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In this study, we tested the tolerance of tissue balls (TBs, 100–400 μm in diameter) from the coral Pocillopora damicornis produced using mechanical excision to exposure to cryoprotectant (CPA) solutions. TBs were treated for 20 min at room temperature with individual, binary, ternary or quaternary CPA solutions with a total molarity from 2.0 to 5.0 M. Four CPAs were used: ethylene glycol (EG), dimethylsulfoxide (Me 2 SO), methanol (Met) and glycerol (Gly). In some experiments, the molarity of the CPA solutions was increased and decreased in a stepwise manner. The tolerance of TBs following CPA treatment was evaluated using two parameters. The Tissue Ball Regression (expressed in μm/h) measured the diameter regression of TBs over time. The % Undamaged TBs quantified the proportion of TBs, which remained intact over time after the CPA treatment. TBs tolerated exposure to binary solutions with a total molarity of 4.0 M containing 2.0 M EG + 2.0 M Met and 2.0 M EG + 2.0 M Gly. TBs displayed tolerance to ternary solutions with a total molarity up to 3.0 M, containing each CPA at 1.0 M. Quaternary solutions with a total molarity of 4.0 M containing each CPA at 1.0 M were not tolerated by TBs. When the molarity of the CPA solutions was increased and decreased in a stepwise manner, TBs withstood exposure to a CPA solution with a total molarity of 4.5 M, containing 1.5 M EG + 1.5 M Gly + 1.5 M Me 2 SO. This study confirmed the interest of using TBs to test CPA solutions, with the objective of developing a vitrification-based cryopreservation protocol.

• Background and Aims The purpose of this study was to investigate the basis of the optimal hydration status for cryopreservation of intermediate oily seeds using Citrus as a model. • Methods The relationships between equilibrium relative humidity (RH), seed water content, presence of freezable water as determined by DSC analysis, and germination percentage after immersion in liquid nitrogen (LN) were investigated in Citrus aurantifolia, C. grandis, C. madurensis and C. reticulata. The relationship between the lipid content of seeds and their unfrozen water content was also investigated. • Key Results Independent of their level of seed desiccation tolerance, the optimal desiccation RH for seed tolerance to LN exposure was 75–80 % in the four species studied. This optimal hydration status always coincided with that at which presence of frozen water could not be detected in seed tissues during the cooling/thawing process. The unfrozen water content of seeds was variable between species and negatively correlated to seed lipid content. Using the present data, those obtained previously in seven coffee species and those reported by other authors for five other species, a significant linear relationship was found between the lipid content and the unfrozen water content of seeds. • Conclusions This study provides additional evidence that intermediate oily seeds do not withstand the presence of freezable water in their tissues during the cooling/warming process. Moreover, it offers two important applied perspectives: (1) independent of their level of desiccation tolerance, testing germination of seeds of a given oily seed species after equilibration in 75–80 % RH at 25 °C and LN exposure, gives a rapid and reliable evaluation of the possibility of cryopreserving whole seeds of this given species; (2) it is now possible to calculate the interval of water contents in which non-orthodox oily seeds of a given species are likely to withstand LN exposure as a function of their lipid content.

Cryopreservation (liquid nitrogen, −196°C) represents the only safe and cost-effective option for long-term conservation of germplasm of non-orthodox seed species, vegetatively propagated species, and of biotechnology products. Classical cryopreservation techniques, which are based on freeze-induced dehydration, are mainly employed for freezing undifferentiated cultures and apices of cold-tolerant species. New cryopreservation techniques, which are based on vitrification of internal solutes, are successfully employed with all explant types, including cells suspensions and calluses, apices, and somatic and zygotic embryos of temperate and tropical species. The development of cryopreservation protocols is significantly more advanced for vegetatively propagated species than for recalcitrant seed species. Even though its routine use is still limited, there are a growing number of examples where cryopreservation is employed on a large scale for different types of materials, including seeds with orthodox and intermediate storage behaviour, dormant buds, pollen, biotechnology products, and apices sampled from in vitro plantlets of vegetatively propagated species. Cryopreservation can also be employed for uses other than germplasm conservation, such as cryoselection, i.e., the selection through freezing of samples with special properties, or cryotherapy, i.e., the elimination of viruses from infected plants through apex cryopreservation. Because of its high potential, it is expected that cryopreservation will become more frequently employed for long-term conservation of plant genetic resources.

The genetic determinism of seed desiccation sensitivity was studied using a cross between two coffee species exhibiting a large difference for this trait, Coffea pseudozanguebariae (tolerant) and C. liberica (sensitive). Throughout the whole study, seed desiccation tolerance was quantified both in terms of water content and water activity. Whatever the parameter used, the level of seed desiccation tolerance in F1 hybrids corresponded to that of the mid-parent, thus indicating an additive inheritance of seed desiccation tolerance at the F1 level. A broad variation was observed among hybrids backcrossed to C. liberica (BCs) for seed desiccation tolerance, independent of the parameter used to quantify it. This variation was continuous and BCs showed transgression in the direction of the most desiccation sensitive parent, indicating (i) that desiccation tolerance is a polygenic trait in coffee species, and (ii) that C. pseudozanguebariae does not present the most favourable alleles for all the genes involved in seed desiccation tolerance. No significant difference was observed between the two reciprocal backcrosses, F1xC. liberica and C. libericaxF1, for the level of desiccation tolerance of their seeds, showing the absence of a maternal effect on this trait. There was no significant effect of the number of seeds harvested from each BC on the level of desiccation tolerance of its seeds. Moreover, there was no significant correlation within BCs between seed size, seed viability, and water content before desiccation and desiccation tolerance.

For the first time, regenerated plantlets were obtained from immature zygotic embryos of mango (Mangifera indica L.) through direct somatic embryogenesis. Pro-embryogenic mass (PEM)-like structures, which are differentiated as clusters of globular structures, were easily induced directly from the abaxial side of cotyledons from immature fruits, 2.0–3.5 cm diameter by a 2-wk culture period on a modified Murashige and Skoog medium with 5 mgl−1 (25μM) indole-3-butyric acid (IBA). Conversion of somatic embryos into plantlets was achieved after 4 wk of culture on the conversion medium containing 5mgl−1 (23 μM) kinetin. Secondary somatic embryogenesis could also be obtained directly from the hypocotyls of mature primary somatic embryos cultured on the conversion medium. In our experimental system, only minor problems were noted with browning of cultures.

