Plant Vitrification Solution Research Articles

Y2SK2- and SK3-type dehydrins from Agapanthus praecox act as protectants to improve plant cell viability during cryopreservation

The cryopreservation of plants through vitrification prevents ice damage by bringing the plants to a glassy state for cryogenic storage. Most angiosperm seeds are dehydrated to extremely low levels of their original water content, accumulate late embryogenesis abundant (LEA) proteins, and greatly increase their cytoplasmic viscosity, thereby reaching a glassy state during maturation. In this study, two LEA family recombinant dehydrin (DHN) proteins (Y2SK2 and SK3) from Agapanthus praecox, when added to the plant vitrification solution, were found to double the survival rate of Arabidopsis thaliana seedlings after cryopreservation. Evans blue staining revealed that the Y2SK2 and SK3 proteins decreased the damage to the plasma membranes of plant cells during cryopreservation. Furthermore, the Y2SK2 and SK3 proteins significantly decreased the malondialdehyde and H2O2 contents, increased the glutathione (GSH) content, and downregulated NADPH oxidase (RbohA) in A. thaliana seedlings. Thus, these DHNs may reduce excessive reactive oxygen species production and membrane lipid peroxidation damage under the complex stresses of cryopreservation. Additionally, Y2SK2 can effectively enhance peroxidase activity and catalase2 (CAT2) expression levels; SK3 obviously improved ascorbic acid (AsA) content, as well as Cu/Zn superoxide dismutase (Cu/Zn-SOD) and ascorbate peroxidase 5 (APX5) expression in plant cells and enhanced H2O2 scavenging capacity by promoting the activity of the AsA-GSH cycle. These findings suggested that Y2SK2 and SK3, the DHNs added at the dehydration step, can relieve cell cryoinjury by inducing high antioxidant levels and positive oxidative stress responses and act as protectants, improving plant cell viability after cryopreservation. Application of dehydrins optimization plant cryopreservation protocol, dehydrins can significantly improve complex stress damage and enhance the cell viability from cryopreservation.

Cocoa (<i>Theobroma cacao</i> L.) Somatic Embryos Tolerate Some Ice Crystallization during Cryopreservation

Secondary somatic embryos (SSEs) of cocoa, a recalcitrant tropical, seed-producing species, were cryopreserved using a vitrification approach and Differential Scanning Calorimetry (DSC) was employed to optimise sucrose preculture and Plant Vitrification Solution 2 (PVS2) incubation. The objective of the study was to evaluate the influence of sucrose preculture and PVS2 dehydration on water content of SSE that will enable it to survive cryostorage. SSEs were precultured for 3 or 5 days on media containing 0.5 M or 0.75 M sucrose and cryoprotected in loading solution (2 M glycerol and 0.4 M sucrose in medium) for 20 min before they were dehydrated with cold PVS2 for 0 - 90 min. Thermal analysis revealed the occurrence of ice crystallization in the SSEs with the extent declining with increasing PVS2 exposure. Maximal survival of SSEs was promoted by preculture on 0.5 M sucrose medium and dehydration with PVS2 for 45 - 60 min, which was characterised by small ice crystallization. Exposure of SSEs beyond 60 min leads to excessive dehydration as characterized by no change in the thermograms. Based on these findings, preculture of SSEs on 0.5 M sucrose medium and dehydration with cold PVS2 for 60 min has been adopted for the successful cryopreservation of cocoa germplasm.

Droplet-vitrification for shoot tip cryopreservation of shallot (Allium cepa var. aggregatum): effects of PVS3 and PVS2 on shoot regrowth

