Cryopreservation of Plant Genetic Resources.

Cryopreservation encompasses several interconnect disciplines including physiology and cryophysics. This chapter reviews the current techniques for cryopreservation of plant genetic resources (PGRs). Vitrification is an effective ice crystal avoidance mechanism for hydrated cells and tissues. With any cryopreservation method, whole or partial parts of specimens which are sufficiently dehydrated can be vitrified by rapid cooling in liquid nitrogen (LN). Techniques discussed are the vitrification protocol, encapsulation-vitrification protocol, droplet vitrification protocol (DV), vitrification protocol using cryo-plates (V cryo-plate), and air dehydration protocol using cryo-plates (D cryo-plate). In these DV, V, and D cryo-plate protocols, specimens to be cryopreserved are immersed directly into LN on aluminum foil strips or cryo-plates; removal from LN to rewarming solution results in a high level of plant regrowth with ultrarapid cooling and warming. The protocols were applied to a wide array of plant species including wild and multi-ploid species, although fine tuning of the protocols was required for successful application to specific plant species and lines. These three protocols efficiently complement each other and appear highly promising to facilitate large-scale cryobanking of PGRs in genebanks. Cryo-scanning electron microscopy makes it possible to examine the cellular and water behavior in plant tissues when immersed in LN. It has been verified that tissues cryopreserved by the process of vitrification and the cryo-plate protocols are cryopreservation methods for reliable long-term preservation of PGRs.

Freeze-substitution transmission electron microscopy of gentian shoot tips cryopreserved at ultra low temperatures.

Transmission electron microscopy (TEM) combined with freeze substitution was employed to examine the ultrastructure of cells of gentian shoot tips cooled to the ultra-low temperature of slush nitrogen and liquid nitrogen. When shoot tips were cooled in ultra-low temperature without plant vitrification solution 2 (PVS2) treatment, massive ice formation was observed throughout the cells, indicating that severe injury occurred during cooling. In contrast, when shoot tips were treated with PVS2 and subsequently cooled to ultra- low temperatures, no ice crystals were observed in the cells. In addition, the cells of PVS2-treated shoot tips exhibited considerable plasmolysis and formation of small vesicles in cytoplasm. These results clearly demonstrate that the PVS2 treatment is essential for preventing damage caused by ice formation and for successful cryopreservation of plant shoot tips.

Cryopreservation of blueberry shoot tips derived from in vitro and current shoots using D cryo-plate technique.

Cryopreservation is an important tool for long-term storage of plant germplasm that is currently used for plant germplasm storage at many institutes worldwide. Recently, novel cryogenic procedures (V and D cryo-plate methods) have been developed. In this study, the most suitable conditions for preserving blueberry shoot tips derived from in vitro and current shoots using the D cryo-plate method were investigated. The D cryo-plate method has advantages such as higher regrowth after cryopreservation and a more user-friendly process compared with conventional cryogenic methods. The optimum duration of desiccation for regrowth of shoot tips from each shoot type was 1 h. To induce dehydration tolerance for the shoot tips, the effects of two cryoprotection treatments (sucrose preculture and loading solution [LS] treatment) on shoot regrowth after cryopreservation were investigated. The combined effect of both treatments significantly increased percentage regrowth (approximately 90%). No regrowth of shoot tips was attained without the two treatments. Thus, preculture and LS treatment were effective to induce dehydration tolerance for cryopreservation of blueberry shoot tips. The optimized conditions for blueberry shoot tips using the D cryo-plate technique were: preculture with 0.3 M sucrose for 1 day, LS treatment (2 M glycerol +0.4-1.0 M sucrose) for 30 min, and air dehydration for 1 h. This optimized procedure was applied to additional blueberry cultivars shoot tips derived from in vitro shoots (regrowth 46.7-100%) and current shoots (regrowth 17.2-62.7%). Furthermore, in vitro shoot tips were suitable material for the D cryo-plate method in blueberry.

Cryopreservation for preservation of potato genetic resources.

Cryopreservation is becoming a very important tool for the long-term storage of plant genetic resources and efficient cryopreservation protocols have been developed for a large number of plant species. Practical procedures, developed using in vitro tissue culture, can be a simple and reliable preservation option of potato genetic resources rather than maintaining by vegetative propagation in genebanks due their allogamous nature. Cryopreserved materials insure a long-term backup of field collections against loss of plant germplasm. Occurrence of genetic variation, in tissue culture cells during prolonged subcultures, can be avoided with suitable cryopreservation protocols that provide high regrowth, leading and facilitating a systematic and strategic cryo-banking of plant genetic resources. Cryopreservation protocols for potato reviewed here, can efficiently complement field and in vitro conservation, providing for preservation of genotypes difficult to preserve by other methods, wild types and other species decided as priority collections.

Cryopreservation of sugarcane using the V cryo-plate technique.

BACKGROUND: Sugarcane is a tropical crop of major importance primarily for its high sucrose content. It is difficult to conserve it in the field or in vitro because of biotic and abiotic stresses. Cryopreservation of sugarcane germplasm is an appropriate approach for conserving its genetic diversity. OBJECTIVE: This study was carried out to develop an efficient and practical cryopreservation protocol for sugarcane with high post-cryopreservation recovery. MATERIALS AND METHODS: Factors affecting regrowth after cryopreservation using the V cryo-plate method including preculture medium, size of shoot tips, sucrose concentration in loading solution, exposure time to PVS2, light conditions after liquid nitrogen exposure, presence and absence of alginate gel and recovery medium composition were studied. RESULTS: Shoot tips with a length of 1.5 to 2.0 mm, precultured on semi-solid 1/2 MS medium for 1 day and semi-solid MS medium with 0.5 M sucrose for 1 day, treated with LS containing 2.0 M glycerol + 1.6 M sucrose for 30 min and exposed to PVS2 for 30 min showed maximum (100%) recovery after cryopreservation. It was also observed that removing the alginate gel and keeping the cultures in the dark for 7 days after cryopreservation significantly improved recovery. After optimizing the cryopreservation conditions using sugarcane variety Ni-1, 10 additional varieties were cryopreserved using the optimized protocol, with regrowth ranging from 56.7% to 100%. CONCLUSION: This study showed that V cryo-plate is an efficient and practical method for cryopreservation of sugarcane shoot tips in genebanks.

