Studies on the development of transformation in Phalaenopsis.

Transgenic Phalaenopsis plants have been obtained by particle bombardment with a pneumatic particle gun device.All promoters of cauliflower mosaic virus(CaMV)35S，maize ubiquitin and rice actin genes could be expressed in Phalaenopsis using transient assay of beta-glucuronidase(Gus)gene as a reporter.Bialaphos resistance gene(Bar)which confers tolerance to herbicide bialaphos，was used as a selectable marker。Protocorm-like bodies(PLBs)of Phalaenopsis derived from the leaf segment culture were bombarded by gold particles coated with pMSP38and pWI-GUS DNA containing the bar gene and the gus gene respectively driven by 35S promoter.Newly-formed PLBs were selected on the medium including bialaphos。Finally seven transgenic plants resistant to bialaphos were obtained，and one of them contained and expressed gus gene.PCR analysis confirmed their transgenic nature and western analysis and histochemical GUS assay showed the expression of bar and gus gene respectively.New PLBs(T_1…More)were formed from the segments obtained by the division of the transgenic PLB(T_0)。The bisected segments of T_1 PLBs formed T_2 PLBs on the segments。From the detection of amplified DNA by PCR，all plantlets of the T_1and T_2 generation contained the bar gene.The western analysis showed that the bar gene expressed in all of the clonal progenies as well T_0generation.To use the callus of Phalaenopsis for transformation，the callus formation and plant regeneration in Phalaenopsis Richard Shaffer‘Santa Cruz’were examined。PLB segments formed calli in Vacin and Went(1949)medium with sucrose.The optimal concentration of sucrose was40g·1^&lt；-1&gt；。Media containing200ml·1^&lt；-1&gt；coconut water were effective for the callus formation。The media solidified with gellan gum was suitable for the callus induction as compared with those with agar.The calli formed PLBs easily after transferring to media without sucrose。No variation was observed in the flowering plants regenerated through somatic embryogenesis.The calli bombarded the gus gene showed the expression of the gene through the histochemical assay，suggesting the availability of calli for the transformation of Phalaenopsis。Less：Less

Transgenic Phalaenopsis plants have been obtained by particle bombardment with a pneumatic particle gun device. All promoters of cauliflower mosaic virus (CaMV) 35S,maize ubiquitin and rice actin genes could be expressed in Phalaenopsis using transient assay of beta-glucuronidase (gus) gene as a reporter. Bialaphos resistance gene (bar) which confers tolerance to herbicide bialaphos, was used as a selectable marker. Protocorm-like bodies (PLBs) of Phalaenopsis derived from the leaf segment culture were bombarded by gold particles coated with pMSP38 and pWI-GUS DNA containing the bar gene and the gus gene respectively driven by 35S promoter. Newly-formed PLBs were selected on the medium including bialaphos. Finally seven transgenic plants resistant to bialaphos were obtained, and one of them contained and expressed gus gene. PCR analysis confirmed their transgenic nature and western analysis and histochemical GUS assay showed the expression of bar and gus gene respectively.New PLBs (T_1 … More ) were formed from the segments obtained by the division of the transgenic PLB (T_0). The bisected segments of T_1 PLBs formed T_2 PLBs on the segments. From the detection of amplified DNA by PCR,all plantlets of the T_1 and T_2 generation contained the bar gene. The western analysis showed that the bar gene expressed in all of the clonal progenies as well T_0 generation.To use the callus of Phalaenopsis for transformation, the callus formation and plant regeneration in Phalaenopsis Richard Shaffer 'Santa Cruz' were examined. PLB segments formed calli in Vacin and Went (1949) medium with sucrose. The optimal concentration of sucrose was 40 g・1^<-1>. Media containing 200 ml・1^<-1> coconut water were effective for the callus formation. The media solidified with gellan gum was suitable for the callus induction as compared with those with agar. The calli formed PLBs easily after transferring to media without sucrose. No variation was observed in the flowering plants regenerated through somatic embryogenesis.The calli bombarded the gus gene showed the expression of the gene through the histochemical assay, suggesting the availability of calli for the transformation of Phalaenopsis. Less