Factors affecting the transformation efficiency of Arabidopsis thaliana plant and its application in analyzing the oil palm MT3-B gene promoter

Ramli, Zubaidah and Abdullah, Siti Nor Akmar (2003) Factors affecting the transformation efficiency of Arabidopsis thaliana plant and its application in analyzing the oil palm MT3-B gene promoter. [Proceedings Paper]

Arabidopsis thaliana is a small flowering plant that is widely used as an experimental model plant for genetic, biochemical and molecular biological studies. Agrobacterium-mediated transformation of Arabidopsis is relatively simple and well established. Since tissue culture system is not required for regeneration, it becomes a very popular heterologous system for testing plant genes, gene constructs and promoters. In this study, the isolated MT3-B gene promoter of the oil palm was cloned into binary promoter less plasmid (pBI101) containing β-glucuronidase gene (GUS) as reporter gene to produce a recombinant plasmid designated pBI102b. Restriction digestion and PCR analysis confirmed the expected insert size of about 750bp. pBI102b was then mobilised into Agrobacterium tumefaciens(C58) via electroporation method and successful introduction of the plasmid confirmed by PCR analysis. Agrobacterium-mediated transformation of Arabidopsis was carried out using C58 (pB1102b) via floral dip method. T1 seeds were harvested in bulk and transformed seeds were selected in MS (Murashige and Skoog 1962) plates containing 50mg/l kanamycin. T2 seeds were harvested individually. Analysis of the expression of the reporter gene in the transgenic Arabidopsis plant is being carried out using homozygous seeds in order to determine the strength and specificity of the promoter. PCR analysis using a pair of primers from the 3 ' and 5 ' rerminal sequence of the oil palm MT3-B gene promoter confirmed the presence of this ?romoter in the antibiotic resistant Arabidopsis plants.

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Abstract

Arabidopsis thaliana is a small flowering plant that is widely used as an experimental model plant for genetic, biochemical and molecular biological studies. Agrobacterium-mediated transformation of Arabidopsis is relatively simple and well established. Since tissue culture system is not required for regeneration, it becomes a very popular heterologous system for testing plant genes, gene constructs and promoters. In this study, the isolated MT3-B gene promoter of the oil palm was cloned into binary promoter less plasmid (pBI101) containing β-glucuronidase gene (GUS) as reporter gene to produce a recombinant plasmid designated pBI102b. Restriction digestion and PCR analysis confirmed the expected insert size of about 750bp. pBI102b was then mobilised into Agrobacterium tumefaciens(C58) via electroporation method and successful introduction of the plasmid confirmed by PCR analysis. Agrobacterium-mediated transformation of Arabidopsis was carried out using C58 (pB1102b) via floral dip method. T1 seeds were harvested in bulk and transformed seeds were selected in MS (Murashige and Skoog 1962) plates containing 50mg/l kanamycin. T2 seeds were harvested individually. Analysis of the expression of the reporter gene in the transgenic Arabidopsis plant is being carried out using homozygous seeds in order to determine the strength and specificity of the promoter. PCR analysis using a pair of primers from the 3 ' and 5 ' rerminal sequence of the oil palm MT3-B gene promoter confirmed the presence of this ?romoter in the antibiotic resistant Arabidopsis plants.

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Item Type:	Proceedings Paper
Additional Information:	Available at Perpustakaan Sultan Abdul Samad, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. TP684 P3I61 2003 Call Number
AGROVOC Term:	oil palm > oil palm Prefer using Elaeis guineensisElaeis guineensis
AGROVOC Term:	genetic transformation
AGROVOC Term:	data analysis
AGROVOC Term:	plasmids
AGROVOC Term:	transgenic plants
AGROVOC Term:	gene expression
Geographical Term:	Malaysia
Uncontrolled Keywords:	Arabidopsis thaliana
Depositing User:	Nor Hasnita Abdul Samat
Date Deposited:	22 Jul 2024 06:22
Last Modified:	22 Jul 2024 06:22
URI:	http://webagris.upm.edu.my/id/eprint/840

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