Optimisation of gusA gene transfer into cocoa (Theobroma cacao) via agrobacterium tumefaciens-mediated transformation

This study describes the optimization of parameters (pH, temperature, periods of co-cultivation and age of explant) for gusA gene transfer into two cocoa clones (PBC 123 and BR 25) via Agrobacterium tumefaciens-mediated transformation by using staminode cocoa bud flower collected one day before anth...

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Main Author:	Anisah Savantil
Format:	Thesis
Language:	English English
Published:	2011
Subjects:	QK474.8-495 Spermatophyta Phanerogams
Online Access:	https://eprints.ums.edu.my/id/eprint/41848/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/41848/2/FULLTEXT.pdf
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spelling	my-ums-ep.418482024-12-05T03:51:16Z Optimisation of gusA gene transfer into cocoa (Theobroma cacao) via agrobacterium tumefaciens-mediated transformation 2011 Anisah Savantil QK474.8-495 Spermatophyta. Phanerogams This study describes the optimization of parameters (pH, temperature, periods of co-cultivation and age of explant) for gusA gene transfer into two cocoa clones (PBC 123 and BR 25) via Agrobacterium tumefaciens-mediated transformation by using staminode cocoa bud flower collected one day before anthesis as explants. Super avirulent A. tumefaciens strain AGL 1 harbouring the binary vector pGPTVKan/ GUS was used. The binary vector contains a CaMV 35S-driven β-glucuronidase (GUS) gene and a complete neomycin phosphotransferase II (nptII) gene for conferring plant resistance to the antibiotic paromomycin. Callus induction medium (IM) for clone PBC 123 consisted of Driver and Kuniyuki Walnut (DKW) minerals, glucose, vitamins, 2 mg l-1 2, 4-dichlorophenoxyacetic acid (2,4-D) and 0.005 mg l-1 Thidiazuron (TDZ) pH5.3. Callus IM for clone BR 25 was identical to IM for clone PBC 123 except the TDZ was replaced with 100 mg l-1 2-iP. Callus was allowed to proliferate on semi-solid callus inducing medium for 14 days, 21 days, and 28 days prior co-cultivation with A. tumefaciens on semi-solid co-cultivation media for one, two, and three days at various temperatures (190C, 210C, 230C, and 250C), and pHs (4.8, 5.3, and 5.8). Each set of parameter was design in triplicate plate. Half amount of treated calli in each tested parameter was examined for GUS expression, while another half was selected on selective medium containing 100 μg ml-1 paromomycin. GUS expression analyses were done after 18 weeks selection. Based on the GUS expression analyses, up to 86% of calli had at least one blue sector after 18 weeks on selection media containing 100 μg ml-1 paromomycin. The best transformation frequency (86.1%) for clone BR 25 was obtained when explant was 21 days old and transformation condition was with pH 5.8, 250C, and three days cocultivation period. For clone PBC 123, the best transformation frequency (83.3%) was obtained when explant was 14 days old and transformation condition was with pH 4.8, 190C, and three days co-cultivation period. A PCR-walking method (Cottage et al. 2001) with modification was used to verify the integration of the T-DNA into genome of the putatively transformed cocoa calli. Based on the sequencing results, this method worked with purified plasmid pGPTV-KAN/GUS but the sensitivity too low for putatively transformed cocoa calli. 2011 Thesis https://eprints.ums.edu.my/id/eprint/41848/ https://eprints.ums.edu.my/id/eprint/41848/1/24%20PAGES.pdf text en public https://eprints.ums.edu.my/id/eprint/41848/2/FULLTEXT.pdf text en validuser masters Universiti Malaysia Sabah School of Science and Technology
institution	Universiti Malaysia Sabah
collection	UMS Institutional Repository
language	English English
topic	QK474.8-495 Spermatophyta Phanerogams
spellingShingle	QK474.8-495 Spermatophyta Phanerogams Anisah Savantil Optimisation of gusA gene transfer into cocoa (Theobroma cacao) via agrobacterium tumefaciens-mediated transformation
description	This study describes the optimization of parameters (pH, temperature, periods of co-cultivation and age of explant) for gusA gene transfer into two cocoa clones (PBC 123 and BR 25) via Agrobacterium tumefaciens-mediated transformation by using staminode cocoa bud flower collected one day before anthesis as explants. Super avirulent A. tumefaciens strain AGL 1 harbouring the binary vector pGPTVKan/ GUS was used. The binary vector contains a CaMV 35S-driven β-glucuronidase (GUS) gene and a complete neomycin phosphotransferase II (nptII) gene for conferring plant resistance to the antibiotic paromomycin. Callus induction medium (IM) for clone PBC 123 consisted of Driver and Kuniyuki Walnut (DKW) minerals, glucose, vitamins, 2 mg l-1 2, 4-dichlorophenoxyacetic acid (2,4-D) and 0.005 mg l-1 Thidiazuron (TDZ) pH5.3. Callus IM for clone BR 25 was identical to IM for clone PBC 123 except the TDZ was replaced with 100 mg l-1 2-iP. Callus was allowed to proliferate on semi-solid callus inducing medium for 14 days, 21 days, and 28 days prior co-cultivation with A. tumefaciens on semi-solid co-cultivation media for one, two, and three days at various temperatures (190C, 210C, 230C, and 250C), and pHs (4.8, 5.3, and 5.8). Each set of parameter was design in triplicate plate. Half amount of treated calli in each tested parameter was examined for GUS expression, while another half was selected on selective medium containing 100 μg ml-1 paromomycin. GUS expression analyses were done after 18 weeks selection. Based on the GUS expression analyses, up to 86% of calli had at least one blue sector after 18 weeks on selection media containing 100 μg ml-1 paromomycin. The best transformation frequency (86.1%) for clone BR 25 was obtained when explant was 21 days old and transformation condition was with pH 5.8, 250C, and three days cocultivation period. For clone PBC 123, the best transformation frequency (83.3%) was obtained when explant was 14 days old and transformation condition was with pH 4.8, 190C, and three days co-cultivation period. A PCR-walking method (Cottage et al. 2001) with modification was used to verify the integration of the T-DNA into genome of the putatively transformed cocoa calli. Based on the sequencing results, this method worked with purified plasmid pGPTV-KAN/GUS but the sensitivity too low for putatively transformed cocoa calli.
format	Thesis
qualification_level	Master's degree
author	Anisah Savantil
author_facet	Anisah Savantil
author_sort	Anisah Savantil
title	Optimisation of gusA gene transfer into cocoa (Theobroma cacao) via agrobacterium tumefaciens-mediated transformation
title_short	Optimisation of gusA gene transfer into cocoa (Theobroma cacao) via agrobacterium tumefaciens-mediated transformation
title_full	Optimisation of gusA gene transfer into cocoa (Theobroma cacao) via agrobacterium tumefaciens-mediated transformation
title_fullStr	Optimisation of gusA gene transfer into cocoa (Theobroma cacao) via agrobacterium tumefaciens-mediated transformation
title_full_unstemmed	Optimisation of gusA gene transfer into cocoa (Theobroma cacao) via agrobacterium tumefaciens-mediated transformation
title_sort	optimisation of gusa gene transfer into cocoa (theobroma cacao) via agrobacterium tumefaciens-mediated transformation
granting_institution	Universiti Malaysia Sabah
granting_department	School of Science and Technology
publishDate	2011
url	https://eprints.ums.edu.my/id/eprint/41848/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/41848/2/FULLTEXT.pdf
_version_	1818611435946639360

Optimisation of gusA gene transfer into cocoa (Theobroma cacao) via agrobacterium tumefaciens-mediated transformation

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