In vitro culture of single node microcuttings is often employed for producing large amounts of homogeneous plant material for cryopreservation experiments. In this work, we investigated the effect of culture duration of grapevine microcuttings on medium with zeatine riboside (ZR) on shoot tip recovery after cryopreservation. Single node microcuttings of grapevine cultivar Portan were placed on ½ MS medium + 1 µM ZR with transfers to new medium every 4 weeks, and shoot tips were excised after 4, 8, 12 and 16 weeks of culture. Control explants consisted of shoot tips sampled from microcuttings cultured for two months on ½ MS devoid of ZR. The droplet-vitrification technique was employed for cryopreservation of shoot tips. After excision, shoot tips were placed for 24 h on medium containing 0.3 M sucrose, treated for 20 min with loading solution containing 1.2 M glycerol + 0.4 M sucrose, dehydrated with the PVS2 vitrification solution for 25, 50 or 75 min, cooled rapidly in PVS2 droplets placed on aluminium foils, rewarmed rapidly by immersion of foils in unloading solution with 1.2 M sucrose for 20 min, then transferred on standard medium for recovery. After cryopreservation, 30-33 % recovery was obtained for shoot tips sampled from microcuttings cultured for 0, 4 and 8 weeks on medium with ZR. No recovery was noted with shoot tips sampled from microcuttings cultured for longer periods on ZR medium. These results underline the importance of selecting explants in an optimal physiological state for cryopreservation experiments.

UMR 1334 AGAP, Equipe DAVEM = Diversité et Adaptation de la Vigne et des Espèces Méditerranéennes; Cryopreservation using aluminium cryo-plates was successfully applied to in vitro-grown shoot tips of two [i]Prunus[/i] genotypes, cherry plum ([i]Prunus cerasifera Ehrh.[/i]) and the plum cultivar ‘Požegača’ ([i]Prunus domestica L.[/i]). Shoot tips were dissected from the shoots and precultured for 1 day at 23 °C in the dark on Murashige and Skoog medium containing 0.3 M sucrose. The precultured shoot tips were placed on aluminium cryo-plates containing 10 or 12 wells and embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates in two types of loading solution (LS1 − 2 M glycerol + 0.4 M sucrose or C4 − 1.9 M glycerol + 0.5 M sucrose) for 30 min at room temperature. In the V cryo-plate protocol, dehydration was performed for 30 min at room temperature in a plant vitrification solution containing 37.5% (w/v) glycerol, 15% (w/v) dimethylsulfoxide, 15% (w/v) ethylene glycol and 22.5% (w/v) sucrose. In the D cryo-plate protocol, dehydration was performed by placing the cryo-plates for 2, 2.5 or 3 h under the air current of the laminar flow cabinet or in closed glass containers over silica gel. In both protocols, cooling was performed by placing the cryo-plates in uncapped 2 mL cryotubes, which were immersed in liquid nitrogen. Rewarming was done by direct plunging of cryo-plates in liquid MS medium containing 0.8 M sucrose at room temperature for 30 min (plum cultivar) or 60 min (cherry plum). All experiments were conducted twice in two different laboratories (IRD Montpellier, France and FRI Čačak, Serbia). In the V cryo-plate procedure regrowth (calculated as average values for two laboratories) of cryopreserved shoot tips loaded with C4 solution was 41.7% (cherry plum) and 34.2% (plum cultivar ‘Požegača’), while in those loaded with LS1 solution regrowth was 56.1% and 44.6%, respectively. As for the D cryo-plate procedure, the average regrowth of cryopreserved explants ranged between 57.7% and 77.5% in cherry plum and between 28.5% and 47.5% in plum cultivar ‘Požegača’. Although during the first subculture after regrowth, the multiplication capacity of cryopreserved explants was lower compared with those originating from dissection controls, by the third subculture they regained and even exceeded the multiplication capacity of shoots regenerated from explants placed on regrowth directly after dissection. The results obtained clearly indicate that both cryopreservation procedures using aluminium cryo-plates can facilitate efficient cryostorage of [i]Prunus[/i] germplasm.

Chrysanthemum (Dendranthema x grandiflora (Ramat.) Kitamura) is an ornamental plant that responds well to in vitro growth conditions. This receptivity makes it a particularly attractive target for low-temperature storage and cryopreservation studies. This review examines in detail the protocols thus far used to achieve the short- to long-term low-temperature and cryostorage of important chrysanthemum germplasm. Occasionally, medicinal chrysanthemum species have also been cryostored, and these studies are also examined in detail. Since chrysanthemum is sensitive to both osmotic stress and chemical toxicity of vitrification solutions, a generalized protocol for the cryopreservation of chrysanthemum apical or axillary shoot tips is proposed: excision of apical or axillary shoot tips after 4 or 7 weeks, respectively, from final subculture; progressive preculture with 10 % sucrose for 31 h, 17.5 % sucrose for 17 h, then 25 % sucrose for 7 h; osmoprotection with 17.5 % glycerol + 17.5 % sucrose for 40 min; cryoprotection with PVS3 (50 % glycerol + 50 % sucrose) vitrification solution for 60 min (axillary) or 90 min (apical); cooling and warming using aluminium foil strips; unloading with 30 % sucrose for 40 min. When smaller axillary shoot tips are used, cryoprotection of samples with 37.5 % glycerol + 15 % DMSO + 15 % ethylene glycol + 22.5 % sucrose at 0 A degrees C for about 60 min can be applied.

This report highlights the first successful cryopreservation of in vitro shoot tips of Cleome rosea, achieved by the vitrification technique using aluminum cryo-plates (V cryo-plate). The effects on survival and recovery of C. rosea shoot tips of different plant vitrification solutions (PVS2 and PVS3), light intensity (standard and dim light), and 6-benzyladenine (BA) supplementation (0.10, 0.25, or 0.50 mg L−1 for 1 or 3 wk) in the recovery medium were investigated. Cryopreserved shoot tips showed high regeneration frequencies when treated with PVS2 and PVS3, reaching survival frequencies of 97 and 70%, respectively. When placed onto medium without growth regulators, recovery of cryopreserved shoot tips did not exceed 33% (obtained with PVS2) or 23% (PVS3). Supplementation of recovery medium with 0.5 mg L−1 BA for 3 wk increased both survival and recovery to 100% after cryopreservation following treatment with either PVS2 or PVS3. These values were maintained even when shoot tips were cultured in the presence of 0.10 mg L−1 BA for 1 wk. Maintenance under dim light resulted in better phenotypic characteristics of plants produced from cryopreserved shoot tips.

In this study, the tolerance of tissue balls (TBs, 100–300 μm in diameter) from the coral Pocillopora damicornis produced using mechanical excision to exposure to cryoprotectant (CPA) solutions was tested. TBs were treated for 20 min at room temperature with solutions of ethylene glycol (EG), methanol (Met), glycerol (Gly) or dimethyl sulfoxide (Me2SO) at concentrations between 1.0 and 4.5 M. Two parameters were used to evaluate the survival of TBs following CPA treatment. The Undamaged Duration of Tissue Balls (expressed in h) corresponded to the time period during which the membrane surface of TBs remained smooth and their motility was preserved. Tissue Ball Regression (expressed in μm/h) corresponded to the size reduction of TBs over time. TBs tolerated exposure to all CPAs tested at the three lower concentrations employed (1.0 M, 1.5 M and 2.0 M). No survival was achieved following exposure to a 4.5 M CPA solution. At concentrations of 3.0 and 4.0 M, higher Undamaged Duration of Tissue Balls and lower Tissue Ball Regression were obtained following treatment with EG compared to the other three CPAs. Our experiments show that TBs constitute a good experimental material to evaluate CPA toxicity on corals using large numbers of samples. Performing preliminary experiments with TBs may allow reducing the number of tests carried out with less easily available coral forms such as planulae, thereby preserving larval stocks.

