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Aims: This study aims to identify the best surface sterilization and evaluate the effect of haustorium suppression on in vitro germination of coconut palm (Cocos nucifera L.) zygotic embryos. Study Design: Survival rate and contamination rate of zygotic embryos after different surface sterilization treatments, regeneration rate and organogenesis through the number of leaves and the length of shoots after haustorium suppression were determined. For data processing, the Analysis of Variance was used to compare the means which were separated according to Tukey test (P = 0.05). Place and Duration of Study: Coconut fruits (hybrid PB121) were collected 12 to 14 months after controlled pollination from CRAPP (Centre de Recherches Agricoles Plantes Pérennes), station of Sèmè-kpodji in Benin. Experiments were done in Central Laboratory of Plant Biotechnology and Plant Improvement, University of Abomey-Calavi and conducted from june to december in 2019. Methodology: For the zygotic embryos surface sterilization, four treatments combining three concentrations (3%, 6% and 15%) of commercial bleach (Javel la Croix© containing 12° active chlorine) and immersion durations (5 min, 10 min and 20 min) were tested and the survival rate were determined for each treatment after two months culture. The zygotic embryos were then divided in two sets (haustorium excised embryos set and the whole embryos set) and cultured in modified Y3 medium supplemented with 7 g L-1 agar, 2.5 g L-1 activated charcoal, 5% sucrose, 6.10-3 mM 2.4 D (2.4-dichlorophonoxyacetic acid), gibberellic acid and 0.3 mM BAP(6-benzylaminopurine). After five months culture, the regeneration rate, the number of leaves and the length of shouts were recorded. Results: The high survival rate (80%) was obtained with 6% of bleach and 20 min for the immersion duration without pre-disinfection. The suppression of haustorium have significantly increased the number of leaves (4.3 ± 0.02) and the length of shoots (16.2 ±0.7cm) compared to the whole zygotic embryos. Conclusion: This protocol can help to ensure better surface sterilization of zygotic embryos before their in vitro culture and the development of vigorous plantlets in order to improve the slow growth of plantlets, when transferred to the greenhouse or field.

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.

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

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.

Genetic improvements in plant breeding are dependent upon having access to novel plant genetic resources that are available in plant genebanks. Many crops that are vegetatively-propagated are maintained as plants in the field or greenhouse, making them vulnerable to biotic and abiotic threats. Increasingly, plant genebanks are using cryopreservation technologies to secure vegetatively propagated collections at secondary locations. Droplet vitrification and cryo-plate cryopreservation methods have been used to successfully cryopreserve the shoot tips of many plant species. New propagule types, including small leaf square-bearing adventitious buds, stem disc-bearing adventitious buds, microtubers and rhizome buds are alternative explants for use in cryopreservation. This review describes new technologies for in-vitro based cryopreservation systems that have advanced the field of plant cryopreservation. Future advances will allow even more diverse germplasm to be successfully preserved in cryobanks. New technologies for in-vitro based cryopreservation systems have advanced the field of plant cryopreservation since the twenty first century. Further advances will certainly facilitate even more diverse germplasm to be successfully preserved in cryobanks.

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.

We studied the effects of liquid nitrogen storage of maize, common bean and soybean seeds on their germination, electrolyte leakage, levels of chlorophylls, phenolics, aldehydes, proteins and peroxidase activity. After storage for 28 days, seeds were retrieved from liquid nitrogen, some were set to germinate and others were analyzed biochemically. No phenotypic modifications were observed visually 5 days after beginning of germination, although percentage of seed germination was reduced by LN in maize and soybean. Moreover, numerous significant effects of seed cryopreservation were recorded at the biochemical status. In maize seeds, the most important and statistically significant modifications were observed in the increased levels of chlorophyll b and total chlorophyll pigments and in the decreased contents of free phenolics after 28 days of exposure to LN, compared to the control treatment. In common bean, relevant changes were observed in the increased electrolyte leakage and in the reduced levels of chlorophyll pigments (b, total) and free phenolics. In soybean, modifications were observed in the increased levels of chlorophyll pigments (a, b, total), malondialdehyde and electrolyte leakage, and in the decreased peroxidase activity. We have shown for the first time that immersion of maize, common bean and soybean seeds in liquid nitrogen modified the levels of different biochemicals.

