PVS2 Vitrification Research Articles

Ice crystals and oxidative stress affect the viability of Areca catechu seeds following cryopreservation

This study aimed to examine the effects of ice crystals and oxidative stress on seed viability in the context of cryopreservation, with the ultimate goal of identifying potential solutions to address the persistently low regeneration rate observed in recalcitrant medicinal plant seeds following cryopreservation. Using differential scanning calorimetry technology alongside seeds germination at different moisture levels after cryopreservation helped determine the best moisture content and freezing process for Areca catechu seeds. Seeds with 17–21 % moisture content and treated with PVS2 vitrification solution showed higher survival rates after cryopreservation. The oxidative markers of A. catechu seed embryos exhibited alterations due to vitrification freezing. However, there was no substantial association between seed viability and oxidative markers, such as reactive oxygen species (ROS), suggesting that oxidative damage mediated by ROS is not the primary factor contributing to the diminished viability of A. catechu seeds following cryopreservation. The inclusion of vitamin E, reduced glutathione (GSH), and 2-nitrobenzoic acid in the vitrification cryoprotectant achieved some improvement in seed viability of approximately 10 times, with GSH exhibiting the most pronounced optimizing effect.

Comparative Cryopreservation of Indian Wild Orange (Citrus Indica Tanaka) Embryonic Axes

BACKGROUND: Indian Wild Orange (Citrus indica Tanaka) is an endangered and endemic species from northeast India for which effective ex situ conservation strategies, including embryo cryopreservation, are urgently needed. MATERIALS AND METHODS: Desiccation tolerance and cryopreservation ability for embryonic axes of Citrus indica was determined using three techniques (air desiccation-freezing, PVS2 vitrification-freezing and encapsulation-dehydration-freezing). Success was assessed as survival and recovery in vitro. RESULTS: Successful cryopreservation of embryonic axes was achieved using all three methods, with the highest survival achieved when using air desiccationfreezing (90%) followed by encapsulation-dehydration (85%) and PVS2 vitrification cryopreservation (80%). Regeneration levels were lower than survival levels for all three proceedures. Post-cryo regeneration success was: encapsulation-dehydration (64%) > air desiccation-freezing (55%) > PVS2 vitrification (52%). CONCLUSION: Although there was relatively high post-cryopreservation recovery growth obtained using all the three techniques, the air desiccation-freezing technique is preferred, as it is a simple, practical and reproducible technique for the long-term cryobanking of this important wild species.

Cryopreservation of Malus and Pyrus Wild Species in the 'Fruit Genebank' in Dresden-Pillnitz, Germany.

A unique and valuable diversity of the Malus and Pyrus wild species germplasm is maintained ex situ in field collections in the 'Fruit Genebank' in Dresden-Pillnitz, Germany. (1) Background: The establishment of a duplicate collection is necessary to preserve this material safely from abiotic and biotic stress factors. (2) Methods: Two different techniques, cryopreservation using dormant buds and PVS2 vitrification using in vitro shoot tips, were applied and compared. (3) Results: In Malus altogether 180 accessions belonging to 32 species were processed with an average recovery rate of 39% within ten years using the dormant bud method. Accessions, 116 in number, achieved the criterion of 40% recovery which was 64.44% of all accessions tested. In the case of Pyrus germplasm a total of 35 accessions of 21 species and both techniques for cryopreservation were tested. In the results of these experiments the PVS2 method led to better results compared to the dormant bud method. (4) Conclusions: In Malus the dormant bud technique will be the method of choice for the future to build up the duplicate collection. Further experiments using both techniques are planned in the future to make a final decision for Pyrus.