International audience; In this study, we compared the growth of Dioscorea cayenensis-rotundata (African yam) nodal segments, using semisolid medium in test tubes and liquid medium in 1-L Recipient for Automated Temporary Immersion (RITA((R))) temporary immersion bioreactors (TIB), and the application of various culture parameters. The addition of activated charcoal (AC) had a positive effect on the growth of nodal segments, both in semisolid medium and in liquid medium in RITA((R)) bioreactors. After 2 mo culture in the presence of AC, plantlets were 6.4-6.6 cm long compared to 3.2-3.8 cm in absence of AC, with no significant difference observed between the culture systems. In the range of inoculation densities tested (5-20 nodal segments per RITA((R)) bioreactor), there was no effect on the number of buds produced per nodal segment, the moisture content of plantlets (fresh weight basis), or on net fresh weight gain. By contrast, the individual leaf surface area of plantlets decreased in line with increasing inoculation density. Among the range of benzylaminopurine (BAP) concentrations tested (0-17.6 mu M), 0.44 mu M induced the highest number of buds (3.8 buds per nodal segment) in the TIB. However, comparable numbers of buds could be produced with media devoid of BAP, either by increasing the frequency of 1-min daily immersion cycles in RITA((R)) bioreactors from one every 12 h to one every 4 h or by using semisolid medium containing AC.

Currently cryopreservation is applied mainly for safe long-term germplasm conservation of seedless and thus vegetatively propagated crops like banana (Musa spp.). Three cryopreservation methods for shoot-tip cultures of banana are currently available. The first method relies on rapid freezing of highly proliferating meristem cultures precultured for 2 weeks on 0.4 M sucrose. The second method is based on vitrification of tiny meristems excised from rooted in vitro plants. The third, and until now most successful protocol, is a combination of the previous ones; vitrification of highly proliferating, sucrose-precultured meristem cultures. Postthaw regeneration rates are up to 75 %, depending on the cryopreservation protocol and the cultivar. Besides its traditional application for germplasm storage, cryopreservation of meristem cultures can also result in virus eradication of BSV and CMV from infected plants. Only the most meristematic, and thus the least virusinfected, part of these cultures regenerates after freezing. Finally, cryopreservation can also be applied to plant material with specific characteristics, such as medicinal and alcohol producing cell lines, genetically transformed tissues and transformation competent tissues. The safe storage of transformation competent embryogenic cell suspensions of banana is of utmost importance since their initiation is difficult and time-consuming and their morphogenic capacity decreases with time. Cell cultures were recovered after 4 years of storage in liquid nitrogen. We showed that viability and regeneration capacity remainded intact, as well as competence to Agrobacterium mediated transformation. INTRODUCTION Bananas (Musa spp.) are grown in approximately 120 countries where they provide sustenance to millions of people. With a yearly production of around 88 million metric tons they are the most important fruit crop in the world. The export trade, which deals almost exclusively with one variety, ‘Cavendish’, accounts for only 13 % of world production. The remaining production is locally consumed and consists of a large range of varieties with different eating or cooking qualities; local dessert bananas, true plantains, highland bananas and cooking bananas etc. Banana plantations are extremely vulnerable to pests and diseases due to clonal propagation and monoclonal cultivation of this crop. Besides dealing with phytopathologic constraints, bananas could also be improved for tolerance to wind, salinity, cold and water stress, and increased yield, shelf life and fruit quality. Germplasm collections can provide a source of such ‘superior’ genetic material. Moreover, the availability of a wide variety of germplasm is essential for both classical and modern breeding. Seed preservation, the most convenient method to preserve plant germplasm, is not applicable to edible (seedless) banana cultivars. Field collections lose germplasm 51 Proc. IInd IS on Biotech. of Trop & Subtrop. Species Eds: W.-C. Chang and R. Drew Acta Hort 692, ISHS 2005 (genetic erosion) because of pests, diseases and adverse weather conditions and their maintenance is labour-intensive and expensive. Therefore, in vitro collections were established. However, the maintenance of such in vitro banana collections is labourintensive, and even at reduced growth conditions, there is always the risk of losing accessions due to contamination or human error. Moreover, in vitro material is subject to somaclonal variation. Cryopreservation or freeze-preservation at ultra-low temperatures (-196°C) is the method of choice since at these conditions biochemical and most physical processes are arrested. As such, plant material can be stored for unlimited periods. Currently, three cryopreservation protocols are available for shoot-meristematic tissues of banana; (i) simple freezing of proliferating meristem culture using a sucrose preculture (Panis et al., 1996) (ii) vitrification of apical meristems (Thinh et al., 1999) and (iii) vitrification of sucrose-precultured meristem cultures (Panis et al., 2000). Further optimisation of the latter protocol is presented below. Until now, the availability of embryogenic cell suspensions has been an essential requirement for genetic engineering of Musa spp. (Sagi et al., 1995: Hernandez et al., 1998). However, the initiation of transformation competent banana embryogenic suspensions remains a difficult and time-consuming endeavour. Moreover, once initiated, they are subject to somaclonal variation, loss of regeneration capacity and contamination (Schoofs et al., 1999). Their safe, long-term storage through cryopreservation is therefore recommended. Recently, it was discovered that cryopreservation is not only applicable as a conservation tool but can also be applied to eradicate plant viruses (Helliot et al., in press). Successful eradication of Cucumber Mosaic Virus (CMV) and Banana Streak Virus (BSV) from proliferating shoot cultures of banana through cryopreservation is reported. MATERIALS AND METHODS Cryopreservation of Meristem Clumps through Vitrification 1. Starting Material and Preculture. All varieties were obtained from the in vitro INIBAP Musa germplasm collection situated at K.U.Leuven, Belgium. In order to prepare ‘Cauliflower-like’ meristem clumps, shoot-tips were transferred to semi-solid Murashige and Skoog (1962) medium supplemented with 100 μM BA and 1 μM IAA (p4 medium). Every 1 to 2 months, the material was subcultured but only small clumps of 'cauliflowerlike' meristems were selected and transferred to fresh medium. As soon as ‘cauliflowerlike’ clumps were obtained, white meristematic clumps of about 4 mm diameter, each containing at least four apical domes were excised and transferred onto a preculture medium. The preculture medium consisted of Murashige and Skoog (1962) elements supplemented with 10 μM BA, 1 μM IAA and 0.4 M sucrose. These cultures were kept for 2 weeks under normal growth conditions after which meristem clumps of 1.5 to 3 mm diameter (containing 3 to 5 meristematic domes) were excised. 2. Loading, Dehydration with PVS2 and Freezing. Loading solution was added to the meristem clumps and kept for 20 min at room temperature. The filter sterilised loading solution contained 2 M glycerol and 0.4 M sucrose dissolved in MS medium (pH 5.8). Ice-cooled PVS2 solution was then added. The PVS2 solution consisted of 30 % (3.26.M) glycerol, 15 % (2.42 M) ethylene glycol (EG), 15 % (1.9.M) DMSO and 0.4 M sucrose (Sakai et al., 1990). These compounds were dissolved in MS medium, pH adjusted to 5.8 followed by filter sterilisation. The meristems were subjected to the PVS2 solution for 60, 120, 150 or 180 min at 0°C. During this period, the meristems were transferred from the PVS2 in plastic containers into cooled 2 ml sterile Cryo Vials which contained 1 ml of fresh ice-cold PVS2. After the PVS2 treatment, cryotubes were plunged into liquid nitrogen. 3. Thawing, Deloading and Regeneration. The tubes were rapidly thawed by stirring the cryotubes in a warm water bath (40°C) for 1 min 20 sec. Directly after thawing, the toxic PVS2 solution was removed and replaced by the deloading solution for 15 min at room