The present study described a droplet-vitrification cryopreservation for shoot tips of shallot (Allium cepa var. aggregatum), a small bulb onion. Shoot tips taken from in vitro stock shoots were precultured with 0.3 M and 0.5 M of sucrose, with 1 day for each concentration. Precultured shoot tips were treated with a loading solution containing 2 M glycerol and 0.6 M sucrose for 20 min and then exposed to plant vitrification solution 3 (PVS3) at 24 °C for 3 h of dehydration. Following exposure to PVS3, shoot tips were moved onto 5.0 µl PVS3 droplets on aluminum foil strips, followed by direct immersion into liquid nitrogen for 1 h. Frozen shoot tips were thawed by incubation in liquid MS medium containing 1.2 m sucrose for 20 min at room temperature, and then post-thaw cultured for shoot regrowth. Exposure of the shoot tips to PVS3 produced shoot regrowth (58%). Differential scanning calorimetry (DSC) detected 1.8% of freezable water in the shoot tips that had been dehydrated by PVS2, and no freezable water in those by PVS3 treatment. Exposure to PVS3 provided a broader safe temperature range (− 196 °C to − 88 °C), compared to that (− 196 °C to − 116 °C) of PVS2, for cryopreserved samples. Histological observations found that PVS3 dehydration allowed many cells in the apical dome and in the leaf primordia to survive following freezing in LN, while PVS2 dehydration resulted in much fewer surviving cells in the apical dome. The droplet-vitrification cryopreservation produced 56%, 72% and 32% shoot regrowth in cryopreserved shoot tips taken from in vitro shoots, adventitious buds regenerated from stem discs and field-grown bulbs, respectively. Advantages and disadvantages of the use of different source explants for cryopreservation were discussed. The droplet-vitrification cryopreservation produced 45% and 70% shoot regrowth in the additional two shallot genotypes ‘Kverve’ and ‘Lunteviga’. The results obtained in this study provide technical supports for setting-up cryo-bankings of genetic resources of shallots and other Allium species. Establishment of a droplet-vitrification cryopreservation of shallot shoot tips provided technical supports for long-term preservation of diverse genetic resources and cryotherapy for virus eradication in shallot plants.

Modification of the droplet-vitrification method of cryopreservation to enhance survival rates of adventitious roots of Panax ginseng

Panax ginseng Meyer is an important medicinal plant producing bioactive compounds. A droplet-vitrification method was developed for cryopreserving adventitious root cultures of mountain ginseng, and variations of this procedure were tested to determine their effects on regrowth rates and osmotic stress responses. Root regrowth rates were examined after exposing root segments to two pre-culture treatments, three loading solutions, and two vitrification solutions with different exposure periods. Pre-culturing excised segments of adventitious roots with 0.3 M sucrose produced the highest rate of regrowth after cryopreservation. Loading for 20 min with 17.5% (w/v) sucrose and 17.5% (w/v) glycerol at room temperature increased the regrowth rate of cryopreserved adventitious roots fourfold, compared with non-loaded samples. Treatments involving different vitrification solutions and exposure periods were compared, and the highest rate of regrowth (15%) after cryopreservation was achieved by incubating adventitious roots in modified plant vitrification solution 3 containing 40% (w/v) glycerol and 40% (w/v) sucrose for 10 min at room temperature, suggesting that ginseng adventitious root tips were sensitive to osmotic stress. Further study is necessary to develop optimal vitrification solutions that enhance the survival rate of cryopreserved adventitious roots of mountain ginseng.

The cryoprotectant PVS2 plays a crucial role in germinating Passiflora ligularis embryos after cryopreservation by influencing the mobilization of lipids and the antioxidant metabolism

Cryopreservation is a process whereby biological structures are preserved in liquid nitrogen (−196 °C) without losing their viability. Many cryopreservation techniques use the Plant Vitrification Solution 2 (PVS2) for cryoprotection. This study will therefore evaluate the influence of different exposure times to the cryoprotectant PVS2 and discuss the importance of the mobilization of reserves and the antioxidant metabolism during the germination of cryopreserved Passiflora ligularis embryos. The composition of P. ligularis seeds was analytically determined. We tested the germination capacity and the Germination Speed Index (GSI) of embryos (that is, seeds without external tegument) which were exposed to different PVS2 exposure times (0, 30, 60 and 120 min) at 30 days after thawing. Proline content, hydrogen peroxide, activity of isocitrate lyase (ICL), malate synthase (MSy), lipid peroxidation and antioxidant enzyme activities (SOD, CAT, APX) were measured at 7, 14 and 21 days after cryopreservation. The germination from cryopreserved embryos was maximal (85%) after 60 min PVS2 exposure with a GSI of 0.6. At 60 min, the highest activity of the enzymes involved in the glyoxylate cycle, ICL and MSy were recorded. We hypothesize that a 60 min exposure to PVS2 accelerates the reserve mobilization which correlates positively with germination. Until 60 min, there was a positive correlation between the PVS2 exposure time and the proline content, as well as the activity of antioxidant enzymes (SOD, CAT, APX), and a negative correlation with the lipid peroxidation. This study enables us to optimize the long-term conservation of this species. In conclusion, fundamental research is necessary to optimize the cryopreservation procedure, and this study offers an effective and efficient workflow which can be extrapolated to other (oil-rich) species.