[EN] Conservation of pineapple (Ananas comosus L. Merr.) genetic resources - including cryopreservation in liquid N-2 at -196 degrees C - is essential for future breeding programmes to develop new varieties with improved agronomic performance. However, the potentially deleterious effects of cryopreservation on subsequent plant regrowth should be evaluated before large-scale development of cryobanks is implemented. This paper describes the histological analysis of pineapple plantlets regenerated from cryopreserved shoot tips. Two controls were included in the study: i) conventional micropropagation-derived plantlets, and ii) plants from shoot tips subjected to pre-cryostorage conditioning treatments but never exposed to liquid N-2. Histological studies of roots, leaves and stems were conducted after 45 days of hardening. No statistically significant differences with the controls were observed in any of the histological parameters evaluated, which supports the practical value of cryopreservation of pineapple germplasm., This research was supported by the Bioplant Centre (Cuba), the Escuela Superior Politecnica Agropecuaria de Manabi Manuel Felix Lopez (Ecuador), the IRD Representation in Benin, and the Universitat Politecnica de Valencia (Spain).

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.

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 chapter, we describe a cryopreservation (liquid nitrogen, -196 °C) protocol developed for long-term storage of date palm pro-embryonic masses (PEMs), which uses the recently established D cryo-plate technique. Clumps of PEMs (3-5 mm in size) were dissected from PEM cultures and placed on pretreatment medium containing 171 g/L sucrose for 3 days. Clumps were placed in the wells of aluminum cryo-plates in which they were made to adhere using droplets of 3% calcium alginate. PEMs were treated for 20 min with a loading solution containing 184 g/L glycerol and 136.8 g/L sucrose. They were then dehydrated for 90-120 min in the air current of a laminar airflow cabinet and immersed directly in liquid nitrogen. For rewarming, the cryo-plates holding the PEMs were immersed for 15 min in an unloading solution containing 410.4 g/L sucrose. The PEMs were then detached from the cryo-plates, placed for 3 days in the dark on posttreatment medium containing 102.6 g/L sucrose, and transferred on recovery medium under light conditions. Using this protocol, 74.6 and 95.8% recovery were achieved with the PEMs of the two cultivars tested, Sukkari and Sultany.

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 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.

Advances in plant biotechnology provide new options for collection, multiplication and short- to long-term conservation of plant biodiversity, using in vitro culture techniques. Significant progress has been made for conserving endangered, rare, crop ornamental, medicinal and forest species, especially for non-orthodox seed and vegetatively propagated plants of temperate and tropical origin. Cell and tissue culture techniques ensure the rapid multiplication and production of plant material under aseptic conditions. Medium-term conservation by means of in vitro slow growth storage allows extending subcultures from several months to several years, depending on the species. Cryopreservation (liquid nitrogen, −196 °C) is the only technique ensuring the safe and cost-effective long-term conservation of a wide range of plant species. Cryopreservation of shoot tips is also being applied to eradicate systemic plant pathogens, a process termed cryotherapy. Slow growth storage is routinely used in many laboratories for medium-conservation of numerous plant species. Today, the large-scale, routine application of cryopreservation is still restricted to a limited number of cases. However, the number of plant species for which cryopreservation techniques are established and validated on a large range of genetically diverse accessions is increasing steadily.

