Genetic engineering of Musa sapientum L. CV. nangka (AAB) for tolerance to fusarium wilt

Bananas and plantains are the fourth most important crop in the world as well as Malaysia. In recent years, their production has been seriously threatened by Fusarium wilt, caused by Fusarium oxysporum f.sp. cubense fungus, and it is one of the most important destructive diseases of banana (Musa spp...

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Main Author:	Mahdavi Darvari, Fatemeh
Format:	Thesis
Language:	English English
Published:	2011
Subjects:	Plant genetic engineering Bananas - Diseases and pests Fusarium wilt of banana
Online Access:	http://psasir.upm.edu.my/id/eprint/26785/7/ITA%202011%208RR.pdf
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spelling	my-upm-ir.267852022-01-26T05:39:46Z Genetic engineering of Musa sapientum L. CV. nangka (AAB) for tolerance to fusarium wilt 2011-04 Mahdavi Darvari, Fatemeh Bananas and plantains are the fourth most important crop in the world as well as Malaysia. In recent years, their production has been seriously threatened by Fusarium wilt, caused by Fusarium oxysporum f.sp. cubense fungus, and it is one of the most important destructive diseases of banana (Musa spp.). Genetic engineering offers the greatest opportunity to the breeders to increase tolerance to Fusarium wilt. Hence, this study aims to produce a new banana cultivar tolerant to F. oxysporum f.sp. cubense race 4 through genetic engineering. Male inflorescence of Pisang Berangan (AAA), Pisang Nangka (AAB), Pisang Rastali (AAB) and Pisang Abu (ABB) as a potential explant for rapid micropropagation were used for in vitro propagation study. Considering the importance of cooking banana in the diet of the local people, Pisang Nangka (AAB), a cooking banana, was chosen for the genetic engineering study. Single cauliflower-like bodies cluster induced from male flowers, in MS medium containing 8 mg/L of BAP, was used as target tissue for gene transfer. Hygromycin was applied as a selection agent since plasmids used in this study contain the hygromycin resistance gene. Physical and biological parameters which affect DNA delivery into the Pisang Nangka (AAB) single cauliflower-like bodies’ have been optimised using gfp as reporter gene. The optimised bombardment parameters for the ‘CLBs’ clusters was 1100 psi, 9 cm target distance, 28 mmHg, 1 μm gold particle size, two times bombardment, 60 μg/μL amount of the gold particles per bombardment, 1.5 μg/μL DNA per bombardment and three days pre-culture prior to the bombardment. Rice thaumatin-like protein gene was cloned in pCambia 1304 binary vector by NcoI and PmlI restriction enzymes. Transformation of cloned rice thaumatin-like protein (tlp) gene was performed using particle bombardment. Integration of transgene was assessed by PCR amplification of tlp gene using its specific primer. Genomic southern blot hybridization confirmed the incorporation of the tlp gene in the host genome. RT-PCR revealed the expression of transgene in leaf tissue as well as root tissue in transformants.The 28 day old spores of F. oxysporum f.sp. cubense (Race 4) with 66% germinating capacity were used to estimate the transgenic Pisang Nangka (AAB) plantlets tolerance against Fusarium wilt. The percentage of disease incidence occurred in control plantlets was 89.1% after four weeks of infection while it was 29.4 % in transgenic plants. The results demonstrated that expression of rice thaumatin-like protein in transgenic banana plants enhanced resistanceto Fusarium wilt significantly. Plant genetic engineering Bananas - Diseases and pests Fusarium wilt of banana 2011-04 Thesis http://psasir.upm.edu.my/id/eprint/26785/ http://psasir.upm.edu.my/id/eprint/26785/7/ITA%202011%208RR.pdf application/pdf en public doctoral Universiti Putra Malaysia Plant genetic engineering Bananas - Diseases and pests Fusarium wilt of banana Institute of Tropical Agriculture English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
