Droplet-vitrification Technique Research Articles

Development of Cryoconservation Protocol for In-vitro Shoot Bases of Allium ampeloprasum L. for Long-term Conservation

Critical factors for the development of cryo conservation protocol for Allium ampeloprasum L. conserved in the in-vitro Genebank (IVGB) at ICAR-NBPGR of India were investigated. Shoot bases (1.0 x 1.5 mm) excised from 8-wk-old shoot cultures grown on growth media (MS + 0.1 mg/l NAA + 0.02 mg/l 2iP + 0.3 M sucrose) and pregrown at 22/ 5ºC for 16/8 hours alternate temperature regime, precultured on MS medium supplemented with 0.3 M sucrose at standard culture conditions (SCC) for 16 hours followed by loading solution treatment for 60 minutes, PVS2 dehydration for 40 minutes were successfully cryoconserved using vitrification and droplet-vitrification technique. Droplet-vitrification technique improved post-thaw regrowth by ~58.33% as compared to vitrification. Thus, a standardized protocol was used for cryobanking of shoot bases of A. ampeloprasum

Cryoconservation of In Vitro Grown Shoot Tips of Cicer Microphyllum: A Crop Wild Relative of Chickpea

BACKGROUND: Cicer microphyllum Benth. is a crop wild relative (CWR) of chickpea (C. arietinum L.), that possess useful genes for cold and drought tolerance. The species is being conserved in the In Vitro Active Genebank for short- to medium – term conservation. Cryopreservation would be a useful complementary approach for its long-term conservation. OBJECTIVE: The current work aimed to develop an efficient cryoconservation protocol for cryobanking of C. microphyllum shoot tips. MATERIALS AND METHODS: In vitro shoot tips excised from 4-month old shoot cultures grown on B5 + 0.5 mg L-1 KIN + 0.1 mg L -1 NAA + 10 mg L -1 AgNO 3medium were cryoconserved using a droplet-vitrification technique. Post-thaw regrowth was evaluated after: (i) preculture medium (B5 basal, B5 + 3, 4, 6 and 10% sucrose), (ii) preculture incubation temperature (25 ± 2, 10, 8 and 22/5°C), (iii) PVS2 duration (10, 20, 30. 40, 50 and 60 min) and (iv) regrowth medium (B5) supplemented with 0.5 mg L-1 KIN + 0.1 NAA mg L-1 ; 0.5 mg L-1 KIN + 0.1 mg L-1 NAA + 10 mg L-1 AgNO3; 0.2 mg L-1 BAP + 10 mg L-1 AgNO3 ; 0.2 mg L-1 BAP + 20 mg L-1 AgNO3 and 0.2 mg L-1 BAP + 30 mg L-1 AgNO3,. RESULTS: In vitro shoot tips grown on B5 + 0.5 mg L-1 KIN + 0.1 mg L-1 NAA + 10 mg L-1 AgNO3, precultured on B5 + 6% sucrose at 10°C for 3 days, followed by PVS2 treatment for 20 min, unloading solution for 60 min and regrowth on B5 + 0.2 mg L-1 BAP + 20 mg L-1 AgNO3 resulted in highest survival (57%) and regrowth (40%) after cryoconservation. CONCLUSION: The standardized protocol was successfully used for cryobanking of in vitro shoot tips of C. microphyllum in the In Vitro Base Genebank of ICAR-NBPGR, New Delhi.

Transcriptome Profiling during Sequential Stages of Cryopreservation in Banana (Musa AAA cv Borjahaji) Shoot Meristem.

