Agrobacterium tumefaciens - mediated transformation of nicotiana benthamiana with dehalogenase gene resistant to monochloroacetic acid

Weeds give adverse effects to crops because of the competition to get nutrients, light and moisture. Many farmers used broad-spectrum herbicide such as monocloroacetic acid (MCA) which is effective at killing a wide range of weeds. Unfortunately, broad-spectrum of herbicide can also kill valuable cr...

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Main Author:	Mohamed, Elizah
Format:	Thesis
Language:	English
Published:	2017
Subjects:	QH426 Genetics
Online Access:	http://eprints.utm.my/id/eprint/79281/1/ElizahMohamedPFBME2017.pdf
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spelling	my-utm-ep.792812018-10-14T08:41:56Z Agrobacterium tumefaciens - mediated transformation of nicotiana benthamiana with dehalogenase gene resistant to monochloroacetic acid 2017 Mohamed, Elizah QH426 Genetics Weeds give adverse effects to crops because of the competition to get nutrients, light and moisture. Many farmers used broad-spectrum herbicide such as monocloroacetic acid (MCA) which is effective at killing a wide range of weeds. Unfortunately, broad-spectrum of herbicide can also kill valuable crops and cause significant losses in agricultural productivity. One of the solutions to this problem is by developing herbicide resistant plant using dehalogenase D (dehD) gene isolated from Rhizobium sp. RC1. A dehD gene encoding dehalogenase enzyme that has the capability to degrade monochloroacetic acid (MCA) was isolated and characterized from Rhizobium sp. RC1. dehD gene was used as herbicide resistance gene and selectable marker gene in Nicotiana benthamiana plant transformation. The 798 bp dehD gene was inserted into pCAMBIA 1305.2 under the control of the Cauliflower Mosaic Virus 35S (CaMV35S) promoter and designated as pCAMdehD, with a total size of 10,592 bp. A few parameters of Agrobacterium tumefaciens-mediated transformation were optimized including hygromycin concentration (40 μg/mL of hygromycin), and the MCA toxicity level to N. benthamiana at tissue culture (60 μg/L of MCA) and whole plant stage (2.0 g/L of MCA). pCAMdehD was introduced into N. benthamiana via Agrobacterium mediated transformation method. Based on the screening of the transformants on MS media containing 60 μg/L MCA, the results showed that N. benthamiana was successfully transformed with dehalogenase D gene with 50 % of transformation efficiency. The integration and expression of dehD gene in N. benthamiana were confirmed by PCR, Southern Blotting and reverse transcription PCR. Analysis of leaf-painting assay revealed that transgenic N. benthamiana (T1) was resistant to 4.0 g/L MCA compared to 2.0 g/L for non-transformed plants control. The Chi Square analyses of five transgenic plants (T1), suggested that the dehD gene was segregated according to Mendelian 3:1 ratio. These findings showed that transgenic N. benthamiana plant resistant to MCA herbicide was successfully produced. 2017 Thesis http://eprints.utm.my/id/eprint/79281/ http://eprints.utm.my/id/eprint/79281/1/ElizahMohamedPFBME2017.pdf application/pdf en public phd doctoral Universiti Teknologi Malaysia, Faculty of Biosciences and Medical Engineering Faculty of Biosciences and Medical Engineering
institution	Universiti Teknologi Malaysia
collection	UTM Institutional Repository
language	English
topic	QH426 Genetics
spellingShingle	QH426 Genetics Mohamed, Elizah Agrobacterium tumefaciens - mediated transformation of nicotiana benthamiana with dehalogenase gene resistant to monochloroacetic acid
description	Weeds give adverse effects to crops because of the competition to get nutrients, light and moisture. Many farmers used broad-spectrum herbicide such as monocloroacetic acid (MCA) which is effective at killing a wide range of weeds. Unfortunately, broad-spectrum of herbicide can also kill valuable crops and cause significant losses in agricultural productivity. One of the solutions to this problem is by developing herbicide resistant plant using dehalogenase D (dehD) gene isolated from Rhizobium sp. RC1. A dehD gene encoding dehalogenase enzyme that has the capability to degrade monochloroacetic acid (MCA) was isolated and characterized from Rhizobium sp. RC1. dehD gene was used as herbicide resistance gene and selectable marker gene in Nicotiana benthamiana plant transformation. The 798 bp dehD gene was inserted into pCAMBIA 1305.2 under the control of the Cauliflower Mosaic Virus 35S (CaMV35S) promoter and designated as pCAMdehD, with a total size of 10,592 bp. A few parameters of Agrobacterium tumefaciens-mediated transformation were optimized including hygromycin concentration (40 μg/mL of hygromycin), and the MCA toxicity level to N. benthamiana at tissue culture (60 μg/L of MCA) and whole plant stage (2.0 g/L of MCA). pCAMdehD was introduced into N. benthamiana via Agrobacterium mediated transformation method. Based on the screening of the transformants on MS media containing 60 μg/L MCA, the results showed that N. benthamiana was successfully transformed with dehalogenase D gene with 50 % of transformation efficiency. The integration and expression of dehD gene in N. benthamiana were confirmed by PCR, Southern Blotting and reverse transcription PCR. Analysis of leaf-painting assay revealed that transgenic N. benthamiana (T1) was resistant to 4.0 g/L MCA compared to 2.0 g/L for non-transformed plants control. The Chi Square analyses of five transgenic plants (T1), suggested that the dehD gene was segregated according to Mendelian 3:1 ratio. These findings showed that transgenic N. benthamiana plant resistant to MCA herbicide was successfully produced.
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Mohamed, Elizah
author_facet	Mohamed, Elizah
author_sort	Mohamed, Elizah
title	Agrobacterium tumefaciens - mediated transformation of nicotiana benthamiana with dehalogenase gene resistant to monochloroacetic acid
title_short	Agrobacterium tumefaciens - mediated transformation of nicotiana benthamiana with dehalogenase gene resistant to monochloroacetic acid
title_full	Agrobacterium tumefaciens - mediated transformation of nicotiana benthamiana with dehalogenase gene resistant to monochloroacetic acid
title_fullStr	Agrobacterium tumefaciens - mediated transformation of nicotiana benthamiana with dehalogenase gene resistant to monochloroacetic acid
title_full_unstemmed	Agrobacterium tumefaciens - mediated transformation of nicotiana benthamiana with dehalogenase gene resistant to monochloroacetic acid
title_sort	agrobacterium tumefaciens - mediated transformation of nicotiana benthamiana with dehalogenase gene resistant to monochloroacetic acid
granting_institution	Universiti Teknologi Malaysia, Faculty of Biosciences and Medical Engineering
granting_department	Faculty of Biosciences and Medical Engineering
publishDate	2017
url	http://eprints.utm.my/id/eprint/79281/1/ElizahMohamedPFBME2017.pdf
_version_	1747818190136672256

Agrobacterium tumefaciens - mediated transformation of nicotiana benthamiana with dehalogenase gene resistant to monochloroacetic acid

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