tip dissected from 2-years-old pot-grown plants was cultured on Murashige and Skoog (MS) medium supplemented with 50 combinations of BAP, IBA and NAA at concentrations of 0.00, 0.50, 1.00, 1.50 and 2.00mgl−1. Here, we present a new protocol for sterilization of shoot tip. This shoot tip sterilization protocol minimized bacterial and fungal contamination. Multiple shoot formation (6.433/plantlet) was obtained through the use of 1.00mgl−1 BAP. Also, the optimum plant growth regulators (PGRs) combination for the largest number of root (7.633/plantlet) was 0.50mgl−1 BAP plus 1.00mgl−1 NAA. Plantlets were hardened-off and transferred to soil for further growth. About 95% of the acclimatized plantlets were established successfully. Regenerated plantlets were morphologically identical. For the third experiment, B. hyrcan shoot tip was cryopreserved by direct immersion in liquid nitrogen (LN) using encapsulation-dehydration method. After rapid rewarming, encapsulated and non-encapsulated shoot tips were cultured on MS medium enriched with 0.00, 0.50, 1.00, 1.50 and 2.00mgl−1 of BAP, IBA and NAA for rapid germination and growth. The highest percentage of re-growing the cryopreserved shoot tip was 60% for those grown on medium containing 0.50mgl−1 BAP along with 1.50mgl−1 NAA. No survival was observed for non-encapsulated shoot tip after storage in LN and through cultivation on regeneration medium.

Cryopreservation of somatic embryos from Petiveria alliacea L. by different techniques based on vitrification

Petiveria alliacea L. is a medicinal plant originating from the Amazon region. This study describes an efficient cryopreservation protocol for somatic embryos (SEs) produced from roots of P. alliacea based on the comparison of vitrification, encapsulation-dehydration, and D cryo-plate techniques. With the vitrification technique, SEs treated with PVS2 solution (0.4 M sucrose, 3.3 M glycerol, 2.4 M ethylene glycol, and 1.9 M DMSO) for 30 min displayed high viability (85%) and intermediate proliferation recovery (about 12 adventitious SEs produced from original SEs [SEs/SE] after 90 d of culture). With the encapsulation-dehydration technique, lower viability (70%) and very low proliferation recovery (about two SEs/SE) were achieved with cryopreserved SEs dehydrated for 10 min in a laminar air flow cabinet. The D cryo-plate technique led to high viability (85%) and proliferation recovery (19 SEs/SE) of cryopreserved SEs after 90 min dehydration. In the experimental conditions tested, the D cryo-plate method was the most efficient technique for cryopreservation of P. alliacea SEs.

Cryopreservation of Cat’s Claw (&amp;lt;i&amp;gt;Uncaria tomentosa&amp;lt;/i&amp;gt;)

Uncaria tomentosa presents tomentum that resembles cat’s claws, hence its common name, is a plant that produces various secondary metabolites that are traditionally used in alternative medicine. The natural distribution of this species has been affected by indiscriminate harvesting from its habitat. In the present research, cryopreservation (liquid nitrogen, LN, -196&degC) was evaluated as an option for ex situ conservation of this species. The following techniques were evaluated: vitrification and encapsulation-dehydration of apices, vitrification of cell suspensions, and seed desiccation and vitrification. Preculture conditions and exposure times to LS and PVS2 were evaluated. Apex survival was the highest (82%) with preculture in 0.25 M sucrose followed by incubation for 20 and 30 min in LS and PVS2, respectively, prior to cooling in LN. The encapsulation-dehydration technique was evaluated by using sucrose preculture and different capsule moisture contents. Survival of apices cooled in LN was not significantly different between treatments and varied from 31.8% to 52.9% for capsule moisture contents between 22.7% and 20.3%. For cell suspensions precultured in 0.5 M sucrose, cell multiplication and formation of calli with very good appearance were observed in 61.1% of the cultures following vitrification. For cryopreservation of seeds, germination was 89.5% using the desiccation technique and 67.6% to78.1% using vitrification.