Large differences in seed desiccation sensitivity have been observed previously among ten coffee species (Coffea arabica, C. brevipes, C. canephora, C. eugenioides, C. humilis, C. liberica, C. pocsii, C. pseudo-zanguebariae, C. sessiliflora and C.stenophylla). Of these species,C. libericaandC. humiliswere the most sensitive to desiccation andC. pseudozanguebariaethe most tolerant. A study was carried out using the same seed lots to investigate if these differences in desiccation tolerance could be correlated with differences in soluble sugar content. Soluble sugars were extracted from dry seeds and analysed using high performance liquid chromatography. The seed monosaccharide (glucose and fructose) content was very low (1.5 to 2 mg g-1dry weight [dw]) in all species studied. The sucrose content ranged from 33 mg g-1dw inC. libericaseeds to 89 mg g-1dw in seeds ofC. pocsii. Raffinose was detected in the seeds of only five species (C.arabica, C.brevipes, C.humilis, C.sessiliflora, C.stenophylla), among which only three species (C.arabica, C.sessilifloraandC.brevipes) also contained stachyose. Both raffinose and stachyose were present in very low quantities (0.3–1.4 mg g-1dw and 0.1–0.7 mg g-1dw, respectively). Verbascose was never detected. No significant relationship was found between seed desiccation sensitivity and: (i) the sugar content; (ii) the presence/absence of oligosaccharides; and (iii) the oligosaccharide:sucrose ratio.

A broad variability for seed desiccation sensitivity, as quantified by the water content and the water activity at which half of the initial viability is lost, has been previously observed within nine African coffee species. In order to investigate if these different degrees of desiccation sensitivity correspond to an adaptive trait, additional data, such as the duration of seed development and seed water content at maturity, were measured for these species, and the relationships between these parameters and some climatic characteristics of their specific native environments were investigated. Since flowering in all coffee species occurs only a few days after the main rainfall marking the end of the dry season, simulations could be made, based on the continuous sequences of rainfall data compiled in databases of nine climatic stations, chosen for their appropriate location in the collecting areas. The simulations revealed a highly significant correlation between the duration of seed development and that of the wet season. Consequently, mature seeds are shed at the beginning of the following dry season. Moreover, the mean number of dry months that seeds have to withstand after shedding was significantly correlated with the parameters used to quantify seed desiccation sensitivity. By contrast, seed moisture content at maturity was not correlated with the level of seed desiccation tolerance. All these results are discussed on the basis of more detailed descriptions of the natural habitats of the coffee species studied.

Seed desiccation sensitivity was studied in nine species of the genus Coffeaby measuring seed viability after equilibration over various saturated salt solutions. A quantal response model based on the logistic distribution was developed in order to describe the typical S-shaped patterns observed. The closeness of fit of the desiccation sensitivity model was shown, and the assumption that seed desiccation sensitivity follows a continuous distribution within species was verified. For each species, the water content at which 50% of initial viability was reached, WC50, and a specific parameter describing the intra-specific variability, β, were calculated using a non-linear regression. A simplified water sorption model was developed which allowed easy calculation of water activity and water potential corresponding to WC50 (aw50 and Ψ50) for relative humidities ranging between 10 and 100%. Distribution of WC50and Ψ50 (or aw50) in the genus Coffea was homogeneous within the following intervals: from 0.05 to 0.38 g H2O.g−1dw for WC50 and from −168 to −11 MPa for Ψ50. Different classifications of the coffee species studied as regards to their desiccation sensitivity were obtained depending on whether WC50 or Ψ50was used for classification. The continuum for desiccation sensitivity observed within the nine species studied confirmed that coffee is an appropriate material for studying desiccation sensitivity.

In vitro techniques have great potential for collecting, exchange and conservation of: i) genetic resources of recalcitrant-seed and vegetatively propagated species as well as of endangered species; ii) elite genotypes which are multiplied on a large scale in production laboratories; iii) cultures with special attributes, eg, metabolite-producing cell lines and genetically engineered material. In vitro collecting techniques, which consist of introducing embryos or vegetative tissues in vitro under field conditions, have been developed for collection of germplasm of various problem species. In vitro cultures are routinely used for exchange of plant genetic resources of a number of species, due to their advantages in terms of phytosanitary status and reduced cost. Slow growth techniques have been developed for medium-term conservation of numerous species but their routine use is still restricted to a limited number of crop species. Routine use of cryopreservation is mostly restricted to conservation of cell lines in research laboratories. However, simple and efficient freezing protocols have been developed recently for apices and embryos, and can be considered operational for an increasing number of species. Current research priorities include: i) the development of in vitro collecting techniques for additional species; ii) demonstration of the flexibility, simplicity and practicality of slow growth storage to increase its utilisation; iii) experimentation of existing cryopreservation techniques on a large scale in a genebank context and the development of cryopreservation protocols for additional species.

No single conservation technique can adequately conserve the full range of genetic diversity of a target species or genepool. To optimize the efficiency of germplasm conservation, a range of conservation techniques needs to be applied simultaneously. In this paper we review the progress made in the application of both in situ and ex situ complementary conservation strategies and techniques to the conservation of the genus Coffea and identify areas where more research work is required. Traditionally, Coffea species have been conserved ex situ as living plants in field genebanks due to their intermediate seed storage behaviour. However, progress made in the field of biotechnology in recent years have made it possible to envisage the use of in vitro, slow growth and cryopreservation for medium- and long-term conservation of Coffea germplasm, respectively. In situ conservation is also an important option for long-term conservation, but there are no genetic reserves specifically set up for conserving Coffea. Protected areas and managed reserves offer the best opportunities for conserving Coffea. A case study for Mascarene Coffea in Mauritius is presented and a strategy for their conservation is discussed.

International audience; 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.

