Droplet-vitrification Protocol Research Articles

Comparative Studies for Cryopreservation of Agave Shoot Tips by Droplet-Vitrification.

The objective of this work was to assess the suitability of the Droplet-vitrification protocol previously developed with Agave peacockii shoot tips for the cryopreservation of six Agave species. Shoot tips were precultured for 1 day on a medium with 0.3 M sucrose in the dark, loaded in a solution with 1.6 M glycerol and 0.4 M sucrose for 20 min, and dehydrated by exposure to Plant Vitrification Solution 2 (PVS2) at 0 °C for 20 min. Complementary studies using histological analysis, Differential scanning calorimetry (DSC), and evaluation of morphological characteristics in cryo-derived plants were performed. Survival rates ranged from 84% to 100% and from 76% to 97% before and after cryopreservation regardless of the Agave species belonging to two taxonomic subgenera. Thermal analysis of shoot tips subjected to the successive steps of the Droplet-vitrification protocol identified ice crystal formation after loading treatment and glass transition after osmotic dehydration with PVS2. The average glass transition temperature (Tg) was -55.44 °C based on the results of four Agave species. The histological studies showed the anatomical differences that could be found in the meristematic structures depending on the loss of apical dominance. This is the most advanced research on cryopreservation of Agave shoot tips.

Osmo- and cryotherapy of sugarcane (Saccharum spp. L.) shoot-tips infected with sugarcane mosaic virus (SCMV).

In this work, we investigated the effect of different osmoprotective treatments and of cryopreservation using a droplet-vitrification (D-V) protocol to eliminate sugarcane mosaic virus (SCMV) of shoot-tips excised from in vitro propagated infected plantlets. Shoot-tips of sugarcane (Saccharum spp. L.) were precultured on semisolid MS medium supplemented with 0.3M sucrose for 1day, loaded in solution with 0.4M sucrose and 2M glycerol for 30min and exposed to plant vitrification solution 2 for 15min at room temperature prior to ultra-rapid cooling in liquid nitrogen. Virus indexing was performed by the DAS-ELISA immunoenzymatic test. The presence of SCMV was confirmed in the donor-plantlets derived of infected field material. No virus was detected in the regenerated plantlets from shoot-tips subjected to cryopreservation protocol. The progressive decrease in absorbances occurred from the first preculture treatment and no significant differences (P ≤ 0.05) were found with respect to following steps of D-V protocol. These results indicate that the osmotic dehydration treatments (osmotherapy) and cryopreservation (cryotherapy) may be potentially effective strategies to remove the SCMV from infected plants.

Development of a fast and user-friendly cryopreservation protocol for sweet potato genetic resources

Sweet potato (Ipomoea batatas) is one of the ten most important staple crops and provides a livelihood for many people around the globe. To adapt to ever-changing circumstances farmers and breeders need to have access to a broad diversity of germplasm. This study focuses on the development of a cryopreservation protocol that allows the long term storage of different sweet potato cultivars. For this, a droplet vitrification protocol was optimized, comparing several parameters; preculture method (0.3 M sucrose vs no preculture); meristem position (axillary vs apical); plant age (3 to 9 weeks); regeneration medium (MS + 2.22 µM BA, Hirai and MS); and length of loading solution treatment (20 to 360 min). Two months after cryopreservation, the regeneration rates of the meristems were compared, which resulted in significant differences for the preculture method, meristem position and loading solution. With these new insights an optimized droplet vitrification protocol was developed with the following parameters: use of 3–9 week old axillary meristems, no preculture phase, 20 min LS treatment, 30 min PVS2 treatment, exposure to liquid nitrogen by droplet vitrification, warming treatment in RS for 15 min, 1 day 0.3 M sucrose recuperation culture, 1 month MS + 2.22 µM BA followed by 1 month of MS cultures. This protocol was subsequently tested on 10 representative accessions resulting in a post cryopreservation regeneration rate of more than 40% for 70% of the tested cultivars, showing that this protocol could be implemented for a large portion of existing sweet potato collections.