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 chapter, we describe a cryopreservation (liquid nitrogen, -196 °C) protocol developed for long-term storage of date palm pro-embryonic masses (PEMs), which uses the recently established D cryo-plate technique. Clumps of PEMs (3-5 mm in size) were dissected from PEM cultures and placed on pretreatment medium containing 171 g/L sucrose for 3 days. Clumps were placed in the wells of aluminum cryo-plates in which they were made to adhere using droplets of 3% calcium alginate. PEMs were treated for 20 min with a loading solution containing 184 g/L glycerol and 136.8 g/L sucrose. They were then dehydrated for 90-120 min in the air current of a laminar airflow cabinet and immersed directly in liquid nitrogen. For rewarming, the cryo-plates holding the PEMs were immersed for 15 min in an unloading solution containing 410.4 g/L sucrose. The PEMs were then detached from the cryo-plates, placed for 3 days in the dark on posttreatment medium containing 102.6 g/L sucrose, and transferred on recovery medium under light conditions. Using this protocol, 74.6 and 95.8% recovery were achieved with the PEMs of the two cultivars tested, Sukkari and Sultany.

Equipe DAVEM = Diversité et Adaptation de la Vigne et des Espèces Méditerranéennes; Proline has been shown to accumulate in plants in response to biotic and abiotic stresses. Exogenous proline has thus been used for improving some plant cryopreservation protocols. Further enhancement of cryopreservation efficiency for in vitro grapevines could be expected if stresses linked to cryopreservation procedures could be reduced. We therefore studied the possible beneficial effect of proline in grapevine cryopreservation. Single-node explants from in vitro grown grapevine plantlets (Vitis vinifera L. cv Portan) were cultured on shooting media (half-strength MS + 1 μM BAP) con- taining no proline (control) or 50, 500, or 2000 μM filter-sterilized L-proline. Shoot tips excised from these micro- shoots were subjected to a PVS2-based droplet-vitrification procedure. Control and rewarmed explants were grown on a recovery medium containing 1 μM BAP. Shoot development on control medium and lower proline concentrations did not notably differ whereas the highest concentration of proline inhibited shoot development. Carry-over effects were observed since lower survival and regrowth were obtained both for non-frozen or LN-treated explants excised from micro-shoots obtained on the 2000 μM proline medium. No significant differences in survival and regrowth were ob- served for non-frozen explants subjected to pretreatment without LN exposure. A slightly enhancing effect (although non-significant) on post-cryopreservation survival was observed for explants derived from shoots developed on 50 or 500 μM proline, but no significant improvement of regrowth percentage was observed for these two conditions. Al- though a slight increase in survival could be observed, no significant beneficial effect of proline pretreatment on post- cryoconservation regrowth could be evidenced in our conditions. However, the 2-week period before explant excision could have allowed at least partial metabolism and catabolism of exogenous proline; the results observed could thus have been the consequence of complex interactions. Shorter proline treatments applied closer to the actual LN exposure step might produce different results and allow for clearer interpretation.

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%.

Depuis le debut des annees 1990, les semences des cafeiers ont ete adoptees par de nombreux laboratoires internationaux comme le systeme modele pour l’etude de la physiologie des semences de type « intermediaire ». A la difference des semences orthodoxes, dont la longevite augmente lorsqu’elles sont deshydratees et placees a basse temperature, les semences intermediaires ne sont que partiellement tolerantes a la dessiccation et au froid. Dans les plages etroites de teneurs en eau et de temperatures qui peuvent ainsi etre utilisees, la longevite des semences intermediaires est tres reduite, ce qui constitue une contrainte majeure pour la conservation de la biodiversite de ces especes. Le developpement de nouvelles techniques de chimie analytique a permis d’imputer la faible longevite des semences de cafeiers a des dommages cellulaires de plusieurs natures : une hydrolyse des lipides neutres conduisant a une accumulation d’acides gras libres destabilisant les membranes ; la perte selective d’une classe de phospholipides ; une perte et une oxydation des deux principaux antioxydants hydrophiles des cellules, l’acide ascorbique et le glutathion. Grâce a ces nouvelles connaissances sur les processus impliques dans le vieillissement des semences de cafeiers, nous proposons pour la premiere fois dans cet article des recommandations techniques tres precises pour la preparation et le stockage a court terme des lots de semences. Parallelement, grâce a des approches biophysiques, utilisant notamment la microcalorimetrie, la comprehension des mecanismes impliques dans la tolerance des semences de cafeiers a une exposition aux tres basses temperatures a progresse de maniere significative. Sur la base de ces connaissances, une technique de cryoconservation tres efficace a ete mise au point pour ces semences, permettant d’envisager desormais la sauvegarde a long terme des ressources genetiques du genre Coffea.