Cryopreservation of papaya seeds cv. Sukma, Callina, and Caliso: Effect of loading treatment and immersion time in plant vitrification solution-2

Abstract. Wardani FF, Efendi D, Dinarti D, Witono JR. 2019. Cryopreservation of papaya seeds cv. Sukma, Callina, and Caliso: Effect of loading treatment and immersion time in plant vitrification solution-2. Nusantara Bioscience 11: 71-78. Sukma, Callina, and Caliso are papaya cultivars released by the Center for Tropical Horticulture Studies, IPB University, Bogor, West Java, Indonesia. In general, papaya seeds cannot be stored for a long time with a conventional GenBank storage system, even though some of them consider as “orthodox” class of seed. Cryopreservation, storage at an ultra-low temperature (-196 0C) of liquid nitrogen, could be possible for long-term storage of papaya seeds. The experiment conducted to get the necessary of loading treatment and the best immersion time in PVS2 for cryopreservation of papaya seeds cv. Sukma, Callina, and Caliso, so they still had considerable viability. We conducted experiment as factorial in completely randomized design, with the first factor was loading treatment with two levels (with and without loading treatment), and the second factor was immersion time in PVS2 with three levels (15, 30 and 45 min). So, there was six treatment and we used 50 seeds for each treatment. Results showed that the three papaya cultivars gave different responses to the treatment before cryopreservation proved that papayas seed had different characteristics, depending on genotype. For papaya seeds cv. Sukma, loading treatment was not needed and the best immersion time in PVS2 was 15 min. The viability of papaya seeds cv. Callina was low, so we should try another treatment before cryopreservation. For papaya seeds cv. Caliso, loading treatment was needed and the best immersion time in PVS2 was 30 min.

Y2SK2 and SK3 type dehydrins from Agapanthus praecox can improve plant stress tolerance and act as multifunctional protectants

Two dehydrins from Agapanthus praecox (ApY2SK2 and ApSK3) show important protective effects under complex stresses. Both ApY2SK2 and ApSK3 contain one intron and consist of a full-length cDNA of 981 bp and 1057 bp encoding 186 and 215 amino acids, respectively. ApY2SK2 and ApSK3 transgenic Arabidopsis thaliana show reduced plasma membrane damage and ROS levels and higher antioxidant activity and photosynthesis capability under salt, osmotic, cold and drought stresses compared with the wild-type. ApY2SK2 and ApSK3 are mainly located in the cytoplasm and cell membrane, and ApY2SK2 can even localize in the nucleus. In vitro tests indicate that ApY2SK2 and ApSK3 can effectively protect enzyme activity during the freeze-thaw process, and ApY2SK2 also exhibits this function during desiccation treatment. Furthermore, ApY2SK2 and ApSK3 can significantly inhibit hydroxyl radical generation. These two dehydrins can bind metal ions with a binding affinity of Co2+> Ni2+> Cu2+> Fe3+; the binding affinity of ApSK3 is higher than that of ApY2SK2. Thus, ApY2SK2 has a better protective effect on enzyme activity, and ApSK3 has stronger metal ion binding function and effect on ROS metabolism. Moreover, plant cryopreservation evaluation tests indicate that ApY2SK2 and ApSK3 transformation can enhance the seedling survival ratio from 23% to 47% and 55%, respectively; the addition of recombinant ApY2SK2 and ApSK3 to plant vitrification solution may increase the survival ratio of wild-type A. thaliana seedlings from 24% to 50% and 46%, respectively. These findings suggest that ApY2SK2 and ApSK3 can effectively improve cell stress tolerance and have great potential for in vivo or in vitro applications.