A safeguard measure of endemic and endangered plant species: cryostorage of Dianthus taxa

The aim of the present study was to determine the optimum parameters for high regrowth following cryostorage (− 196 °C) of seven endemic and endangered Dianthus species. A cryopreservation approach based on a droplet-vitrification protocol was successfully applied using explants (shoot apices and axillary buds) from a collection of in vitro grown Dianthus species. The plants were micropropagated for two years prior using them as explant donors. Osmoprotection in different sucrose concentrations and various dehydration durations in the plant vitrification solution (PVS2) were tested to assess survival and regrowth following cryostorage. The regrowth rates after cryostorage ranged between 63% (D. glacialis ssp. gelidus) and 73% (D. nardiformis) and were achieved after osmoprotection in 0.5 M sucrose and 30 min dehydration in PVS2 for D. glacialis ssp. gelidus and osmoprotection in 0.25 M sucrose and 30 min dehydration in PVS2 for D. nardiformis. The morphogenetic response to liquid nitrogen storage was direct multiple shoot formation for both non-cryopreserved and cryopreserved explants for all species. This biotechnological approach can be efficiently applied for the ex situ conservation of endemic and endangered Dianthus species to ensure the long-term conservation of biodiversity.

Comparison of shoot tip culture and cryotherapy for eradication of ampeloviruses associated with Pineapple mealybug wilt in wild varieties

The Pineapple mealybug wilt-associated virus (PMWaV) provokes a disease that causes considerable losses to pineapple growers due to the reduced leaf tissue turgescence, resulting in leaf necrosis and death in severe cases. In this work, we describe the comparison of shoot tip culture and cryotherapy methods to eradicate the pineapple wilt disease-associated ampeloviruses. Plants from the accessions Ananas comosus var. comosus (AGB-009), var. bracteatus (AGB-119), and var. parguazensis (AGB-376) were indexed by RT-PCR, confirming mixed infections of PMWaV-1, PMWaV-2, and PMWaV-3. The accessions were cultured in vitro and their shoot tips treated by cryotherapy following a droplet vitrification protocol. The regeneration rate from shoot tip culture was 93% for AGB-376 and 100% for the other two accessions. After freezing, AGB-376 had 100% regeneration subsequent to exposure to PVS2 for 45 min, followed by 95% for AGB-009, while for AGB-119 the optimal exposure time was 60 min, with plant regeneration from nearly 80% of the shoot tips. For the accessions AGB-009 and AGB-376, all the recovered plants were virus free by the two methods, while 50% of the plants from accession AGB-119 remained infected. These results indicate that shoot tip culture alone or in association with cryotherapy is a promising routine method for virus removal from pineapple plant tissues and is useful to ensure that backup reserves of pineapple germplasm, conserved by in vitro bank and cryobank, are formed with virus-free plants.

Cryopreservation of shoot tips, evaluations of vegetative growth, and assessments of genetic and epigenetic changes in cryo-derived plants of Actinidia spp.

A droplet-vitrification protocol was described for cryopreservation of shoot tips of kiwifruit ‘Yuxiang’ (Actinidia chinensis var. deliciosa). No significant differences were found in root formation and shoot growth between the in vitro-derived shoots (the control) and cryo-derived ones when cultured in vitro. No significant differences were detected in survival and vegetative growth between the in vitro-derived plants (the control) and cryo-derived ones after re-establishment in greenhouse conditions. Inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) did not detect any polymorphic bands in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-establishment in greenhouse conditions. These data indicate rooting ability, vegetative growth and genetic stability are maintained in the cryo-derived kiwifruit plants recovered from the droplet-vitrification cryopreservation. Methylation sensitive amplification polymorphism (MSAP) detected 12.8% and 1.6% DNA methylation in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-established in greenhouse conditions, respectively. This droplet-vitrification was applied to five cultivars and three rootstocks belonging to A. chinensis var. deliciosa, A. chinensis var. chinensis, A. macrosperma, A. polygama and A. valvata. The highest (68.3%) and lowest (22.5%) shoot regrowth were obtained in A. macrosperma and A. chinensis var. chinensis ‘Jinmi’, respectively, with an average of 46.4% shoot regrowth obtained across the eight genotypes. The droplet-vitrification protocol described here can be considered the most applicable cryopreservation method so far reported for the genus Actinidia. Results reported here provide theoretical and technical supports for setting up cryo-banks of genetic resources of Actinidia spp.