Cryopreservation approaches have been implemented in gene banks as a strategy to back up plant genetic resource collections that are vegetatively propagated. Different strategies have been employed to effectively cryopreserve plant tissue. There is little information on the cellular processes and molecular adjustments that confer resilience to the multiple stresses imposed during a cryoprotocol. In the present work, the cryobionomics of banana (Musa sp.), a non-model species, was investigated through the transcriptomic approach using RNA-Seq. Proliferating meristems of in vitro explants (Musa AAA cv 'Borjahaji') were cryopreserved using the droplet-vitrification technique. Transcriptome profiling analysis of eight cDNA libraries including the bio-replicates for T0 (stock cultures (control tissue), T1 (high sucrose pre-cultured), T2 (vitrification solution-treated) and T3 (liquid nitrogen-treated) meristem tissues was carried out. The raw reads obtained were mapped with a Musa acuminata reference genome sequence. A total of 70 differentially expressed genes (DEGs) comprising 34 upregulated and 36 downregulated were identified in all three phases as compared to control (T0). Among the significant DEGs (>log FC 2.0), during sequential steps, 79 in T1, 3 in T2 and the 4 in T3 were upregulated and 122 in T1, 5 in T2 and 9 in T3 were downregulated. Gene ontology (GO) enrichment analysis showed that these significant DEGs were involved in the upregulation of biological process (BP-170), cellular component (CC-10) and molecular function (MF-94) and downregulation of biological process (BP-61), cellular component (CC-3) and molecular function (MF-56). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEGs were involved in the biosynthesis of secondary metabolites, glycolysis/gluconeogenesis, MAPK signaling, EIN 3-lke 1 protein, 3-ketoacy-CoA synthase 6-like, and fatty acid elongation during cryopreservation. For the first time, a comprehensive transcript profiling during four stages of cryopreservation in banana were carried out, which will pave the way for devising an effective cryopreservation protocol.

Effects of droplet vitrification on the regeneration of apical shoot tips of mangabeira accessions

The mangabeira (Hancornia speciosa Gomes) is a fruit tree of high potential that is distributed in several regions of the Brazil, but which is of great social economic importance for the Northeast region. Due to the wide possibility of using its fruits that can be consumed in natura or industrialized, and that represent a financial source for traditional communities that practice the extractivism of this species. The objective of this study was to evaluate the efficiency of the droplet vitrification technique for the long-term conservation of five mangabeira accessions, as well as to verify if there is any different behavior among the accessions to the possible effects of the cryopreservation process. The apical shoot tips were subjected to different periods (30 and 50 min for test I; 25, 50 and 75 min for test II) of exposure to plant vitrification solution 2 (PVS2) before plunging into liquid nitrogen. To evaluate the effects of cryoprotective solutions and exposed times on apical shoot tips exposed or not exposed to liquid nitrogen, the percentage of regeneration were observed at 30th and 60th day of in vitro cultivation. It was possible to cryopreserve the apical shoot tips from five accessions studied through the droplet vitrification technique. Adjusts must be made in the droplet vitrification technique steps in the pre-culture and post-culture media so that there is a mitigation of the damage caused during the cryopreservation.

Comparative Study of Three Vitrification Solutions for an Effective Droplet-vitrification Cryopreservation Procedure for Pfaffia Glomerata (Spreng.) Pedersen

Aims: The objective of this research was to establish a cryopreservation protocol for shoot tips (ST) of in vitro P. glomerata using the droplet-vitrification technique.
 Study Design: The experimental design was a factorial, with four factors, arranged in a completely randomized design. Three vitrification solutions (PVS2, PVS3, PVS4), three times (20, 40, 60 min) and two temperatures (25 ± 2 °C and 0 °C) of treatment with the solutions, followed by freezing (LN+) or not (LN-) with liquid nitrogen (LN) were tested. All tests were performed using six replicates and the results analysed using Two-way ANOVA and Tukey’s tests and expressed as the mean ± the standard error of the means (SEM) deviation.
 Place and Duration of Study: Laboratory of Plant Cryobiology, Embrapa Genetic Resources and Biotechnology, over a two-year period.
 Methodology: ST excised from in vitro plantlets were pre-cultured overnight (19h), treated with a loading solution (LS) and three different vitrification solutions (PVS2, PVS3, PVS4) prior to freezing in LN. Treatment with the vitrification solutions was carried out at 0 or 25°C, for 20, 40 or 60 min. For freezing, drops of the vitrification solutions containing a single ST were dispensed on aluminum foil strips and the strips were submerged in LN (-196°C). For thawing, foil strips were submerged into unloading solution (US) at 40 ± 2°C, for three min. Thawed ST were transferred to regeneration medium and cultured in vitro.
 Results: Highest regeneration percentages after cryopreservation were 82% for ST treated with PVS3, at 0°C, for 60 min; 32% for ST treated with PVS4 at 25°C for 60 min or 0°C for 40 min and 22% for those treated with PVS2 at 0°C for 60 min.
 Conclusion: Droplet-vitrification is a suitable technique to ensure survival of P. glomerata ST after cryopreservation. This procedure can be applied to establish germplasm collections of this medicinal species in gene banks.