Hydrogen peroxide and nitric oxide are involved in programmed cell death induced by cryopreservation in Dendrobium protocorm-like bodies

Programmed cell death (PCD) plays a key role in animal tissue cell death following cryopreservation. However, there are few studies evaluating the plant PCD characteristics induced by cryopreservation and the possible role of signal molecules in PCD. Here, we performed cell ultrastructural observation and evaluation of PCD-regulating gene expression, caspase-3-like activity, and hydrogen peroxide (H2O2) and nitric oxide (NO) contents occurring in Dendrobium protocorm-like bodies (PLBs) cryopreserved by vitrification. The main changes in cell ultrastructure were noted at the plant vitrification solution 2 (PVS2) dehydration stage with severe plasmolysis, chromatin condensation and vesicle formation. Liquid nitrogen exposure increased autophagy activity with the appearance of many autophagic vesicles. Expression of the autophagy-related protein 8C gene (Atg 8C) and reticulon-like protein B8 gene (Rtnl B8) was increased at the preculture and cooling-rewarming stages, while bax inhibitor 1-like gene (BIL 1) expression was decreased at the preculture and PVS2 dehydration stages compared with expression levels in fresh PLBs (CK). Caspase-3-like activity was low at the preculture stage but increased significantly with the treatment of loading and PVS2 dehydration and finally reached a peak at the stage of cooling-rewarming. Both H2O2 and NO began to increase at the preculture stage, reached the highest value at the loading stage, and then decreased at the late stage of cryopreservation. Catalase (CAT) and 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) addition at the preculture stage significantly increased survival and decreased caspase-3-like activity in cryopreserved PLBs. These results indicated that the cryopreservation protocol, especially PVS2 dehydration and cooling-rewarming, induced PCD in D. nobile PLBs. H2O2 and NO produced at the preculture stage may also be involved. Cryopreservation protocol by vitrification, especially the PVS2 dehydration and cooling-rewarming, induced programmed cell death in PLBs. Inhibition of H2O2 and NO produced at the preculture stage can decrease caspase-3-like activity and improve PLBs viability.

Eradication of latent viruses from apple cultivar ‘Monalisa’ shoot tips using droplet-vitrification cryotherapy

In apple, virally transmitted diseases cause economic losses as a result of their direct effect on production and yield and by causing an increased susceptibility to other phytopathogenic agents. This study evaluated the effectiveness of a droplet-vitrification cryopreservation technique in eradicating latent viruses: Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) from in vitro axillary shoot tips excised from ‘SC417 Monalisa’ apple cultivar shoot tips. In vitro stock cultures, four weeks after subculture, infected with ASGV, ASPV and ACLSV were used as source material. Axillary shoot tips were excised from stock cultures, precultured in MS + 2 M glycerol + 0.8 M sucrose, and then exposed to plant vitrification solution 2 (PVS2) for between 0 and 80 min (six treatments) at 0 °C or room temperature prior to liquid nitrogen (LN) exposure. Shoot tips were then warmed and recovered on growth medium. The survival and regrowth of cryopreserved (+LN) and non-cryopreserved (-LN) 'Monalisa' shoot tips were evaluated and the efficiency of virus eradication was determined using RT-PCR after recovered plants were grown in a greenhouse for 6 months. With this protocol, the highest degree of regrowth (45%) were obtained after 20 to 40 min PVS2 exposure at room temperature. After 6 months of growth in a greenhouse, all of the plants regenerated after cryopreservation were free of ASPV, 95% were free of ACLSV, and 35% were free of ASGV. This promising cryotherapy procedure may facilitate the production of virus-free plants.