Developing coconut cryopreservation protocols and establishing cryo-genebank at RDA; a collaborative project between RDA and Bioversity International

Coconut (Cocos nucifera L.) is one of the most important palms in the world. Storing coconut for a long term in a conventional seed genebank is not possible because its seeds are recalcitrant. Also field genebanks are not effective as they are prone to unknown undesirable climatic factors and diseases. Cryopreservation has been defined as a welcome alternative for field genebanks for the conservation of coconut germplasm. To develop and validate robust cryopreservation protocols for coconut and exploring the potential for other priority species, a collaborative project between the Rural Development Administration of Korea (RDA) and Bioversity International was initiated. In the framework of this project, three letters of agreement (LOAs) were finalised between Bioversity International and (i) The Sunchon National University (SNU, Korea), (ii) University Leuven (KU Leuven, Belgium) and (iii) the Philippine coconut authority (PCA, the Philippines). In this report, we present an overview of some of the activities, as well as some preliminary results. It showed that coconut embryos can be effectively transported with normal air courier services and normal plants can be grown therefrom. Also small meristem tips excised from in vitro plant can grow into normal plants. Finally, the 1-mm sized meristems excised from plants can survive the droplet-vitrification protocol.

Cold, antioxidant and osmotic pre-treatments maintain the structural integrity of meristematic cells and improve plant regeneration in cryopreserved kiwifruit shoot tips.

Cryopreservation is a reliable and cost-effective method for the long-term preservation of clonally propagated species. The number of vegetatively propagated species conserved by cryopreservation is increasing through development of vitrification-based methods; droplet vitrification in particular is becoming the preferred method for many species, as it ensures fast freezing and thawing rates. This research investigated if cold, antioxidant and osmotic pre-treatments could maintain the structural integrity of cells, thence aid in developing a droplet vitrification protocol for kiwifruit using Actinidia chinensis var. chinensis 'Hort16A' as a model. Cold acclimation of donor plantlets at 4°C for 2weeks followed by sucrose pre-culture of shoot tips and supplementation of ascorbic acid (0.4mM) in all media throughout the procedure registered 40% regeneration after cryopreservation. Transmission electron microscope imaging of meristematic cells confirmed sucrose and ascorbic acid pre-treatment of shoot tips from cold acclimated plantlets following treatment in vitrification solution exhibited severe plasmolysis and some disruption of membrane and vacuoles. In contrast cells without cold acclimation or sucrose and ascorbic acid pre-treatments exhibited minimal change after exposure to vitrification solution. After cryopreservation and recovery, all cells of untreated shoot tips showed rupture of the plasma membrane, loss of cytoplasmic contents and organelle distortions. By comparison, most pre-treated shoot-tip cells from cold acclimated plantlets retained their structural integrity, showing that only those cells that have been dehydrated and plasmolysed can withstand cryopreservation by vitrification.

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.

Cryopreservation of red raspberry cultivars from the VIR in vitro collection using a modified droplet vitrification method

The goal of the present study was to analyze the post-cryogenic recovery of 12 red raspberry cultivars from the N.I. Vavilov All-Russian Institute of Plant Genetic Resources in vitro collection. The 1.1–1.8 mm shoot tips of microplants were subjected to cryopreservation using the modified droplet vitrification method. The current modifications to the droplet vitrification protocol included the elimination of the initial pretreatment stage of the microplants, and the use of modified media at the stages of initial micropropagation, explant isolation, and post-cryogenic regeneration. The optimized method reduced the duration of some cryopreservation stages compared to the initial protocol, and reduced the total procedure from 14 to 11 wk. This modified cryopreservation method also demonstrated a relatively high level of post-cryogenic regeneration. Depending on the genotype, the shoot recovery of explants after rewarming varied from 24.2–89.3% and averaged 58.8 ± 5.3%. There was a statistically significant influence of the genotype on the shoot recovery after rewarming. No differences in inter simple sequence repeats and in start codon targeted marker spectra were found between post-cryopreservation microplants and donor in vitro plants from two red raspberry cultivars.

Cryopreservation of Jerusalem artichoke cultivars using an improved droplet-vitrification method

