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Key message Establishment of a cryopreservation protocol for Passiflora suberosa shoot tips with the V-Cryo-plate technique, and assessment of the oxidative stress for the determination of the critical stages of the protocol. Passiflora suberosa L. is a wild species of Passiflora, with great agronomic, ornamental and medicinal potential. In spite of this, there are few biotechnological studies aiming at its in vitro propagation and conservation. Thus, the development of cryopreservation protocols is considered of great relevance for this species. However, cryopreservation may be associated with oxidative damages, which cause injuries and may result in low recovery frequencies. The goal of this work was the establishment of a cryopreservation protocol for P. suberosa shoot tips with the V-Cryo-plate technique, evaluating the influence of the age of the explant and exposure to the vitrification solutions PVS2 and PVS3. In addition, the occurrence of oxidative stress at the different stages of the protocol was evaluated by monitoring lipid peroxidation and the activity of antioxidant enzymes. Plant recovery from cryopreserved shoot tips occurred at distinct frequencies, according to explant age and exposure to the vitrification solutions. Highest post-freezing recovery was observed in 40-day old shoot tips treated with PVS2 for 60 min (45%) or PVS3 for 45 to 90 min (50-60%). The occurrence of oxidative stress was evaluated by the quantification of lipid peroxidation through malondialdehyde detection, total protein content, and activity of the antioxidant enzymes superoxide dismutase, catalase and ascorbate peroxidase. These assays revealed that oxidative stress mainly occurred at the osmoprotection and PVS3 dehydration stages, which were considered as the most critical of the V-Cryo-plate protocol for the cryopreservation of P. suberosa shoot tips.

Main conclusion This paper reviews the cryopreservation of the ornamental, carnation (Dianthus caryophyllus L.), as an important method for the long-term preservation of this plant's germplasm. Carnation (Dianthus caryophyllus L.) is an important ornamental plant that is used as a potted plant as well as a cut flower. Important Dianthus germplasm would benefit from long-term strategies such as cryopreservation. Unlike the in vitro tissue culture literature of this ornamental, which has been studied in considerable detail, and with several genetic transformation protocols, surprisingly, the literature on its cryopreservation is still fairly scant, with barely two dozen or so studies, mostly having employed shoot tips. Early (< 2007) and more recent (2007-2020) cryopreservation techniques for carnation, including ultra-rapid cooling, encapsulation-vitrification, and encapsulation-dehydration, efficiently replaced programmed slow cooling processes used in early studies in the 1980s. Two large gaps (1997-2006, and 2016-2020) in which no carnation cryopreservation studies were published, requires future studies to cover new knowledge to fill gaps in information. Carnation cryopreservation research would benefit from testing a wide range of in vitro explants, new techniques such as the cryo-mesh, improved regeneration protocols for post-cryopreserved material, and the use of low-temperature storage as a mid- to long-term complementary germplasm storage strategy. This mini-review provides details of what has been achieved thus far and future objectives that could fortify cryopreservation research of this ornamental, as well as provide a robust long-term germplasm repository.

Optimized cryopreservation protocols are essential for the safe, cost-effective and long-term conservation of germplasm of great economic interest, such as Guinea (Petiveria alliacea L.), a medicinal species that synthesizes a great diversity of bioactive substances, among them polysulfides with antitumor activity. Somatic embryos (SEs) produced from in vitro roots were cryopreserved using the V-cryoplate technique. Their viability was measured using the triphenyltetrazolium test and their recovery by counting the number of secondary SEs produced per cryopreserved SE 90 days after liquid nitrogen (LN) exposure. Structural alterations were evaluated qualitatively and quantitatively during the successive stages of the protocol. SEs were dehydrated in 0.5 M sucrose, attached to cryoplates using sodium alginate solution (3%) and treated with PVS2 for different periods. After immersion in LN, SEs were rewarmed in unloading solution (1.2 M sucrose) at room temperature (25 °C) for 20 min. Viability based on the triphenyltetrazolium test was 100% after 15 min of treatment with PVS2 and LN exposure, and recovery, based on multiplication rate, was 21 somatic embryos produced per cryopreserved somatic embryo after 90 days of culture. The histological analysis of cryopreserved somatic embryos showed plasmolysis in the different cell types after treatment with PVS2, with meristematic and parenchymatic cells presenting a higher plasmolysis level. However, after rewarming, the level of plasmolysis decreased over cultivation time, reaching only 2% in meristematic cells after 30 days, indicating the good ability of Petiveria alliacea SEs to develop cryotolerance under the experimental conditions tested.