Cryopreservation of an endangered pharmaceutically important orchid, Cymbidium finlaysonianum Lindl. using vitrification technique

Cryopreservation is an in vitro conservation method which has become an important tool for long-term storage of plant genetic resources. New protocorm-like bodies (PLBs) (about 4-5 mm in diameter) of Cymbidium finlaysonianum Lindl. were isolated individually from 2-month-old proliferating PLB clusters which had been cultured in VW liquid medium (VW; Vacin and Went, 1949) supplemented with 8.84 µM 6-benzyl-aminopurine were successfully cryopreserved using a vitrification method. In this cryogenic procedure, PLBs were precultured in MS liquid medium (MS; Murashige and Skoog, 1962) supplemented with 0.5 M sucrose at 25±2°C for 2 d on an orbital shaker at 110 rpm. The PLBs were treated with loading solution (2 M glycerol plus 0.4 M sucrose) for 20 min at 25±2°C to make the precultured PLBs tolerant to plant vitrification solution 2 (PVS2). Subsequently, the selected PLBs were subjected to PVS2 (30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% (w/v) dimethyl sulfoxide and 0.4 M sucrose in MS medium, pH 5.8) treatment at various exposure times (0-120 min) at 0°C and plunged into liquid nitrogen (LN) for 1 d. After storage in LN, the PLBs were rewarmed and washed by MS liquid medium containing 0.5 mL of 1.2 M sucrose for 20 min. One week after rewarming PLBs, viability was determined by TTC reduction and regrowth assessed. The results showed that the PLBs precultured with 0.5 M sucrose for 2 d, followed by dehydration with vitrification solution for 60 min had the highest post rewarming viability in terms of TTC reduction (40%) and regrowth (33.5%). No survival rate of PLBs was found without vitrification treatment. Regenerated plants showed the same morphological characteristics as the control.

Effect of immersion time in PVS2 and mesotesta removing on cryopreservation of papaya (Carica papaya L. ‘Sukma’) seeds

Cryopreservation of papaya seeds is recommended because these seeds, although tolerant to desiccation, cannot be stored for long-term at the low temperatures of standard seed banks (i.e., seeds are classified as “intermediate”). Cryopreservation is a method of germplasm conservation using liquid nitrogen. This study aimed to compare the viability of seeds with and without mesotesta after being stored by cryopreservation. This research was conducted at the Seed Storage Laboratory of Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University, from August to December 2015. The experiment was designed as a factorial in completely randomized design with two factors. The first factor was the immersion duration in plant vitrification solution 2 (PVS2) with five incubation times, i.e., 0, 15, 30, 45, and 60 min. The second factor was seed condition either with or without mesotesta. The results showed that seeds without mesotesta tend to give the best germination rate, and the best immersion duration in cryoprotectants was 30 min, with the results of the germination rate and growth rate being 48.0 and 2.9%, respectively.

Cryopreservation of vanilla (Vanilla planifolia) root-tips: a new alternative for in vitro long-term storage of its germplasm

Vanilla planifolia (Orchidaceae) is the natural source of vanillin, which is the most widely appreciated flavour compound in the world. Although vanilla is cultivated throughout the tropics, the species' areas of natural distribution are being severely reduced due to anthropogenic effects. As a result, ex situ preservation actions are necessary to safeguard the threatened diversity of this species. In that sense, cryopreservation of vanilla shoot-tips has been previously reported using the droplet-vitrification technique. However, survival and plant regeneration following this protocol were low and not very reproducible. In this study, we evaluated a new alternative for the cryopreservation of vanilla germplasm by using root-tips as explants and following the vitrification and droplet-vitrification protocols. Survival (~60%) and further regeneration (~40%) were obtained using the droplet-vitrification technique by preconditioning root-tips from in vitro propagated plants on semi-solid MS with 0.3 M sucrose (7 days), exposing to loading solution consisting of 0.4 M sucrose plus 2 M glycerol (20 min) followed by glycerol-sucrose plant vitrification solution PVS3 (45 min on ice), and direct plunging into liquid nitrogen in droplets of PVS3 deposited on aluminium foils strips. Tissues were rewarmed by plunging the aluminium foils directly in liquid MS enriched with 1.2 M sucrose (15 min) at room temperature. Growth recovery and bud induction were efficiently achieved by culturing cryostored root-tips on MS added with 1 mg L-1 KIN or BAP. Plant regeneration was reached by transferring the induced buds to MS media. This protocol has great potential for long-term conservation of V. planifolia germplasm and of other vanilla relatives.

