INTRODUCTION OF THE RS-AFP2 GENE INTO EUCALYPTUS UROPHYLLA FOR RESISTANCE TO PHYTOPHTHORA CAPSICI

• Published in: Journal of tropical forest science Vol. 24; no. 2; pp. 198 - 208
• Main Authors: Ouyang, LJ, He, WH, Huang, ZC, Zhao, LY, Peng, SH, Sha, YE, Zeng, FH, Lu, XY
• Format: Journal Article
• Language: English
• Published: Kuala Lumpur Forest Research Institute Malaysia 01.04.2012
• Subjects: Adventitious shoots; Agrobacterium; Coculture techniques; Disease resistance; Eucalyptus urophylla; Forestry; Hypocotyls; Inoculation; Phytophthora capsici; Plants; Polymerase chain reaction; Transgenic plants; Tropical forests
• ISSN: 0128-1283; 2521-9847

We developed an Agrobacterium tumefaciens-mediated transformation system for Eucalyptus urophylla using hypocotyl explants. Antibiotic concentrations, pre-culture times, pH of the inoculation medium and co-culture times were optimised. Pre-cultured hypocotyl explants were co-cultured with A. tumefaciens strain EHA105 harbouring the binary vector pPBR-2 containing the Rs-AFP2 gene, which encodes an antifungal protein, under the control of the prp1-1 promoter, for six days and were then transferred to selective callogenesis-inducing medium containing kanamycin and cefotaxime. Calluses developed shoots and were cultured in an elongation medium and finally multiplied. The integration of T-DNA into the genome of transgenic E. urophylla was confirmed by polymerase chain reaction (PCR). The reverse transcription (RT)-PCR results showed that Rs-AFP2 gene expression could be detected only after the transformed plants were inoculated with Phytophthora capsici 60 hours after inoculation. These results indicated that the prp1-1 promoter was inducible and Rs-AFP2 could enhance the resistance of E. urophylla to P. capsici. This protocol enabled effective transformation and regeneration of E. urophylla. Kami membangunkan sistem pengubahan berantarakan Agrobacterium tumefaciens bagi Eucalyptus urophylla menggunakan eksplan hipokotil. Kepekatan antibiotik, masa prakultur, pH medium inokulasi dan masa pengkulturan bersama dioptimumkan. Eksplan hipokotil dikultur bersama-sama dengan A. tumefaciens jenis EHA105 selama enam hari dan kemudiannya dipindahkan ke medium pengaruh pengkalusan terpilih yang mengandungi kanamisin dan sefotaksim. Baka ini menyimpan vektor perduaan pPBR-2 yang mengandungi gen Rs-AFP2 yang mengekod protein antikulat di bawah kawalan penggalak prp1-1. Kalus menghasilkan pucuk dan dikultur dalam medium pemanjangan dan kemudiannya digandakan. Integrasi T-DNA ke dalam genom E. urophylla yang transgenik disahkan oleh reaksi rantai polimerase (PCR). Keputusan trankripsi (RT)-PCR yang bertentangan menunjukkan yang gen Rs-AFP2 terperi boleh dikesan hanya selepas tumbuhan terubah telah diinokulasi dengan Phytophthora capsici (60 jam selepas inokulasi). Keputusan menunjukkan yang penggalak prp1-1 boleh diaruh dan Rs-AFP2 boleh meningkatkan kerintangan E. urophylla terhadap P. capsici. Protokol ini membolehkan pengubahan dan pertumbuhan semula E. urophylla yang berkesan.