topic	Plant genetic engineering Bananas - Diseases and pests Fusarium wilt of banana
spellingShingle	Plant genetic engineering Bananas - Diseases and pests Fusarium wilt of banana Mahdavi Darvari, Fatemeh Genetic engineering of Musa sapientum L. CV. nangka (AAB) for tolerance to fusarium wilt
description	Bananas and plantains are the fourth most important crop in the world as well as Malaysia. In recent years, their production has been seriously threatened by Fusarium wilt, caused by Fusarium oxysporum f.sp. cubense fungus, and it is one of the most important destructive diseases of banana (Musa spp.). Genetic engineering offers the greatest opportunity to the breeders to increase tolerance to Fusarium wilt. Hence, this study aims to produce a new banana cultivar tolerant to F. oxysporum f.sp. cubense race 4 through genetic engineering. Male inflorescence of Pisang Berangan (AAA), Pisang Nangka (AAB), Pisang Rastali (AAB) and Pisang Abu (ABB) as a potential explant for rapid micropropagation were used for in vitro propagation study. Considering the importance of cooking banana in the diet of the local people, Pisang Nangka (AAB), a cooking banana, was chosen for the genetic engineering study. Single cauliflower-like bodies cluster induced from male flowers, in MS medium containing 8 mg/L of BAP, was used as target tissue for gene transfer. Hygromycin was applied as a selection agent since plasmids used in this study contain the hygromycin resistance gene. Physical and biological parameters which affect DNA delivery into the Pisang Nangka (AAB) single cauliflower-like bodies’ have been optimised using gfp as reporter gene. The optimised bombardment parameters for the ‘CLBs’ clusters was 1100 psi, 9 cm target distance, 28 mmHg, 1 μm gold particle size, two times bombardment, 60 μg/μL amount of the gold particles per bombardment, 1.5 μg/μL DNA per bombardment and three days pre-culture prior to the bombardment. Rice thaumatin-like protein gene was cloned in pCambia 1304 binary vector by NcoI and PmlI restriction enzymes. Transformation of cloned rice thaumatin-like protein (tlp) gene was performed using particle bombardment. Integration of transgene was assessed by PCR amplification of tlp gene using its specific primer. Genomic southern blot hybridization confirmed the incorporation of the tlp gene in the host genome. RT-PCR revealed the expression of transgene in leaf tissue as well as root tissue in transformants.The 28 day old spores of F. oxysporum f.sp. cubense (Race 4) with 66% germinating capacity were used to estimate the transgenic Pisang Nangka (AAB) plantlets tolerance against Fusarium wilt. The percentage of disease incidence occurred in control plantlets was 89.1% after four weeks of infection while it was 29.4 % in transgenic plants. The results demonstrated that expression of rice thaumatin-like protein in transgenic banana plants enhanced resistanceto Fusarium wilt significantly.
format	Thesis
qualification_level	Doctorate
author	Mahdavi Darvari, Fatemeh
author_facet	Mahdavi Darvari, Fatemeh
author_sort	Mahdavi Darvari, Fatemeh
title	Genetic engineering of Musa sapientum L. CV. nangka (AAB) for tolerance to fusarium wilt
title_short	Genetic engineering of Musa sapientum L. CV. nangka (AAB) for tolerance to fusarium wilt
title_full	Genetic engineering of Musa sapientum L. CV. nangka (AAB) for tolerance to fusarium wilt
title_fullStr	Genetic engineering of Musa sapientum L. CV. nangka (AAB) for tolerance to fusarium wilt
title_full_unstemmed	Genetic engineering of Musa sapientum L. CV. nangka (AAB) for tolerance to fusarium wilt
title_sort	genetic engineering of musa sapientum l. cv. nangka (aab) for tolerance to fusarium wilt
granting_institution	Universiti Putra Malaysia
granting_department	Institute of Tropical Agriculture
publishDate	2011
url	http://psasir.upm.edu.my/id/eprint/26785/7/ITA%202011%208RR.pdf
_version_	1747811561478553600

Genetic engineering of Musa sapientum L. CV. nangka (AAB) for tolerance to fusarium wilt

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