PVS2 Solution Research Articles

Cryopreservation of zygotic embryos derived from BBTV resistant accessions of wild banana Musa acuminata

The wild banana species Musa acuminata encompasses numerous subspecies, some of which serve as ancestors of edible bananas. Preserving these subspecies is critical for food security, especially in regions where bananas are staple crops. While cryopreservation of M. acuminata subsp. burmanicoides zygotic embryos using air dehydration have shown high success rates, this method has not been effective for other subspecies. With at least 15 subspecies displaying various morphological traits, including seed morphology, alternative cryopreservation methods warrant exploration. In a recent study, successful cryopreservation using the droplet-vitrification method was achieved for mature zygotic embryos of accession of M. acuminata subspecies tomentosa, rutilifes, and malaccensis, all of which are resistant to the Banana bunchy top virus (BBTV). Using this approach, subjecting embryos to dehydration for 20 min in PVS2 solution and employing a regeneration medium comprising MS medium supplemented with benzyladenine and thidiazuron resulted in a success rate of 77.4 % (86 % survival rate × 90 % regrowth rate) for subsp. tomentosa and 50.4 % (70 % survival rate × 72 % regrowth rate) for subsp. malaccensis. Conversely, for subsp. rutilifes, dehydration for 10 min led to a success rate of 15.6 % (30 % survival rate × 52 % regrowth rate). Subspecies emerged as the primary factor influencing embryo regrowth, suggesting the need for protocol optimizations tailored to other subspecies. These findings highlight the potential of droplet-vitrification as a long-term preservation method for certain subspecies, while underscoring the necessity for further optimization for the other subspecies of M. acuminata.

A droplet vitrification cryopreservation protocol for conservation of hops (Humulus lupulus) genetic resources

Hops (Humulus lupulus L.) is essentially used in the brewing industry as it contributes to flavor, and aroma of beer. However, the genetic diversity of hops is increasingly threatened by diseases, environmental changes, and urbanization. Cryopreservation has emerged as a pivotal strategy for safeguarding and maintaining the genetic diversity of hops. The present work presents a comprehensive study on the cryopreservation of hops, focusing on the development and optimization of a droplet vitrification based cryopreservation protocol. Shoot tips excised from one month old in vitro cultures were precultured on 0.3 M sucrose, dehydrated in a loading solution followed by treatment with PVS2 solution for different durations. Significant effect of PVS2 dehydration was observed on post-thaw survival and regeneration after cryoconservation with maximum 50% post-thaw regeneration observed in shoot tips dehydrated in PVS2 for 30 min. Genetic fidelity of the regenerated plants was confirmed using 30 ISSR markers. Reproducibility of the developed protocol was tested on seven other accessions and post thaw regeneration ranging from 43 to 70% was observed across the accessions. The present study reports a highly efficient protocol for conservation of hops germplasm. The results indicate that droplet vitrification can be used as a reliable and sustainable approach for hop genetic preservation, with high survival rates and minimal genetic alterations observed in cryopreserved samples. To the best of our knowledge, this is the first report on DV based cryopreservation of hops germplasm.

Cryopreservation of highbush blueberry, strawberry, and saskatoon using V and D cryo-plate methods and monitoring of multiplication ability of regenerated shoots