Petiveria alliacea L. is a medicinal plant originating from the Amazon region. This study describes an efficient cryopreservation protocol for somatic embryos (SEs) produced from roots of P. alliacea based on the comparison of vitrification, encapsulation-dehydration, and D cryo-plate techniques. With the vitrification technique, SEs treated with PVS2 solution (0.4 M sucrose, 3.3 M glycerol, 2.4 M ethylene glycol, and 1.9 M DMSO) for 30 min displayed high viability (85%) and intermediate proliferation recovery (about 12 adventitious SEs produced from original SEs [SEs/SE] after 90 d of culture). With the encapsulation-dehydration technique, lower viability (70%) and very low proliferation recovery (about two SEs/SE) were achieved with cryopreserved SEs dehydrated for 10 min in a laminar air flow cabinet. The D cryo-plate technique led to high viability (85%) and proliferation recovery (19 SEs/SE) of cryopreserved SEs after 90 min dehydration. In the experimental conditions tested, the D cryo-plate method was the most efficient technique for cryopreservation of P. alliacea SEs.

Adventitious root cultures of Tarenaya rosea were successfully cryopreserved using the encapsulation-vitrification technique. Histological analysis revealed useful information on the successive steps of cryopreservation. Coupled with complementary histochemical approaches, these studies provided cellular and tissue descriptions of T. rosea root cultures during cryopreservation and contributed to an understanding of cellular stress responses, as well as characterization of the anatomical pattern of root regeneration. The effects of exposure duration to PVS3 solution (0–120 min), unloading treatment (direct and gradual), and recovery medium (liquid and solid) on recovery of cryopreserved roots were investigated. The highest recovery (91%) after cooling in liquid nitrogen (LN) was reached with PVS3 treatment for 90 min, gradual rehydration in unloading solution, and recovery on solid MS medium. The cryopreserved roots showed high multiplication capacity, which was maintained for up to four subcultures. The effect of cryopreservation on root structure was investigated by histological and histochemical studies. Plasmolysis intensified during exposure to loading and PVS3 solutions, but decreased after unloading treatment. The proportion of intercellular spaces increased progressively throughout the cryopreservation protocol, culminating in root cortex disruption. Histochemical analyses revealed polysaccharides, proteins, and both lipidic and pectic substances in intercellular spaces. The vascular cylinder remained intact, ensuring the formation of new roots from the pericycle, showing that proliferative capacity of cryopreserved roots had not diminished.

International audience; Cryopreservation stands out as the main strategy to ensure safe and cost efficient long-term conservation of plant germplasm, especially for biotechnological materials. However, the injuries associated with the procedure may result in structural damage and low recovery rates after cooling. Histological analysis provides useful information on the effects of osmotic dehydration, LN exposure, and recovery conditions on cellular integrity and tissue organization, allowing the determination of the critical steps of the cryopreservation protocol and, thus, the use of optimized treatments. Passiflora pohlii Mast. (Passifloraceae) is a native species from Brazil with potential agronomic interest. Recent studies showed the presence of saponins in its roots, which presented antioxidant activity. The goal of this work was to develop a cryopreservation technique for root tips of in vitro-derived plants of P. pohlii using the V-Cryo-plate technique and to characterize the anatomical alterations that occurred during the successive steps of the protocol. Root tips were excised from in vitro plants and precultured before adhesion to cryo-plates and then treated for different periods with the plant vitrification solutions PVS2 or PVS3. Treatment with PVS2 for 45 min resulted in higher recovery (79%) when compared with PVS3 (43%). The greatest number of adventitious roots per cryopreserved explant was also observed after a 45-min exposure to PVS2. Plasmolysis levels were higher in cortical cells of cryopreserved explants treated with PVS2, while pericycle and central cylinder cells were not damaged after this treatment. Thirty days after rewarming, no plasmolysis could be detected, regardless of the experimental conditions.

