MSc

The present investigation on “Exploitation of in vitro cultures of Indian
Madder (Rubia cordifolia L.) for anticancerous compounds” was carried out at the
Plant Tissue Culture Laboratory of the Centre for Plant Biotechnology and
Molecular Biology, College of Horticulture, Vellanikkara and Amala Cancer
Research Centre, Thrissur
during the period 2006-2008. The study was
undertaken with the objective to standardize the in vitro techniques for initiation
and proliferation of static and suspension cultures of Rubia cordifolia and to
screen the in vitro cultures for synthesis of naphthoquinone and quantify it. It was
also envisaged to enhance the level of product synthesis in in vitro cultures and to
assess the anticancerous activity of in vitro and in vivo extracts in terms of
cytotoxicity, antioxidant and prooxidant activities in vitro.
Leaf, nodal and root derived callus cultures of Rubia cordifolia were
established in vitro. Explants were pre treated with the fungicide, Bavistin 2.5 per
cent for 15 minutes. Surface sterilization with mercuric chloride (HgCl2) at 0.1 per
cent for 1 min and 30 sec was effective for yielding healthy, contamination free
cultures from nodal segments and leaves, respectively. MS medium at full
strength, supplemented with NAA at 2 mg l-1 along with BA at 0.5 mg l-1 was
observed ideal for initiation and proliferation of calli. The auxin synergist
phloroglucinol, when supplemented to the medium, did not not yield encouraging
results, with respect to callusing in the experimental species. Root derived
cultures were inferior with respect to callus initiation and proliferation, registering
low values for all the parameters studied. Incubating in vitro cultures under
illuminated condition at 26 ± 2
C was superior to dark incubation, with respect
to callus initiation and proliferation.
Chloroform – methanol at 8.5 :1.5 ratio was indentified as the appropriate
solvent system for detection of naphthoquinone on thin layer chromatograms in
the test extracts of the experimental species, with alcoholic KOH (10 per cent) as
the spray reagent.
Ms medium at full strength, fortified with NAA and BA at 2.0 mg l-1 and
0.5 mg l-1 respectively, which recorded maximum naphthoquinone synthesis, was
standardized as the production medium.
Enhancing concentration of sucrose to 5 per cent in the production
medium, did not elicit a positive response on naphthoquinone production in vitro.
Reducing nitrate concentration of the production medium, to half and one fourth
the original concentration, resulted in enhanced in vitro synthesis of the target
compound. Supplementing the production medium with yeast extract (1 per cent
and 2 per cent) as well as precursor feeding with phenyl alanine and tyrosine each
at levels of 50 mg l-1, 100 mg l-1 and 150 mg l-1 exerted a favourable influence on
synthesis of naphthoquonines, in vitro. Incubation in dark resulted in marginal
increase in in vitro production of naphthoquinones.
Incorporation of autoclaved mycelia of Pythium aphanidermeatum at
levels of 2.0 per cent and 5.0 per cent resulted in enhanced in vitro production of
naphthoquinone. The abiotic elicitor, salicylic acid at concentration of 10 μM and
100 μM resulted in maximum synthesis of naphthoquinones in in vitro root
cultures (8.76 units g -1 calli) of Rubia cordifolia. Immobilization of test calli with
sodium alginate – calcium chloride complex as well as subjecting the in vitro
cultures to stress conditions, as imposed by sorbitol failed to bring about an
enhancement in the in vitro production of naphthoquinones. None of the explants
employed in the study induced hairy roots, when co- cultured with the
Agrobacterium rhizogenes strains, MTCC 2364 and MTCC 532. Based on cell
count, subculturing intervals of leafs, nodal and root derived suspension were
fixed as 24, 27 and 27 days respectively with the respective packed cell volume as
0.93 per cent, 0.83 per cent and 0.80 per cent.
Naphthoquinone was detected, in ex vitro and in vitro test extracts at all
levels of maturity tested. Both ex vitro and in vitro root extracts exihibited
maximum cytotoxicity, as revealed by the percentage of cell death on DLA and
EAC cell lines as well as their IC50 values. As compared to whole plant extract, in
vitro systems of the experimental species exhibited least antioxidant action.
Extent of pro-oxidant activity was higher in in vitro root extract of the
experimental species.