Evidence for improved Agrobacterium tumefaciens-mediated transformation of cultivated tomato (L. esculentum) and the transfer of the oat polyamine oxidase (PAO) gene into tomato.

摘要

GUS reporter gene and polyamine oxidase (PAO) gene were transformed into cultivated tomato (L. esculentum UC82) via Agrobacterium tumefaciens gene transformation. The two experiments were conducted to improve transformation efficiency and study plant drought-resistance.; In the first experiment unproved transformation efficiency was obtained by evaluating regeneration responses from cotyledon explants (regeneration media and wounding degree), preculture condition (tobacco cell feeder), inoculation media and inoculation plasmids, cocultivation condition (tobacco cell feeder), and transformant recovery (antibiotics for controlling Agrobacterium overgrowth and transformant selection). Transformation efficiency of 25% was obtained using AZ medium for bud initiation and MS 119 medium for shoot elongation, medium wounding degree on cotyledon explant, tobacco cell feeder for preculture and cocultivation, inoculation with C58C1 strain and pBI121 plasmid in LB medium pH 7.5, 250 mg/L claforan + 250 mg/L ticarcillin antibiotics for controlling Agrobacterium overgrowth, 50 mg/L kanamycin for bud initiation selection and then 150 mg/L kanamycin for shoot elongation. Also, transformant “purification” was established for uniform (nonchimeric) transformant recovery through regeneration of cotyledon explants after several subcultures on kanamycin selection media. Finally, 77% of the F1 progeny plants showed Mendelian inheritance ratios.; In the second experiment, the improved transformation protocol was used to transfer oat Polyamine oxidase (PAO) gene into cultivated tomato ( L. esculentum UC82). PAO transformed plants were confirmed by PCR test and PAO enzyme activity. These transgenic plants will be used to study the possible adaptive value of the PAO gene in conferring abiotic stress tolerance to crop plants.