We recorded the crypreservation effects (direct immersion) on various parameters of early germination stages of maize seeds (0, 7 and 14 days). Percentages of germination; fresh mass of different seedling parts; levels of chlorophyll pigments (a, b); carotenoids; malondialdehyde; other aldehydes; phenolics (cell wall-linked, free) and proteins were determined. Various statistically significant effects of seed exposure to liquid nitrogen (LN) were recorded. Maize seeds did not seem to be affected by LN exposure either visually or regarding fresh weight or germination rate. However, delayed growth was observed in seedlings recovered from cryopreserved seeds. This trend indicated an increase in the effect of seed cryopreservation on growing plants. The most significant effects of LN exposure were recorded in the combined fresh weight of stems and leaves at day 7 of germination and in fresh weights of roots, stems and leaves at day 14. At the biochemical level, numerous indicators varied following LN exposure, but the most significant effects were recorded in carotenoids, malondialdehyde and other aldehyde contents. LN exposure modified 50.0% of indicators in cotyledons, 48.1% in stems and leaves, 38.8% in roots and 11.1% in seeds. LN storage modified 11.1% of the variables measured at day 0 of germination, 37.0% at day 7, and 52.7% at day 14. Field performance of cryostored seed-derived plants should be evaluated to measure the durability of the changes observed.

In this study, we applied the vitrification (V) cryo-plate protocol for cryopreservation of Clinopodium odorum in vitro-derived shoot tips. We studied the recovery of shoot tips after modification of various parameters of the V cryo-plate protocol, including sucrose concentration in the preconditioning medium, composition of the loading solution (LS) and vitrification solution (VS) and duration of treatment with PVS2 VS, which contained (w/v) 30% glycerol, 15% dimethylsulphoxide, 15% ethylene glycol and 13.7% sucrose. We also compared the efficiency of the V cryo-plate protocol with the dehydration (D) cryo-plate protocol. The optimal conditions determined for the V cryo-plate protocol included a 24-h preconditioning treatment on medium with 0.3 M sucrose; treatment for 20 min at room temperature with LS containing 2.0 M glycerol and 0.4 M sucrose; and dehydration with PVS2 for 60 min at 0A degrees C. Under these conditions, 71.0% recovery of cryopreserved shoot tips was achieved. Only 29.2% regeneration was noted with the D cryo-plate protocol.

Jatropha curcas is a species with a variety of uses. It is grown primarily for oil for biodiesel, but also has agronomic and medicinal applications. Two methods were evaluated for cryopreservation of seeds and zygotic embryos of J. curcas: desiccation followed by rapid immersion of seeds and embryos in liquid nitrogen (LN, -196°C), and vitrification of zygotic embryos. Prior to cryo-preservation, seeds were manually scarified and the moisture content (MC) of seeds and embryos was determined. Explants were disinfected after cryopreservation. Seed germination after LN exposure was 100%. Plantlet development was better in sand substrate than that in vitro. Survival of zygotic embryos after cryopreservation was also 100%, without significant differences between treatments. Optimal development (100%) and plantlet length (51.77 mm) were observed with embryos dried for 60 min to 9.4% MC under laminar flow prior to cryopreservation. Zygotic embryos subjected to the vitrification procedure did not withstand LN exposure. Survival data for non-cryopreserved embryos after each step of the vitrification procedure provided information about embryo tolerance to cryoprotectants.

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.

In this work we compared the effect of the growth regulator content of the culture medium on the growth of in vitro shoot tips of five yam accessions belonging to four yam species (one Dioscorea alata, one D. rotundata, one D. cayenensis and two D. trifida). Medium S contained 0.6 µM benzyl adenine, 1.07 µM naphthalene acetic acid and 0.23 µM gibberellic acid while medium EBR contained 0.23 µM gibberellic acid and 0.1 µM 24-epibrassinolide. After 2 months of culture, oxidation level was significantly reduced on medium EBR compared to medium S for four of the five accessions tested. By contrast, medium EBR did not have any positive effect on shoot length since length of shoots produced after 2 months of culture on medium S and EBR were similar, except with accession 3-45T, for which shoot length was shorter on medium S compared to medium EBR. These results underline the potential of 24-epibrassinolide to reduce oxidation phenomena during in vitro culture and call for its utilization for regeneration of cryopreserved yam shoot tips, which is often impeded by oxidation phenomena.