Jerusalem artichoke (Helianthus tuberosus L.) cultivars are conserved in genebanks for use in breeding and horticultural research programs. Jerusalem artichoke collections are particularly vulnerable to environmental and biological threats because they are often maintained in the field. These field collections could be securely conserved in genebanks if improved cryopreservation methods were available. This work used four Jersualem artichoke cultivars (‘Shudi’, ‘M6’, ‘Stampede’, and ‘Relikt’) to improve upon an existing procedure. Four steps were optimized and the resulting procedure is as follows: preculture excised shoot tips (2–3 mm) in liquid MS medium supplemented with 0.4 M sucrose for 3 days, osmoprotect shoot tips in loading solution for 30 min, dehydrate with plant vitrification solution 2 for 15 min before rapid cooling in liquid nitrogen, store in liquid nitrogen, rapidly rewarm in MS liquid medium containing 1.2 M sucrose, and recover on MS medium supplemented with 0.1 mg L−1 GA3 for 3–5 days in the dark and then on the same medium for 4–6 weeks in the light (14 h light/10 h dark). After cryopreservation, Jerusalem artichoke cultivar ‘Shudi’ had the highest survival (93%) and regrowth (83%) percentages. Cultivars ‘M6’, ‘Stampede’, and ‘Relikt’ achieved survival and regrowth percentages ranging from 44 to 72%, and 37–53%, respectively. No genetic changes, as assessed by using simple sequence repeat markers, were detected in plants regenerated after LN exposure in Jerusalem artichoke cultivar ‘Shudi’. Differential scanning calorimetry analyses were used to investigate the thermal activities of the tissues during the cryopreservation process and it was determined that loading with 2.0 M sucrose and 0.4 M sucrose dehydrated the shoot tips prior to treatment with PVS2. Histological observations revealed that the optimized droplet vitrification protocol caused minimal cellular damage within the meristem cells of the shoot tips.

Development of cryopreservation methods for cherry birch (Betula lentaL.), an endangered tree species in Canada

Cherry birch (Betula lenta L.) is an endangered species in Canada, with only a single natural population of 18 trees found in the Niagara region of Ontario. The tree was reportedly used for medicinal purposes by First Nations and Native American peoples. The current study describes a cryopreservation method for seeds and in vitro shoot tips of B. lenta in hopes of conserving available germplasm. Cryopreservation of mature seeds was successful, with 24% of seeds germinating in the greenhouse and 12% of seeds germinating in vitro on medium supplemented with 10 μmol·L–1 thidiazuron or 6-benzylaminopurine. Postcryopreservation regrowth of in vitro shoot tips was achieved using a droplet-vitrification protocol after preculture in 0.3 mol·L–1 sucrose for 24 h followed by osmoprotection in loading solution for 20 min and treatment with vitrification solution A3 composed of glycerol, sucrose, ethylene glycol, and dimethyl sulfoxide at 0 °C for 60 min. The highest plant regeneration (52%) was obtained after unload...

A droplet-vitrification protocol enabled cryopreservation of doubled haploid explants of Malus x domestica Borkh. ‘Golden Delicious’

High throughput cryopreservation is a key step in the development of an apple t-DNA mutant library. The aim of this study was to develop an easy, efficient and rapid droplet vitrification protocol to cryopreserve doubled haploids of Malus x domestica Borkh. ‘Golden Delicious’. The doubled haploid genotype proved to be less tolerant to cryopreservation than ‘Golden Delicious’. Apices improved the quality of regeneration and the facility of dissection compared to meristems. About 50% of the explants treated with PVS2 for 40min survived after immersion in liquid nitrogen, and 10% showed sustainable development. Phytotoxicity was observed mostly after immersion in the vitrification solution. Hyperhydricity was regularly observed in these experiments and decreased final efficiency. The use of meta-topolin instead of 6-benzylaminopurine (BAP) reduced hyperhydricity by almost 50%. We demonstrated that doubled haploid apple apices can be cryopreserved, but further improvements are needed before this technique can be used at high throughput.

Identification of a highly successful cryopreservation method (droplet-vitrification) for petunia

Petunia (Petunia × hybrida Vilm.) is an important horticultural crop conserved in the National Genebank of China. Here, a droplet-vitrification cryopreservation protocol was developed for petunia shoot tips. An orthogonal array experiment and additional one-factor experiments were performed to optimize key variables, including the age of in vitro plants, the concentration of sucrose in the preculture solution, the preculture duration, the duration of osmoprotection (2.0 M glycerol and 0.4 M sucrose), the length of exposure to and concentration of plant vitrification solution 2 (30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.4 M sucrose), and the recovery medium. By using the combined results of the orthogonal and one-factor experiments, the droplet-vitrification procedure for petunia cultivar Niu 2 was formulated efficiently and effectively. The highest regrowth levels were obtained using the following procedure: shoot tips were dissected from in vitro plantlets that were 20 d old, precultured in MS liquid medium with 0.2 M sucrose solution for 2 d, incubated with osmoprotection solution for 30 min at 25°C, cryoprotected with PVS2 for 30 min at 0°C, and rapidly immersed in liquid nitrogen. Cryopreserved shoot tips were then diluted in MS liquid medium with 1.2 M sucrose for 20 min at 25°C and regrown on solidified MS basal medium with half concentration of NH4NO3, KH2PO4, KNO3, and sucrose. Regrowth levels were as high as 80%. No morphological changes were observed and amplification of 15 simple sequence repeats revealed no genetic alterations after cryopreservation.