Morphoanatomical aspects of the starting material for the improvement of pineapple cryopreservation by the droplet-vitrification technique.

Cryopreservation of pineapple shoot tips has been established from various protocols, including droplet vitrification. Thus, this work aimed to evaluate the morphoanatomical conditions of the starting material over different times (30, 45 and 60 days) of culture before freezing and its correlation with the survival percentage of the cryopreserved shoot tips. Four accessions, Ananas comosus var. comosus (BGA-009); var. bracteatus (BGA-119); var. parguazensis (BGA-376), var. erectifolius (BGA-750) from the Pineapple Active Germplasm Bank (BGA Pineapple) and two hybrids from the Embrapa Genetic Breeding Program, FIB-ROX1 (var. bracteatus X var. erectifolius) and FIB-ROX2 ( var. erectifolius X var. bracteatus), recently introduced in the field from in vitro storage, were used. Histological sections before freezing and the percentages of survival after freezing were obtained taking into account the different times of cultivation of the donor plants. The results showed a significative interaction between genotypes (accessions and hybrids) and the culture period. The accessions BGA-009 and BGA-119 showed the highest survival rates, with 95% and 90% respectively for the 30-day culture time. Different results were obtained for each genotype, showing the need for improvements in the standardization of starting material, which would allow better repeatability of the protocol.

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.

Droplet vitrification technique for cryopreservation of a large diversity of blackcurrant (Ribes nigrum L.) cultivars

The aim of plant gene banks is to preserve genetic resources selected based on their phenotypic, agronomic, historical or other cultural values for future utilization. In the present study the modified PVS2 droplet vitrification technique was tested and optimized for cryopreservation of a large diversity of blackcurrant (R. nigrum L.) accessions propagated in vitro and selected into a national gene bank core collection. Out of four accessions tested to optimize the method, three recovered and regenerated by 89–97% on average, but one recalcitrant in vitro line only by 25%. The tested post-cryopreservation recovery media with different macronutrient and growth regulator levels showed no generalized effect on regenerated shoots, but the effect of recovery media was different between cultivars. When the whole regeneration chain from cryopreservation via micropropagation to greenhouse conditions was tested, shoots at least 1 cm in length were found necessary for successful transfer ex vitro. The long-term cryopreservation of 22 blackcurrant accessions was finally conducted, with practices slightly modified from the tested protocol. The estimated recovery of shoot tips after 9 weeks in vitro was 17–94% with at least 75% recovery in seven accessions and at least 40% recovery in 19 out of 22 accessions. Only one accession had no cryopreservation success. The results demonstrated that the modified droplet vitrification technique is appropriate for a large diversity of blackcurrant accessions. However, cultivar-related differences and recovery procedures are to be considered for success in regeneration and ex vitro adaptation.

Cryopreservation of Brazilian green dwarf coconut plumules by droplet-vitrification

ABSTRACT: This study evaluated the effect of vitrification solutions and exposure time on the cryopreservation of Brazilian green dwarf coconut plumules (BGD) using the droplet vitrification technique. Explants were excised from BGD mature fruits from the Active Germplasm Bank of Embrapa Tabuleiros Costeiros, Sergipe, Brazil. Firstly, embryos were disinfected, and after excision, plumules were pre-cultivated for 72 hours in Y3 + 0.6 M sucrose + 2.2 g L-1 Gelrite® culture medium. Plumules were exposed to PVS2 and PVS3 solutions for 15 and 30 minutes and rapidly immersed in liquid nitrogen (-196 ºC). After cryopreservation, they were thawed in culture medium solution (Y3 + 1.2 M sucrose) and cultured in regeneration medium. The experimental design was completely randomized in a 2x2 factorial scheme (vitrification solutions per exposure times), with five replicates per treatment. Data were compared by the Tukey’s test at 5% probability. Significant differences were observed in the callogenesis percentage for the solutions x exposure time interaction for non-cryopreserved cultures (-NL) and for exposure time after cryopreservation (+NL). PVS2 and PVS3 combined with 15 minutes of exposure promoted the highest callus formation (70 and 100%, respectively) in control cultures. The exposure time of 30 min, regardless of vitrification solution, resulted in 30% embryogenic callus formation after cryopreservation. These results contributed to the long-term conservation of coconut palm.