The objective of this work was to evaluate if cryostorage of Phaseolus vulgaris L. seeds induced variations in regenerated plants at the phenotypic and molecular levels. A series of agricultural traits was measured on plants grown from control, non-cryopreserved and cryopreserved seeds, and the genetic stability of plants of the second generation was analysed at selected microsatellite loci. The phenotype of the second generation plants was evaluated as well. No statistically significant phenotypic differences were observed for the parameters measured, neither in the first nor in the second generations. Averaging both treatments, about 76% of the seeds had germinated 10 days after sowing. At harvest we recorded plants with about 73 cm in height, 13 stem internodes, 25 fruits, 103 grains and 4 grains per fruit. One hundred seeds weighted about 26 g. The genetic analyses performed on the second generation plants using six nuclear Simple Sequences Repeats (SSR) markers revealed no changes in microsatellite length between control and cryopreserved samples, implying that there was no effect of seed liquid nitrogen exposure on genome integrity. The phenotypic and molecular results reported here confirm that cryostorage is an efficient and reliable technique to conserve P. vulgaris seeds and regenerate true-to-type plants.

Oil palm (Elaeis guineensis Jacq.), a tropical plant, is the leading source of edible oil. This review deals with the cryopreservation of oil palm as a way to preserve this important tropical germplasm. Somatic embryos have been the most popular source of material for cryopreservation as they are propagules that are effectively produced during micropropagation. In contrast, fewer studies exist on the cryopreservation of pollen, zygotic embryos, seeds, kernels and embryogenic cell suspensions. This review highlights the ideal protocols, in detail, in a bid to offer guidance for further advances in oil palm cryopreservation.

International audience; Cryopreservation is the only alternative, safe and cost-effective method for long-term storage of plant genetic resources, particularly for stone fruits (Prunus spp.). In this study, an efficient cryopreservation protocol was developed for sour cherry (Prunus cerasus L.). In vitro shoot tips of two varieties (Montmorency and Schattenmorelle) were successfully cryopreserved using the vitrification technique. Our study showed the possibility of replacing the 3-week cold hardening treatment of mother-plants with preculture of apices on medium enriched with sucrose and/or glycerol. The highest recovery percentages after liquid nitrogen exposure were obtained after a cold hardening treatment followed by a 3-day preculrure on 0.8 M sucrose medium or by replacing cold hardening with a 7-day preculture on the following media: 0.4 M glycerol or sucrose, 0.4 M sucrose + 0.4 M glycerol or 0.8 M glycerol. Under these conditions, recovery after cryopreservation ranged between 41 and 63%, These results complement the range of Prunus species successfully cryopreserved using in vitro explants. Our protocol, which is simplified in comparison with the original one, since cold hardening of mother-plants in a cold chamber is replaced by pretreatment of apices on medium with high sugar concentration, may facilitate the application of vitrification for cryopreservation of additional Prunus species

An efficient protocol for cryopreservation of madder hairy root cultures has been developed using droplet-vitrification. In previous study, combining loading solution C4 (35% PVS3) and vitrification solution B5 (80% PVS3) was the most effective method. In this study, we tried three types of vitrification solution, B5, A3 (90% PVS2, on ice), and A5 (70% PVS2, on ice). Combining loading solution C4 and vitrification solution A5 (on ice) showed the best regeneration rate in this study. Histological changes of the cells within the hairy root of madder were also observed in different steps. The cells from the hairy roots of the control treatment were full and intact with different size of vacuoles and obvious cell nucleus having a dark nucleolus. After the stage of preparing for cryopreservation (after preculturing, loading, followed by dehydration solution A5 or B5), intercellular spaces had become distinct, and within cells, the cytoplasms had become denser and week plasmolyses had appeared. The cell plasmolyses were much more apparent and we measured the degree of plasmolysis by calculating, the area of cell/the area of cytoplasm. The value of plasmolysis degree was the highest in the combination of preculture, loading solution C4, and dehydration solution A5, 1.97. Because the highest regeneration rates appeared in the treatment of A5 for 20 min, we could assume that the optimal degree of plasmolysis for cryopreservation might be around 1.97. The changes in cell structure during cryopreservation might be a useful basis for the development of a proper long-term preservation method for madder germplasms.