Development of an Efficient ‘One-Step Freezing’ Cryopreservation Protocol for a Georgian Provenance of Chestnut (Castanea sativa Mill.) Zygotic Embryos

Experiments were performed to determine the influence of various dehydration and vitrification treatment times on the ‘one-step freezing’ cryopreservation of embryonic axes (EAs), composed of zygotic embryos and cotyledon residuals, from mature seeds of a Georgian provenance of chestnut (Castanea sativa Mill.). Dehydration was carried out in laminar flow hood from 1 to 5 h, and vitrification experiments were carried out by immersion of EAs in PVS2 vitrification solution up to 120 min, both followed by direct immersion in liquid nitrogen. Both systems resulted in inducing specimen tolerance to ultra-rapid freezing, although to a different extent. Full germination of cryo-stored EAs after 5 h of dehydration (reducing moisture content from initial 66% to 21%) has been increased from 0% to 66.7%. A pre-treatment of EAs in PVS2 vitrification solution for 30 min produced fully developed plantlets at a rate of 55.6% in post-cryopreservation. Plantlet regrowth from cryopreservation was faster in EAs that underwent the dehydration/‘one-step freezing’ procedure. All the plantlet from cryopreserved EAs could be easily acclimatized, producing healthy potted plants. Finally, the TTC test showed to be useful for a fast evaluation of specimen survival after thawing and, as a consequence, to speed up the development of optimized cryo-protocols.
 
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 In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 4, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue.
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Optimization of cryopreservation protocols for zygotic embryos of Citrus reticulata

Citrus seeds are known to have short life spans in conventional seed banks. Seed of most Citrus species show a typical intermediate storage behaviour where they survive desiccation to c. 7-10%. However, longevity is compromised at this low moisture content at sub-zero temperatures. Cryopreservation of zygotic embryos is an option for the long-term storage of germplasm of this species and we investigated its feasibility for Citrus reticulata var. Mandarin. The experimental strategies evaluated included: seed and embryo desiccation sensitivity assessment, naked embryo cryopreservation, encapsulation-dehydration cryopreservation, and cryopreservation of embryos following the PVS2 vitrification technique. Differential scanning calorimetry was used at every desiccation step to determine the critical water content for cryopreservation. Desiccated and cryopreserved naked embryos with a moisture content of 13% (fresh weight basis) showed the highest regrowth (60%). Maximum regrowth of about 58 and 52% was recorded for encapsulated embryos with a moisture content of ~22% and for embryos treated with PVS2 vitrification for 120 min, respectively. Although encapsulated, PVS2 treated and naked embryo cryopreservation had almost similar regrowth percentages, the seedlings obtained from naked embryo cryopreservation appeared more vigorous, probably because of the lack of mechanical contraints imposed by the alginate encapsulation and no exposure to cryo-protectant, a known plant stressor. We conclude that naked embryo desiccation is the optimum technique for cryopreservation of C. reticulata zygotic embryos.

La coltura in vitro per la conservazione della biodiversità orticola

Today, in vitro culture is a strategic tool to support medium and long-term conservation of plant genetic resources by using the slow growth storage of shoot cultures and the cryopreservation of organs and tissues. Over the last 30 years, considerable progresses were made in the development of both techniques that are nowadays considered as ex situ conservation strategies complementary to traditional seed banks and in-field clonal collections. Efficient protocols were developed for the conservation of a large number of crops, including important vegetables of the temperate environment (garlic, artichoke, asparagus, mint, potato, sweet potato, tomato, red chicory, thyme). Conservation in slow growth storage consists in modifying the medium and/or culture conditions to reduce the growth of plant material without affecting the viability and regrowth potential of shoots when moved back to standard culture conditions. The technique allows medium-term crops conservation, with a storage time of vegetables ranging from a few months to two years and more, without recurring subculturing typical of micropropagation. Cryopreservation preserves plant organs and tissues at ultra-low temperature, as liquid nitrogen temperature (-196°C). Currently, various techniques are available, based on cold tolerance induction by cell cytoplasm vitrification during the fast ultra-freezing through in liquid nitrogen immersion of explants. The term “vitrification” refers to the solidification of a liquid without crystallization. If induced in plant cells, it avoids the formation of lethal intra-cellular ice crystals during the ultra-freezing process, keeping tissues at stopped metabolism condition but vital. Working with vegetables, the techniques based on direct immersion of specimens in liquid nitrogen (“one-step freezing”, such as the “droplet-method”, the “PVS2 vitrification”, the “encapsulation-based” and the “cryo plate-based” procedures) are the most used. Further, the “two-step freezing”, i.e. the slow explants cooling before immersion in liquid nitrogen, still finds some applications. The experimental activity mainly focused on three economically important species, i.e., Allium sp., potato and sweet potato. Currently, almost 38,000 accessions of garlic, cassava, mint, potato, sweet potato, taro, yam are maintained in 17 genetic resources conservation centers, located in 12 Countries and 5 Continents (Europe, Asia, Africa, North and South America). Approximately 4/5 of these accessions are maintained in vitro by means of slow growth storage of shoot cultures, but more recent cryopreservation is constantly growing, and over 7,500 accessions from vegetables are stored at -196 °C. The germplasm of potato (Solanum sp.) is by far the most collected in the world, with almost 17,700 accessions presently maintained in in vitro banks and cryobanks.

