Utilisation of in vitro cultures of Tinospora cordifolia Miers (chittamrithu) for berberine
Loading...
Files
Date
2002
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Department of Plantation Crops, College of Horticulture, Vellanikkara
Abstract
The present investigation on "Utilisation of in vitro cultures of
Tinospora cordifolia Miers. (Chittamrithu) for berberine" was carried out in the
Plant Tissue Culture and Biochemistry Laboratories, College of Horticulture,
Vellanikkara, Thrissur during the period 1999-2001. The study was undertaken
with the objective to standardise the in vitro techniques for initiation and
proliferation of static and suspension cultures of T cordifolia and to screen the in
vitro cultures for synthesis of berberine and quantify it. It was also envisaged to
enhance the level of product synthesis in in vitro cultures.
Leaf, petiole and stem derived callus cultures of Vellanikkara and
Madurai ecotypes were established in vitro. Surface sterilisation with mercuric
chloride (HgCh) at 0.1 per cent for 8 min was most effective in all the explants.
MS medium at full strength supplemented with NAA at 4 mg r ' was observed
ideal for initiation and proliferation of calli. Kinetin at 3 mg r' and BA at 4 mg r'
enhanced the callus inducing property of NAA. Both the ecotypes responded
equally for most of the parameters observed, with respect to callusing. The auxin
synergist, phloroglucinol at levels of 100.0 mg r' and 125 mg r' and casein
hyrdolysate at 100, 200 and 300 mg r' registered favourable influence on
callusing. Incubating leaf and stem cultures under illuminated conditions at
26±1 QC was significantly superior to incubation in dark.
Successful regeneration of roots from leaf and stem calli of the
experimental ecotypes was achieved on MS medium at half strength supplemented
with NAA or lAA each at 2 mg r'. Calli derived from Madurai ecotype performed
better with respect to root regeneration. None of the treatments tried resulted in a
positive response with respect to shoot initiation from callus cultures of both the
ecotypes. Substituting sucrose with lactose in proportions of 2: 1 in MS medium at
full strength fortified with NAA at 1 mg r ' and Kin at 4 mg r' initiated embryoids
in both the ecotypes.
MS media at full strength supplemented with NAA and BA or NAA and
Kin each at 2 mg r', was standardised as the production medium, which recorded
maximum berberine synthesis. Butanol-glacial acetic acid-water at 7:1:2 was
identified as the appropriate solvent system for detecting the alkaloid with
Dragendorff's reagent as the localizing spray.
Substituting sucrose with lactose maintaining a proportion of 2: 1 and
reducing the phosphorus level in basal medium to half the original strength
resulted in increased levels of berberine synthesis. The precursor phenyl alanine at
100, 150 and 200 mg r' elicited synthesis of berberine. Addition of osmoregulants,
polyethylene glycol at 2.0 and 3.0 per cent and mannitol at 1.5 per cent exerted a
favourable influence on synthesis of berberine in Tinospora.
Incorporation of autoclaved mycelia of Pythium aphanidermatum at 0.5,
1.0 and 1.5 g r' and immobilisation of calli with sodium alginate-calcium chloride
complex revealed a positive influence on synthesis of berberine.
Liquid suspensions of Vellanikkara and Madurai ecotypes registered
0.92 and 0.87 per cent of packed cell volume. Based on critical cell density, the
liquid suspension were subcultured at 16 days interval. As compared to static
cultures, suspensions synthesized lesser quantity of berberine.
Berberine was detected only in stem extracts of ex vitro plants. When
compared to ex vitro samples, in vitro cultures yielded higher quantities of
berberine.
The highest berberine yield (23.176 ug/g of callus) was obtained from
stem cultures maintained in solid MS media supplemented with NAA 2 mg r' +
BA 2 mg r ' and autoclaved mycelia of P. aphanidermatum at 0.5 g r'.
Madurai ecotype performed better with respect to berberine synthesis
with a mean value of 17.565 ug of berberine/gram of callus whereas Vellanikkara
ecotype synthesized 16.051 ug/g of callus under positively responding treatments.
Description
PG
Keywords
null