Development of transgenic papaya with delayed ripening characteristics containing the ACC [1-aminocyclopropane-1-carboxylic acid] oxidase gene via agrobacterium-mediated transformation

The transformation cassette containing the antisense ACC oxidase gene was constructed into the pGAG43 plasmid vector. The transformation of the plasmid vector pGAG43 containing the antisense ACC oxidase gene to Agrobacterium LBA4404 was established at 2.46KV using the Transporator BTX plus electroporator. The ACC oxidase gene were characterized by PCR analysis and gel electrophoration. The ACC oxidase gene and 'Davao Solo' papaya was sent to sequenced by the Waikato Sequencing Facility in New Zealand. Through blast search, the pSOLO ACO 2 was found 98% homologous to the other ripening-induced ACC oxidase of Papaya based on the gaps and length of the nucleotide sequences. The ACO2 of 'Davao Solo' is closely related to the Cuban, Indonesian and Taiwan ACO. The CDNA sequence of 1-Aminocyclopropane-1-carboxylate oxidase 2 (ACO2) was submitted to the Genebank under accession number AY570545. Seventeen thousand one hundred forty-five (17,145) somatic embros were produced and used un Agrobacterium transformation. The plasmid vector pGAG43 containing the ACC oxidase gene 2 was introduced to the somatic embryos and multishoots of papaya by Agrobacterium-Twelve thousand nine hundred forty-seven (912,947) proliferating somatic embryos and callus were transformed using Agrobacterium carrying the pGAG43 plasmid of antisense ACO2. Two thousand nine hundred twenty-five (2,925) somatic embryo clusters and calli that passed Kanamycin selection were regenerated. Different types of regeneration medium were tested to promote growth of transformed tissues. The nurse culture technique was identified promising for promoting regeneration of tissues. Addition of brassinolide at 0.08% (200 µl/250 mL) in the De fossard medium in the presence of 0.5 µM NAA and 0.5 µM BA promoted regeneration by inducing the formation of green shoots and roots of the transformed somatic embryo. A total of 1332 T sub 0 cultures of putatively transformed regenerants were produced. A total of 92 T sub 0 regenerants were micropropagated. 1080 shoot cuttings were produced. There are 252 putatively transformed papaya plantlets.