La coltura in vitro per la conservazione della biodiversità orticola

Today, in vitro culture is a strategic tool to support medium and long-term conservation of plant genetic resources by using the slow growth storage of shoot cultures and the cryopreservation of organs and tissues. Over the last 30 years, considerable progresses were made in the development of both techniques that are nowadays considered as ex situ conservation strategies complementary to traditional seed banks and in-field clonal collections. Efficient protocols were developed for the conservation of a large number of crops, including important vegetables of the temperate environment (garlic, artichoke, asparagus, mint, potato, sweet potato, tomato, red chicory, thyme). Conservation in slow growth storage consists in modifying the medium and/or culture conditions to reduce the growth of plant material without affecting the viability and regrowth potential of shoots when moved back to standard culture conditions. The technique allows medium-term crops conservation, with a storage time of vegetables ranging from a few months to two years and more, without recurring subculturing typical of micropropagation. Cryopreservation preserves plant organs and tissues at ultra-low temperature, as liquid nitrogen temperature (-196°C). Currently, various techniques are available, based on cold tolerance induction by cell cytoplasm vitrification during the fast ultra-freezing through in liquid nitrogen immersion of explants. The term “vitrification” refers to the solidification of a liquid without crystallization. If induced in plant cells, it avoids the formation of lethal intra-cellular ice crystals during the ultra-freezing process, keeping tissues at stopped metabolism condition but vital. Working with vegetables, the techniques based on direct immersion of specimens in liquid nitrogen (“one-step freezing”, such as the “droplet-method”, the “PVS2 vitrification”, the “encapsulation-based” and the “cryo plate-based” procedures) are the most used. Further, the “two-step freezing”, i.e. the slow explants cooling before immersion in liquid nitrogen, still finds some applications. The experimental activity mainly focused on three economically important species, i.e., Allium sp., potato and sweet potato. Currently, almost 38,000 accessions of garlic, cassava, mint, potato, sweet potato, taro, yam are maintained in 17 genetic resources conservation centers, located in 12 Countries and 5 Continents (Europe, Asia, Africa, North and South America). Approximately 4/5 of these accessions are maintained in vitro by means of slow growth storage of shoot cultures, but more recent cryopreservation is constantly growing, and over 7,500 accessions from vegetables are stored at -196 °C. The germplasm of potato (Solanum sp.) is by far the most collected in the world, with almost 17,700 accessions presently maintained in in vitro banks and cryobanks.

In Vitro Proliferation and Cryoconservation of Banana and Plantain Elite Clones

Abstract Agriculture and modern biotechnology are increasingly becoming interdependent, and many new techniques have brought new opportunities for enhancing production and marketing. Germplasm storage is an alternative for the conservation of plant genetic diversity, contributing to the improvement and maintenance of propagation programs for species of interest. In this work, banana corms were collected as plant material from relatively young commercial plantations of three different cultivars: ‘Williams’, Valery (AAA genome; Cavendish subgroup), and ‘Barraganete’ (AAB genome; Plantain subgroup). Their shoot tips were introduced into in vitro conditions, and subcultured monthly to obtain the required number of shoots. The shoots were subsequently rooted and stimulated to invigoration in order to extract apical meristems (0.8–1.0 mm), which were prepared for cryopreservion in liquid nitrogen (−196 °C) following pre-conditioning in PVS2 vitrification solution. Thereafter, the explants were rapidly thawed and then recovered and regenerated using two different methods – by Panis (2009) and Korneva et al. (2009) – consisting of two different sets of recovery and subsequent regeneration media. Statistical analysis of the results showed that the banana cultivar ‘Williams’ demonstrated higher survival and regeneration rates after cry-opreservation using the Korneva method, whereas in cultivars ‘Valery’ and ‘Barraganete’, there were no significant differences between the tested methods. The ‘Barraganete’ cultivar had the lowest survival and regeneration rates, regardless of the applied method.