International audience; In this work, we performed qualitative and quantitative observations of the cytological changes occurring in cells of yam (Dioscorea alata) in vitro shoot tips cryopreserved using the encapsulation-dehydration (E-D) technique. Shoot tip osmoprotection for 24 h in 1.25 M sucrose medium induced drastic changes in cellular cytological features, including high plasmolysis in all three cellular areas studied, the external cell layer (L1), one to three (L1-3) and seven to nine (L7-9) cell layers from the surface of the meristematic dome, pyknotic nuclei in meristematic area cells and disappearance of nucleoli. Nucleus size decreased significantly in all cellular areas studied. Nucleocytoplasmic ratio decreased significantly in L1-3 and L7-9 cells. Nuclear protein content increased, particularly in L1 and L1-3 cells. After physical dehydration, plasma membrane of numerous basal part cells was broken and intracellular soluble protein leakage was observed. Nucleus area and nucleocytoplasmic ratio decreased significantly in L7-9 cells. One week after cryopreservation, shoot tips showed regrowth and living cells had recovered their original morphology. In all cellular areas studied, nuclei had retrieved their original staining and nucleoli were visible. Original nucleus area values were recovered in L1-3 and L1 cells. The nucleocytoplasmic ratio retrieved its initial value in L1 cells but remained at levels observed after osmoprotection for L1-3 and L7-9 cells. The nuclear protein content had retrieved its original level. This investigation provided new insights in changes occurring in D. alata apices throughout an E-D protocol.

This chapter presents different technical aspects related to the development and large-scale application of cryopreservation techniques, as a biotechnological approach for the long-term storage of plant biodiversity. The main cryogenic procedures and the key steps for their successful adaptation to diverse forms of germplasm are described. Some representative examples of cryopreservation of different plant species are presented to illustrate the significant progress achieved in the practical utilization of cryopreservation as a complementary alternative for germplasm conservation. In addition, other potential uses of this technology to support genetic breeding programs, and its recent utilization to eliminate systemic plant pathogens through cryotherapy are discussed.

In this study, we investigated the tolerance of Pocillopora damicornis apexes to treatments with solutions containing penetrating and non-penetrating cryoprotective agents (CPAs). CPAs were employed individually or in binary, tertiary or quaternary solutions. In some experiments apexes were treated successively with two CPA solutions with increasing total concentration. P. damicornis apexes withstood exposure for up to 30 min to solutions containing 0.6-0.8 M sucrose (Suc) or trehalose (Tre). When apexes were treated with binary cryoprotectant solutions containing Suc and ethylene glycol (EG), methanol (Meth), dimethyl sulfoxide (Me2SO) or glycerol (Gly), the CPAs employed in combination with Suc could be ranked in the following order of decreasing tolerance: EG > Meth > Me2SO > Gly. P. damicornis apexes tolerated exposure to complex CPA solutions containing Suc, Me2SO, EG and/or Meth with a total molarity of 2.45 M. In experiments where two successive CPA solutions were employed, apexes withstood treatment with the second, more concentrated solution at 0 degrees C for up to 10 min. These preliminary results pave the way to the development of a cryopreservation protocol for P. damicornis apexes.

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).

This paper presents the advances made over the last decade in cryopreservation of economically important vegetatively propagated fruit trees. Cryopreservation protocols have been established using both dormant buds sampled on field-grown plants and shoot tips sampled on in vitro plantlets. In the case of dormant buds, scions are partially dehydrated by storage at -5 degrees C, and then cooled slowly to -30 degrees C using low cooling rates (c.a. 1 degrees C/h) before immersion in liquid nitrogen. After slow rewarming and rehydration of samples, regrowth takes place either through grafting of buds on rootstocks or excision of apices and inoculation in vitro. In the case of shoot tips of in vitro plantlets, the cryopreservation techniques employed are the following: controlled rate cooling procedures involving slow prefreezing followed by immersion in liquid nitrogen or vitrification-based procedures including encapsulation-dehydration, vitrification, encapsulation-vitrification and droplet-vitrification. The current status of cryopreservation for a series of fruit tree species including Actinidia, Diospyros, Mobs, Olea, Prunus, Pyrus and Vitis is presented. Routine application of cryopreservation for long-term germplasm storage in genebanks is currently limited to apple and pear, for which large cryopreserved collections have been established at NCGRP, Fort Collins (USA), using dormant buds and in vitro shoot tips, respectively. However, there are a growing number of examples of pilot scale testing experiments under way for different species in various countries. Progress in the further development and application of cryopreservation techniques will be made through a better understanding of the mechanisms involved in the induction of tolerance to dehydration and ctyopreservation in frozen explants.

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.

In the present study, the efficiency of two cryopreservation strategies for oil palm (Elaeis guineensis Jacq.) was compared. If extracted from rehydrated kernels, 65% of the embryos desiccated to around 0.3 g H2O/g DW developed into plantlets after cryopreservation. In contrast, only 25% of embryos (0.12 g H2O/g DW) extracted from cryopreserved dry kernels developed into plantlets. However, this value was increased to 63% if kernels were partially rehydrated before freezing until the water content of embryos reached 0.3 g H2O/g DW. This study emphasizes the importance of partial rehydration of oil palm embryos before cryopreservation.

The various strategies and methods available for in situ and ex situ conservation of plant diversity should be used in a complementary manner to optimize and secure the conservation of a given genepool. A lot of collecting remains to be performed before valuable germplasm is lost, both for crops and their wild relatives. Despite the fact that numerous challenges still exist, a number of new tools are now available to improve collecting efficiency. In situ/on farm conservation is of high relevance for tropical plant species, especially for those with non-orthodox seeds and vegetatively propagated ones. In situ/on farm conservation will become more efficient when communities, farmers and local organizations are more directly involved in research and implementation activities, and when researchers and funding agencies fully realize the importance and relevance of this conservation approach. Conservation of orthodox seeds is the most widely practised method of ex situ conservation of plant genetic resources. Yet, it is faced with important challenges, which relate to the observation that longevity of seeds of a large number of species stored under standard genebank conditions is shorter than anticipated previously. This calls for research in seed thermodynamics at low temperature and strongly advocates for the systematic cryogenic storage of one sub-sample of every genebank accession. For in vitro slow growth storage, the main challenges are to improve the introduction and maintenance of contaminant-free plants in culture, and to find the appropriate growth media for suitable micropropagation, which may have to be customized to improve the response to storage of unique plant groups. Cryopreservation is much more advanced for vegetatively propagated plants, in comparison with recalcitrant species, because of many of their characteristics and because most of them are wild species, with no or little information on their biology or storage behaviour. Challenges to improve the cryopreservation of recalcitrant species include the prerequisite development of in vitro culture protocols for the explants used, the necessity to dehydrate the tissues as rapidly as possible before immersion in liquid nitrogen and to reduce the damage caused by the burst of reactive oxygen species, which occurs during dissection and desiccation. The range of analytical tools, which allow better understanding of the biological and physical processes occurring during cryopreservation of plant tissues and organs, is increasing. Such tools will not only increase our knowledge but also lead to more efficient cryopreservation protocols for problem materials. It should be realized that countries rely on each other for access to genetic resources for agricultural development. This interdependency calls for increased collaboration among countries to fully support, participate in and implement the various treaties, conventions and agreements, which aim at the effective conservation and sustainable use of biological diversity essential for food and agriculture at the global level.

