PhD 1997

Investigations on 'Standardisation of in vitro techniques for rooting,
hardening and micrografting in cocoa (Theobroma cacao L.)' were carried out at the
Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara
utilising the existing facilities of the Plant Tissue Culture Lahoratory of the
Department of Plantation Crops and Spices during the period from 1993-1996.
Studies were conducted to identify the best rooting protocol and also to refine the
technique of micrografting.
The nodal segments taken from the field were pretreated with Bavistin
(0. 1 %) for half an hour and then surface sterilized in chlorine water for four
minutes. The explants were then cultured in the medium, WPM + 2ip 5 + AgNO3
5 + CCC 0.75 + AdSO4 + PG 200 mg l-1 and incubated at 28 + 2oC under 4000
lux light intensity for shoot bud release. For getting sufficient number of elongated
shoots, subculturing was done in WPM supplemented with 0.5 % activated charcoal
and 200 mg l-1 streptomycin sulphate. Observations of three different genotypes
revealed that they responded differently to in vitro contidition and the genotype
S 44.1 exhibited a very good growth.
Rooting was very poor under In vitro condition and was completely
absent under ex vitro condition. Among the different basal media for in vitro
rooting, 1/2 MS supplemented with activated charcoal was the best. Maximum
rooting was obtained when the shoots were pretreated in IBA 5000 mg l-1 for 3
seconds followed by culturing in the basal medium. Optimum concentration of
sucrose for rooting was 3 per cent and that of agar was 0.6 per cent.


For rooting, the cultures should be kept at a temperature of 28 + 20 C
under dark condition. The genotypes differed in their response to rooting. Among
the three genotypes - S 44.1, G VI 67 and G IV 4.1 tested, S 44.1 recorded better
rooting.
The rooted shoots should be potted in a medium containing a mixture of
soilrite and potting mixture in equal proportion for establishment. The plantlets
should be covered with polybags for 2-3 weeks and then exposed to ambient condi-
tions periodically for hardening. Plantlet survival rate decreased up to the second
month of planting out and after that it became static.
In vitro micrografting and ex vito micrografting were possible in cocoa.
The best rootstock for in vitro micrografting was axenic seedlings cultured in half
MS liquid medium devoid of sucrose. These seedlings were ready for grafting in two
weeks when raised under high light intensity (4000 lux) and high temperature
(28 + 2°C).
In vitro shoots from nodal segments were found to be a very good scion
material for grafting. Among the different grafting techniqes, side grafting was the
most ideal one. Success was the highest when scions with two or more hardened
leaves were grafted 4 cm below the cotyledons in 4-5 weeks old axenic seedlings.
Anatomical studies revealed that the graft union was complete in about a month.
Grafted seedlings showed profuse growth after planting out.
Ex vitro micrografting recorded lower percentage of success than in vitro
micrografting. Older plants with a few hardened leaves were the most ideal root-



stock and the scions should have at least one hardened leaf. Rapid and extensive
scion elongation was observed in ex vitro micrografting. The most significant
achievement of the present investigation was the standardisation of the technique of
in vitro micrografting by which the rooting problem can be surmounted to a great
extent.