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 24 h 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.

Coffee (Coffea spp.) is one of the world?s most valuable agricultural export commodities produced by small-scale farmers. Its germplasm, which holds useful traits for crop improvement, has traditionally been conserved in fi eld genebanks, which presents many challenges for conservation. New techniques of in vitro and cryopreservation have been developed to improve the Long-term conservation of coffee. But a question remains as to whether these new techniques are more cost effective than fi eld collections and more effi cient at reducing genetic erosion. This study compared the costs of maintaining one of the world?s largest coffee fi eld collections with those of establishing a coffee cryo-collection at the Centro Agronómico Tropical de Investigación y Enseñanza (CATIE) in Costa Rica. The results indicate that cryopreservation costs less (in perpetuity per accession) than conservation in fi eld genebanks. A comparative analysis of the costs of both methods showed that the more accessions there are in cryopreservation storage, the lower the peraccession cost. In addition to cost, the study examined the advantages of cryopreservation over fi eld collection and showed that for species that are diffi cult to conserve using seeds, and that can only be conserved as live plants, cryopreservation may be the method of choice for long-term conservation of genetic diversity. Made available in DSpace on 2011-04-09T22:27:55Z (GMT). No. of bitstreams: 1 Costefficiency.pdf: 871770 bytes, checksum: 8bd257ccdbf65f45dffbcc9248c7172a (MD5) Previous issue date: 2011-03-10

In this work, we observed the effect of hormonal content of four culture media on the growth of Dioscorea trifida shoot tips. Medium S included 0.6 µM 6-benzylaminopurine, 1.07 µM naphthalene acetic acid, and 0.23 µM gibberellic acid (GA3), medium EBR 0.1 µM 24-Epibrassinolide and 0.23 µM GA3, medium T 25 μM meta-topolin and 0.23 µM GA3 and medium ZR 25 μM zeatin riboside and 0.23 µM GA3. After 2 months in culture, shoot length was highest on medium EBR and ZR, with values of 11.63 and 11.30 mm, respectively, intermediate on medium S (9.70 mm) and lowest on medium T (3.07 mm). Oxidation reached a similar level on medium S, T and ZR (2.17 - 2.40) while it was only 1.63 on medium EBR. Well-developed shoots were obtained on medium EBR with an average of 1.93 leaves and 0.70 roots per shoot. Shoots were less developed on medium S and ZR, with an average of 1.30 leaves and 0.20 roots per shoot on medium S and 1.60 leaves and 0.00 roots per shoot on medium ZR. On medium T, buds showed a tuberized aspect and no leaves or roots were produced. In conclusion, medium EBR proved superior to the three other media tested based on the parameters tested.Keywords: Yam, Dioscorea trifida, shoot tip growth, 6-benzylaminopurine, 24-Epibrassinolide, meta-topolin, zeatin ribosideAfrican Journal of Biotechnology, Vol 13(15), 1645-1649

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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)

