Genetic Transformation Of A Pollen Lysine-rich Gene From Tomato To Thinopyrum Intermedium And Regeneration Of Transgenic Plants

The study in this paper, a Pollen-specific Lysine-rich Protein cDNA (TSB), from tomato by RT-PCR was obtained. A plant expression plasmid pBIUB-TSB was constructed and lysgene was under the control of maize ubiquitin promoter which is the highest efficient monocotyledon promoter. With that, the tisse culture and plant regeneration was constructed using the mature seeds of gramineous forage-Thinopyrum intermedium. the plasmid pBIUB-TSB was translate into the callus of the Thinopyrum intermedium by the way of Microprojectile bombardment and Laser Microbeam Puncture Technique.Regenerated fertile transformed plants were obtained by selecting for Kanamycin resistance then polarizated the new plant translation. The experimental results showed that: The callus inducing medium: (1) MS+2.4-D 4.5mg/L+BA 0.1mg/L+Sucrose 30g/L+ Agar 6.5g/L pH:5.8~6.0; (2) Subculture medium: MS+2.4-D 4mg/L+BA 0.1mg/L+Vc 5mg/L+ Sucrose 30g/L+Agar 6.5g/L+Inositol 100mg/L pH:5.8~6.0; (3) Differentiation medium: MS+BA 2mg/L+NAA 1mg/L +CoCl2 5mg/L+ZT 0.5mg/L+ Sucrose 30g/L+ Inositol 100mg/L+ Agar 6.5g/L pH:5.8~6.0; (4) the rooting medium: 1/2MS+ IAA0.5mg/L +NAA0.5mg/L+VC 0.1%+ Sucrose 30gL+ Agar 6.5g/L pH:5.8~6.0.Some regenerated plants were obtained by selecting for Kanamycin resistance of 300mg/L. Genomic PCR, Southern blotting analysis showed that the construct was integrated into the Thinopyrum intermedium genome, we got four transgenic plants, two of it by Microprojectile bombardment, the rest by Laser Microbeam Puncture Technique.