Freeze-substitution transmission electron microscopy of gentian shoot tips cryopreserved at ultra low temperatures.

Transmission electron microscopy (TEM) combined with freeze substitution was employed to examine the ultrastructure of cells of gentian shoot tips cooled to the ultra-low temperature of slush nitrogen and liquid nitrogen. When shoot tips were cooled in ultra-low temperature without plant vitrification solution 2 (PVS2) treatment, massive ice formation was observed throughout the cells, indicating that severe injury occurred during cooling. In contrast, when shoot tips were treated with PVS2 and subsequently cooled to ultra- low temperatures, no ice crystals were observed in the cells. In addition, the cells of PVS2-treated shoot tips exhibited considerable plasmolysis and formation of small vesicles in cytoplasm. These results clearly demonstrate that the PVS2 treatment is essential for preventing damage caused by ice formation and for successful cryopreservation of plant shoot tips.

Shoot tip cryotherapy for efficient eradication of grapevine leafroll‐associated virus‐3 from diseased grapevine in vitro plants

We describe a droplet‐vitrification cryotherapy method for the eradication of grapevine leafroll‐associated virus‐3 (GLRaV‐3) from diseased in vitro shoots of Vitis plants. The procedure involved pre‐culture of 1.0‐mm shoot tips containing five to six leaf primordia (LPs) for 3 days with a pre‐culture medium containing 0.3 M sucrose, 0.16 mM glutathione and 0.14 mM ascorbic acid, treatment of the pre‐cultured shoot tips for 20 min at room temperature with a loading solution composed of 2 M glycerol and 0.4 M sucrose and exposure to plant vitrification solution 2 (PVS2), prior to freezing in liquid nitrogen of dehydrated shoot tips contained in 2.5‐μL PVS2 droplets on aluminium foil strips. Virus localisation showed GLRaV‐3 was not present in apical dome (AD) and LPs 1–4, but it was detected in the basal shoot tip region, approximately 0.5 mm from the AD, as well as in LP 5 and more mature tissues. Histological observations identify that only freezing in liquid nitrogen results in the death of all cells in areas of shoot tips harbouring virus, whereas PVS2 treatment does not. Thus, freezing in liquid nitrogen is a necessary step that eradicates GLRaV‐3. This cryotherapy procedure produced shoot regrowth levels that ranged from 43% to 59%, and all plants recovered after cryotherapy were free of GLRaV‐3 in two wine, one table and one rootstock cultivars. Thus, this procedure can be considered to be efficient and wildly applicable for eradication of GLRaV‐3 from Vitis spp.

Droplet-vitrification cryopreservation of in vitro-grown shoot tips of grapevine (Vitis spp.)

An efficient and broad-spectrum protocol for cryopreservation of Vitis spp. shoot tips by droplet-vitrification is reported. Shoot tips (1.0 mm) containing 5–6 leaf primordia (LPs) were precultured for 3 d with a preculture medium containing 0.3 M sucrose, 0.16 μM glutathione, and 0.14 μM ascorbic acid. Precultured shoot tips were treated for 20 min at 24°C with a loading solution composed of 2 M glycerol and 0.4 M sucrose, followed by exposure at 0°C to half-strength plant vitrification solution 2 (PVS2) for 30 min, and then full-strength PVS2 for 50 min. Dehydrated shoot tips were transferred into 2.5-μL PVS2 carried on aluminum foil, prior to a direct immersion in liquid nitrogen. With this method, an average shoot regrowth level of 50.5% was obtained from cryopreserved shoot tips in six V. vinifera genotypes (three wine cultivars, two table cultivars, and one rootstock) and two V. pseudoreticulata genotypes. Vegetative growth of the regenerants recovered from cryopreservation, significantly increased as the number of subculture cycles increased and was greater than the control after the third subculture following cryopreservation. Inter-simple sequence repeats (ISSR) and random amplification of polymorphic DNA (RAPD) analyses did not detect any polymorphic loci in the plants of V. vinifera L. cv. ‘Cabernet Sauvignon’ from cryopreserved shoot tips compared to the original cultures. This droplet-vitrification cryopreservation method provides a technical platform to set up cryobanks of Vitis spp.