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.

Development of a common PVS2 vitrification method for cryopreservation of several fruit and vegetable crops

Many cryopreservation techniques are currently available, and it is common for new modifications to be developed for individual crops or specific genotypes. In this study, results of variations of the PVS2 cryopreservation protocol are compared to provide evidence for the suitability of a standard form of this technique for cryopreservation of a range of fruit, berry crops, and potato. Shoot cultures of Malus, Solanum, Lonicera, and Berberis were tested with variations of cold acclimation, pretreatment media, and PVS2 exposure times. A general protocol with some modifications was produced that was suitable for all four genera. The regenerative capacity of shoot tips after cryopreservation by this method exceeded a mean of 50% for Malus, Solanum, Lonicera, and Berberis, which is sufficient for setting storage in a cryobank. After liquid nitrogen storage, the shoot cultures that survived had a healthy appearance and developed rapidly. For each species tested, the only optimization required was the preparation of donor plants by cold acclimation and pretreatment. The choice of one common method simplifies the methodology for conducting experiments and storing a range of germplasm. The use of the PVS2 vitrification method with a 0.3-M sucrose pretreatment is multiuse and can be recommended as the most effective method for the cryopreservation of shoot tips from many plant species.

C-2019: Cryopreservation of chrysanthemum using aluminium Cryo-plate protocol

Two cryopreservation procedures using aluminium cryo-plates were successfully developed using in vitro-grown chrysanthemum shoot tips. Shoot cultures were cold-hardened at 5 °C on 1/2 MS medium for 30 days. Shoot tips with basal plate (1.5–2.0 × 1.0 mm) were excised from shoot cultures and precultured at 5 °C for 3 days on solidified 1/2 MS medium containing 0.3 M sucrose. Precultured shoot tips were placed on aluminium cryo-plate with 10 wells (diameter 1 mm) and embedded in calcium alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 min at 25 °C in a loading solution (2 M glycerol + 1.0 M sucrose). In the D-cryo-plate procedure, the shoot tips were dehydrated by placing the cryo-plates in the air current of a laminar flow cabinet for 30–150 min. In the V-cryo-plate procedure, buds were dehydrated by immersing the cryo-plates in PVS2 vitrification solution for 40 min at 25 °C. In both procedures, cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, shoot tips attached to the cryo-plates were rewarmed by immersion in a petri dish containing 1.0 M sucrose solution at 25 °C. Regrowth of cryopreserved shoot tips using D-Cryo-plate and V-Cryo-plate procedures, was 90% and 80%, respectively. The two procedures were applied to 20 additional chrysanthemum lines. This protocol appears to be a promising technique for cryopreservation of chrysanthemum genetic resources.

Comparison of droplet-vitrification and D-cryoplate for cryopreservation of date palm (Phoenix dactylifera L.) polyembryonic masses

In this work we tested the efficiency of two techniques, droplet-vitrification (DV) and dehydration (D) cryo-plate, for cryopreservation of polyembryonic masses (PEMs) of two date palm varieties, Sokary and Sultany. With DV, recovery of non-precultured, cryopreserved PEMs was nil without treatment with the PVS2 vitrification solution, which contained 3.3M glycerol+2.4M ethylene glycol+0.4M sucrose+1.9M dimethylsulfoxide. Following PVS2 treatments between 15 and 120min, it was comprised between 90.9–98.6% and 85.6–88.0% for varieties Sokary and Sultany, respectively. Sucrose preculture (3 days, 0.5M) led to 21.1% recovery of cryopreserved PEMs of variety Sokary only without PVS2 treatment, and slightly reduced recovery in all other experimental conditions with both varieties, compared with non-cryopreserved PEMs. Regrowth intensity of cryopreserved PEMs was generally lower with variety Sultany compared to variety Sokary. With the D cryo-plate technique, no recovery of cryopreserved PEMs was achieved without sucrose preculture. Sucrose preculture (3 days, 0.5M) had a positive effect on recovery of cryopreserved PEMs. For variety Sokary, the highest recovery (92.0–95.8%) was achieved for desiccation periods between 60 and 120min. Recovery was between 67.0 and 74.6% after desiccation periods of 90–120min for variety Sultany. With the D cryo-plate technique, regrowth intensity of cryopreserved PEMs was higher with variety Sokary compared to variety Sultany.

