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

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....

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Main Author:	Maniam, Kalai Vani
Format:	Thesis
Language:	English
Published:	2012
Subjects:	Oil palm Rice Oil palm - Somatic embryogenesis
Online Access:	http://psasir.upm.edu.my/id/eprint/48907/8/FBSB%202012%2054R.pdf
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spelling	my-upm-ir.489072016-11-16T01:19:52Z Development of oil palm (Elaeis guineensis Jacq) RNAi constructs and transformation of cDNA candidates into rice (Oryza sativa L.) 2012-08 Maniam, Kalai Vani 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. Oil palm Rice Oil palm - Somatic embryogenesis 2012-08 Thesis http://psasir.upm.edu.my/id/eprint/48907/ http://psasir.upm.edu.my/id/eprint/48907/8/FBSB%202012%2054R.pdf application/pdf en public masters Universiti Putra Malaysia Oil palm Rice Oil palm - Somatic embryogenesis
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	Oil palm Rice Oil palm - Somatic embryogenesis
spellingShingle	Oil palm Rice Oil palm - Somatic embryogenesis Maniam, Kalai Vani Development of oil palm (Elaeis guineensis Jacq) RNAi constructs and transformation of cDNA candidates into rice (Oryza sativa L.)
description	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.
format	Thesis
qualification_level	Master's degree
author	Maniam, Kalai Vani
author_facet	Maniam, Kalai Vani
author_sort	Maniam, Kalai Vani
title	Development of oil palm (Elaeis guineensis Jacq) RNAi constructs and transformation of cDNA candidates into rice (Oryza sativa L.)
title_short	Development of oil palm (Elaeis guineensis Jacq) RNAi constructs and transformation of cDNA candidates into rice (Oryza sativa L.)
title_full	Development of oil palm (Elaeis guineensis Jacq) RNAi constructs and transformation of cDNA candidates into rice (Oryza sativa L.)
title_fullStr	Development of oil palm (Elaeis guineensis Jacq) RNAi constructs and transformation of cDNA candidates into rice (Oryza sativa L.)
title_full_unstemmed	Development of oil palm (Elaeis guineensis Jacq) RNAi constructs and transformation of cDNA candidates into rice (Oryza sativa L.)
title_sort	development of oil palm (elaeis guineensis jacq) rnai constructs and transformation of cdna candidates into rice (oryza sativa l.)
granting_institution	Universiti Putra Malaysia
publishDate	2012
url	http://psasir.upm.edu.my/id/eprint/48907/8/FBSB%202012%2054R.pdf
_version_	1747812006792003584

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

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