Cryotherapy of shoot tips is a new method for pathogen eradication based oncryopreservationtechniques.Cryopreservationreferstothestorageofbiologicalsamplesatultra-lowtemperature,usuallythatofliquidnitrogen(2196 C),andis considered as an ideal means for long-term storage of plant germplasm. Incryotherapy, plant pathogens such as viruses, phytoplasmas and bacteria areeradicated from shoot tips by exposing them briefly to liquid nitrogen. Unevendistribution of viruses and obligate vasculature-limited microbes in shoot tipsallowseliminationoftheinfectedcellsbyinjuringthemwiththecryo-treatmentand regeneration of healthy shoots from the surviving pathogen-free meriste-maticcells. Thermotherapyfollowed bycryotherapy ofshoot tipscan beused toenhance virus eradication. Cryotherapy of shoot tips is easy to implement. Itallows treatment of large numbers of samples and results in a high frequency ofpathogen-free regenerants. Difficulties related to excision and regeneration ofsmall meristems are largely circumvented. To date, severe pathogens in banana(Musa spp.), Citrus spp., grapevine (Vitis vinifera), Prunus spp., raspberry (Rubusidaeus), potato (Solanum tuberosum) and sweet potato (Ipomoea batatas) havebeen eradicated using cryotherapy. These pathogens include nine viruses(banana streak virus, cucumber mosaic virus, grapevine virus A, plum poxvirus, potato leaf roll virus, potato virus Y, raspberry bushy dwarf virus, sweetpotato feathery mottle virus and sweet potato chlorotic stunt virus), sweetpotato little leaf phytoplasma and Huanglongbing bacterium causing ‘citrusgreening’. Cryopreservation protocols have been developed for a wide varietyof plant species, including agricultural and horticultural crops and ornamentalplants, and can be used as such or adjusted for the purpose of cryotherapy.Conservation of plant genetic resourcesfor food securityThe world population today is 6.6 billion and is predictedtoincreaseby50%before2050(UNFPA,2007).Providingfood security for such a rapidly growing population willrequire a dramatic increase in crop production. Obvi-ously, the challenge for further development of cropquality and quantity is much greater than ever (Hobbs,2007). Availability of plant genetic resources of cultivatedcrops and their wild relatives is a prerequisite for breedingof elite cultivars via classical breeding and biotechno-logical techniques. Sustainable agricultural productionthat ensures both crop quality and quantity largely

In this paper, we described the direct somatic embryogenesis from both immature cotyledon cuts and nucelli in the same mango cultivar (Mangifera indica L. var Zihua), studied the effect of growth conditions of embryogenic cultures (EMs) on cryopreservation and compared the cryopreservation response of EMs induced from these two different explants. Histological studies demonstrated that EMs derived from nucelli could be induced directly from epidermal cells of both sides of nucelli, whereas EMs derived from cotyledon cuts were induced only from epidermal cells of the adaxial side of the cotyledons. EMs from either nucelli or cotyledon cuts could be maintained in liquid medium or on solid medium and cryopreserved using a vitrification procedure. Success of cryopreservation of EMs depended on the dehydration treatment and the defined growth conditions during culture but not on their origins. When EMs were sampled during their exponential growth phase in liquid medium and dehydrated with PVS(3) solution for 5 min, survival of the EMs induced from cotyledon cuts and nucelli reached 77.7 and 80%, respectively, after cryopreservation in liquid nitrogen for 24 h. Furthermore, when dehydrated with PVS(3) solution for 30 min, all EMs induced from cotyledon cuts and 96.7% of EMs induced from nucelli could survive after cryopreservation. Cryopreservation did not affect the plant regeneration potential of EMs through somatic embryogenesis. The protocols of somatic embryogenesis and cryopreservation of mango EMs established in this study may offer potential ways to improve mango germplasm conservation and genetic improvement.

The effect of exposure to ultra-low temperature (liquid nitrogen, LN) on viability of seeds desiccated to various water contents was investigated in 9 coffee species. Three groups of species could be distinguished based on seed survival after LN exposure. In group 1 species, no seedling production could be obtained after LN exposure due to endosperm injury. In group 2 species, recovery was very low or nil after rapid cooling, and only moderate after slow cooling. In group 3 species, very high percentages of seedling development were observed after both rapid and slow cooling. A high interspecific variability for the high moisture freezing limit was observed within the species of groups 2 and 3, since it ranged from 0.14 to 0.26 g H2O g-1 dry weight. A very highly significant correlation was found for those species between the unfreezable water content, as determined from DSC analysis, and the high moisture freezing limit of their seeds. No significant correlation was found between seed lipid content, which varied from 9.8 to 34.6% dry weight, and survival after LN exposure. However, a negative relationship was found between seed unfreezable water content and lipid content. Interspecific differences in fatty acid composition of seed lipids resulted in a high variability in the percentage of unsaturated fatty acids, which ranged from 28.7 to 54.4% among the 9 species studied. For all species studied, a highly significant correlation was found between the percentage of unsaturated fatty acids and the percentage of seedling recovery after rapid or slow cooling.