In this work, we studied the effects of cryopreservation on various parameters of early stages of germination of Phaseolus vulgaris seeds (0, 7 and 14 days). Percentages of germination, fresh mass of different plant parts, levels of chlorophyll pigments (a, b, total), malondialdehyde, other aldehydes, phenolics (cell wall-linked, free, and total) and protein were determined. No phenotypic changes were observed visually in seedlings recovered from cryopreserved seeds. However, several significant effects of seed liquid nitrogen exposure were recorded at the biochemical level. There was a significant negative effect of cryopreservation on shoot protein content, which decreased from 3.11 mg g(-1) fresh weight for non-cryopreserved controls to 0.44 mg g(-1) fresh shoot weight for cryopreserved seeds. On the other hand, cryopreservation significantly increased levels of other aldehydes than malondialdehyde in shoots at day 7, from 56.47 mu mol g(-1) for non-cryopreserved controls to 253.19 mu mol g(-1) fresh shoot weight for cryopreserved samples. Liquid nitrogen exposure significantly reduced phenolics contents (free, cell-wall linked, total) in roots at day 7 after onset of germination. In general, roots were more affected by cryostorage compared with other plant parts, while leaves were the least affected. The effects of seed cryopreservation seem to decline progressively along with seedling growth.

Sugarcane (Saccharum sp. hybrids) is a crop of major importance, which is cultivated on a large scale in tropical and subtropical regions primarily for its high sucrose content. Cultivated pineapple (Ananas comosus (L.) Merrill, which is now called Ananas comosus var comosus) belongs to the family Bromeliaceae. It is economically the fourth most important crop worldwide in terms of tropical fruit production and follows banana, mangoes and citrus. One of the main drawbacks faced by sugarcane and pineapple agriculture worldwide is the vegetative (i.e. asexual) nature of its conventional propagation. The consequence is that plants in the field must be replaced at intervals ranging from 1 to 5 years, a process that is costly, tedious and time-consuming. Furthermore, if the planting material is of low quality, yields decrease and more tillage is needed. The crops are exposed to natural disasters, while the propagation system leads to systemic disease transmission, and natural selection and plagues also take their toll. Moreover, the industry is in dramatic need of planting material, which cannot be produced in sufficient quantities to meet the demand using classical macropropagation techniques.

Shoot-tips isolated from two transgenic lines of chrysanthemum (Dendranthema grandiflorum Kitam.) var. Indianapolis in vitro plantlets with induced capacity to biosynthesize trehalose, and from a non-transformed line, were subjected to cryopreservation using a vitrification procedure. After dissection, apices were precultured on semi-solid MS medium with 0.3 M sucrose for 4 days, loaded in a 0.4 M sucrose + 2 M glycerol solution for 20-30 min and exposed to PVS2 or PVS3 vitrification solutions for 0, 20, 40 or 60 min at room temperature prior to rapid immersion in liquid nitrogen. The highest shoot regeneration after cryopreservation was obtained with exposure to either PVS solution for 40 min. Plant regeneration from cryopreserved shoot-tips ranged between 48 percent and 67 percent for transgenic lines and between 33 percent and 36 percent for non-transgenic lines. No polymorphic loci were detected in plantlets regenerated from cryopreserved and non-cryopreserved shoot-tips with RAPD techniques using eight primers that amplified 101 monomorphic loci.

Publisher Summary An appropriate conservation strategy for a particular plant gene pool requires a holistic approach, combining the different ex situ and in situ. This chapter presents the new biotechnological possibilities for improving ex situ conservation of plant biodiversity in genebanks and botanical gardens. Two basic conservation strategies, in situ and ex situ, each composed of various techniques, are employed for conservation of plant biodiversity. Ex situ conservation means the conservation of components of biological diversity outside their natural habitat. In situ conservation means the conservation of ecosystems and natural habitats and the maintenance and recovery of viable populations of species, and in the case of domesticated or cultivated species, in the surroundings where they have developed their distinctive properties. Selection of the appropriate methods should be based on a range of criteria, including the biological nature of the species in question and practicality and feasibility of the particular methods chosen (which depend on the availability of the necessary infrastructures) as well as the cost-effectiveness and security afforded by their application. The complementarity between genebanks and botanical gardens should be fully recognized and capitalized on to optimize plant biodiversity conservation.

During a 7-day conditioning treatment of clumps of oil palm (Elaeis guineensis Jacq.) somatic embryos on a medium containing 0.75 M sucrose, the fructose and glucose concentrations remained constant, whereas a ten-fold and twenty-fold increase were noted for sucrose and starch concentrations, respectively. The only new sugar detected was arabinose which remained at a low concentration. After conditioning on media supplemented with various sugars and polyols at similar osmolarities, recovery of clumps of embryo growth was satisfactory except with ribose. After an additional desiccation period, survival was optimal with fructose, galactose, sucrose and glucose, intermediate with maltose and lower with other compounds. When embryos were cryopreserved without previous desiccation, survival was noted after conditioning treatment with sucrose only. In contrast, when freezing was performed after dehydration, survival could be obtained with several substances. It was optimal with sucrose, fructose, galactose and raffinose but was possible also with sorbitol and glucose. Intensity of recovery of proliferation was highest with embryos conditioned with sucrose.

An efficient protocol for the cryopreservation of madder (Rubia akane Nakai) hairy root cultures was developed using droplet-vitrification and alternative loading and vitrification solutions formulated previously in our laboratory. Among eight preculture treatments tested, the highest post-cryopreservation regeneration was obtained for explants incubated in liquid half-strength MS medium with progressively increased sucrose concentration (0.3 M for 54 h, then 0.5 M for 16 h). Loading of precultured explants improved their post-cryopreservation regeneration by 50-75% compared with non-loaded control. Combining loading solution C4 (35% PVS3) and vitrification solution B5 (80% PVS3) was the most effective, while applying six PVS2-based solutions at room temperature resulted in low post-cryo regeneration. Treatment duration was optimized to 30 min for loading and to 10-20 min for vitrification solution. Apices of primary and secondary hairy roots showed similar post-cryo regeneration (88 and 95%, respectively), which was significantly higher than regeneration of root sections without apices (65%). Droplet-vitrification produced higher post-cryo regeneration than 'classical' vitrification in cryovials. Our results suggest that droplet-vitrification using alternative loading and vitrification solutions is an efficient method for cryopreservation of R. akane hairy root cultures.

Coconut is a very important crop for millions of people in tropical countries. With coconut, in vitro culture protocols have been developed with two main objectives, viz. the large scale production of particular types of coconuts and the international exchange and conservation of coconut germplasm. The methods described in this chapter have been developed in the framework of collaborative activities between research institutes in Côte d'Ivoire and France. Two coconut embryo in vitro collecting protocols have been established, one consisting of storing the disinfected embryos in a KCl solution until they are brought back to the laboratory, where they are re-disinfected and inoculated in vitro under sterile conditions, and the other including in vitro inoculation of the embryos in the field. For international germplasm exchange, zygotic embryos inoculated in vitro in plastic test tubes or endosperm cylinders containing embryos in plastic bags are used. For in vitro culture, embryos are inoculated on semi-solid medium supplemented with sucrose and activated charcoal and placed in the dark, and then transferred to light conditions with the same (solid or liquid) medium once the first true leaf is visible and the root system has started developing.