AbstractThe purpose of this work was to apply V and D cryo-plate methods for cryopreservation of Vaccinium corymbosum ʻToroʼ, Fragaria × ananassa ʻCleryʼ, and Amelanchier alnifolia (Nutt.) M. Roem. and to monitor the multiplication capacity of shoots regenerated from cryopreserved explants. Shoot tips pre-cultured for 1 d at 23°C in the dark on medium containing 0.3 M sucrose were used as explants. Loading was performed in a solution containing 2 M glycerol and 0.8 M sucrose (30 min at room temperature). In the V cryo-plate, dehydration was carried out at room temperature (20 to 50 min) using the following plant vitrification solutions: original PVS2, 90% PVS2 solution, and PVS3. Regarding the D cryo-plate, dehydration was performed in closed glass containers over silica gel for 2, 2.5, or 3 h. In both protocols, rewarming was carried out in a 1.0 M sucrose solution (15 min at 25°C). Regenerated shoots were multiplied and multiplication parameters were monitored after the second subculture. Using the V cryo-plate method, the highest regrowth in highbush blueberry was obtained following 50-min treatment with all three VSs (61.7 to 80.9%). The D cryo-plate method was even more suitable with maximum regrowth of 89.4% achieved after 2.5 h of desiccation. For strawberry, 62.5% was the highest regrowth recorded using PVS3-based V cryo-plate method while 83.3% of regrowth was observed using D cryo-plate protocol. Regrowth of saskatoon reached a maximum of 50% after 50-min treatment with PVS3 while it did not exceed 40% in other treatments. By the second subculture, shoots regenerated from cryopreserved explants regained and even exceeded the multiplication capacity of shoots regenerated from non-cryopreserved explants. This study is the first to present the successful application of the V cryo-plate method in highbush blueberry, as well as the utilization of both V and D cryo-plate methods in saskatoon.

Survival and State of Garlic Explants of Two Lithuanian Cultivars after Cryopreservation

Cryopreservation features of Lithuanian garlic (Allium sativum L.) cultivars ‘Žiemiai’ and ‘Dangiai’ were investigated. The survival rate and state of explants, depending on the type of explant, and dehydration conditions were evaluated in the experiment. Explants isolated from cloves and bulbils were dehydrated with a plant vitrification solution 3 (PVS 3) containing 50% w/v glycerol and 50% w/v sucrose in liquid MS medium. Three different exposure durations of 1.5, 2, and 3 h in PVS3 solution were applied at 0 °C. Unloaded stem domes were planted in Petri dishes with MS medium supplemented with 1 mg L−1 naphthaleneacetic acid (NAA) and 6–dimethylallylamino purine (2-iP) on 0.8% plant agar. The obtained results showed that the cryopreservation in liquid nitrogen reduced the survival rate of explants by 20–40%. The average number of surviving explants after freezing reached 64.3% for both cultivars. Cultivar ‘Žiemiai’ showed a 24% higher capability for survival than ‘Dangiai’. The total number of surviving explants of ‘Žiemiai’ reached 76.1%. Explants from bulbils were 23% more effective for cryopreservation compared to cloves. Evaluation of the effect of dehydration duration showed that the survival rate of the explants from bulbils of both cultivars was highest after treatment with PVS3 for 3 h and reached 91%. Treatment in PVS3 solution for 2 h was sufficient for survival of explants from cloves. Obtained results confirm that the efficiency of garlic cryopreservation depends on complex factors.

Cryopreservation of raspberry in vitro cultures by PVS3 solution

Cryopreservation of raspberry in vitro cultures by PVS3 solution

Cryopreservation of coconut plumule using droplet vitrification

In the present investigation, four types of explants from mature zygotic embryos of coconut, viz., whole upper cotyledonary region without haustorium, half of the upper cotyledonary region without haustorium, plumule with a portion of radicle and exclusively plumular tissue, were cultured in 12 different media combinations to find a suitable explant which could be regenerated after cryopreservation. Explants were pre-cultured in medium with 0.4 and 0.5 M sucrose for three days followed by dehydration in PVS3 solution for different durations on a sterile aluminum strip after treating with loading solution. Strips were treated with liquid nitrogen inside a cryoflask until bubbling stopped and quickly transferred to a cryovial and stored for a minimum period of 24 hours in liquid nitrogen. It was observed that plumule alone or with a small portion of outer tissue was ideal for fast in vitro growth and recovery of whole plantlets of coconut in a medium supplemented with NAA alone. Addition of glutamine (5 mg L-1), TDZ (1 mg L-1) and NAA (18 mg L-1) aided the vigorous growth of plantlets. In control, the survival rate ranged from 60 to 90 per cent in plumule pre-grown in media containing 0.5 M sucrose after dehydration with PVS3 for various durations, whereas it was 14 to 75 per cent in cryopreserved ones. Considering the high survival (75%) and regrowth (35%) of cryopreserved plumule in the present study, there is much scope for further improvement of the procedure to find the right combination of factors so as to enhance complete recovery of plantlets without much injury to plumules during cooling and rewarming.