A simple way to overcome the recalcitrance of the water fern Ceratopteris thalictroides (L.) Brongn. to cryopreservation

&lt;em&gt;Ceratopteris thalictroides&lt;/em&gt; is a water fern very sensitive to both dehydration and low temperature. This study focuses on the cryopreservation of this species by encapsulation-dehydration technique, in particular on the effects of pre-culture step, alginate bead size and the physical conditions of culture on the cryopreservation efficiency. Encapsulated and non-pre-cultured gametophytes did not survive cooling with liquid nitrogen. When cryopreservation was preceded by a 2-week period of pre-culture, regrowth reached 42.1%. Reduction in the size of the alginate bead, and culture in total darkness resulted in improved gametophyte regrowth capacity (75.5% or 81.7%, respectively). The best results (91.3%) were obtained when all factors tested occurred simultaneously. The gametophytes recovered very quickly and sporophytes were formed within 4 weeks after rewarming. These simple improvements can be used, not only for the cryopreservation of gametophytes in cryptogams but also for some recalcitrant species of seed plants.

English

Plumules excised from zygotic embryos through the largest representative diversity of four of the five different areas of coconut cash and food crops were used in a cryopreservation process using encapsulation-dehydration technique. Five accessions of coconut trees were used [Panama Tall (PNT/GPA), Brazilian Green Tall (BGD/NVB), Cameroon Red Dwarf (CRD/NRC), Vanuatu Tall (VTT/VNT/GVT), and Tagnanan Tall (TAGT/GTN)] in addition to the accession model [Malayan Yellow Dwarf (MYD)] from which an optimal protocol was obtained. A great variability of response was observed depending on accessions with survival and growth recovery rates varying from 6 to 66% and 0 to 24% after 2 and 7 months of culture, respectively. &nbsp; Key words: Coconut, accessions, germplasm, plumules, cryopreservation, encapsulation-dehydration.

Establishment of encapsulation-dehydration technique for in vitro fragmented explants of Rosa hybrida L. cv. Helmut Schmidt

Establishment of encapsulation-dehydration technique for in vitro fragmented explants of Rosa hybrida L. cv. Helmut Schmidt

Cryopreservation of in vitro-grown shoot tips of chrysanthemum by encapsulation-dehydration

ABSTRACT Chrysanthemums are amongst the most economically important flowers in the world. The protection and storage of these valuable genetic resources is of great importance. Today, cryopreservation, or the storage of biological material at the temperature of liquid nitrogen (-196°C), is believed to be the most promising long-term storage method. To optimise the cryopreservation protocol, the shoot tips of Chrysanthemum × grandiflorum /Ramat./ Kitam. ‘Lady Orange’ and ‘Lady Salmon’ mutants were cryopreserved using the encapsulation-dehydration technique. During the experiment, the influence of sucrose concentration (2, 3 and 6%) during preculture and the concentration of kinetin (0.25, 0.5, 0.75 and 1.0 mg dm-3) in the regrowth medium were tested. A higher survival rate was observed for ‘Lady Salmon’. In general, the media with higher sucrose levels provided the best survival and recovery rates (35-40%). Kinetin had no influence on the survival rate; however, it influenced the morphogenesis of the plants. The lowest number of explants forming multiple shoots was observed on the medium with the lowest sucrose (during preculture) and kinetin (in the recovery medium) concentration. On the other hand, the best rhizogenesis efficiency was observed when 0.25 mg dm-3 kinetin was added. In conclusion, the composition of both preculture and recovery media need to be adjusted to single cultivars. The use of 3% sucrose (preculture) and 0.25 mg dm-3 kinetin (recovery) seems reasonable, since it guarantees a satisfying recovery rate of the explants and at the same time prevents the formation of callus and multiple shoots, stimulating the rooting instead.

