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

Abstract

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 μE m⁻² s⁻¹) for 2 weeks before being transferred to conditions of high light intensity (60 μE 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⁻¹BAP and 0.1 mg l⁻¹ indole-3-butyric acid. The embryos, however, germinated when pre-exposed to a low temperature of 4°C for 2 weeks. If they were transferred directly to room temperature under conditions of high light intensity (60 μE m⁻² s⁻¹), shoot buds were produced, whereas under conditions of low light intensity (15 μE 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 β-glucuronidase as a reporter gene.