Cryopreservation of shoot tips of Gentiana kurroo Royle – a critically endangered medicinal plant of India

Gentiana kurroo Royle, a critically endangered medicinal plant species of India is conserved as in vitro slow growing cultures in the In Vitro Genebank (IVGB) at ICAR-National Bureau of Plant Genetic Resources (NBPGR), New Delhi, India. Shoot tips (about 1 mm in length) excised from 8-weeks-old stock cultures subjected to cold hardening for four weeks at 8 °C (dark) were precultured on Murashige and Skoog (MS) medium supplemented with 5% Dimethylsulfoxide (DMSO) at 8 °C (dark) for two days. Thereafter, shoot tips were dehydrated with PVS2 solution (30 min) at 0 °C and cryopreserved using conventional vitrification (V) and droplet-vitrification (DV) techniques. Average post-thaw regeneration after 4 weeks was ~ 35% by V technique and ~ 60% by DV in all the three accessions tested. Genetic stability assessment on the basis of 40 Inter Simple Sequence Repeats and 30 Expressed Sequence Tagged-Simple Sequence Repeats markers revealed no variation between in vitro-cryopreserved plants and controls, in any of the genotypes. Thus, standardized DV method has the potential for long-term conservation of germplasm of G. kurroo and the three accessions are now safely cryobanked at ICAR-NBPGR. Gentiana kurroo is a critically endangered medicinal plant endemic to North-western Himalayas, India. A protocol to cryopreserve in vitro shoot-tips was developed using droplet vitrification technique with high recovery rates and genetically stable regnerants. The protocol is being applied for cryobanking of Gentiana germplasm at ICAR-NBPGR.

Long-term conservation of Tarenaya rosea (Cleomaceae) root cultures: histological and histochemical analyses during cryopreservation using the encapsulation-vitrification technique.

Adventitious root cultures of Tarenaya rosea were successfully cryopreserved using the encapsulation-vitrification technique. Histological analysis revealed useful information on the successive steps of cryopreservation. Coupled with complementary histochemical approaches, these studies provided cellular and tissue descriptions of T. rosea root cultures during cryopreservation and contributed to an understanding of cellular stress responses, as well as characterization of the anatomical pattern of root regeneration. The effects of exposure duration to PVS3 solution (0-120min), unloading treatment (direct and gradual), and recovery medium (liquid and solid) on recovery of cryopreserved roots were investigated. The highest recovery (91%) after cooling in liquid nitrogen (LN) was reached with PVS3 treatment for 90min, gradual rehydration in unloading solution, and recovery on solid MS medium. The cryopreserved roots showed high multiplication capacity, which was maintained for up to four subcultures. The effect of cryopreservation on root structure was investigated by histological and histochemical studies. Plasmolysis intensified during exposure to loading and PVS3 solutions, but decreased after unloading treatment. The proportion of intercellular spaces increased progressively throughout the cryopreservation protocol, culminating in root cortex disruption. Histochemical analyses revealed polysaccharides, proteins, and both lipidic and pectic substances in intercellular spaces. The vascular cylinder remained intact, ensuring the formation of new roots from the pericycle, showing that proliferative capacity of cryopreserved roots had not diminished.

Cryopreservation of sievers wormwood (Artemisia sieversiana Ehrh. Ex Willd.) seeds by vitrification and encapsulation

Artemisia sieversiana is being used for the medicinal purpose, there are not many conservation objects, and the species is registered as a rare plant in Korea. There is also no research on the preservation of A. sieversiana seeds. In this paper, we investigated the preservation of seeds using cryopreservation. The vitality and germination rate of seeds were investigated with pretreatment, vitrification, and encapsulation. The initial germination rate of the seeds was 95%. PVS3 solution treated for 60 min showed the highest vitality, and germination rate. When encapsulation, both vitality and germination rate decreased. Changes in vitality rate during storage period were measured. As a result, the seed-maintained vitality when the vitrification solution used. Encapsulation is unavailable because of too much change. Therefore, seeds can be safely preserved without deterioration of vitality at cryopreservation by using PVS 3 solution and it will be helpful in future studies.

