Direct cryopreservation of winter buds of nine cultivars of blackcurrant (Ribes nigrum L).

Direct cryopreservation of overwintering, dormant buds has been applied to nine blackcurrant cultivars, using a 7 day dehydration period at c.-20° C before plunging directly into liquid nitrogen. The buds on shoots thawed from -20° C showed normal development simply by standing them in water and all the cultivars could be successfully recovered (> 58%) by grafting. None of the shoots thawed from liquid nitrogen showed any development after standing in water and all of the grafts failed. Shoots thawed from liquid nitrogen showed significant damage to xylem transport, and the cortical tissues necessary fro successful grafting showed significant loss of membrane semipermeability. However, buds excised from shoots immediately after thawing from liquid nitrogen were viable and could be recovered using in vitro culture. Survival ranged from 88 to 55%, depending upon cultivar.

Cryopreservation of plant cell suspensions.

The cryopreservation of dedifferentiated cells, grown in suspension culture, is one of the portfolio of techniques employed for the long-term conservation of higher plant germplasm. Suspension cultures are also important in biotechnology, particularly in transformation studies and for the production of specific metabolites, and, here, there is also a pressing need for genetically stable, long-term storage of cell lines. Cryopreservation of suspension cell cultures can be exploited by either slow, or rapid, cooling techniques. During slow cooling the extracellular solutions are nucleated and the cells cryodehydrate during controlled cooling as a consequence of extracellular ice, to the point where their intracellular fluids will vitrify on subsequent transfer to liquid nitrogen. In the rapid cooling protocols, the cells are prepared by extreme osmotic dehydration, with cryoprotection, before plunging the samples directly into liquid nitrogen to achieve vitrification. Extensive success has been achieved with both techniques but rapid cooling is, currently, widely favored because of its simplicity.

HPLC analysis of plant DNA methylation: a study of critical methodological factors.

HPLC analysis of nucleosides is important for determining total DNA methylation in plants and can be used to help characterise epigenetic changes during stress, growth and development. This is of particular interest for in vitro plant cultures as they are highly susceptible to genetic change. HPLC methodologies have been optimised for mammalian and microbial DNA, but not for plants. This study examines critical methodological factors in the HPLC analysis of plant DNA methylation using in vitro cultures of Ribes ciliatum. HPLC revealed that complete removal of RNA from plant DNA extractions is difficult using RNase (A and T1) digestions and LiCl precipitation. This suggests that base analysis should be avoided when using these RNA removal techniques, as bases from residual RNA fragments will inflate peak areas for DNA-derived bases. Nucleoside or nucleotide analysis is therefore recommended as a more suitable option as RNA and DNA constituents can be readily separated. DNA digestion was also a critical factor as methylation was under-estimated following incomplete nuclease digestion and over-estimated following incomplete phosphatase digestion. The units of enzyme required for complete DNA digestion was optimised and found to be 20-200 times less for nuclease P1 and 15 times less for alkaline phosphatase as compared with previous protocols. Digestion performance was conveniently monitored using marker peaks that indicate incomplete digestion products. This study identifies critical components of HPLC analysis and offers a comprehensive guide for the stringent analysis of DNA methylation in plants.