MSc

The present investigations were carried out in the Plant Tissue Culture
and Biochemistry Laboratories, College of Horticulture, Vellanikkara, Thrissur
during the year 1997-1999. The study was undertaken with the objective to
standardise the in vitro techniques for callus induction, proliferation and
regeneration. It was also envisaged to identify and quantify the active principle in
in vitro cultures.
Surface sterilisation treatments were standardised for the different types
of explants from the field. For immature fruits and leaves, the sterilisation treatment
standardised were found to be with 0.05 per cent cetrimide immersion for 5 minutes
followed by 0.1 per cent HgCh for 5 minutes with reference to least percentrage of
contamination and higher rate of establishment and growth of the explants obtained.
The exudation of polyphenols from the cut surfaces of shoot tip, nodal
and internodal segments was minimised by pre-treatment with antioxidants. The
pre-treatment followed was with Cetrimide 0.1 per cent for 5 minutes, Emisan 0.1
per cent for 5 minutes, ascorbic acid 0.01 per cent + citric acid 0.01 per cent for 10
minutes and mercuric chloride 0.1 per cent for 3 minutes.
Though calli were obtained from leaf segments as well as leaf segments
with petiole bases, leaf segments were found most suitable for callus cultures and
produced profuse calli. The best treatment for callus induction was found to be
solid Vz MS medium for leaf bit cultures. The treatment with IAA 2 mg rl and BA 1
mg r' on Vz MS solid medium was the best for callus induction from leaf segments
and leaf segments with petiole bases. Similarly a combination of auxins such as
IAA 2 mg rl and 2,4-D 1 mg rl on Vz MS solid medium also readily induced


callusing from leaf segments and leaf segments with petiole bases. The above
treatments were superior with respect to callus index and the number of days taken
for callus initiation.
Callusing was obtained from immature fruits when cultured on solid Y2
MS medium with phosphate ions reduced to 25 per cent supplemented with lAA 2
rng rl and BA 1 mg r'.
The shoot tips, nodal and internodal segments exhibited recalcitrancy
and there was no cell growth from these explants.
There was neither any proliferation nor any noticeable cell growth of
calli in the liquid medium.
Light stimulated the growth of yellowish friable callus and berberine
synthesis in the initial stages. But in the later stages, continuous illumination proved
to be detrimental and its growth was retarded.
The calli did not respond to regeneration treatments being neither
organogenic nor embryogenic.
The wavelength of marker berberine was recorded as 228 nm.
Berberine was detected in calli produced from leaf explants of different treatments
in solid Y2 MS media supplemented with growth regulators such as BA 0.25 mg i',
BA 0.5 mg r', Kin 0.25 rng r', lAA 2 mg rl + BA 1 mg r' and lAA 2 mg r' + 2,
4-D 1 mg r'. Age of the callus had a profound influence on berberine production.
Employing special techniques for synthesis of berberine in in vitro
cultures such as administration of osmoregulants, incorporation of media additives,

increasing concentration of agar, addition of stress inducers and modification of
carbon source in solid Yz MS medium failed to sustain the growth of callus.
Incorporation of abscissic acid at low concentration of 0.25 mg r'
sustained the callus development and berberine was detected by thin layer
chromatography. The quantity of berberine recovered was 0.095 ug/g callus tissue.
Administration of spermidine to liquid Yz MS medium neither caused any
noticeable cell browning nor any cell growth. The medium became deep yellow due
to the release of the alkaloid. Spermidine at 60 ug concentration in the liquid Yz MS
production medium had a berberine yield of 5.0 15 ug/g callus.
Berberine was detected from the field grown plant samples also. The
quantity was the highest in the tender leaves, that is 0.320 ug from 25 g tender
leaves which is equivalent to 0.013 ~g/g leaf. The berberine content in the stem was
found to be 0.010 ug/g stem, that is 0.304 ~g/30 g stem. The stem extract also
contained another compound whose Rfvalue was 0.66 and it gave a brownish spot
under visible light.
The highest berberine recovery obtained was in callus obtained from half
strength MS liquid medium than in half strength MS solid medium.
The in vitro derived callus had higher amounts of berberine than the
samples from the field grown plant. The highest berberine yield was obtained when
phosphorus ion sources were reduced to 25 per cent in the Yz MS liquid medium
supplemented with IAA Zmgl" and BA lrngl'. The recovery was 10.079 ~g/g
callus tissue. The next highest yield of berberine (5.015 ug/g callus) was obtained
•.
when 60 ug of spermidine was added to the 25 ml of the Yz MS liquid medium
supplemented with IAA Zmgl" and BA Irngl".