ISHS Acta Horticulturae 528: VII International Symposium on Grapevine Genetics and Breeding
FIRST STEPS TO USE THE PROTOPLAST TECHNIQUE FOR BREEDING PURPOSES |
| Authors: | G.M. Reustle, A. Matt |
| Keywords: | Vitis sp., somaclonal variation, selection, gene transfer |
| Abstract:
Spontaneous arising somaclonal variation in plants regenerated from grapevine (Vitis sp. cv. Seyval blanc) protoplasts (protoclones) was investigated. Using Flow Cytometry 5 out of 105 tested clones showed about the double DNA content compared to control plants and to other protoclones. The tetraploid character was confirmed by counting the chromosomes in root tips. Using RAPD-PCR technique, three out of 47 clones, analyzed with 60 different primers showed different band pattern compared to control plants and to other protoclones. Phenotype of the protoclones was uniform after transfer into greenhouse except of three clones, which showed an increased formation of axillary shoots. A selection system on protoplast level was established using a filtrate of Botrytis cinerea (BCF) as selecting agent. With increasing concentrations of the filtrate (0 to 100 mg/1), the formation of somatic embryos on the protoplast derived callus and the regeneration of plants was reduced by the filtrate. Plant regeneration succeeded from control treatment, 0.1 and 1.0 mg/L filtrate concentration. To increase somaclonal variation, freshly isolated protoplasts were treated with UV-light and 1-methyl-3-nitro-1- nitroso-guanidine (MNNG) and subsequently selected on regeneration media containing BCF. Direct transfer of DNA to protoplasts was performed using PEG. Transient expression of the transferred genes (GUS, Endochitinase) depends on concentration of plasmid DNA and the use of carrier DNA. | |
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