Development of the droplet-vitrification cryopreservation technique as a viable alternative to the dormant bud technique for recalcitrant pears

The French INRA Pip Fruit Biological Resource Center is in charge of the maintenance, management, characterization and promotion of the traditional and scientific genetic resources of apple, pear, quince and related species. In 2010, the first cryopreservation trials were performed on apple and pear using the dormant bud technique with the main aim of cryopreserving a major part of the INRA Malus and Pyrus collections. However, some accessions, mainly from the Pyrus genus, sometimes responded badly to the dormant bud technique. To optimize the ability to cryopreserve the largest number of accessions in our collections, the droplet-vitrification technique was tested on two cultivars of Pyrus: ‘Williams’ and ‘Conference’, used as controls since 2010. Our objective was to evaluate this method under our experimental conditions and to compare it with the dormant bud technique. Experiments were carried out using samples from a batch of budsticks collected in January 2015, in order to test the impact of several critical steps in the process. With both techniques, the immersion in liquid nitrogen step appeared to have the most important impact on the final results, much more than dehydration and slow-freezing for dormant bud technique or LS and PVS2 steps for droplet-vitrification technique. Regenerated plants were obtained after droplet-vitrification. With some improvements, this method could be a viable alternative to the dormant bud technique and opens up the possibility of new development paths for the optimization of long-term preservation systems of our genetic resources collections.

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.

Implementation of Citrus shoot tip cryopreservation in the USDA-ARS National Plant Germplasm System

The USDA-ARS National Plant Germplasm System (NPGS) maintains a citrus genetic resource collection of 1522 accessions of Citrus cultivars and crop wild relatives. Of these, 540 are maintained as pathogen-free duplicate clones in a screenhouse at the National Clonal Germplasm Repository for Citrus and Dates (NCGRCD) in Riverside, California. An additional 815 accessions of citrus and citrus relatives are maintained in greenhouses or orchards in Riverside until sanitation for pathogens can be completed. This paper describes progress in backing up the 540 pathogen-tested accessions using cryopreservation techniques and methods to accelerate processing and viability testing. Vegetative budwood was harvested from trees in the screenhouse and sent to NLGRP. At least 170 shoot tips from each accession were excised from surface-sterilized budwood, and then cryopreserved using a PVS2 droplet-vitrification technique. Viability was assessed by micrografting 10 thawed shoot tips onto in vitro grown 'Carrizo' seedling rootstock. The remaining shoot tips are held in long-term liquid nitrogen storage. A total of 451 of the pathogen-tested citrus accessions were cryoprocessed: 354 accessions had regrowth levels of 40% or greater, 17 had 0% regrowth, 47 had viability levels between 10 and 30%, and 33 had endogenous contaminants. Technical staff familiar with shoot-tip cryoprocessing could be fully trained for specific applications of citrus in about 2 months and thereafter processed a single accession of at least 170 shoot tips in about 16 h. This large-scale effort has revealed that shoot tip cryopreservation can be successfully scaled-up to secure the NPGS Citrus collection.

Cryopreservation of Pineapple Shoot Tips by the Droplet Vitrification Technique.

Cryopreservation is a technique that allows the conservation of many species for long periods. Among the protocols used for cryopreservation, droplet vitrification has shown efficient results in preserving shoot tips of various wild and cultivated pineapple genotypes. The method consists of extraction of shoot tips from plants grown in vitro, dehydration for a period of 48 h in a preculture medium supplemented with a high concentration of sucrose, treatment in a plant vitrification solution (PVS2), and immersion in liquid nitrogen. The method described in this chapter has produced survival and regeneration indices of around 70%, depending on the genotype and physiological conditions of the initial explants. The objective of this chapter is to describe in detail a droplet vitrification protocol for shoot tips that is easy to perform for cryopreservation of pineapple germplasm.