Cryopreservation of Dendrobium cruentum Rchb. f. Seeds by D Cryo-plate and V Cryo-plate Techniques

Dendrobium cruentum Rchb. f. is a native Thai orchid species that has faced extinction because of its attractive characteristics. Consequently, conservation of this species is urgently needed. In this study, cryopreservation technique was applied to D. cruentum Rchb. f. seeds for long-term conservation. A successful protocol for D. cruentum Rchb. f. seed cryopreservation was developed by using D cryo-plate and V cryo-plate techniques. Seed viability was tested by TCC solution and 93.8 % of dyed seeds were shown. For cryo-plate technique, seeds were encapsulated over the cryo-plate by using 2 % (w/v) sodium alginate and polymerized with 100 mM CaCl2. Encapsulated seeds were desiccated by using a laminar airflow and PVS2 solution treatment with the same exposure time (0, 30, 60, 90, and 120 min). After cryopreservation, encapsulated seeds were cultured on modified VW agar medium. From the results, the maximum germination and regrowth percentage were observed; D cryo-plate technique with 60 min of dehydration time gave the highest germination (68.9 %) and regrowth (57.8 %). Thus, the excess of dehydration may cause the reduction of germination and plant regeneration. In conclusion, D cryo-plate technique proved to be appropriate for D. cruentum Rchb. f. seed cryopreservation.

Cryopreservation: A tool to conserve date palm in Saudi Arabia.

The cryostoring of embryogenic tissue of the date palm (Phoenix dactylifera L. cv. Sagai) was examined through dehydrated-encapsulation, vitrification, and vitrification-encapsulation. The most extreme regeneration rate (53.33%) of epitomized, cryostored liquid nitrogen (+LN) treated embryos was observed when pre-embryonic masses were hatched with 0.5 M sucrose for 48 h pursued by 6 h air drying out. The most noteworthy survival rate (80.0%) of epitomized, cryopreserved embryonic cluster came about when calli were hatched with 0.3 or 0.7 M sucrose for 48 h pursued by four hours of lack of hydration, or with 0.5 M sucrose for 48 h without air drying out or with 2 h of air drying out. Following cryopreservation utilizing the embodiment vitrification convention, the most astounding survival (86.7%) as well as the greatest growth (46.7%) was accomplished when the typified vitrified, cryopreserved calli were treated with Vitrification Solution 2 for plants (PVS2) for 60 min at 25 °C. Cryopreservation utilizing the vitrification convention brought about the most extreme recuperation of 53.3%, when vitrified-cryopreserved calli were subjected to PVS2 solution for 30 min at 25 °C. Most extreme (40%) regeneration of vitrified, cryopreserved embryonic calli was seen when these calli were treated with PVS2 solution for 60 min at 25 °C. The outcome got amid this investigation of regrowth after cryopreservation of the cv. Sagai was over the base suitable for a cryo-germplasm bank. Recovery and regrowth were above 30% for all the techniques developed for the cv. Sagai.

Efficiency of aluminum cryo-plates for cryopreservation of Dendrobium signatum Rchb. f. pollinia

The efficiency of V cryo-plate and D cryo-plate methods for cryopreservation of pollinia of Dendrobium signatum Rchb. f., a Thai orchid was investigated. Pollinia were collected from flowers and then placed on the aluminum cryo-plate containing 12 wells embedded with 3% sodium alginate gel. In V cryo-plate method, cryo-plates with pollinia were immersed in 0.6 M sucrose + 2 M glycerol loading solution (LS) for 15 min, then dehydrated with PVS2 solution for 40 min at room temperature (29±2°C). In D cryo-plate method, cryo-plates with pollinia were treated with LS for 15 min, then dehydrated in a laminar air-flow cabinet for 3 h at room temperature (29±2°C). In both methods, cryo-plates were directly plunged into liquid nitrogen for 40 min, and rapidly warmed in 1.2 M sucrose for 15 min. The cryopreserved and non-cryopreserved pollinia were used to pollinate flowers of the same species. The results showed that cryopreserved pollinia retained fertilizing ability, and had similar pod formation as those pollinated with non-cryopreserved pollinia. The pod formation from cryopreserved pollinia in V cryo-plate and D cryo-plate methods were 55.6 and 50%, respectively. Seeds from non-cryopreserved and cryopreserved pollinia were successfully produced and germinated into plantlets with well-formed leaves and roots. Multiple stems (4.8 stems plant(-1)) produced from one plantlet when cultured on modified VW agar medium (1949) supplemented with 100 g L(-1) banana, 150 mL coconut water, 50 g L(-1) potato, and 20 g L(-1) sucrose for 120 d at 25±2°C. Cryopreserved pollinia using aluminum cryo-plate can be used successfully for pollination.