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.

The large majority of species for which cryopreservation protocols are necessary are grown in tropical countries. It is therefore necessary to develop cryopreservation research activities in these countries, and to promote the dissemination of relevant scientific and technical information as a prerequisite to the initiation of such research activities. Central America, South America and the Caribbean host several world biodiversity hotspots. Many of the plant species found in these areas produce recalcitrant seeds or are vegetatively propagated. They are thus relevant candidates for the development and application of cryopreservation protocols. At this moment, cryopreservation research activities are limited in Latin American and Caribbean (LAC) countries. Their development is hampered notably by the absence of specialized literature in Spanish. Specialists from Mexico, Argentina, Bolivia, Colombia, Costa Rica, Cuba, Ecuador, Peru, as well as representatives of international institutions (FAO, Bioversity International) are significantly contributing to the publication of the first technical book in Spanish on plant cryopreservation, that summarises the research activities developed in the region and will hopefully lead in the near future to the establishment of a collaborative network of Latin American scientists.

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.

In vitro techniques are very useful for conserving plant biodiversity, including (a) genetic resources of recalcitrant seed and vegetatively propagated species, (b) rare and endangered plant species and (c) biotechnology products such as elite genotypes and genetically engineered material. Explants from recalcitrant seed and vegetatively propagated species can be efficiently collected under field conditions using in vitro techniques. In vitro culture techniques ensure the production and rapid multiplication of disease-free material. Medium-term conservation is achieved by reducing growth of plant material, thus increasing intervals between subcultures. For long-term conservation, cryopreservation (liquid nitrogen, -196 degrees C) allows storing plant material without modification or alteration for extended periods, protected from contaminations and with limited maintenance. Slow growth storage protocols are routinely employed for a large number of species, including numerous endangered plants, from temperate and tropical origin. Cryopreservation is well advanced for vegetatively propagated species, and techniques are ready for large-scale experimentation in an increasing number of cases. Research is much less advanced for recalcitrant species due to their seed characteristics, viz., very high sensitivity to desiccation, structural complexity and heterogeneity in terms of developmental stage and water content at maturity. However, various technical approaches should be explored to develop cryopreservation techniques for a larger number of recalcitrant seed species. A range of analytical techniques are available, which allow understanding physical and biological processes taking place in explants during cryopreservation. These techniques are extremely useful to assist in the development of cryopreservation protocols. In comparison with crop species, only limited research has been performed on cryopreservation of rare and endangered species. Even though routine use of cryopreservation is still limited, an increasing number of examples where cryopreservation is used on a large scale can be found both in genebanks for crops and in botanical gardens for endangered species.

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

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

Shoot-tips and somatic embryos are the explants of choice for the in vitro long-term storage of ex situ plant genetic resources in liquid nitrogen. Cryopreservation of organized structures has significantly progressed, especially for species of tropical origin, with the development of several vitrification-based procedures such as encapsulation-dehydration, vitrification and droplet-vitrification approaches. They have allowed improvements in survival and recovery after cryopreservation compared with conventional crystallization-based protocols, proving their effectiveness for large scale application with embryos and shoot-tips of different plants. This review addresses the main physical and technological aspects involved in plant cryopreservation methods, illustrating the development of research with three cases: citrus, cassava and potato. These studies demonstrate how cryopreservation strategies are increasingly applied for their successful employment in the genebanks.

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