In vitro plant regeneration and cryopreservation of Arachis glabrata (Fabaceae) using leaflet explants

Fil: Dolce, Natalia Raquel. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Nordeste. Instituto de Botanica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botanica del Nordeste; Argentina

Comparison of two PVS2-based procedures for cryopreservation of commercial sugarcane (Saccharum spp.) germplasm and confirmation of genetic stability after cryopreservation using ISSR markers

Conservation of Saccharum spp. germplasm as ex situ collections of plants has a high cost, and in natural conditions, the plants remain exposed to pests, pathogens, and natural disasters. Long-term preservation of plant germplasm is important for agricultural biodiversity and food safety, so the aim of this study was to develop a cryogenic procedure for cryopreservation of sugarcane germplasm. The first study compared droplet vitrification and encapsulation-vitrification techniques for cryopreservation of in vitro shoot tips of Saccharum spp. variety Halaii. The best regeneration rate (70.9%) was obtained from 45-min PVS2 vitrification solution-treated shoot tips via the droplet vitrification technique. This technique was tested on two other Saccharum sp. varieties, and the best regeneration rates for varieties NG 57-024 and H 83-6179 were 63.3 and 76.3%, respectively. Shoots derived from cryopreserved shoot tip buds developed well-formed roots, and were easily acclimated to greenhouse conditions. The second study evaluated genetic stability of the cryopreserved varieties using ten inter-simple sequence repeat primers. A total of 211 (Halaii), 198 (H83-6179), and 201 (NG 57-024) reproducible bands, ranging from 125 to 5500 bp, were scored with this technique. One hundred genetic stability was detected from Halaii and H 83-6179 whereas 98.5% genetic stability was detected from varieties of NG 57-024. The PCR reactions showed that there was no crucial variation on genetic stability for all cryopreserved varieties.

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.

Droplet-vitrification and morphohistological studies of cryopreserved shoot tips of cultivated and wild pineapple genotypes

Germplasm conservation of pineapple [Ananas comosus (L.) Mer.] is crucial to preserve the genus’ genetic diversity, to secure material for genetic improvement and to support innovative and new research. Long-term conservation is accomplished through cryopreservation, that is done by storing cells or tissues at ultra-low temperature in liquid nitrogen (−196 °C). Droplet-vitrification, a combination of droplet freezing and solution-based vitrification, was used to establish a protocol for cryopreservation of pineapple genetic resources. This protocol was tested on cultivated and wild pineapple genotypes to establish a long-term germplasm security duplicate as well as to investigate cryo-injuries in the tissues by means of histological techniques. Excised shoot tips (0.5–1 mm with one primordial leaf) of different pineapple genotypes were precultured for 48 h on solid MS medium containing 0.3 M of sucrose. Three PVS2 exposure times (30, 45 and 60 min) were tested. The results showed high post cryopreservation survival for all genotypes evaluated. The best PVS2 exposure time varied according to genotype, although 45 min gave the best survival for the majority of genotypes. The technique was highly efficient in cryopreserving meristem shoot tips of different pineapple genotypes, and was also less laborious than techniques previously reported. This is a first report on application of the droplet-vitrification technique to diverse genotypes of cultivated and wild pineapples and the first report on histological changes occurring in cryopreserved Ananas tissue.