Cryopreservation of non-precultured protocorms of Acampe rigida (Buch.-Ham. ex Sm.) P.F. Hunt using V cryo-plate and D cryo-plate methods

Non-precultured protocorms of Acampe rigida (Buch.-Ham. ex Sm.) P.F. Hunt were used to find the suitable duration time for dehydration on V cryo-plate and D cryo-plate methods. For V cryo-plate method, non-precultured protocorms were dehydrated with PVS2 solution for 0, 20, 40, 60 and 120 min. The results showed that protocorms dehydrated with PVS2 solution for 40 min gave the highest survival score at 0.08 and gave the highest percentage of survival at 16.67% which was higher than the control (+LN; 0 min), but most protocorms showed dark green color and developed slower than the control (-LN; 0 min). The suitable treatment for V cryo-plate in this study is exposing to PVS2 solution for 40 min. For D cryo-plate method, non-precultured protocorms were dehydrated with silica gel for 0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 h. The results showed that protocorms dehydrated with silica gel for 1.0 h gave the highest survival score at 0.56 and gave the highest percentage of survival up to 74.19%. Most protocorms with dark green developed slower than control (-LN; 0 min). The suitable treatment for D cryo-plate method in this study is dehydrating with silica gel for 1.0 h. An unpaired t-test was used to compare the data of survival score between suitable treatments from V cryo-plate and D cryo-plate methods. The suitable treatment for cryopreservation of protocorms of A. rigida is cryopreserving with D cryo-plate method dehydrated with silica gel for 1.0 h.

Long-term preservation of Lotus tenuis adventitious buds

Encapsulation-dehydration, encapsulation-vitrification, and vitrification were tested for cryopreservation of Lotus tenuis (Fagaceae) adventitious buds clusters (ABCs) obtained by a direct regeneration system from leaves cultures. Among them, the PVS3-based vitrification procedure was found to be useful for survival and regrowth of the preserved explants. For vitrification, the ABCs were dehydrated in a solution containing 2 M glycerol + 0.4 M sucrose for 25 min at room temperature, submerged in PVS3 solution for 1 h at 0 °C, then immersed in liquid nitrogen for 48 h and rapidly rewarmed. Afterword, the explants were unloaded in MS liquid medium with 1.2 M sucrose for 30 min. The washed tissues were dried superficially on filter paper and cultured in semisolid hormone-free MS medium containing 0.1 M sucrose. All cultures were maintained at 25 °C in the dark for 10 days and transferred to the light conditions. With this procedure, 79 ± 5.3% survival and more than 80% of the plantlets displaying a phenotype similar to the non-treated control after acclimatization. The data settled from ISSR showed no genetic dissimilarities between in vitro regenerants derived from cryopreserved tissues and the non-treated plants. Thus, our results indicate that the use of vitrification-based PVS3 solution offers a simple, accurate, and appropriate procedure for the cryopreservation of L. tenuis adventitious buds.

