Development of oil palm (Elaeis guineensis Jacq) RNAi constructs and transformation of cDNA candidates into rice (Oryza sativa L.)

• Main Author: Maniam, Kalai Vani
• Format: Thesis
• Language: English
• Published: 2012
• Online Access: http://psasir.upm.edu.my/id/eprint/48907/8/FBSB%202012%2054R.pdf; http://psasir.upm.edu.my/id/eprint/48907/

The current rate of oil palm embryogenesis in the industry ranges from 3 % to 6 %,and is an acknowledged obstacle in scaling up tissue culture production. Isolation of cDNA candidates that may have potential involvement in the oil palm somatic embryogenesis has been carried out in previous studies. In this study, four oil palm cDNA candidates (EgPER1, EgHOX1, OPSC10 and EgPK1) were chosen for functional analysis studies. Construction of RNAi vectors and rice transformation using the overexpression vectors were performed. The PCR products were amplified from full length cDNA candidates that were previously cloned into the intermediate vector, pDONR221 and cloned into pANDA vector with LR clonase enzyme. The positive clones obtained from the LR reaction were screened with PCR in the sense and antisense direction and verified by sequencing. All four cDNA candidates which have been cloned into the overexpression vector, pMDC32 driven by a double cauliflower mosaic virus (CAMV) were transformed into Taipei 309 rice. The calli transformed with pMDC32/OPSC10 failed to regenerate on normal regeneration medium. The calli had slow growth rate and was stunted, leading to phenotypic aberrations. Modifications of the regeneration medium by completely removing sucrose and adding high cobalt concentration (100 μM) promoted regeneration of the stunted calli. Although several calli were obtained from the transformation, only one plantlet survived while others displayed albinism and failed to revert to normal growth on the modified regeneration medium. The plantlet had a drastic increase in height in 14 days once transferred onto the modified regeneration medium. However, it did not survive outside the tissue culture environment. The putative transformants obtained from the subsequent transformation were screened with PCR using four different sets of primers (nosT, hygromycin, 35 S and gene specific forward). Only one line transformed with pMDC32/EgPK1 showed consistent results with all four primers. Southern blot analysis of PCR products generated using gene specific primers confirmed that the EgPK1 was successfully integrated into the rice genome. This transformant was phenotypically normal. The results obtained were preliminary but will provide guidance for further analysis of EgPK1 and OPSC10 to verify their functions in oil palm somatic embryogenesis.