Cryopreservation of an endangered Hladnikia pastinacifolia Rchb. by shoot tip encapsulation-dehydration and encapsulation-vitrification

The objective of the present study was the cryopreservation of monotypic endemic Hladnikia pastinacifolia Rchb. shoot tips from an in vitro culture, via encapsulation-dehydration (ED) or encapsulation-vitrification (EV). For all tested genotypes, the highest rates of shoot regrowth and multiplication were obtained after overnight preculture in 0.4 M sucrose, encapsulation in Murashige and Skoog (MS) medium with 0.4 M sucrose and 1 M glycerol, followed by polymerization in 3% (w/v) Na-alginate in MS with 0.4 M sucrose. Optimal osmoprotection was achieved for ED with 0.4 M sucrose plus 1 M glycerol and for EV with 0.4 M sucrose plus 2 M glycerol. The best dehydration time for ED was 150 min in a desiccation chamber with silica gel, and the best vitrification time for EV was 85 min in plant vitrification solution 2 (PVS2). For ED, dehydration for 150 min resulted in explant water content of 22%. When the encapsulation method was combined with ED, 53% regrowth was achieved, and when it was combined with EV, 64% regrowth was achieved. Both methods could become applicable for the long-term cryopreservation of H. pastinacifolia germplasm, although EV was faster and resulted in better final regrowth success. Genetic stability analysis of cryopreserved plant samples was carried out for two genotypes, using random amplified polymorphic DNA (RAPD) markers to compare the two different cryopreservation protocols. Significant genetic differences between the genotypes were detected and a low level of genomic variation was observed.

Critical vitrification steps towards survival of Garcinia hombroniana (Clusiaceae) shoot tips after cryopreservation

A cryopreservation protocol was developed for in vitro shoot tips of Garcinia hombroniana using the vitrification technique. Four critical steps in the technique were investigated, namely preculture, loading, dehydration with Plant Vitrification Solution 2 (PVS2), and unloading. Shoot tips precultured for 48 hr gave significantly higher survival (75 %) compared to 24 hr preculture (50 %) after cryopreservation. Treatment with 1 M glycerol plus 0.4 M sucrose as a loading solution gave higher survival (45.83 %) compared to the other treatments (0.4 M sucrose + 2 M glycerol; 0.4 M sucrose). Shoot tips dehydrated with PVS2 for 25 min gave the highest survival after immersion in liquid nitrogen. Stepwise PVS2 treatment for 15 min with 50 % PVS2 followed by 10 min with 100 % PVS2 solution improved survival of the shoot tips after cryopreservation (41.67 %). Murashige and Skoog medium with 0.4 M sucrose gave significantly higher survival (66.67 %) than MS with 1.2 M sucrose (25 %) as an unloading solution. Water content was shown to decrease throughout the whole vitrification steps from 6.83 ± 1.66 g g-1 dw for fresh shoot tips down to 2.93 ± 0.28 g g-1 dw after PVS2 treatment. Further study on each step including recovery medium is required to improve the survival. Nevertheless, the present study showed the potential of using the vitrification technique for cryopreservation of G. hombroniana.