Cryopreservation of Serbian autochthonous Prunus spp. by droplet-vitrification

AbstractIn vitro shoot tips of Serbian autochthonous plums ‘Sitnica’ (Prunus domestica L.) and ‘Crvena Ranka’ (Prunus insititia L.) were tested for recovery after cryopreservation using the droplet-vitrification technique. After 30-min loading with 1.9 M glycerol and 0.5 M sucrose, explants were dehydrated at room temperature for 10, 20, 30, 40 and 50 min with PVS A3 solution (37.5% glycerol, 15% dimethylsulfoxide, 15% ethylene glycol and 22.5% sucrose) or for 60, 90 and 120 min using PVS3 solution (50% glycerol and 50% sucrose). Rewarming was performed in unloading solution containing 0.8 M sucrose for 30 or 60 min. Duration of PVS3 treatment significantly affected survival (27.3-72.7%) and regrowth (0-18.2%) of cryopreserved explants in plum ‘Sitnica’, with the highest values of both parameters being achieved with the 90-min treatment. Also, survival of explants dehydrated with PVS A3 solution significantly varied between 18.2-73.9%, depending on duration of both dehydration and unloading treatments. However, cryopreserved explants displayed a very low regrowth capacity, the highest being 10% for 30-min dehydration in combination with 60-min unloading. ‘Crvena Ranka’ exhibited a higher regrowth capacity after cryopreservation. Duration of PVS3 treatment significantly affected survival (22.2-77.8%) and regrowth (0-30.0%) of cryopreserved explants, with the highest values of both parameters being achieved with the shortest treatment duration. As regards PVS A3 treatments, both survival and regrowth significantly varied between 27.3- 81.8%, and 0-36.4%, respectively. The highest regrowth was achieved with 20- and 30-min treatment durations combined with 30-min unloading. Further optimization of the protocol is necessary to improve recovery after cryopreservation.

Unravelling the effect of sucrose and cold pretreatment on cryopreservation of potato through sugar analysis and proteomics

Apical shoot tips were dissected from donor plants (cultured in several conditions) and cryopreserved using the droplet-vitrification technique. The effect of two preculture treatments (sucrose pretreatment medium or cold-culturing during two weeks) on donor plants of four potato species (Solanum commersonii, S. juzepcukii, S. ajanhuiri, and Solanum tuberosum) was studied. Post-cryopreservation meristem growth and plant recovery were influenced by the treatments, but the effect on the regeneration was strongly genotype-dependent. The highest post-rewarming plant recovery percentage was obtained using meristems dissected from donor plants of S. commersonii cultured on sucrose pretreatment medium or cold-cultured. Both preculture conditions also enhanced plant recovery in S. juzepcukii compared to control cultures. Cold preculture, however, proved to be undesirable for S. tuberosum whereas sucrose pretreatment had a positive impact on the plant regeneration of this species. The determination of changes in the concentration of soluble sugars revealed sugar accumulation, especially of sucrose and the raffinose family of oligosaccharides (RFOs), which can be linked to tolerance towards the cryopreservation. Additionally, a study of the proteome of the donor plantlets after the pretreatments by 2D-fluorescence difference gel electrophoresis (DIGE) was carried out to identify differentially abundant proteins. Carbon metabolism-related proteins, together with stress-response and oxidative-homeostasis related proteins were the main class of proteins that changed in abundance after the pretreatments. Our results suggest that oxidative homeostasis-related proteins and sugars may be associated with the improved tolerance to cryopreservation and the ability to cold acclimate by S. commersonii in contrast to the other genotypes. The increased accumulation of sucrose and RFOs play a fundamental role in the response to stress in potato and may help to acquire tolerance to cryopreservation.

<b>Cryopreservation of the mangaba tree (<em>Hancornia speciosa</em> Gomes): a protocol for long-term storage

The aim of this study was to evaluate the efficiency of vitrification and droplet vitrification for the cryopreservation of Hancornia speciosa shoot tips. The shoot tips were subjected to four different periods of exposure (15, 30, 45 and 60 min.) to plant vitrification solution 2 (PVS2) before plunging into liquid nitrogen. We evaluated the regrowth of H. speciosa shoot tips that were cryopreserved by the classical vitrification technique and by the droplet vitrification technique. Shoot tips were submitted to different periods of pre-culture (absence, 24 or 48h) in a medium containing 0.3 M sucrose prior to cryopreservation. With a PVS2 exposure period of 60 min., significant differences were observed between the two techniques used in this study with respect to regrowth rates; however, both cryopreservation methods allowed for a shoot tip regrowth rate of over 70%. Use of a preculture step increased the regrowth rate of cryopreserved shoot tips. Shoot tip cryopreservation techniques employing vitrification and droplet vitrification proved to be viable for the long-term storage of mangaba tree samples.