Encapsulation-vitrification and encapsulation-dehydration cryopreservation of Cattleya labiata Lindley, a threatened Brazilian orchid

Orchid populations have been decreasing due to habitat loss and over-collection for ornamental use. Consequently, there is an urgent need to conserve germplasm resources. Cryopreservation of the threatened orchid Cattleya labiata L. was successfully achieved using encapsulation-vitrification and encapsulation-dehydration cryopreservation methods. Protocorms, of 0.1 cm in diameter, developed from 2-months-old germinating seeds and encapsulated in sodium alginate-beads, were precultured in half-strength Murashige and Skoog liquid medium containing 0.4 M sucrose on a shaker (110 rpm) at 25°C for 24 h. For encapsulation-dehydration, beads were treated in the loading solution (2 M glycerol and 0.4 M sucrose in half-strength Murashige and Skoog liquid medium) for 20 min and then exposed to a sterile air-flow from the laminar air-flow cabinet for 0, 1, 2, 4 or 6 h. For encapsulation-vitrification, beads were treated in the loading solution for 20 min and then exposed to PVS2 solution (30% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) dimethyl sulfoxide in half-strength Murashige and Skoog liquid medium containing 0.4 M sucrose at pH 5.7) for 0, 10, 20, 30 and 40 min. Comparing the two methods tested, it was observed that regeneration of cryopreserved protocorms using encapsulation-dehydration was higher (42%) with 4 h dehydration in laminar air-flow cabinet, whereas using the dehydration-vitrification procedure it was 27% after dehydration for 10 min in PVS2 solution. Seedlings developed from these methods did not show any abnormal characteristics of growth in vitro, and were successfully hardened in greenhouse showing 80% survival after 120 days of acclimatization.

Cryopreservation of Protocorm-like Bodies of Vanda lilacina Teijsm. & Binn., a Thai Orchid Species, by V-cryo-plate and D-cryo-plate Methods

Protocorm-like bodies (PLBs) of Vanda lilacina Teijsm. & Binn. were used to study two new cryopreservation methods, the V-cryo-plate and D-cryo-plate methods, for survival score and percentage of survival. PLBs were dehydrated with PVS2 solution (0, 20, 40, 60, and 120 min) and silica gel (0, 0.5, 1, 1.5, 2, 2.5, and 3 h) for the V-cryo-plate and D-cryo-plate methods, respectively. The results showed that the V-cryo-plate method, dehydration with PVS2 solution for 20 min, was a suitable method. It gave a survival score of 0.31, higher than control (+LN; 0 min) and gave a percentage of survival of 33.33 %. For the D-cryo-plate method, the suitable method for cryopreserved PLBs of V. lilacina was dehydration with silica gel for 1 h. It gave the highest survival score of 0.78 and percentage of survival was up to 83.78 %. Then, an unpaired t-test was used to compare data of survival scores of cryopreserved V. lilacina PLBs between the V-cryo-plate and D-cryo-plate methods. The best suitable cryopreservation method of V. lilacina PLBs is the D-cryo-plate method using silica gel for 1 h in dehydration step.

Cryoprotectants and components induce plasmolytic responses in sweet potato (Ipomoea batatas (L.) Lam.) suspension cells

Plant genebanks often use cryopreservation to securely conserve clonally propagated collections. Shoot tip cryopreservation procedures may employ vitrification techniques whereby highly concentrated solutions remove cellular water and prevent ice crystallization, ensuring survival after liquid nitrogen exposure. Vitrification solutions can be comprised of a combination of components that are either membrane permeable or membrane impermeable within the timeframe and conditions of cryoprotectant exposure. In this study, the osmotic responses of sweet potato [Ipomoea batatas (L.) Lam.] suspension cell cultures were observed after treatment with plant vitrification solution 2 [PVS2; 15% (v/v) dimethyl sulfoxide (DMSO), 15% (v/v) ethylene glycol, 30% (v/v) glycerol, 0.4 M sucrose], plant vitrification solution 3 (PVS3; 50% (v/v) glycerol, 50% (w/v) sucrose), and their components at 25 and 0°C, as well as cryoprotectant solution, PGD (10% (w/v) PEG 8000, 10% (w/v) glucose, 10% (v/v) DMSO) at 25°C. At either 25 or 0°C, sweet potato cells plasmolyzed after exposure to PVS2, PVS3, and PGD solutions as well as the PVS2 and PVS3 solution components. Cells deplasmolyzed when the plasma membrane was permeable to the solutes and when water re-entered to maintain the chemical potential. Sweet potato suspension cells deplasmolyzed in the presence of 15% (v/v) DMSO or 15% (v/v) ethylene glycol. Sweet potato plasma membranes were more permeable to DMSO and ethylene glycol at 25°C than at 0°C. Neither sucrose nor glycerol solutions showed evidence of deplasmolysis after 3 h, suggesting low to no membrane permeability of these components in the timeframes studied. Thus, vitrification solution PVS2 includes components that are more membrane permeable than PVS3, suggesting that the two vitrification solutions may have different cryoprotectant functions. PGD includes DMSO, a permeable component, and likely has a different mode of action due to its use in two-step cooling procedures.

