Cloning and characterization of partial 1-aminocyclopropane-1-carboxylate oxidase gene and antisense transformation into yellow 'Solo' papaya via Agrobacterium tumefaciens
Issue Date
1-2013
Abstract
A partial 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase) gene from Philippine yellow 'Solo' papaya was cloned using reverse transcription-polymerase chain reaction (RT-PCR) from fully ripened fruits and subcloned into a binary vector in an antisense orientation and used for Agrobacterium-mediated transformation of the yellow 'Solo' papaya to create putative transgenic papaya. The partial fragment of 800 bp was characterized, sequenced and subjected to similarity analysis with other papaya ACC oxidase genes. The sequence has very high homology (99.0%) with the ACC oxidase mRNA of the Hawaiian 'Sunset Solo' papaya and also highly related to the sequence of the ACC oxidase gene of the papaya varieties namely: 'Semangka' (Indonesia), 'Maradol Roja' (Cuba) and 'Tainung 2' (Taiwan). The 800 bp partial ACC oxidase fragment was ligated into pGA643 in an antisense orientation and plasmid vector was transformed to Agrobacterium tumefaciens strain LBA 4404 by electroporation. One-month old somatic papaya embryos derived from zygotic embryo explants cultured on somatic embryo induction medium (SEIM) supplemented with 2,4-D (10 mg L-1) were transformed using this bacteria. Putatively transformed somatic embryos and calli were selected for a three-month period in a full strength De Fossard medium supplemented with carbenicillin (500 mg L-1) and kanamycin (150 mg L-1). The selected tissues were regenerated in a full-strength De Fossard medium supplemented with benzylaminopurine (0.5 μM), α-napthalene acetic acid (0.5 μM) and brassinolide (0.08%, v/v) eleven months after transformation. One hundred seventy-seven putative transformed plantlets were produced and further multiplied by micropropagation using nodal cuttings.
Source or Periodical Title
Journal of the International Society for Southeast Asian Agricultural Sciences
ISSN
0859-3132
Volume
19
Issue
1
Page
63-76
Document Type
Article
Language
English
Subject
1-aminocyclopropane-1 carboxylate oxidase, Agrobacterium-mediated transformation, Antisense, Delayed ripening, Ethylene, Papaya, Somatic embryos
Recommended Citation
Magdalita, P.B., Laurena, A.C., Perez, M.T.M.(2013). Cloning and characterization of partial 1-aminocyclopropane-1-carboxylate oxidase gene and antisense transformation into yellow 'Solo' papaya via Agrobacterium tumefaciens. Journal of the International Society for Southeast Asian Agricultural Sciences, 19 (1), 63-76.
Digital Copy
yes