TY  - BOOK
AU  - Sindhu M 
AU  - Kesavachandran R (Guide)
TI  - In Vitro Callus Induction And Its Exploitation In Coscinium Fenestratum (Gaertn.) Colebr
U1  - 633.8 
PY  - 1999///
CY  - Vellanikkara 
PB  - Department of Plantation Crops, College of Horticulture 
N2  - 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". 


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