COMPARISON OF DIFFERENT PRECONDITIONING AND LOADING TREATMENTS WITH VANILLA (VANILLA PLANIFOLIA JACK.) APICES CRYOPRESERVED USING THE DROPLET-VITRIFICATION PROCEDURE

In order to improve post-cryopreservation survival of vanilla (Vanilla planifolia) apices, different preconditioning and loading treatments were studied comparing the effect of using sucrose or trehalose. In this work, vanilla apices were isolated from in vitro grown plants and subjected to a direct preculture on MS Semisolid medium supplemented with 0.3 M sucrose or trehalsoe for one or seven days. Additonally, apices were also pre-conditioned after dissection by transferring them first to standard MS semisolid medium for seven days, followed by the same MS medium but containing 0.3 M sucrose or trehalose for one or seven days. After all pre-conditioning treatments, apices were loaded in solutions containing 0.4 M sucrose or trehalose mixed with 2 M glycerol, or in 0.8 M sucrose or trehalose mixed with 1 M glycerol, exposed to PVS2 or PVS3 vitrification solutios for 30 min, and then directly plunged into liquid nitrogen on droplets of PVS solution placed o aluminium foil strips. Dehydration with PVS2 solution produced the best results after cryopreservation with apices were subjected to the pre-conditioning treatments with trehalose, and loading with sucrose-glycerol solutions. Nevertheless, the highest survival (57 and 62%) rates of cryopreserved aices were achieved using trehalose both in preconditioning and in loading solutions, followed by exposure to PVS3. The droplet-vitrification protocols which allowed improvements (from 30 to 60%) in survival of crypopreserved vanilla apices comprised: preconditioning and in loading solutions, followed by exposure to PVS3. The droplet-vitrification protocols which allowed improvements (from 30 to 60%) in survival of cryopreserved vanilla apices comprised: preconditioning of dissected apices on standard MS semisolid medium for seven days, followed by one or seven days on MS medium supplemented with 0.3 M trehalose, loadin in 0.4 M trehalose +2 M glycerol, and exposure to PVS3 for 30 min.

<i>In vitro</i> Conservation and Cryopreservation of <i>Nandina domestica</i>, an Outdoor Ornamental Shrub

The study focused on an economically-important ornamental outdoor shrub, Nandina domestica, with the aims to (i) optimize an effective in vitro conservation method, and (ii) develop a cryopreservation protocol for shoot tips by the PVS2 vitrification and droplet-vitrification techniques. For in vitro conservation of shoot cultures, the tested parameters were sucrose content in the storage medium (30, 45, 60 g/L) and storage temperature (4 °C or 8 °C). Cryopreservation was performed by applying the PVS2 vitrification solution, in 2-ml cryovials or in drops over aluminum foil strips, for 15, 30, 60 or 90 min at 0 °C, followed by the direct immersion in liquid nitrogen of shoot tips. Results show that N. domestica shoots can be conserved successfully for 6 months at both the temperatures tested, especially when 60 g/L sucrose is used in the storage medium. However, conservation at 4 °C showed to be more appropriate, as hyperhydricity was observed in post-conservation of shoots coming from storage at 8 °C. As for cryopreservation, a daily gradual increase of sucrose concentration (from 0.25 to 1.0 M) produced better protection to the samples that were stored in liquid nitrogen. Indeed, with this sucrose treatment method, a 30-min PVS2 incubation time was enough to produce, 60 days after thawing, the best recovery (47% and 50%) of shoot tips, cryopreserved with PVS2 vitrification and droplet-vitrification, respectively.