Histocytological analysis of yam (Dioscorea alata) shoot tips cryopreserved by encapsulation–dehydration.

In this work, we performed qualitative and quantitative observations of the cytological changes occurring in cells of yam (Dioscorea alata) in vitro shoot tips cryopreserved using the encapsulation-dehydration (E-D) technique. Shoot tip osmoprotection for 24 h in 1.25 M sucrose medium induced drastic changes in cellular cytological features, including high plasmolysis in all three cellular areas studied, the external cell layer (L1), one to three (L1-3) and seven to nine (L7-9) cell layers from the surface of the meristematic dome, pyknotic nuclei in meristematic area cells and disappearance of nucleoli. Nucleus size decreased significantly in all cellular areas studied. Nucleocytoplasmic ratio decreased significantly in L1-3 and L7-9 cells. Nuclear protein content increased, particularly in L1 and L1-3 cells. After physical dehydration, plasma membrane of numerous basal part cells was broken and intracellular soluble protein leakage was observed. Nucleus area and nucleocytoplasmic ratio decreased significantly in L7-9 cells. One week after cryopreservation, shoot tips showed regrowth and living cells had recovered their original morphology. In all cellular areas studied, nuclei had retrieved their original staining and nucleoli were visible. Original nucleus area values were recovered in L1-3 and L1 cells. The nucleocytoplasmic ratio retrieved its initial value in L1 cells but remained at levels observed after osmoprotection for L1-3 and L7-9 cells. The nuclear protein content had retrieved its original level. This investigation provided new insights in changes occurring in D. alata apices throughout an E-D protocol.

Cryopreservation of Cyrtopodium hatschbachii Pabst (Orchidaceae) immature seeds by encapsulation-dehydration

The aim of the present study was to investigate the efficiency of the encapsulation-dehydration technique for cryopreservation of Cyrtopodium hastchbachii Pabst seeds. Immature seeds of this species were cryopreserved by an encapsulation-dehydration technique. Seeds of five immature pods, 120 days after pollination, were encapsulated in 3% calcium alginate matrix and pretreated in liquid medium supplemented with 0.08 M sucrose (24 h), 0.15 M sucrose (24 h), 0.25 M sucrose (48 h), 0.5 M sucrose (24 h) and 0.75 M sucrose (24 h) in shaker at 60 rpm. Alginate beads were dehydrated 5 h in silicagel and immersed in liquid nitrogen for 12 h. Cryopreserved beads were thawed at 30 degrees C for 1 min, rehydrated using the same liquid mediums [0.75 M sucrose (24 h), 0.5 M sucrose (24 h), 0.25 M sucrose (48 h) and 0.15 M sucrose (24 h)] and cultivated in half strength Murashige & Skoog medium (1962) with the addition of 2 g/L activated charcoal. Sixty four percent of seeds survived and developed into acclimatized plants after being cryopreserved. In this work, the encapsulation-dehydration technique was employed for first time in Cyrtopodium hatschbachii.

Preliminary investigation of cryopreservation by encapsulation-dehydration technique on Brassidium Shooting Star orchid hybrid

Protocorm-like bodies (PLBs) of Brassidium Shooting Star, a new commercial ornamental orchid hybrid, were cryopreserved by an encapsulation-dehydration technique. The effects of PLB size, various sucrose concentrations in preculture media and sodium alginate concentration for encapsulation were the main parameters assessed. Four-week old PLBs (1 to 2 and 3 to 4 mm) were precultured in half strength semi-solid Murashige and Skoog (MS) media supplemented with six different sucrose concentrations (0, 0.2, 0.4, 0.6, 0.8 and 1.0 M) for 24 h, followed by encapsulation in 2.5, 3.0 or 3.5% sodium alginate, with 0.1 M calcium chloride been used as the hardening agent. The beads formed were then osmoprotected in half-strength liquid MS media supplemented with 0.75 M sucrose and dehydrated for three hours in 50 g heat-sterilized silica gel before cryostorage in sterile cryovials. The beads were thawed in a 40 ± 2°C water bath and then directly placed in recovery media for two weeks under tissue culture conditions. After two weeks of recovery, the survival rates of the encapsulated PLBs were evaluated by the 2,3,5-triphenyltetrazolium chloride (TTC) assay. The best conditions for the encapsulation-dehydration of Brassidium Shooting Star were discovered to be the preculture of 3 to 4mm PLB in half strength semi-solid MS media supplemented with 0.8 M sucrose, followed by encapsulation in 3.5% sodium alginate. Further biochemical analysis (chlorophyll, total soluble protein and peroxidases activities) were conducted to investigate the physiological responses of the PLBs after cryopreservation.

