Hyoscyamine and scopolamine production in transformed root cultures of Datura metel L

The transformed root cultures of Datura metel L (kecubung) was successfully established via Agrobacterium rhizogenes-mediated, which contained the pBI 121 plasmid harbouring the GUS and kanamycin coding genes. The transformation was biochemically confirmed with GUS assay indicated by the presence...

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Main Author:	Ahmad, Aziz
Format:	Thesis
Language:	English English
Published:	2000
Subjects:	Atropine Scopolamine Datura meteloides
Online Access:	http://psasir.upm.edu.my/id/eprint/9185/1/FSAS_2000_40_IR.pdf
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id	my-upm-ir.9185
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spelling	my-upm-ir.91852024-02-02T03:28:29Z Hyoscyamine and scopolamine production in transformed root cultures of Datura metel L 2000-12 Ahmad, Aziz The transformed root cultures of Datura metel L (kecubung) was successfully established via Agrobacterium rhizogenes-mediated, which contained the pBI 121 plasmid harbouring the GUS and kanamycin coding genes. The transformation was biochemically confirmed with GUS assay indicated by the presence of blue spot on roots, Southern blotting and the resistance of transformed root to Kanamycin. Transformed roots were showed a typical character of transformed root, which is sensitive to exogenous IAA (auxin). Sustained root cultures appeared to produce hyoscyamine and scopolamine ten times higher than that produced in the intact plant. The ability of the transformed roots to produce hyoscyamine and scopolamine in different types of basal media used was examined. It was ubserved that Gamborg's B 5 basal medium was the best medium for root growth as well as the hyoscyamine and scopolamine production. Gamborg's B5 medium was used for subsequent studies. Among the carbon source tested, sucrose appeared to be the best carbon source for root growth. Consequently, the effect of Gamborg's B5 medium ionic strength and sucrose concentration was examined. Gamborg's BS medium was used in quarter, half and full strength, and supplemented with sucrose concentration in the range of 1 - to 8% (w/v). Full strength Gamborg' s B5 medium with 4% sucrose was observed to enhance the root growth as well as hyoscyamine and scopolamine production. Studies were also carried out to examine the effect of various concentrations of macro and microelements on root growth, hyoscyamine and scopolamine production. The macro elements used were nitrogen (ammonium and nitrate balance), magnesium, calcium and phosphate. Amongst the macro element tested, nitrogen, which is in the form of nitrate and/or ammonium, was found to have a significant effect on the hyoscyamine and scopolamine production. Meanwhile, the microelements that have been studied were copper, ferric, manganese, zinc and boron. Ferric and copper appeared to have the greater effect on hyoscyamine and scopolamine production than other elements. Roots cultured in medium with lower concentration of microelement than that present in Gamborg's B5 medium was also observed to enhance the hyoscyamine and scopolamine content. Feeding of each precursors i. e. putrescine, I-ornithine, arginine, 1-phenylalanine, hyoscyamine and scopolamine at lower concentration (less than 0.2 mM) into treatment medium was observed to reduce the root growth as well as the hyoscyamine and scopolamine production. Combination of l-phenylalanine with putrescine appeared the best precursor for both hyoscyamine and scopolamine production. Atropine Scopolamine Datura meteloides 2000-12 Thesis http://psasir.upm.edu.my/id/eprint/9185/ http://psasir.upm.edu.my/id/eprint/9185/1/FSAS_2000_40_IR.pdf text en public doctoral Universiti Putra Malaysia Atropine Scopolamine Datura meteloides Faculty of Science and Environmental Studies Mahmood, Marziah English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
advisor	Mahmood, Marziah
topic	Atropine Scopolamine Datura meteloides
spellingShingle	Atropine Scopolamine Datura meteloides Ahmad, Aziz Hyoscyamine and scopolamine production in transformed root cultures of Datura metel L
description	The transformed root cultures of Datura metel L (kecubung) was successfully established via Agrobacterium rhizogenes-mediated, which contained the pBI 121 plasmid harbouring the GUS and kanamycin coding genes. The transformation was biochemically confirmed with GUS assay indicated by the presence of blue spot on roots, Southern blotting and the resistance of transformed root to Kanamycin. Transformed roots were showed a typical character of transformed root, which is sensitive to exogenous IAA (auxin). Sustained root cultures appeared to produce hyoscyamine and scopolamine ten times higher than that produced in the intact plant. The ability of the transformed roots to produce hyoscyamine and scopolamine in different types of basal media used was examined. It was ubserved that Gamborg's B 5 basal medium was the best medium for root growth as well as the hyoscyamine and scopolamine production. Gamborg's B5 medium was used for subsequent studies. Among the carbon source tested, sucrose appeared to be the best carbon source for root growth. Consequently, the effect of Gamborg's B5 medium ionic strength and sucrose concentration was examined. Gamborg's BS medium was used in quarter, half and full strength, and supplemented with sucrose concentration in the range of 1 - to 8% (w/v). Full strength Gamborg' s B5 medium with 4% sucrose was observed to enhance the root growth as well as hyoscyamine and scopolamine production. Studies were also carried out to examine the effect of various concentrations of macro and microelements on root growth, hyoscyamine and scopolamine production. The macro elements used were nitrogen (ammonium and nitrate balance), magnesium, calcium and phosphate. Amongst the macro element tested, nitrogen, which is in the form of nitrate and/or ammonium, was found to have a significant effect on the hyoscyamine and scopolamine production. Meanwhile, the microelements that have been studied were copper, ferric, manganese, zinc and boron. Ferric and copper appeared to have the greater effect on hyoscyamine and scopolamine production than other elements. Roots cultured in medium with lower concentration of microelement than that present in Gamborg's B5 medium was also observed to enhance the hyoscyamine and scopolamine content. Feeding of each precursors i. e. putrescine, I-ornithine, arginine, 1-phenylalanine, hyoscyamine and scopolamine at lower concentration (less than 0.2 mM) into treatment medium was observed to reduce the root growth as well as the hyoscyamine and scopolamine production. Combination of l-phenylalanine with putrescine appeared the best precursor for both hyoscyamine and scopolamine production.
format	Thesis
qualification_level	Doctorate
author	Ahmad, Aziz
author_facet	Ahmad, Aziz
author_sort	Ahmad, Aziz
title	Hyoscyamine and scopolamine production in transformed root cultures of Datura metel L
title_short	Hyoscyamine and scopolamine production in transformed root cultures of Datura metel L
title_full	Hyoscyamine and scopolamine production in transformed root cultures of Datura metel L
title_fullStr	Hyoscyamine and scopolamine production in transformed root cultures of Datura metel L
title_full_unstemmed	Hyoscyamine and scopolamine production in transformed root cultures of Datura metel L
title_sort	hyoscyamine and scopolamine production in transformed root cultures of datura metel l
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Science and Environmental Studies
publishDate	2000
url	http://psasir.upm.edu.my/id/eprint/9185/1/FSAS_2000_40_IR.pdf
_version_	1794018797265354752

Hyoscyamine and scopolamine production in transformed root cultures of Datura metel L

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