MSc

Diabetes mellitus is a disorder affecting 100 million people round the globe.
Gymnema sylvestre R.Br. is being used in antidiabetic therapies with good success.
Depletion of forest area has reduced the supply of Gymnema leaves. To bridge the gap
between an ever increasing demand and the dwindling supply of antidiabetic medicines
from Gymnema, this study was undertaken to examine whether these metabolites could be
produced by in vitro techniques.
This study was undertaken at the Department of Plantation Crops and Spices,
College of Horticulture, Kerala Agricultural University, Vellanikkara during May, 1998 to
November, 1999. Callus cultures were initiated in test tubes and were maintained at
26 ± 2 QC temperature and 60 to 80 per cent relative humidity for three months.
Observations on growth of calli were noted at weekly intervals, while saponin production
was noted after each month.
Internode, node, petiole, leaf lamina and root segments were evaluated as explants
to initiate calli. Among them, internode and petiole were found to have maximum potential
to initiate and proliferate calli. Root explants did not produce significant amounts of callus.
The auxins evaluated for stimulating callus production were 2,4-D, NAA, lAA and
.IBA. Among them, 2,4-D was the most potent in callusing followed by NAA. Both, lAA
and IBA performed very poorly. The cytokinins (BA and kinetin) showed statistically
similar performances. The combination of2,4-D with BA produced maximum callus.
MS medium supplemented with 2, 4-D (2 mg rl) and BA (I mg rl) was selected as
the basal medium due to high callusing and high saponin yields. Stress inducing chemicals
were added to it to examine whether they increased the production of saponins. It was
found that addition of mannitol, activated charcoal, peptone and malt extract enhanced the

saponin yields. The highest saponin yield per day per tube was produced from the medium
comprising of MS + 2,4-0 (2 mg rl) + BA (I mg rl) + malt extract (I %).
The cell suspensions were maintained on rotary shaker at 105 rpm for four weeks
and were subcultured every seven days. Observations on growth of cultures and saponin
production were recorded at weekly interval. The production of saponins was highest in
the medium containing MS + 2,4-D (2 mg rl) + BA (l mg rl) + phloroglucinol (50 mg rl).
These saponins need to be identified and charachterisecl.
Both calli and cell suspensions produced new groups of saponins which were not
present in the plant extracts, suggesting that de 1101'0 synthesis occurred in the ill vitro
cultures.
Saponins were extracted from ill vitro samples by 60 per cent ethanol solution and
the extract was fractionated with an equal volume of a mixture of chloroform and methanol
in 1: 1 proportion. The chloroform fraction was spotted on thin layer chromatograms and
eluted at 29 to 30°C temperature and 75 per cent relative humidity. Elution was done in
chloroform : acetone: methanol (5 : I : 1.5) running solvent system. Spraying was done
with three per cent vanillin and five per cent sulphuric acid in ethanol to develop purple,
blue, violet and red spots for saponins. Saponins were fractionated into a profile of distinct
spots by eluting in chloroform and methanol mixture in 80 : 20 ratio followed by repeated
elution in 98 : 2 proportion.