An efficient leaf-disc culture method for the regeneration via somatic embryogenesis and transformation of grape (Vitis vinifera L.)

By manipulating hormone levels, light intensities and temperature, we have developed an efficient leaf-disc method for the regeneration of plants via embryogenesis and for transformation in four genotypes of Vitis vinifera L. In MS basal medium supplemented with 1 mg l(-1) 6-benzylaminopurine (BAP) and 0.1 mg l(-1) 2,4-dichlorophenoxyacetic acid, leaf discs cultured for 2 weeks under dark conditions produced calli in over 80% of the cultures. These subsequently differentiated into pro-embryos and embryos only if kept under conditions of low light intensity (15 muE m(-2) s(-1)) for 2 weeks before being transferred to conditions of high light intensity (60 muE m(-2) s(-1)). If the calli were directly transferred to high light intensity, the differentiation into embryos was blocked and the calli turned pink. The somatic embryos germinated at a frequency of about 10% on NN basal medium and about 32% on NN medium supplemented with 1 mg l(-1)BAP and 0. 1 mg l(-1) indole-3-butyric acid. The embryos, however, germinated when pre-exposed to a low temperature of 4degreesC for 2 weeks. If they were transferred directly to room temperature under conditions of high light intensity (60 muE m(-2) s(-1)), shoot buds were produced, whereas under conditions of low light intensity (15 muE m(-2) s(-1)) secondary embryogenesis was induced. About 90-95% of the in vitro grown plantlets could be successfully transferred to soil. The above method was also applicable for developing transgenic embryos whose transgenic nature was monitored using beta-glucuronidase as a reporter gene.