Cryopreservation (liquid nitrogen, -196°C) is the only safe and cost-effective option for long-term -conservation of genetic resources of non-orthodox seed species. Cryopreservation protocols have been developed for various materials including seeds, dormant buds, cell suspensions, calli, apices, zygotic, and somatic embryos of numerous plant species. Zygotic embryos or embryonic axes of almost 100 different species and somatic embryos of almost 40 different species from both temperate and tropical climates, comprising crops, fruit, and forest trees as well as wild species, whose seeds displayed orthodox, intermediate, and recalcitrant storage characteristics, have been successfully cryopreserved. With zygotic embryos and embryonic axes, the desiccation technique has been used with the majority of the species tested, leading to highly variable survival and recovery after freezing, especially during earlier experiments. More recently, new cryopreservation techniques viz. encapsulation-dehydration and vitrification have been employed, leading to generally improved results. With somatic embryos, different cryopreservation methods have been used viz. desiccation, pre-growth-desiccation, encapsulation-dehydration, vitrification, encapsulation-vitrification, and droplet-vitrification. There are also a few examples of the utilisation of slow controlled freezing, which correspond to the earlier experiments performed with somatic embryos. The development and application of cryopreservation is significantly more advanced for somatic embryos, in comparison with zygotic embryos, mainly because of the different origin and characteristics of the species treated. In most cases, zygotic embryos originate from tropical, wild species, for which knowledge and techniques relevant to the development of cryopreservation protocols are limited, or even non-existent. By contrast, somatic embryos are generally produced from cultivated species, which have already been studied extensively and for which propagation techniques are already operational. A number of technical possibilities to explore exist in order to improve the development of cryopreservation protocols for zygotic embryos and embryonic axes. For both categories of materials, the utilisation of analytical techniques has proved to be extremely useful to assist in the development of cryopreservation protocols.

In this study in vitro shoot tips of a Sicilian genotype of Limonium serotinum were successfully cryopreserved using the droplet-vitrification technique. Growth recovery of cryopreserved shoot tips was possible only when samples were pretreated for 16 h in liquid medium with 0.3 M sucrose, then for 5 h in liquid medium with 0.7 M sucrose before performing the cryopreservation protocol. Optimal conditions included treatment for 20 min in a loading solution containing 1.9 M glycerol + 0.5 M sucrose, treatment with vitrification solution B5 (glycerol 40.0%, sucrose 40.0%, w/v) for 60 and 90 min or vitrification solution A9 (glycerol 30.0%, dimethylsulfoxide 20.0%, ethylene glycol 20.0%, sucrose 15.0%) for 20 min, rapid cooling in minute droplets of vitrification solution, rapid rewarming by immersion for 20 min in unloading solution containing 1.2 M sucrose. Under these conditions, 37% recovery of cryopreserved shoot tips was achieved. Regrowth of cryopreserved samples was slow but always direct, without callus formation.

Cryopreservation (liquid nitrogen, -196°C) is the only safe and cost-effective option for long-term -conservation of genetic resources of non-orthodox seed species. Cryopreservation protocols have been developed for various materials including seeds, dormant buds, cell suspensions, calli, apices, zygotic, and somatic embryos of numerous plant species. Zygotic embryos or embryonic axes of almost 100 different species and somatic embryos of almost 40 different species from both temperate and tropical climates, comprising crops, fruit, and forest trees as well as wild species, whose seeds displayed orthodox, intermediate, and recalcitrant storage characteristics, have been successfully cryopreserved. With zygotic embryos and embryonic axes, the desiccation technique has been used with the majority of the species tested, leading to highly variable survival and recovery after freezing, especially during earlier experiments. More recently, new cryopreservation techniques viz. encapsulation-dehydration and vitrification have been employed, leading to generally improved results. With somatic embryos, different cryopreservation methods have been used viz. desiccation, pre-growth-desiccation, encapsulation-dehydration, vitrification, encapsulation-vitrification, and droplet-vitrification. There are also a few examples of the utilisation of slow controlled freezing, which correspond to the earlier experiments performed with somatic embryos. The development and application of cryopreservation is significantly more advanced for somatic embryos, in comparison with zygotic embryos, mainly because of the different origin and characteristics of the species treated. In most cases, zygotic embryos originate from tropical, wild species, for which knowledge and techniques relevant to the development of cryopreservation protocols are limited, or even non-existent. By contrast, somatic embryos are generally produced from cultivated species, which have already been studied extensively and for which propagation techniques are already operational. A number of technical possibilities to explore exist in order to improve the development of cryopreservation protocols for zygotic embryos and embryonic axes. For both categories of materials, the utilisation of analytical techniques has proved to be extremely useful to assist in the development of cryopreservation protocols.

Coconut is a very important crop for millions of people in tropical countries. With coconut, in vitro culture protocols have been developed with two main objectives, viz. the large scale production of particular types of coconuts and the international exchange and conservation of coconut germplasm. The methods described in this chapter have been developed in the framework of collaborative activities between research institutes in Cote d'Ivoire and France. Two coconut embryo in vitro collecting protocols have been established, one consisting of storing the disinfected embryos in a KCl solution until they are brought back to the laboratory, where they are re-disinfected and inoculated in vitro under sterile conditions, and the other including in vitro inoculation of the embryos in the field. For international germplasm exchange, zygotic embryos inoculated in vitro in plastic test tubes or endosperm cylinders containing embryos in plastic bags are used. For in vitro culture, embryos are inoculated on semi-solid medium supplemented with sucrose and activated charcoal and placed in the dark, and then transferred to light conditions with the same (solid or liquid) medium once the first true leaf is visible and the root system has started developing.

Apical shoot tips excised from in vitro plantlets of blackberry (Rubus fruticosus L. 'Cacanska Bestrna') and cherry plum (Prunus cerasifera Ehrh.) were tested for recovery after cryopreservation using the droplet-vitrification technique. Following treatment for 30 min with a loading solution comprising 1.9 M glycerol and 0.5 M sucrose, explants were dehydrated with a highly concentrated cryoprotectant solution, so called vitrification solution. Shoot tips were dehydrated for 10,20 and 30 min at room temperature with a solution derived from the original PVS2 solution (containing 37.5% (w/v) glycerol, 15% (w/v) dimethylsulfoxide, 15% (w/v) ethylene glycol and 22.5% (w/v) sucrose) and for 60,90 and 120 min using the PVS3 solution (containing 50% (w/v) glycerol and 50% (w/v) sucrose). Explants were cooled by direct immersion in LN in 10 mu l droplets of vitrification solution placed on aluminium foil strips. Rewarming was done by direct plunging of foil strips in a preheated (37 degrees C) unloading solution (0.8 M sucrose) for 30 s, after which an equal volume of unloading solution (at room temperature) was added for further incubation for 30 min. As for regrowth of blackberry, PVS3 proved more effective than the modified PVS2, but the difference was significant (P < 0.05) only for the shortest treatment duration. The duration of PVS3 treatment had no significant effect on regrowth of cryopreserved shoot tips (45.8-70%). By contrast, a 30-min treatment with modified PVS2 solution resulted in a significant increase in regeneration percentage (30%), as compared with a 10-min treatment with the same solution (5%). Cherry plum shoot tips were very sensitive to both vitrification solutions and growth recovery of cryopreserved samples was generally lower (5-20%) than that of blackberry explants. No significant influence of PVS treatment (both type of solution and treatment duration) on regrowth of cryopreserved shoot tips was observed with cherry plum shoot tips. Experiments performed in France and in Serbia produced similar results, thereby showing the robustness and reproducibility of the protocols developed.