Various problems are encountered in the establishment and maintenance of embryogenic cultures. The production of embryogenic material is often a difficult and lengthy process, as observed notably with oilpalm and coconut (Duval et al. 1995; Verdeil and Buffard-Morel 1995). The progressive decrease of embryogenic potential over time is a phenomenon which is commonly observed with embryogenic cultures (Withers 1985). Moreover, risks of somaclonal variation increase in line with the extension of the culture period (Reisch 1984). Finally, it is difficult and costly to maintain large numbers of proliferating cultures in large-scale production laboratories. Tools which would allow to overcome these difficulties are urgently needed to enhance the applicability of somatic embryogenesis processes for the mass propagation of elite material.

In the range of water contents studied (0.1–0.4 g H2O g dw−1),Coffea arabicaseeds were less sensitive to desiccation thanC. costatifructa, C. racemosaandC. sessilifloraseeds. At 0.20 g H2O g dw−1, 53% ofC. arabicaseeds germinated after direct immersion in LN (rapid cooling, 200°C min−1), but none of them developed into normal seedlings. By contrast, inC. costatifructa, C. racemosaandC. sessiliflora, when seeds were dehydrated to the optimal water content (0.19, 0.28 and 0.31 g H2O g dw−1, respectively), the percentages of seeds which developed into normal seedlings after LN exposure were 26, 78 and 31% of the desiccation control, respectively. Normal seedlings could be recovered from cryopreservedC. arabicaseeds only if they were desiccated to 0.20 g H2O g dw−1and precooled slowly to −50°C prior to immersion in LN. Precooling seeds at 2°C min−1allowed 25% of seeds to develop into normal seedlings. The thawing rate had no effect on the survival of cryopreservedC. arabicaseeds. In all cryopreservation experiments, the total germination did not reflect the percentage of seeds which developed into normal seedlings. Examination of excised embryos indicated a partial explanation of this difference since only the shoot apex was destroyed in abnormal embryos, whereas the hypocotyl and radicle were normal.

El chayote (Sechium edule) es una importante fuente de divisas para los países exportadores, entre los que Costa Rica mantiene el liderazgo mundial. Entre los factores más limitantes para su producci ón se encuentra el material de siembra, tradicionalmente la semilla, que por su naturaleza no permite la obtención de una producción morfológicamente homogénea de frutos (color, forma y textura de la epidermis) motivo de los altos porcentajes de rechazo en las plantas empacadoras. El establecimiento de una metodología de propagación vegetativa de la especie ayudaría a resolver los problemas de heterogeneidad. Por lo anterior, la presente investigación se dirigió a desarrollar un método de micropropagación a partir de brotes vegetativos. Se evaluó el efecto de varios desinfectantes y concentraciones de reguladores de crecimiento en las etapas de establecimiento, multiplicación y enraizamiento de dos cultivares comerciales. Se observó que una desinfección con hipoclorito de calcio al 4% permitió el mayor porcentaje de explantes establecidos asépticamente bajo condiciones in vitro (49%). También se observó que durante las etapas posteriores de la micropropagación, los requerimientos de reguladores de crecimiento fueron muy bajos (0,1 mgL-1 de BA en la etapa de brotación de yemas y de 0,1 a 0,2 mgL-1 de AIB durante el enraizamiento) o no se requirieron (etapa de multiplicaci ón de microestacas).

