Search

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.

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.

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.

Cryopreservation has become a very important tool for the long-term storage of plant germplasm. A new cryopreservation protocol based on air dehydration of explants placed on aluminum cryo-plates, termed the D cryo-plate technique, was developed. In this study, the most suitable conditions of cryopreservation for dormant shoot tips of Japanese persimmon (Diospyros kaki Thunb. 'Saijo') using the D cryo-plate technique were investigated. Dormant one-year-old shoots of persimmon were collected from the experimental farm of Shimane University in January 2013 and stored at 2 degrees C until use. After surface sterilization, shoot tips of about 1 mm in size were dissected from the dormant buds and precultured overnight at 25 degrees C on solidified 1/2MS medium containing 0.3 M sucrose. Precultured shoot tips were placed on aluminum cryo-plates and embedded in calcium alginate gel. Osmoprotection of shoot tips was performed by immersing the cryo-plates for 30 min at 25 degrees C in an LS solution containing 2 M glycerol + 1.0 M sucrose in 1/2MS solution. For the D cryo-plate technique, encapsulated shoot tips were dehydrated by placing the cryo-plates in the air current of a laminar flow cabinet for 30-90 min. Cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, the cryo-plates were immersed in 1/2MS medium containing 1.0 M sucrose for 20 min at 25 degrees C. In this study, the preculture did not improve the regrowth after cryopreservation; however, we consider that it should be performed in the D cryo-plate procedure for application of other cultivars and utilization in genebanks. A high regrowth rate of cryopreserved shoot tips (84%) was achieved after dehydration for 30 min. This optimized procedure was applied to 10 additional persimmon cultivars, resulting in regrowth rates ranging between 67 and 97%, with an average of 87%. As shoot tips derived from dormant buds proved to be highly tolerant to liquid nitrogen exposure, the D cryoplate technique may facilitate long-term conservation of persimmon germplasm.

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

We evaluated the response of eight economically important Croatian grapevine cultivars and studied the impact of their sanitary status on in vitro introduction, by comparing the response of healthy and virus-infected genotypes of one cultivar. Nodal explant survival on three media, M1 (half-strength MS), M2 (full-strength MS) or M3 (full-strength MS with 4.4 µM L−1 benzylaminopurine) was measured after 2 weeks and regrowth after 8 weeks. After 8 weeks, average shoot length and node number were significantly higher on M2 compared to M1 and M3. M3 induced significantly shorter average internode length, compared to M1 and M2. Survival of one healthy and of five cultivar Plavac mali genotypes infected with GFLV, GLRaV-1, GLRaV-3, GLRaV-3+GVA and GLRaV-1+GLRaV-3 was 97.5 and 82.8–87.5%, respectively. Regrowth of the healthy genotype reached 95.5%, but dropped to 5.5–31.4% in infected ones. The healthy genotype showed significantly higher shoot length (6.3 cm) and node number (7.3) compared to infected genotypes, with shoot length between 1.2–2.6 cm and node number between 1.2–3.0. By contrast, internode length was not significantly different between the healthy and the infected genotypes. The present work represents the first successful in vitro introduction for three of the eight native Croatian cultivars studied.

In this study, we compared the evolution of morphological and agronomical characteristics of coconut (Cocos nucifera L.) palms produced from in vitro cultured embryos and from seeds over an 8-yr period. At the end of the second year after planting, the height and root collar diameter of plants originating from in vitro cultured embryos were significantly lower than those originating from seeds. However, palms from both categories had the same number of leaves. When palms originating from in vitro plantlets and from seeds were observed later in their development, they were similar for most morphological characteristics measured, except for minor differences in inflorescence morphology, which were still present 8 yr after planting. The flowering pattern, bunch, and fruit production were similar between the two categories of palms. These results indicate that in vitro culture of zygotic embryos does not adversely affect further development of palms in natural conditions.

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

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

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.

Many publications describe cryopreservation techniques but only a few studies have focused on the biochemical and physiological changes occurring in plants regenerated from seeds exposed to liquid nitrogen. This paper aims at describing the effect of common bean seed cryostorage on mineral nutrition of young plantlets. The following elements were measured on leaves of 10-day-old plantlets from non-cryopreserved and cryopreserved seeds: Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, S, Se, Sr and Zn. At 10 days after sowing, both treatments (control and cryopreserved seeds) showed 100% seed germination without any visual phenotypic difference. However, contents of several elements in the leaves were different. Exposure of seeds to liquid nitrogen decreased Cu, Cd and Na uptake and increased absorption of B and Al. Further studies are required to understand the mechanisms underlying the relationship between seed exposure to liquid nitrogen and mineral nutrition during the early stages of plantlet growth. DAAD Leibniz University of Hannover University of Ciego de Avila, Cuba

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

• 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 vitro medium term conservation of tropical plant germplasm is used routinely in many laboratories. Growth reduction is achieved by modifying various parameters, such as temperature, culture medium, gaseous environment. For long term conservation, cryopreservation (i.e. storage in liquid nitrogen, −196°C) is the only current method available. Each successive step of the process requires precise conditions which have to be defined for each material. Cryopreservation protocols have been set up for more than 40 tropical species. Results obtained with various culture systems such as cell suspensions, protoplasts, calluses, meristems and embryos are discussed. The first example of the large scale application of cryopreservation (oil palm somatic embryos) is presented.

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.

Two hypotheses, namely the occurrence of post-thaw oxidative stress or imbibitional damage, were tested to explain the high sensitivity of coffee seeds to liquid nitrogen (LN) exposure. Oxidative stress was studied by measuring primary and secondary products of lipid peroxidation in seeds during the desiccation and rehydration periods. The 4-hydroxynonenal (4-HNE) content of seeds remained constant throughout the desiccation step. No significant difference was observed between desiccated seeds and seeds desiccated and exposed to LN for the evolution of their 4-HNE and hydroperoxide contents during rehydration. In both cases, an increase in 4-HNE and hydroperoxide contents of seeds was observed during the first hours of culture under germination conditions, followed by a progressive decrease down to values comparable to those observed in desiccated seeds. The hydroperoxide composition of frozen seeds was not significantly different from that of control seeds. The (S)/(R) enantiomeric ratios of 9- and 13-hydroxy-octadecadienoic acid extracted from rehydrating seeds were chiral, suggesting that they originated from lipoxygenase activity. These results suggest that the high sensitivity of coffee seeds to LN exposure is not directly associated with the occurrence of an oxidative stress during post-thaw rehydration. The effect on seed viability of different rehydration procedures previously identified to reduce membrane imbibitional injury was studied after desiccation and LN exposure. Desiccation tolerance increased with, by increasing order, seed osmoconditioning, pre-heating and prehumidifying prior to their culture under germination conditions. Among the four combinations of pre-humidification durations (24 or 48 h) and temperatures (25 or 37 � C) tested, pre-humidification for 24 h at 37 � C gave the highest level of desiccation tolerance. This rehydration procedure also dramatically increased seed viability after LN exposure. Seed desiccation sensitivity modelling in combination with the calculation of the decrease in seed water activity during cooling facilitated the explanation of the beneficial effect of controlled rehydration after desiccation and LN exposure. These results support the hypothesis that imbibitional membrane damage is involved in the sensitivity of coffee seeds to LN exposure.

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.

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.