Two efficient and simple cryopreservation methods using aluminium cryo-plates have been developed. In the V cryo-plate method, dehydration is performed using the vitrification solution PVS2, while in the D cryo-plate method, dehydration is achieved using the air current of the laminar flow cabinet or silica gel. To date, more than 20 papers have been published related to both methods. The main advantages of the V and D cryo-plate methods are as follows: handling of specimens throughout the procedure is easy and quick because only the cryo-plates are manipulated. The specimens attached on cryo-plates can be efficiently treated with loading solution (LS) and PVS2/air flow. Cooling and warming are performed easily by immersing the cryo-plates in LN and 1.0 M sucrose solution, respectively, resulting in ultra-rapid cooling and warming rates. High regeneration can be obtained using both methods. For species, which are sensitive to PVS2, the D cryo-plate method can be used. Both methods include preparation of material to be cryopreserved, preconditioning, excision, preculture, mounting of explants on cryo-plates, osmoprotection, dehydration by PVS or air flow, liquid nitrogen storage, rewarming and regeneration. Both protocols appear promising for cryopreservation of both herbaceous and woody plants including tropical plants after appropriate modifications of the procedures. Optimization of the dehydration time, preconditioning of materials and post-cryopreservation regrowth conditions are crucial to achieve high regrowth. These new cryopreservation methods will facilitate the efficient implementation of cryo-storage and long-term maintenance of plant genetic resources in genebanks.

BACKGROUND:Cryopreservation opens new avenues in the field of genetic resource conservation, especially in recalcitrant seeded palms such as arecanut for which field genebanks are exposed to pest and disease attacks and natural calamities. It is only through cryopreservation that the safety of the conserved germplasm can be assured at a relatively low cost for extended periods.OBJECTIVE:The objective of this work was to standardize various aspects of arecanut pollen cryopreservation, viz. collection and desiccation of pollen, in vitro germination, viability and fecundity studies.MATERIALS AND METHODS:Pollens of three arecanut genotypes (Sumangala, Hirehalli Dwarf and Hirehalli Dwarf x Sumangala) were collected in December 2013-February 2014. In vitro viability tests were conducted using fresh and desiccated pollen. Desiccated pollen was cryopreserved by direct immersion in liquid nitrogen and cryostored for different durations (24 hours to 2 years). Viability and fertility studies were conducted using cryopreserved pollen.RESULTS:Pollen extraction was achieved from fully opened male flowers by desiccation at room temperature (33-34 degree C). A medium containing 2.5 g/L sucrose was found to be best for in vitro germination at room temperature. There was no significant difference in germination between desiccated and cryopreserved pollen whereas pollen tube length decreased significantly after cryopreservation. Fertility studies using HD x Sumangala pollen cryostored for various durations (1 month, 1 year and 2 years) showed the setting of 70, 43 and 62%, respectively. Normal nut set was observed using cryopreserved pollen.CONCLUSION:Pollen cryopreservation is a viable option for germplasm conservation and hybridization programmes in arecanut.

Uncaria tomentosa presents tomentum that resembles cat’s claws, hence its common name, is a plant that produces various secondary metabolites that are traditionally used in alternative medicine. The natural distribution of this species has been affected by indiscriminate harvesting from its habitat. In the present research, cryopreservation (liquid nitrogen, LN, -196°C) was evaluated as an option for ex situ conservation of this species. The following techniques were evaluated: vitrification and encapsulation-dehydration of apices, vitrification of cell suspensions, and seed desiccation and vitrification. Preculture conditions and exposure times to LS and PVS2 were evaluated. Apex survival was the highest (82%) with preculture in 0.25 M sucrose followed by incubation for 20 and 30 min in LS and PVS2, respectively, prior to cooling in LN. The encapsulation-dehydration technique was evaluated by using sucrose preculture and different capsule moisture contents. Survival of apices cooled in LN was not significantly different between treatments and varied from 31.8% to 52.9% for capsule moisture contents between 22.7% and 20.3%. For cell suspensions precultured in 0.5 M sucrose, cell multiplication and formation of calli with very good appearance were observed in 61.1% of the cultures following vitrification. For cryopreservation of seeds, germination was 89.5% using the desiccation technique and 67.6% to78.1% using vitrification.