L'embryogenèse somatique s'avère être le seul moyen de multiplication clonale chez le palmier à huile. Le processus est initié à partir d'une étape de callogenèse. Les embryons somatiques sont obtenus à la suite de 3 méthodes de culture : - les cals à croissance rapide (FGC) qui aboutissent à des plantes anormales suite à une dérégulation du métabolisme des hormones de croissance endogènes; - les cals nodulaires sont utilisés en routine à l'ORSTOM / CIRAD, une multiplication à grande échelle n'est pas envisageable, car cette méthode ne convient pas à tous les clones et de nombreuses opérations manuelles sont nécessaires à toutes les étapes du processus; - la culture de suspensions embryogènes en bioréacteur qui est à l'étude

Cryopreservation using encapsulation-dehydration of apices of in vitro plantlets was investigated with sugarcane (Saccharum sp.), cassava (Manihot esculenta) and two coffee species (Coffea racemosa and C. sessiliflora). Maximum survival rates of frozen apices were 91% with sugarcane, 60% with cassava and 38% with coffee. Growth recovery of cryopreserved sugarcane apices was rapid and direct. With cassava and coffee, callusing was often observed during recovery of frozen apices, thus reducing the number of plants regenerated. Restriction fragment length polymorphism analyses of sugarcane revealed several modifications in the band patterns of plants regenerated both from control and cryopreserved apices which were identical in both materials. These variations were therefore not due to freezing but preexisting among the in vitro plantlets used. These results demonstrate that the encapsulation-dehydration technique can be employed for cryopreservation of apices of some tropical species.

The standard cryopreservation process previously developed for oil palm clones using shiny white, finger-like somatic embryos could be applied in some cases to standard cultures. Its efficiency was markedly improved by completing the 7-day pregrowth period on 0.75 M sucrose by an additional dehydration period carried out either by placing the embryos in the air current of the laminar flow cabinet or in an air tight box containing silica gel. This improved process was successfully applied to 7 different clones. It will facilitate the routine uof cryopreservation for oil palm cultures.

A cryopreservation process using encapsulation/dehydration was set up for apices sampled on in vitro plantlets of sugarcane. After dissection, apices were cultured for one day on standard medium and then encapsulated in medium with 3% alginate. Optimal conditions comprised preculture for 2 days in liquid medium with 250 g.l(-1) sucrose, desiccation for 6 hours under the laminar flow or for 10-11 hours with silicagel followed by rapid freezing and slow thawing. Survival after freezing in liquid nitrogen ranged between 38 and 91% for the 5 varieties experimented. Cryopreservation did not modify the electrophoretic profiles for aminoleucine peptidases and amylases with plants of the variety Co 6415.

In France, cryopreservation is currently used for a limited number of forest tree species, but has been applied yet very little to horticultural or other agricultural species. The CRYOVEG project aims at (1) developing or optimizing cryopreservation techniques in a range of selected species; and (2) establishing a national scientific and technical network of plant biological resource centers (CRBs) using cryopreservation. The project has a network organization, with IRD/INRA Montpellier as the cryopreservation expertise centre and partners in continental France and in overseas departments in charge of genetic resource conservation for various species: INRA Petit Bourg, Guadeloupe (yam); INRA San Giuliano, Corsica (Citrus); INRA Bordeaux (Prunus); INRA Angers (apple and pear); INRA Montpellier (grapevine); INRA Ploudaniel (potato, Brassica); IRD La Reunion (coffee); CIRAD Roujol, Guadeloupe (sugarcane); and CIRAD La Reunion (vanilla, garlic). Deliverables will vary depending on the state-of-the-art of cryopreservation for each species: for potato, yam, sugarcane, garlic, Citrus, Brassica and coffee, optimized protocols will be tested on a range of representative accessions. For grapevine, Prunus, apple, pear and vanilla, operational protocols will be developed. The group will work as a think-tank on the integration of cryopreservation in the national plant genetic resource conservation strategy and an action plan for its implementation will be collectively designed. The project will work towards establishing a French research network on plant genetic resource cryopreservation, open to all partners interested in cryopreservation. The group will prepare research and development projects whose results will be disseminated through European plant genetic resource conservation networks e.g. ECP/ GR). Finally, network members will train French and foreign (from North and South countries) researchers and technicians in cryopreservation techniques. (Conflicts of interest: None declared. Source of funding: This project is supported by a French competitive grant (Call for Projects 2008 ''Biological Resource Centers") of the Consultative Committee for Biological Resources/Infrastructures in the Biology, Health and Agronomy sectors (CCRB/IBiSA).). (Texte integral)