The Genetic Transformation Of Pta-CrylAc In Tomato Via Agrobacterium-mediation

An efficient regeneration system and Agrobacterium-mediated transgenic system was established in tomato. A plant expression vector pCAMBIA3300-bt-pta harboring two insecticidal genes was constructed and employed in Agrobacterium-mediated tomato transformation. The molecular assay, genetics analysis and insect-resistance test were performed successfully to tarnsformed tomato variety Micro-Tom. The major results summarized bellow:1 An efficient and rapid Agrobacterium-mediated transformation procedure has beenestablished: (1)The explant in 5 mm long was optimal for callus induction. (2)Some 12 hours' illumination was suitable for callus induction. (3)The optimal time course for inoculation was 8-10 minutes. (4)Optimal concentration of agrobacteria for inoculation was OD₆₀₀=0.15. (5) Acidic solution for inoculation (pH=5.2). (6) Concentration of AS was 50 μmol/L in inoculation solution. (7) Co-cultivation for 3 days. (8) Some 300 mg/1 Carbencillin as inhibitor of agrobacteria. (9) Concentration of PPT was 4 mg/l for the selection.2 We constructed a plant expression vector pCAMBIA3300-bt-pta harboring two insecticidal genes pta gene and bt gene, and used it in Agrobacterium LBA1100 mediated tomato transformation. We harvested 24 independent regenerated plantlets from 325 explants, and regenerative ratio was 8.5% successfully.3 PCR, RT-PCR and Southern hybridization analyses confirmed that the target genes were successfully integrated into 15 tomato transformants and the transformation ratio was 62.5%.4 Genetic analysis in T₁ tomato lines showed that CrylAc gene's segregation ratio was accordant to Mendelian theoretic 3:1, and Pta gene was co- segregated with CrylAc.5 Herbicide resistant analysis in T₁ lines showed that all the transformed tomato plants were resistant to PPT, and the resistance range was from 20mg/lto 75mg/l.6 T₁ tomato lines BP-1 - BP-7 were selected for Plutella xylostella resistance identification. The result showed that the effect of insect-resistance was apparent but different among the lines tested.