This study evaluated the survival and recovery of non-encapsulated and encapsulated shoots of Sequoia sempervirens after storage at 4 °C in the dark for up to 15 months on four different culture media. Survival and regrowth of encapsulated shoots declined within 3 months, regardless of the storage medium composition. By contrast, no significant decrease in survival and regrowth was noted with non-encapsulated shoots after 12 months of storage on Quoirin and Lepoivre medium supplemented, or not, with 1 mg l −1 benzyladenine. Regrowth dropped to 60–61% after 15 months of storage on the same media. Medium-term conservation of S. sempervirens germplasm is therefore possible using in vitro storage of non-encapsulated shoot cultures.

Optimiziranje uvjeta kriprezervacije nužno je za njezino korištenje u očuvanju biljnih genetskih resursa. U ovom radu uspoređuju se dva protokola za krioprezervaciju vegetacijskih vršaka vinove loze, enkapsulacija-dehidratacija (E-D) i droplet-vitrifikacija (D-V). Mikro reznice (nodijski odsječci) s jednim pupom posađene su na 1/2 MS medij s dodatkom 1μM zeatin-ribozida (ZR) te su na tom mediju rasle različito vrijeme prije izolacije vegetacijskog vrška i krioprezervacije. Kod metode E-D testiran je utjecaj predtretmana saharozom (koncentracija i trajanje), trajanje dehidracije i veličina eksplantata. Predtretman s dnevnim povećanjem koncentracije saharoze od 0.25 do 1.0M i dehidracija do 24% sadržaja vode (u svježoj tvari), te veličina eksplantata od 2 i 1 mm rezultirali su regeneracijom od 16% i 36%. Kod metode D-V uspoređene su vitrifikacijske otopine (PVS2 i PVS3). Najveći postotak regeneracija nakon krioprezervacije (50%) postignut je pri izolaciji vegetacijskih vršaka sa mikroreznica nakon 2 tjedna, te vitrificiranih 50 min sa PVS2. Do regeneracije nije došlo kod vršaka izoliranih nakon dužeg vremena na mediju sa ZR, te je stoga potrebno definirati optimalno trajanje kulture nodijskih odsječaka na ZR mediju.

Optimal conditions for the medium-term conservation of zygotic embryos of coconut were developed. After 6 months of storage on a medium devoid of sucrose and containing 2 g l−1 activated charcoal, 100% of the embryos developed into whole plantlets within 5 months after transfer to the recovery medium. After a 12-month storage period on medium containing 15 g l−1 sucrose and devoid of activated charcoal, 51% of the embryos germinated within 2 months after transfer to the recovery medium.

Des microboutures de caféiers (#Coffea arabica$ L. et #Coffea canephora$ Pierre) ont été conservées #in vitro$ pendant 0-20 semaines à différentes températures (12, 17, 22 et 27°C). La survie est généralement élevée, sauf pour #C. canephora$ conservée à 12°C. Après une augmentation transitoire de la concentration en glucose et fructose au début de la période de stockage, le niveau de sucre reste stable. Pendant les premières semaines de conservation, la quantité de proline est multipliée par deux pour les deux espèces, à 12°C. Une chute de la concentration en proline est observée chez #C. canephora$ à la fin de la période de conservation à 12°C. Alors que chez #C. arabica$, aucune modification n'est notée dans la concentration en malonedialdéhyde (MDA), son niveau augmente dans les boutures de #C. canephora$ stockées à 12°C. Cette augmentation est réversible après 10 jours à 27°C, au début de la période de stockage, mais elle devient irréversible après 20 semaines de conservation. La production d'éthylène démarre après transfert à 27°C dès deux semaines de conservation pour #C. canephora$, mais après 12 semaines seulement pour #C. arabica$. Cette synthèse diminue à la fin de la période de stockage chez les boutures de #C. canephora$ stockées à 12°C. Les résultats indiquent que parmi les quatre composés utilisés, le MDA apparaît comme le meilleur indicateur de la sensibilité au froid chez les deux espèces de caféier étudiées. (Résumé d'auteur)

In this work, a morpho-histological study of banana (Musa spp. cv. Grande Naine [AAA]) embryogenic cell suspensions during cryopreservation and regeneration was performed. It was demonstrated that the regeneration process of somatic embryos originating from cryopreserved cell suspensions was different from that of control cell suspensions. Somatic embryos originating from cryopreserved cell suspensions had a unicellular origin. The regeneration process was modified not only by freezing in liquid nitrogen but also by the plasmolyzing effect of the 0.5 M sucrose solution employed during pretreatment. This result explained the high number of embryonic structures formed on M3 medium, compared with the control. Proembryos blocked at the globular stage could pursue their development when they were plated on new culture medium at a lower density after 30 days of culture on M3 medium. The unicellular origin of somatic embryos produced from cryopreserved cell suspensions offers the prospect of using cryopreservation to select non-chimeral transformed plants.

A cryopreservation protocol using the encapsulation-dehydration procedure was established for shoot tips (2-3 mm in length) and meristems (0.3-0.5 mm) sampled from in vitro plantlets of diploid and triploid cytotypes of Arachis pintoi. The optimal protocol was the following: after dissection, explants were precultured for 24 h on establishment medium (EM), encapsulated in calcium alginate beads and pretreated in liquid EM medium with daily increasing sucrose concentration (0.5, 0.75, 1.0 M) and desiccated to 22-23 percent moisture content (fresh weight basis). Explants were frozen using slow cooling (1 C per min from 25C to -30C followed by direct immersion in liquid nitrogen), thawed rapidly and post-cultured in liquid EM medium enriched with daily decreasing sucrose concentrations (0.75, 0.50, 0.1 M). Explants were then transferred to solid EM medium in order to achieve shoot regeneration, then on Murashige and Skoog medium supplemented with 0.05 microM naphthalene acetic acid to induce rooting of shoots. With this procedure, 53 percent and 56 percent of cryopreserved shoot tips of the diploid and triploid cytotypes, respectively, survived and formed plants. However, only 16 percent of cryopreserved meristems of both cytotypes regenerated plants. Using ten isozyme systems and seven RAPD profiles, no modification induced by cryopreservation could be detected in plantlets regenerated from cryopreserved material.