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Alkaloid production from root cultures

Ermayanti, Tri Muji (1993) Alkaloid production from root cultures. PhD thesis, Murdoch University.

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Root culture was used as a method for swainsonine (1,2,8-trihydroxyoctaindolizine) production from the Australian native plant Sxvainsona galegifolia (Darling pea). Root cultures were established either from seedling roots or after infection of plant organs with Agrobacterium rhizogenes. Murashige and Skoog (MS) liquid medium with 5 µM IBA was the best medium for growth of untransformed isolated roots while MS liquid medium with no hormones was best for transformed roots.

Swainsonine produced by root cultures of S. galegifolia reached a maximum 30 - 35 days after subculture, and the identity of the compound was confirmed by its GC retention time and mass spectrum in comparison to swainsonine from intact plants and the swainsonine standard (Sigma Co., USA). Roots and shoots from glasshousegrown plants had a similar swainsonine concentration to shoots of plants grown in vitro.

The concentration of swainsonine in transformed roots was higher than in untransformed roots. Tests of the effect of plant genotype and A. rhizogenes strain LBA 9402 showed that plant genotype influences swainsonine level in transformed roots, but that different transformation events in one genotype resulted in equally large differences in the level of swainsonine.

Polyploid roots might be expected to show higher levels of secondary metabolites than diploids, but attempts to induce polyploidy through use of colchicine, transformation of cotyledons, or differentiation of roots from callus was unsuccessful. Roots from callus showed lowered swainsonine levels concomitant with a reduction in modal chromosome number from 32 to 18.

In normal cultures of untransformed or transformed roots, the level of swainsonine in the medium was negligible. Stimulation of production of swainsonine and its release into the medium was attempted using CUSO4, reduction of medium pH, and supply of swainsonine precursors. Addition of 0.5 - 2.0 mM CUSO4 for 1 - 4 days to the culture medium for transformed roots increased swainsonine level and stimulated release of swainsonine to the medium. The maximum swainsonine concentration was achieved after treatment with 2.0 mM CUSO4 for 2 days. Reduction of medium pH from 5.7 to 2.7 for 1 day also increased swainsonine level in transformed roots and in the medium. The precursors pipecolic acid and malonic acid at 0.005 - 2.0 mM given for 1 - 12 days in MS liquid medium increased root growth and swainsonine level. The maximum swainsonine level was found in transformed roots with 2 mM of pipecolic acid for 6 days. Addition of 1 mM malonic acid for each of 4 successive days also increased growth of transformed roots and swainsonine level but the increase did not exceed that achieved by a single supplementation.

This report adds to the evidence that roots transformed with A. rhizogenes show high and stable levels of secondary metabolite production. Transformed roots of S. galegifolia had a swainsonine production almost 10-fold that measured in intact plants grown under glasshouse condition. The addition of precursors increased the level to almost 20-fold.

Root culture was also attempted for castanospermine production by Castanospermum australe. Growth of untransformed root cultures was unsuccessful. Untransformed roots grew in solid medium but failed to grow in liquid medium. It was not possible to induce transformed roots. No roots were obtained after infection using a range of A. rhizogenes strains or when the bacterium was pre-incubated with acetosyringone in the medium.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Biological and Environmental Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): McComb, Jen and O'Brien, Philip
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