Cryoconservation and regeneration of axillary shoot meristems of Indigofera tinctoria (L.) by encapsulation–dehydration technique

In vitro axillary shoot proliferation was achieved from single-node explants of Indigofera tinctoria on a well-defined medium, Murashige and Skoog (MS) medium supplemented with 1.0 mg l−1 N 6-benzyl adenine (BA) and 0.1 mg l−1 indole-3-acetic acid. Axillary shoot meristems from cultures derived from up to three subcultures were used in the encapsulation–dehydration technique. Preconditioned, calcium alginate-encapsulated, and precultured axillary shoot meristems were subjected to different lengths of desiccation in a laminar flow cabinet. Maximum survival and regeneration rates of 56.7% and 62.2%, respectively, were obtained in half-strength (half the macro- and micronutrients and full-strength vitamins) MS medium supplemented with 0.5 mg l−1 gibberellic acid and 0.2 mg l−1 BA after 4 h of desiccation, during which the moisture content was reduced to 16.0%. According to the analysis of six random amplified polymorphic DNA markers, plantlets derived from cultures initiated with cryopreserved plant material were genetically identical to those derived from nonfrozen (control) tissues.

DESICCATION TOLERANCE AND CRYOPRESERVATION OF IN VITRO GROWN BLUEBERRY AND CRANBERRY SHOOT TIPS

vitro grown shoot tips of two cranberry (Vaccinium macrocarpon) cultivars, Wilcox (PI 614079) and Franklin (PI 554998) and three blueberry (Vaccinium corymbosum) cultivars, Berkeley (PI 554883), O'Neal (PI 554944) and Brigitta Blue (PI 618166) from the tissue culture collections of the USDA-ARS National Clonal Germplasm Repository (NCGR), Corvallis, were tested for recovery from desiccation and following cryopreservation using three protocols. Cold acclimated, encapsulated and sucrose-pretreated blueberry cultivars were tolerant of desiccation under laminar flow of up to 7h while cranberry cultivars were very sensitive to drying by 3h. Cryopreservation screening followed 2 weeks of alternating-temperature cold acclimation. The three blueberry cultivars cryopreserved with the encapsulation-dehydration technique (ED) produced 83% to 92% regrowth. PVS2 vitrification (VIT) (33% to 87%) and controlled rate cooling (CC) (50% to 67%) were also successful for some blueberries. The cranberry cultivars had poor (5% to 37%) recovery with all three techniques and will require further study to improve recovery after desiccation and cryopreservation.

Metabolic stability of plants regenerated from cryopreserved shoot tips of Dioscorea deltoidea – an endangered medicinal plant

Shoot tips of Dioscorea deltoidea, a medicinal yam, were cryopreserved using the vitrification and encapsulation-dehydration technique resulting in high-frequency direct plant regeneration. The biochemical stability of the plants derived from cryopreserved shoot tips was assessed using HPLC analysis. The diosgenin content of plants regenerated from cryopreserved shoot tips was found to be same as that of control plants. Thus plants confirming our cryopreservation experimental conditions ensure the biochemical stability of regenerated D. deltoidea plants.