In the present work the endogenous and exogenous effect of trehalose on plant regeneration of cryopreserved shoot-tips of chrysanthemum (Dendranthema grandiflorum Kitam.) cultivar 'Indianapolis' was evaluated. The cryopreserved shoot-tips of in vitro plantlets representing the same cultivar were isolated from a non-tranformed line (Ctrl), and two transgenic lines (TL1 and TL2) with different intracellular trehalose contents. Shoot-tips were subjected to different preculture conditions using trehalose and/or sucrose, and cryopreserved following the standard vitrification procedure. After dissection, explants were precultured on semi-solid MS medium containing 0.3 M sucrose or trehalose or with both, 0.15 M sucrose and 0.15 M trehalose for four days. Precultured shoot-tips were loaded in 0.4 M sucrose + 2 M glycerol solution for 20-30 min, and then exposed to PVS2 or PVS3 vitrification solutions for 0, 10, 20, 30, 40, 50 or 60 min at room temperature prior to rapid immersion in liquid nitrogen. After cryopreservation, the highest shoot regeneration (45, 63 and 71% for Ctrl, TL1 and TL2, respectively) was achieved after preculture on medium with 0.15 M sucrose + 0.15 M trehalose, and exposure to PVS3 for 40 min. Cryopreserved shoot-tips of both transgenic lines displayed higher plant regeneration than shoot-tips of non-transformed plantlets when preculture was performed using 0.3 M sucrose or 0.15 M sucrose + 0.15 M trehalose, irrespectively of the PVS solution used for 30 or 40 min. However, plant regeneration of non-transgenic shoot-tips was higher than regeneration of transgenic tissues when preculture was performed with 0.3 M trehalose. The 0.3 M trehalose preculture was also the only condition, which allowed survival of transgenic and non-transgenic cryopreserved samples without any exposure to PVS solutions. These results underline the cryoprotective effect provided by the exogenous application of trehalose, and the importance of its endogenous accumulation to improve tolerance to cryopreservation.

BACKGROUND: Coconut genetic resources are threatened by pests and pathogens, natural hazards and human activities. Cryopreservation is the only method allowing the safe and cost-effective long-term conservation of recalcitrant seed species such as coconut. OBJECTIVE: The objective of this work was to test the effect of cryopreservation and of cryostorage duration on coconut pollen germination and fertility. MATERIALS AND METHODS: Pollen of two coconut varieties (West Coast Tall [WCT] and Chowghat Orange Dwarf [COD]) was collected in March-May over three successive years, desiccated to 7.5% moisture content (FW) and cryopreserved by direct immersion in liquid nitrogen. RESULTS: Germination and pollen tube length (PTL) of desiccated and cryopreserved pollen were not significantly different for both WCT and COD over the three harvest months of the three consecutive years of study. Pollen germination ranged from 24 to 32% in desiccated pollen whereas it was between 26 and 29% in cryopreserved COD pollen. In the case of WCT, germination ranged from 30 to 31% in desiccated pollen, while it was between 28 and 32% in cryopreserved pollen. PTL of cryopreserved pollen ranged between 224-390 mu m and 226-396 mu m for COD and WCT, respectively. Germination of COD pollen varied between 29.0 and 44.1% after 4 years and 1.0/1.5 years cryostorage, respectively. Germination of WCT pollen did not change significantly between 0 and 6 years cryostorage, being comprised between 32 (24 h) and 40 % (1.5 years). Germination and vigour of cryopreserved pollen were generally higher compared to that of pollen dried in oven and non-cryopreserved. Normal seed set was observed in COD and WCT palms using pollen cryostored for 6 months and 4 years. Cryopreserved pollen of five Tall and five Dwarf accessions displayed 24-31% and 25-49% germination, respectively. CONCLUSION: These results show that it is now possible to establish pollen cryobanks to contribute to coconut germplasm long-term conservation.

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 trehalose for one or seven days. Additionally, 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 on aluminum foil strips.Dehydration with PVS2 solution produced the best results after cryopreservation when 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 apices 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 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, loading in 0.4 M trehalose + 2 M glycerol, and exposure to PVS3 for 30 min.