Genetic stability assessment of wWasabi plants regenerated from long-term cryopreserved shoot tips using morphological, biochemical and molecular analysis.

This study compared the effect of cryopreserved storage duration of wasabi shoot tips, which derived from the same in vitro mother-plant. We compared the survival of shoot tips and the genetic stability of regenerated plants originating from four experimental groups: shoot tips stored in a -150°C deep-freezer for 10 years; shoot tips stored in liquid nitrogen for 2 h; shoot tips treated with PVS2 vitrification solution; and untreated controls. No significant difference in survival was observed between the four experimental groups. Survival ranged between 93 and 100%. Genetic stability of plants regenerated from cryopreserved shoot tips was assessed over a period of 24 months using morphological, biochemical and molecular markers. While glucose, fructose and glutamic acid concentrations differed slightly between experimental groups after 16 months, these differences disappeared after 24 months. No significant differences were noted for the morphological markers studied (petiole length, shoot number and leaf index). No differences were observed in RAPD profiles obtained with the six primers tested.

Development of a cryopreservation procedure using aluminium cryo-plates.

A cryopreservation procedure using an aluminium cryo-plate was successfully developed using in vitro-grown Dalmatian chrysanthemum (Tanacetum cinerariifolium) shoot tips. Shoot cultures were cold-hardened at 5 degree C on MS medium containing 0.5 M sucrose over a period of 20 to 40 days. Shoot tips with basal plate (1.0-1.5 x 1.0 mm) were dissected from shoot cultures and precultured at 5 degree C for 2 days on MS medium containing 0.5 M sucrose. Precultured shoot tips were placed on aluminium cryo-plates (7 mm x 37 mm x 0.5 mm) with 10 wells (diameter 1.5 mm, depth 0.75 mm) and embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 or 60 min in 25 ml pipetting reservoirs filled with loading solution (2 M glycerol + 1.4 M sucrose). For dehydration, the loading solution was replaced with PVS 7M vitrification solution (30 percent glycerol, 19.5 percent ethylene glycol and 0.6 M sucrose in liquid MS basal medium), which was applied for 40 min. After rapid immersion in liquid nitrogen, shoot tips attached to the cryo-plates were rewarmed by immersion in cryotubes containing 2 ml 1 M sucrose solution. Using this procedure, regrowth of cryopreserved shoot tips of line 28v-75 reached 77 degree. This protocol was successfully applied to six additional lines, with high regrowth percentages ranging from 65 to 90 percent. By contrast, the modified vitrification protocol tested as a reference produced only moderate regrowth percentages. This new method displays many advantages and will facilitate large scale cryostorage in genebank.

Cryopreservation of basal stem buds of in vitro-grown mat rush (Juncus spp.) by vitrification.

An optimal protocol for the cryopreservation of in vitro-grown mat rush (igusa) buds by vitrification has been successfully developed. Established multiple stemmed cultures, which were induced in liquid MS medium containing 8.9 microM BA by roller culture, were cut into small clumps, plated on solid MS medium and cultured for three weeks at 25 degree C. Clumps that grew many buds were cold-hardened at 5 degrees C, with an 8 h photoperiod, for more than 30 d. The basal stem bud (1 to 2 mm long) was dissected from the clumps and precultured at 5 degrees C for 2 d on solid MS medium containing 0.3 M sucrose. The precultured buds were placed in 2 ml plastic cryotubes and osmoprotected with 1 ml loading solution containing 2 M glycerol and 0.6 M sucrose for 30 min at 25 degree C. Then they were dehydrated in 1 ml PVS2 solution at 25 degree C for 30 min and immersed in liquid nitrogen. Using this protocol, the survival level of cryopreserved igusa 'NZ219' buds reached 87 percent. This protocol was successfully applied to 42 different lines from three Juncus species, which had relatively high survival levels ranging from 30 to 90 percent and an average of 63 percent.

Cryopreservation of shoot apices of in-vitro grown gentian plants: comparison of vitrification and encapsulation-vitrification protocols.

Using vitrification and encapsulation-vitrification protocols, we successfully cryopreserved shoot apices from in-vitro plants of different Gentiana cultivars (lines). Although both protocols gave high survival percentages after storage in liquid nitrogen, the encapsulation-vitrification protocol had several distinct advantages over the vitrification protocol: (i) survival was higher under optimal conditions, (ii) the range of optimal exposure periods to the plant vitrification solution 2 (PVS2) was broader, and (iii) regrowth of cryopreserved shoot apices was apparently more vigorous and faster. Shoot apices from ten cultivars/lines of three Gentiana species (G. scabra, G. triflora, and G. pneumonanthe) were successfully cryopreserved using the two protocols with average survival of 49.0 percent and 73.7 percent for vitrification and encapsulation-vitrification, respectively. These results indicate that the two protocols optimized in the present study are promising for cryopreservation of a wide range of Gentiana genetic resources.