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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| Format: | Thesis |
| Language: | English English |
| Published: |
2000
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/9185/1/FSAS_2000_40_IR.pdf |
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| Summary: | 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. |
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