In vitro regeneration of adventitious buds from leaf explants and their subsequent cryopreservation in highbush blueberry

We report a new cryopreservation method for highbush blueberry (Vaccinium corymbosum) using small leaf squares-bearing adventitious buds. Leaf explants were cultured on adventitious bud regeneration medium composed of Woody Plant Medium (WPM) supplemented with 20 µM zeatin. After 21 days of adventitious bud regeneration, small leaf squares (SLSs, 2 × 3 mm), each bearing multiple adventitious buds, were cut from the leaf explant, precultured on WPM containing 0.3 M sucrose for 24 h and were treated for 30 min with a loading solution composed of WPM containing 1.0 M sucrose and 2 M glycerol, followed by exposure to plant vitrification solution 2 at 0 °C for 40 min. Each of dehydrated SLS was then transferred onto an aluminum foil with small holes and PVS2 was dropped until it covered the SLS, prior to a direct immersion in liquid nitrogen. Cryopreserved SLSs were re-warmed in WPM containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for recovery. With this procedure, more than 23 adventitious buds were produced in each leaf explant, and 100% of SLSs were able to survive and resume shoot regrowth, with more than six shoots per SLS obtained following cryopreservation in three highbush blueberry cultivars. In ‘Misty’, the morphology of plantlets regenerated from cryopreserved SLSs was identical to that of the in vitro-derived ones. No polymorphic bands were detected using inter-simple sequence repeat markers and random amplified polymorphic DNA in plantlets of ‘Misty’ recovered from cryopreservation. The use of SLSs-bearing adventitious buds for cryopreservation reported in the present study eliminates shoot tip excision. This cryopreservation method can be considered an efficient cryopreservation of plant shoot tips, and has potential applications to other plant species.

Expanding the toolbox for cryopreservation of marine and freshwater diatoms

Diatoms constitute the most diverse group of microalgae and have long been recognised for their large biotechnological potential. In the wake of growing research interest in new model species and development of commercial applications, there is a pressing need for long-term preservation of diatom strains. While cryopreservation using dimethylsulfoxide (DMSO) as a cryoprotective agent is the preferred method for long-term strain preservation, many diatom species cannot be successfully cryopreserved using DMSO. Therefore, in this study, we studied cryopreservation success in six different diatom species, representing the major morphological and ecological diatom groups, using a range of DMSO concentrations and Plant Vitrification Solution 2 (PVS2) as an alternative cryoprotectant to DMSO. In addition, we tested whether suppressing bacterial growth by antibiotics accelerates the post-thaw recovery process. Our results show that the effects of cryoprotectant choice, its concentration and the addition of antibiotics are highly species specific. In addition, we showed that PVS2 and antibiotics are useful agents to optimize cryopreservation of algae that cannot survive the traditional cryopreservation protocol using DMSO. We conclude that a species-specific approach will remain necessary to develop protocols for diatom cryopreservation and to increase their representation in public culture collections.

Protein changes in the shoot-tips of vanilla (Vanilla planifolia) in response to osmoprotective treatments

Cryogenic storage of vanilla shoot-tips represents the safest biotechnological strategy for the long-term conservation of the vanilla germplasm, but successful cryopreservation depends on its tolerance to both dehydration stress imposed by cryoprotective treatments and thermal stress produced by immersion in liquid nitrogen. In this work, we evaluated the impact of various osmoprotective treatments on protein expression patterns in vanilla (Vanilla planifolia) shoot-tips subjected to successive dehydration steps prior to cryopreservation. Two-dimensional electrophoretic protein profiles of shoot-tips dissected from in vitro grown plants and preconditioned on semisolid media with 0.3 M sucrose for one day, and shoot-tips preconditioned, loaded with a solution of 0.4 M sucrose and 2 M glycerol, and subsequently exposed to plant vitrification solution 3 (50% (w/v) sucrose and 50% (w/v) glycerol), were compared with non-treated dissected shoot-tips. We observed an increase in the expression level of six protein spots (fold change exceeding 1.5) and a decrease (fold change not exceeding 0.6) of ten protein spots after preconditionig treatment, whereas the profiles after preconditioning, loading and exposure to vitrification solution showed an increase in the expression level of 21 protein spots and a decrease in the expression level of 13. Most proteins identified were down-regulated and belonged to groups of biosynthesis, folding, and protein degradation. Many others were related to energetic metabolism, defense, and cell structure. These preliminary results contribute to knowledge of the proteome of this species and partially clarify its sensitivity to osmotic dehydration treatments.