Malaysian Applied Biology Journal

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40-1-07

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Malays. Appl. Biol. (2011) 40(1): 39-45

GENETIC TRANSFORMATION OF ANTISENSE ACC OXIDASE IN Carica papaya L. CV. SEKAKI VIA PARTICLE BOMBARDMENT

CHE RADZIAH, C.M.Z.*, NURUL SHAHNADZ, A.H., NAZIRATUL AIN A.N. and ZAINAL, Z.

School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia,
43600 Bangi, Selangor Darul Ehsan, Malaysia
*E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

ABSTRACT

Papaya (Carica papaya L.) is a very important crop in many tropical countries. Climacteric fruits such as papaya are usually harvested once they have reached maturity, which then undergoes rapid ripening during transit and storage. Hence, papaya is highly susceptible to physiological disorders, mechanical damage and fruit over ripening that can cause postharvest losses of papaya production. Therefore, this study was conducted on genetic transformation of antisense ACC oxidase and its effects on papaya fruit ripening. Through anti-sense technology, down regulation of the ACC oxidase gene results in the suppression of ethylene production, thereby delaying fruit ripening that can produce papaya with better quality. Embryogeniccallus of Sekaki papaya were bombarded with the pCAMBIA 1301 that contains the antisense ACC oxidase gene driven and flanked by constitutively CaMV35S promoter and NOS terminator also hygromycin (hpt) resistance gene as selectable marker. Helium gas pressure (1350 psi), 1.0 um gold particle size and two types of parameters manipulated in the bombardment condition were, the number of bombardment (single or double) and the target position (3 cm, 6 cm and 9 cm). Results showed that double bombardment per sample and 6 cm of target position gave the highest percentage of callus survival which is 88% after 4 weeks in a selection medium containing 50mg/l hygromycin. The PCR analysis for antisense ACC oxidase, hpt and Nad5 genes done on the extracted callus genome gave positive results. The remaining bombarded explants are being maintained to obtain viable regenerates for further analyses.


ABSTRAK

Betik (Carica papaya L.) adalah salah satu tanaman yang penting di kebanyakan negara tropika. Buah klimakterik seperti betik pada kebiasaannya akan dipetik apabila mencapai tahap kematangan, yang mana ia akan melalui proses pemasakan buah dengan cepat semasa penghantaran dan penyimpanan. Oleh yang demikian, betik mempunyai pendedahan yang sangat tinggi terhadap masalah fisiologi, kerosakan luaran dan buah yang terlebih masak yang mana akan menyebabkan kerugian pada penghasilan betik selepas tuai. Oleh itu, kajian transformasi genetik terhadap gen antisense ACC oxidase telah dijalankan di mana ianya memberi kesan terhadap pemasakan buah betik. Melalui teknologi antisense yang dijalankan,penghasilan yang rendah pada gen ACC oxidase akan merencatkan penghasilan etilena yang mana dapat melambatkan proses pemasakan buah seterusnya menghasilkan buah betik dengan kualiti yang baik. Kalus embriogenik betik sekaki yang digunakan telah ditransformasi dengan pCAMBIA 1301 yang mengandungi gen antisense ACC oxidase diapit dengan pencetus konstitutiv CaMV35S dan terminator NOS. Plasmid yang digunakan juga mempunyai gen kerintangan terhadap higromisin (hpt) sebagai penanda pemilihan. Penggunaan tekanan gas helium (1350 psi), saiz partikel emas (1.0 um) dan pemilihan dua parameter yang telah dimanipulasi iaitu bilangan bedilan (1 atau 2 kali) dan juga jarak di antara mikroprojektildengan tisu sasaran (3,6,9 cm) telah dilakukan sewaktu proses pembedilan. Keputusan yang diperolehi menunjukkan bahawa pembedilan sebanyak 2 kali per sampel dan jarak pembedilan 6cm memberikan peratusan kebolehidupan kalus yang paling tinggi iaitu 88% selepas 4 minggu pengkulturan di dalam media pemilihan higromisin (50mg/L). Analisis PCR yangdijalankan terhadap gen antisense ACO, gen hpt dan gen Nad5 hasil daripada ekstraksi kalus menunjukkan keputusan yang positif. Kalus yang masih tinggal disimpan dengan baik untuk regenerasi dan analisis yang seterusnya.

Key words: Carica papaya L., particle bombardment, antisense ACC oxidase, embryogenic callus

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