Sains Malaysiana 46(5)(2017): 703–711

http://dx.doi.org/10.17576/jsm-2017-4605-04

Production of Transgenic Rice (indica cv. MR219) Overexpressing Abp57 Gene through Agrobacterium-Mediated Transformation

(Pengeluaran Padi Transgenik (indica cv. MR219) Lebih Ekspres Gen Abp57 melalui Transformasi Berperantarakan Agrobacterium)

LAY-WEN TAN¹, ZURAIDA A. RAHMAN², HOE-HAN GOH¹, DUK-JU HWANG³,

ISMANIZAN ISMAIL^1,4 & ZAMRI ZAINAL^1,4*

¹Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

²Biotechnology Research Centre, MARDI Headquarters, Persiaran MARDI-UPM

43400 Serdang, Selangor Darul Ehsan, Malaysia

³National Academy of Agricultural Science, RDA, Suwon 441-857, Korea

⁴School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malayasia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

Diserahkan: 18 Januari 2016/Diterima: 24 Oktober 2016

ABSTRACT

Agrobacterium-mediated transformation of indica rice is undoubtedly a challenging task due to the rice recalcitrant nature to transformation process. Therefore, optimization of the transformation protocol is important for specific indica rice cultivar to ensure effectiveness of the transformation. In this study, crucial parameters affecting Agrobacterium-mediated transformation were optimized to obtain transgenic rice of local rice cultivar (indica MR219). Embryogenic calli were chosen for inoculation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pH2GW7-ABP57 containing gene of interest, Auxin binding protein 57 (Abp57). The parameters that have been optimized were the immersion time, co-cultivation period, acetosyringone concentration and co-cultivation temperature. A total of four days co-cultivation period and 30 min immersion of embryogenic callus are optimum for the transformation of MR219 with transformation efficiency of 26.4% and 16.0%, respectively. Acetosyringone at 200 μM and co-cultivation at 28¡C also gave the highest transformation efficiency (14.4 and 18.4%, respectively). Meanwhile, inclusion of 20 g/L maltose+20 g/L sorbitol into the regeneration media has significantly improve the transformed somatic embryos growth and increase the regeneration efficiency up to 40.0%. The results of polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) indicated that the transgene was successfully integrated and overexpressed in transgenic rice of MR219. In conclusion, significant improvement in transformation efficiency for rice cv. MR219 has been obtained by using the optimised protocol for transformation and regeneration developed in this study.

Keywords: Maltose; regeneration; somatic embryo; sorbitol; transformation efficiency

ABSTRAK

Transformasi padi indica berperantarakan Agrobacterium merupakan proses yang mencabar disebabkan oleh sifat semula jadi padi yang rekalsitran terhadap transformasi. Oleh yang demikian, pengoptimuman protokol transformasi adalah amat penting untuk setiap kultivar padi indica yang khusus bagi memastikan keberkesanan transformasi. Dalam kajian ini, beberapa parameter penting yang menjejaskan kecekapan transformasi berperantarakan Agrobacterium telah dioptimumkan untuk mendapatkan padi transgenik daripada kultivar padi tempatan, MR219. Kalus embriogenik dipilih untuk diinokulasikan dengan Agrobacterium tumefaciens strain LBA4404 yang membawa plasmid pH₂GW7-ABP57 dan mengandungi gen yang diminati, Auxin binding protein 57 (Abp57). Parameter transformasi yang dioptimumkan dalam kajian ini adalah masa rendaman, tempoh penanaman bersama, kepekatan asetosiringon, dan suhu penanaman bersama. Tempoh penanaman bersama selama empat hari dan perendaman kalus embriogenik dalam ampaian Agrobacterium selama 30 min adalah optimum untuk transformasi MR219 dengan masing-masing mempunyai kecekapan transformasi sebanyak 26.4% dan 16.0%. Kepekatan asetosiringon dan suhu penanaman bersama yang optimum masing-masing adalah 200 μM (14.4%) dan 28¡C (18.4%). Selain itu, kajian turut mendapati bahawa penggunaan 20 g/L maltosa+20 g/L sobitol dalam media regenerasi telah meningkatkan transformasi pertumbuhan dan keupayaan regenerasi embrio somatik sehingga 40%. Hasil tindak balas berantai polimerase (PCR) dan tindak balas berantai polimerase transkripsi berbalik (RT-PCR) menunjukkan bahawa transgen berjaya diintegrasikan dan diekspreskan dalam padi transgenik MR219. Kesimpulannya, kecekapan transformasi untuk padi cv. MR219 telah ditingkatkan secara signifikan dengan menggunakan protokol transformasi dan regenerasi yang optimum daripada kajian ini.

Kata kunci: Embrio somatik; kecekapan transformasi; maltosa; regenerasi; sorbitol

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*Pengarang untuk surat-menyurat; email: zz@ukm.my