In this study, we investigated the possibility of using the droplet-vitrification technique for cryopreserving nodal segments of in vitro plantlets of the endangered plant species Lithodora rosmarinifolia. Among the three vitrification solutions tested, only solutions B1, containing (w/v) 50 % glycerol and 50 % sucrose, and B3, containing 40 % glycerol and 40 % sucrose, were able to induce cryotolerance in nodal explants, resulting in intermediate survival and recovery after cryopreservation. A three-step vitrification protocol, including an additional dehydration treatment with half-strength vitrification solution for 30 min before the treatment with full-strength vitrification solution, did not lead to any improvement in survival and recovery compared with the two-step protocol. The optimal protocol was the following: preculture of nodal segments in liquid medium with 0.3 M sucrose for 16 h and 0.7 M sucrose for 5 h, treatment for 20 min in loading solution containing 1.9 M glycerol + 0.5 M sucrose, dehydration with vitrification solution B1 (glycerol 50.0 %, sucrose 50.0 %, w/v) for 60 min at room temperature, rapid cooling in minute droplets of vitrification solution, and rapid rewarming by immersion of nodal segments for 20 min in unloading solution containing 1.2 M sucrose. Under these conditions, 33 % recovery of cryopreserved nodal explants was achieved. Regrowth of cryopreserved samples was rapid and direct. These results indicate that long-term storage of L. rosmarinifolia by means of cryopreservation of nodal segments is possible, thereby contributing to securing the diversity of this rare and endangered plant species.

This paper reviews a 10-year experience in establishing a cryopreserved Allium germplasm collection at the genebank of the National Agrobiodiversity Center, Republic of Korea. A systematic approach to Allium cryopreservation included: 1. revealing the most critical factors that affected regeneration after cryostorage; 2. understanding the mechanisms of cryoprotection by analyzing the thermal behavior of explants and cryoprotectant solutions using DSC and influx/efflux of cryoprotectants using HPLC; 3. assessing genetic stability of regenerants; and 4. revealing the efficiency of cryotherapy. Bulbil primordia, i.e. asexual bulbs formed on unripe inflorescences, proved to be the most suitable material for conservation of bolting varieties due to high post-cryopreservation regrowth and lower microbial infection level, followed by apical shoot apices from single bulbs and cloves. A total of 1,158 accessions of garlic as well as some Allium species have been cryopreserved during 2005-2010 using the droplet-vitrification technique with a mean regeneration percentage of 65.9% after cryostorage. These results open the door for large-scale implementation of cryostorage and for simplifying international exchange for clonal Allium germplasm.

The existence of modern cultivated plants is a result of interplaying modification by breeding and maintenance of valuable characters by storing the plant biodiversity. Genebanks were established to maintain and provide the crop diversity for use on the long term. Therefore, all possible methods for maintaining plant germplasm are necessary in order to fulfil this purpose for the various types of germplasm (field culture, seed storage, in vitro culture and cryopreservation). In vitro culture and cryopreservation are the tools with which laboratories can contribute to conservation but also to the improvement of germplasm. They are useful to protect germplasm from threats imposed by environmental hazards, pests and diseases as well as human damage. Some case studies are presented. In case of potato, a crop of high importance, 2700 accessions are maintained in vitro and about 1200 accessions are stored in liquid nitrogen through cryopreservation at the IPK Gatersleben. Also in case of banana, 1250 and 850 accessions are respectively stored in vitro and under cryopreserved conditions at the Bioversity Musa collection, Leuven, Belgium. The interrelationships between field culture, in vitro storage, and cryopreservation are determined by safety and cost factors. Most divers are the collections of Allium and of medicinal plants belonging to the family Lamiaceae, where variable sets of maintenance methods are followed in dependence on the propagation system of the material. Whereas in vitro storage and cryopreservation initially were mainly considered as tools for preservation of clonal crops, they may also contribute to outbreeding populations when they are used complementary to other methods. With the increasing use of these methods on many diverse locations, development of international collaboration becomes a focal point. This involves technology transfer between different partners, joint development of new methods and creation of networks and benefit-sharing larger storage entities. This covers also safety aspects like safety duplication of samples. The COST action 871 "Cryopreservation of crop species in Europe", having joined 21 countries, the European GenRes Project EURALLIVEG, having created a cryobank system of three countries, and the TRUST project on cryopreservation of tropical vegetatively propagated species are discussed as examples. They follow some earlier initiatives such as an EU GenRes project on Allium preservation and the European cryopreservation project CRYMCEPT. Other actions are integrative on a national level such as the CRYOVEG project, recently implemented in France. This project aims at integrating in a rational and coordinated manner cryopreservation in the overall strategies employed for conserving a range of crop species of national interest. In vitro storage and cryopreservation are not simply alternatives designed to replace other methods of conservation but rather valuable complements in an all-inclusive strategy to maintain and use plant germplasm in the long term.