Influence of the cryopreservation technique, recovery medium and genotype on genetic stability of mint cryopreserved shoot tips

This study compared the effect of the cryopreservation protocol, the genotype and the plant growth regulators used in the recovery media on the genetic stability of mint shoot tips. Shoot tips of two micropropagated genotypes of mint were cryopreserved either using an encapsulation-dehydration or droplet-vitrification protocol. Three recovery media were tested, all of them MS based although with different plant growth regulators: (1) 0.5 mg L−1 6-benzylaminopurine (BAP); (2) 0.5 mg L−1 6-dimethylallylamino-purine (2iP) + 0.1 mg L−1 α-naphthalene acetic acid (NAA); and (3) 0.5 mg L−1 BAP + 0.1 mg L−1 NAA. DNA was extracted from three different types of samples: leaves from shoots, callus at the base of shoots and callus. RAPD markers were used to assess the genetic stability. One of the genotypes, ‘MEN 198’, showed higher percentage of stable samples than the other one, ‘MEN 186’ (97 vs. 42 %; considering all media, protocol and type of explant). The material recovered after droplet-vitrification showed higher stability compared to the encapsulation-dehydration recovered samples: 99 versus 87 % in ‘MEN 198’, and 80 versus 24 % in ‘MEN 186’. There were not large differences in the genetic stability obtained from the three types of samples in ‘MEN 196’ (85–100 %), while in ‘MEN 186’ calli were more unstable (53, 44 and 30 % stability for leaves, basal callus and callus, respectively). The effect of the recovery medium on the stability of the samples was noticeable in the more unstable types of explants but not in leaves.

Optimization of droplet-vitrification protocol for carnation genotypes and ultrastructural studies on shoot tips during cryopreservation

This study was carried out to optimize a modified droplet-vitrification procedure for the cryopreservation of shoot tips from different carnation genotypes. The best procedure was developed by applying orthogonal tests to the experimental data and by further investigation of the effects on the regrowth percentage. It consisted in preculturing shoot tips in liquid Murashige and Skoog (MS) medium with 0.3 M sucrose for 2 days, pretreating them in liquid MS medium with 5 % Dimethyl sulfoxide +5 % glycerol + 0.3 M sucrose for 10 min, osmoprotecting in Loading solution for 20 min at 25 °C, cryoprotecting with Plant vitrification solution No.2 (PVS2) for 60 min at 0 °C, transferring in drops of fresh PVS2 over aluminum strips and finally storing them in Liquid nitrogen. With the application of the optimized protocol, four carnation cultivars (‘Master’, ‘Calibra’, ‘Lamour’ and ‘Ofcar’) achieved regrowth percentage after cryopreservation ranging from 41 to 73 %. Ultrastructural observations investigated by using transmission electron microscopy showed that the cells encountered the stress during cryopreservation and the main damages occurred during the dehydration step. For surviving cells, the most of the damaged cells could be repaired after recovery growth. This modified protocol will aid in the long-term conservation of carnation germplasm and the ultrastructural studies will benefit for understanding the damage and recovery of the cells during cryopreservation.

Cold-induced changes affect survival after exposure to vitrification solution during cryopreservation in the south-west Australian Mediterranean climate species Lomandra sonderi (Asparagaceae)

There is limited knowledge of the effects of exposure to low temperatures in the unique Mediterranean climate plant species of Western Australia. We have thus investigated the effect of low temperature on cryogenic tolerance in Lomandra sonderi, an endemic perennial species of southwest Western Australia. Lomandra sonderi plants were preconditioned in tissue culture at constant 23 °C (12 h light/dark cycle) or alternating 20/−1 °C (16 h light and 8 h dark cycle). Shoot tips from both conditions were analysed for their phospholipid, sterol and soluble sugar compositions. Shoot tips were also cryoexposed via a droplet-vitrification protocol. Survival in both preconditioning regimes for cryoexposed and non-cryoexposed samples was the same, but plants from the 20/−1 °C regime displayed an improved tolerance to the overall cryopreservation process in both cryoexposed and non-cryoexposed samples, thereby eliminating exposure to liquid nitrogen as a primary cause of reduced post-cryogenic viability. Preconditioning of in vitro shoots of L. sonderi at 20/−1 °C induced significant increases in phosphatidylcholine (from 7.30 ± 3.46 to 22.2 ± 7.80 ng mg−1 FW) and increases in several soluble sugars (fructose, galactose, glucose, sucrose) compared to shoots incubated at 23 °C—changes consistent with known cold acclimation responses in plant species generally—but sterol content remained largely unchanged. Analysis of electrolyte leakage in shoot tips from both preconditioning regimes generated a significantly lower LT50 value in the 20/−1 °C samples (−5.45 ± 0.53 °C) over the 23 °C samples (−2.5 ± 0.08 °C). Increased tolerance to cryoexposure in L. sonderi appears to lie mainly with acclimation-induced changes in membrane composition and promotion of membrane stability and hence increased resistance to freeze damage.