Cryopreservation of grapevine (Vitis vinifera L.) in vitro shoot tips

Abstract In this work, we compared the efficiency of encapsulation-dehydration and droplet-vitrification techniques for cryopreserving grapevine (Vitis vinifera L.) cv. Portan shoot tips. Recovery of cryopreserved samples was achieved with both techniques; however, droplet-vitrification, which was used for the first time with grapevine shoot tips, produced higher regrowth. With encapsulationdehydration, encapsulated shoot tips were precultured in liquid medium with progressively increasing sucrose concentrations over a 2-day period (12 h in medium with 0.25, 0.5, 0.75 and 1.0 M sucrose), then dehydrated to 22.28% moisture content (fresh weight). After liquid nitrogen exposure 37.1% regrowth was achieved using 1 mm-long shoot tips and only 16.0% with 2 mm-long shoot tips. With droplet-vitrification, 50% regrowth was obtained following treatment of shoot tips with a loading solution containing 2 M glycerol + 0.4 M sucrose for 20 min, dehydration with half-strength PVS2 vitrification solution (30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% dimethylsulfoxide and 0.4 M sucrose in basal medium) at room temperature, then with full strength PVS2 solution at 0°C for 50 min before direct immersion in liquid nitrogen. No regrowth was achieved after cryopreservation when shoot tips were dehydrated with PVS3 vitrification solution (50% (w/v) glycerol and 50% (w/v) sucrose in basal medium).

Cryopreservation of Dendrobium hybrid seeds and protocorms as affected by phloroglucinol and Supercool X1000

This study was conducted to investigate two cryopreservation protocols for Dendrobium Swartz. hybrid ‘Dong Yai’. Vitrification was evaluated for cryopreservation of mature seeds, while droplet-vitrification was evaluated for protocorms of the Dendrobium hybrid. The effectiveness of either phloroglucinol or Supercool X1000® as cryoprotectants was evaluated. In vitro germination, protocorm recovery, and seedling development of cryopreserved seeds and protocorms were assessed, respectively. For seed cryopreservation, the use of the PVS2 vitrification solution for 60min at 0°C containing 1% phloroglucinol resulted in 79% seed germination. For protocorm cryopreservation, pretreatment with sucrose 0.3M for 24h followed by exposure to PVS2 solution containing 1% phloroglucinol for 15min at 0°C resulted in 14% protocorm survival after 75 days, compared to 0–6% survival for controls and other treatments. Seeds were germinated and the seedlings produced showed normal growth and development, with no abnormalities. The addition of 1% Supercool X1000® was not effective as a cryoprotectant for seeds. Phloroglucinol at 1% was effective as a cryoprotectant for Dendrobium hybrid seed cryopreservation using the vitrification method. Phloroglucinol provided limited effectiveness for the cryopreservation of Dendrobium hybrid protocorms using droplet-vitrification, but recovery was enhanced as compared to controls and other treatments.

Phloroglucinol enhances recovery and survival of cryopreserved Dendrobium nobile protocorms

The aim of the present study was to evaluate the effect of phloroglucinol in the recovery and survival of cryopreserved Dendrobium nobile protocorms. The exposure of protocorms to 2 M glycerol osmoprotective solution for 20 min followed by 10 min in PVS2 vitrification solution with 1% phloroglucinol resulted in the highest protocorm recovery and survival (68%). A positive effect of phloroglucinol was observed when combined with glycerol and PVS2. Phloroglucinol added at 1% provided an increase of over 100% in protocorm recovery and survival as compared to the same treatment without phloroglucinol. However, when sucrose was added to treatments, a negative effect was observed with a reduction in survival by 90%. Protocorms that survived cryopreservation were successfully regenerated into plants and acclimatized with 100% survival in greenhouse. Survival of cryopreserved D. nobile protocorms was a determining factor for seedling survival and growth into normal and fully functional plants. This study demonstrated an efficient procedure for cryopreservation and subsequent recovery and survival of cryopreserved D. nobile protocorms using phloroglucinol as a cryoprotectant additive.

