SOMATIC EMBRYOGENESIS OF WALNUT AND RECOVERY OF TRANSGENIC SOMATIC EMBRYOS AFTER AGROBACTERIUM-MEDIATED TRANSFORMATION

Somatic embryos were initiated from the embryonic axes and cotyledons of immature zygotic embryos of English walnut cultivar ‚No. 120‘ (Juglans regia L.) cultured on MS or DKW medium containing 2 mg l^-1 kinetin, 1 mg l^-1 6-benzylaminopurine, 0.01 mg l^-1 indolebutyric acid and 250 mg l^-1 l-glutamine. Embryonic axes were more efficient than cotyledon pieces in producing somatic embryos under the equivalent conditions. MS medium was better for embryonic axes and DKW medium better for cotyledon pieces to undergo somatic embryogenesis while neither embryonic axes nor cotyledon pieces on WPM medium produced somatic embryos. Somatic embryos or embryogenic clusters underwent repetitively somatic embryogenesis on basal MS or DKW medium without plant growth regulators. The frequency of well-developed secondary somatic embryos was improved by using a basal DW medium (½ DKW + ½ WPM). Transformed somatic embryos were recovered by inoculating somatic embryos, 2~4mm in length with a hypervirulent Agrobacterium tumefaciens strain C58C1 ATHV RifR containing the helper plasmid pEHA101 and a binary vector p35S-gus-int. Putative transformed somatic embryos were selected for continued proliferation on a 100 mg l^-1 kanamycin containing DW medium. Histochemical assessments indicated chimeric expression of the gus gene in the 1st and 2nd generations and non-chimeric expression of the gus gene in the 3rd and 4th generations of the transformed somatic embryos.