Coffee seed conservation biology: Fundamental aspects and practical implications. A review Since the early 1990s, the coffee seed has been: designated by several international laboratories as the model system for studying the physiology of seeds of the "intermediate" category. In contrast to orthodox seeds, longevity of which increases when they are dehydrated and stored at low temperature, intermediate seeds are only partially tolerant to desiccation and low temperatures. In the narrow ranges of water contents and temperatures which can thus be used, the longevity of intermediate seeds remains very short, which constitutes a major limitation for the conservation of the biodiversity of these species. The development of new analytical chemistry techniques made it possible to demonstrate that the poor longevity of coffee seeds was associated with several types of cellular damage : neutral lipid hydrolysis leading to the accumulation of free fatty acids(e-) which destabilize membranes, selective loss of a class of phospholipids, loss and oxidation(t-) of the two major hydrophilic antioxidants, ascorbic acid and glutathione. Thanks to the knowledge gained on the processes involved in coffee seed ageing, we propose for the first time in this paper highly accurate technical recommendations for the preparation and the short-term storage of seed lots. Meanwhile, through biophysical approaches including differential scanning calorimetry, the understanding of the mechanisms involved in coffee seed tolerance to ultra-low temperature exposure has significantly progressed. Based on this knowledge, a very effective cryopreservation technique has been developed for coffee seeds, facilitating the long-term conservation of Coffea genetic resources.

A simple and efficient cryopreservation protocol for coconut zygotic embryos has been developed. Embryos were inoculated in Petri dishes on medium containing 3.2 M glucose, which were placed in hermetically closed containers containing 80 or 160 g silica gel for 48 or 24 h. Moisture content of embryos at the end of this treatment varied between 0.25 and 0.65 g g−1 DW, depending on the accession. Embryos were then transferred for cryopreservation by rapid immersion of cryotubes in liquid nitrogen. After rapid re-warming, embryos were transferred to culture medium containing Eeuwens mineral elements for germination. This protocol was applied to ten accessions representative of coconut genetic diversity, with germination percentages of cryopreserved embryos between 13.7% and 74.7%.

In this study, in vitro shoot tips of two sugarcane clones were successfully cryopreserved using encapsulation-dehydration and droplet-vitrification with two vitrification solutions, PVS2 and PVS3. For both clones, encapsulation-dehydration induced significantly higher recovery, reaching 60% for clone H70-144 and 53% for clone CP68-1026, compared with droplet-vitrification in which recovery was 33-37% for clone H70-144 and 20-27% for clone CP68-1026. Optimal conditions included preculture of encapsulated shoot apices for 2411 in liquid medium with 0.75 M sucrose and dehydration with silica gel to 20% moisture content (fresh weight basis) before direct immersion in liquid nitrogen. With both protocols employed, regrowth of cryopreserved samples, as followed by visual observation, was always rapid and direct. (C) 2011 Elsevier B.V. All rights reserved.

In plant vitrification protocols, the "loading" treatment, i.e. exposure of explants to a moderately concentrated cryoprotectant solution, precedes the main dehydration step with highly concentrated vitrification solutions in order to reduce their toxicity. This study aimed at developing alternative loading solutions composed of glycerol and sucrose mixed at various concentrations. The solutions were applied to two model species, garlic and chrysanthemum, cryopreserved via a droplet-vitrification procedure. Differential scanning calorimetry measurements of loading solutions and of loaded and dehydrated explants were performed to assay thermal events occurring during cooling and warming. The response to the loading solutions varied between the two species. In garlic apices, all the loading solutions produced a similar effect, whereas recovery of chrysanthemum shoot tips was significantly influenced by the solution composition. A loading solution comprising of 1.9 M glycerol and 0.5 M sucrose was the most efficient. The loading treatment may thus act as an osmotic stress neutralizer and/or contribute to the physiological adaptation of tissues to both dehydration and freezing.