In vitro conservation of Malaysian biodiversity—achievements, challenges and future directions

Malaysia is fortunate and proud to contain some of the world’s richest biodiversity. In Malaysia, there are an estimated 185,000 species of fauna and 12,500 species of flowering plants, many of which are endemic to tropical forests in this region. Indeed, such diversity is an important and invaluable national asset to safeguard both present and future generations. In vitro conservation offers possible techniques for the preservation of plant germplasm that at present is difficult to maintain or is maintained with limited success. Research at the Universiti Kebangsaan Malaysia (The National University of Malaysia) focuses on the cryopreservation of woody fruit species with seeds that cannot tolerate cryopreservation (recalcitrant or intermediate). Among the plants with recalcitrant seeds are such traditionally important edible tropical fruits as mangosteen, langsat, and rambai (Garcinia mangostana, Lansium domesticum, and Baccaurea motleyana). Citrus aurantifolia, Citrus suhuiensis, Citrus madurensis, Citrus hystrix, and Fortunella polyandra are among the Citrus and Citrus-related species studied. Cryopreservation studies include the Nepenthes species (pitcher plants) of Malaysia. Fundamental research on desiccation and low-temperature tolerance and on the physiology of desiccation are used to understand seed behavior, a prerequisite for the development of successful conservation techniques. At the same time, cryopreservation protocols for several Citrus and forestry species were developed for embryonic axes and adventitious shoots, mainly using rapid dehydration and PVS2 vitrification techniques. There are no successful standard techniques or protocols for species with highly recalcitrant seeds such as Garcinia species. Modification of existing protocols or development of new methods is required, but this can be accomplished only when a detailed understanding of the recalcitrant nature of the seeds or explants is achieved. While we have considerable knowledge concerning the basics of biochemical processes and some molecular data from work on desiccation-tolerant seeds, a great need remains for understanding the cause of the recalcitrance or desiccation sensitivity of these seeds. It may be necessary to use a systems biology approach that exploits the “omics” technologies to generate global molecular data. In combination with bioinformatics for data integration and analyses, this approach would move toward improved modeling of the biological pathways associated with the development of recalcitrant seeds.

Cryopreservation of Malus cultivars: Comparison of two droplet protocols

Two droplet procedures, droplet-vitrification (PVS2) and droplet (DMSO) were applied for cryopreservation of in vitro cultured apple ( Malus domestica Borkh., cvs. Florina, Idared, Colmar and Rebra) plants. The highest post-thaw regrowth rates (70% for cv. Florina, 66% for cv. Idared, 63% for cv. Colmar and 60% for cv. Rebra) were achieved after using the droplet-vitrification (PVS2) protocol. The excised shoot tips (2–3 mm in length) were precultured in 0.5 M sucrose enriched media for 24 h. Subsequently they were transferred in PVS2 vitrification solution for 30 or 40 min (depending on cultivar) at 24 °C and then immersed in liquid nitrogen. Rewarming was performed in liquid MS medium at 24 °C. Plants regenerated from cryopreserved shoot tips did not display any sign of morphological alteration or abnormalities in growth in comparison with control plants.

CRYOPRESERVATION OF STRAWBERRY CV. 'SENGA SENGANA' BY DROPLET VITRIFICATION

Droplet-vitrification method was applied to shoot tips of micropropagated Fragaria ×ananassa Duch. cv. ‘Senga Sengana’. Shoot tips of 2-3 mm in length were precultured in sucrose enriched media for 24 and 48h. Subsequently, shoot tips were transferred into 6 µl droplets of PVS2 vitrification solution for 20 min and plunged afterwards into liquid nitrogen. The effect of sucrose concentration (0.1, 0.25, 0.5, 0.75, 1.0 M) used in preculture of the shoot tips regarding their regrowth, histological changes and the morpholocical stability of the regenerants in the field was analysed. The highest recovering rate (67.7%) was achieved after a preculture with 0.25 M sucrose for 24h. Recovered shoots started to multiply considerably enhancing thereby the number of plantlets ready to transfer for acclimatisation. For field evaluation 22 to 165 plants per sucrose concentration were evaluated. The number of off-types was affected by the sucrose concentration used in preculture. Light and electron microscopy revealed starch accumulation in meristematic cells after preculture in sucrose. Ultra-structure of the meristematic cells was severely affected by PVS2 and liquid nitrogen treatment.