Effect of the Duration of Liquid Nitrogen Storage on the Regrowth of Blackberry Cryopreserved by Droplet Vitrification

Summary In vitro shoot tips of the blackberry cultivar ‘Čačanska Bestrna’ were cryopreserved using the droplet vitrification technique. Upon loading (30 min) in a solution of 1.9 M glycerol and 0.5 M sucrose, the explants were dehydrated for 40 min on ice with the PVS A3 vitrification solution (glycerol 37.5%, dimethyl sulfoxide 15%, ethylene glycol 15% and sucrose 22.5%) and for 40 min at room temperature with the PVS3 solution (glycerol 50% and sucrose 50%). They were subsequently frozen in individual microdroplets of vitrification solution, by direct immersion in liquid nitrogen (LN), and kept therein for 2, 4, 8 and 24 h. The explant rewarming was performed in an unloading solution (0.8 M sucrose) for 30 min at room temperature. The duration of LN exposure did not exert significant effects on the survival and regrowth of explants in both types of vitrification solutions. The survival and regrowth of cryopreserved shoot tips dehydrated with PVS3 solution ranged between 90–95% and 80–90%, respectively. However, dehydration with PVS A3 resulted in a lower survival rate (80–90%) and a considerably lower regrowth rate (55–65%) of explants. Monitoring the shoots regenerated in the in vitro culture revealed their normal capacity for multiplication and rooting in comparison with the controls, which fully confirms the purpose of cryopreservation in the long-term preservation of plant material.

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.

Ex situ conservation of the endangered species Androcalva perlaria (Malvaceae) by micropropagation and cryopreservation

Micropropagation and cryopreservation protocols were developed for the threatened Australian species Androcalva perlaria. Vegetative shoots were brought into culture using a simplified surface sterilisation process with between 26 and 100 % of shoots successfully initiated across all genotypes. Shoots were multiplied on ½ MS basal salts medium (BM) with 1.25 µM 6-furfurylaminopurine (K) + 0.125 µM 6-benzylaminopurine (BAP). Cryopreservation was then developed for a single genotype to facilitate long-term ex situ storage for conservation purposes. Highest survival (>80 %) of shoot tips was achieved by preculture on 1.2 M glycerol for 48 h, incubation in PVS2 solution at 0 °C for 30 min, followed by rapid LN immersion then recovery. Application of this cryogenic approach to shoot tips from a range of genotypes gave variable post-cryopreservation regeneration results; survival for one genotype was only 3 %, while for four other genotypes survival varied between 60 and 80 % which compared favourably with post-cryopreservation regeneration (85 %) of the genotype used to develop the protocol. Callus production was achieved by culturing stem segments on ½ MS BM with 2.5 µM α-naphthaleneacetic acid + 2.5 µM BAP. Adventitious shoots were best regenerated from callus through incubation on BM only. Small callus pieces were successfully cryopreserved from 16 genotypes (1–88 % regeneration). Using a callus tissue pathway plant material was placed into LN storage after 6–8 weeks from the time of collection (compared to ~6 months using shoot tips). Plants derived from cryogenically preserved callus tissues were re-established in soil 28 weeks after removal from LN. This study demonstrates how biotechnology can be effectively utilised for the rapid ex situ conservation of endangered flora while ensuring that a significant range of genetically diverse samples can be conserved for long-term biosecurity.