Cryoconservation of some wild species ofMusaL.

The present work was undertaken to devise suitable medium for induction of proliferating meristems in five wild species of Musa (M. acuminata, M. balbisiana, M. basjo, M. jackeyi and M. textilis) and their subsequent cryopreservation by modification of the standard droplet vitrification protocol. It was found that BAP (100 iM) alone was not sufficient to induce high proliferation rates and inclusion of TDZ (1–10 iM) was essential. The duration of pre-growth desiccation (9–14 d) and dehydration with cryoprotectant (PVS2, 90–120 min) varied with each species. The optimized protocol for each species yielded 46–58% shoot recovery after cryopreservation. In terms of ease of explant generation, percentage shoot recovery and plantlet formation, M. jackeyi > M. basjoo > M. textilis > M. acuminata > M. balbisiana.

Effect of the successive steps of a cryopreservation protocol on the structural integrity of Rubia akane Nakai hairy roots

In this work, we studied the impact of the successive steps of the droplet-vitrification protocol technique employed for cryopreservation of Rubia akane hairy roots on the features of cortical, pericycle and endoderm cells of apical and central root segments, using histology techniques and combining qualitative and quantitative observations. In apical segments, plasmolysis (22-71%, depending on cell type) was observed only after the loading treatment and did not increase after the following steps of the protocol. By contrast, in central segments, plasmolysis (39-45%) was already observed after the sucrose pretreatment; it increased to 54-68%, depending on cell type, after the loading treatment, but no further changes were noted after treatment with the vitrification solution. After liquid nitrogen exposure and unloading treatment, deplasmolysis was more rapid in apical segments, with cortical and pericycle cells having retrieved their original features. In central segments, only cortical cells had retrieved their original features and endoderm and pericycle cells were still highly plasmolysed. Nuclei were more strongly impacted by the cryopreservation protocol in central segments, where they displayed a highly condensed nucleoplasm from the loading treatment onwards and had not retrieved their original aspect after the unloading treatment. By contrast, nuclei had a much less condensed nucleoplasm in cells of apical segments, and they had retrieved their original aspect after the unloading treatment.

Acclimation-induced changes in cell membrane composition and influence on cryotolerance of in vitro shoots of native plant species

Cell membranes are the primary sites of cryopreservation injury and measuring changes to membrane composition arising from cold acclimation may assist with providing a rationale for optimising cryopreservation methods. Shoot tips from two south-west Western Australian species, Grevillea scapigera and Loxocarya cinerea, and Arabidopsis thaliana (reference species) were subjected to cryopreservation using the droplet vitrification protocol. Two pre-conditioning regimes involving a constant temperature (23 °C, CT with a 12 h light/dark cycle) or an alternating temperature (AT) regime (20/10 °C with a 12 h light/dark cycle) were compared. Soluble sugars, sterols and phospholipids present in the shoot tips were analysed. Use of AT pre-conditioning (acclimation) resulted in a modest decrease in cryotolerance in A. thaliana, increased cryotolerance in G. scapigera, and increased survival in the non-frozen control explants of L. cinerea in comparison to CT pre-conditioning. Increased cryotolerance was accompanied by a higher total sugar sterol and phospholipid content, as well as an increase in strong hydrating phospholipid classes such as phosphatidylcholine. The double bond index of bound fatty acyl chains of phospholipids was greater after AT pre-conditioning, mostly due to a higher amount of monoenes in A. thaliana and trienes in G. scapigera and L. cinerea. These findings suggest that AT pre-conditioning treatments for in vitro plants can have a positive influence on cryotolerance for some plant species and this may be related to observed changes in the overall composition of cell membranes. However, alternative factors (e.g. oxidative stress) may be equally important with other species (e.g. L. cinerea).