MSc

The experiment entitled “In vitro studies on morphogenesis and plant regeneration
in elite clones of cocoa (Theobroma cacao L.) was carried out in Tissue culture
laboratory of Department of Plantation Crops and Spices, College of Horticulture,
Vellanikkara during 2016-2018 with an objective to develop a protocol for in vitro
regeneration and also to determine the variation in morphogenesis and regeneration
among elite cocoa clones. The experiment was conducted using five varieties, CCRP
2, CCRP 6, CCRP 8, CCRP 15 and Scavina.
Single nodded cuttings of 2-3 cm collected from the budded plants were used as
explants. The best treatment for surface sterilization of nodal segments was washing in
Teepol followed by shaking in 0.2 per cent Mancozeb for 30 minutes and then,
Streptocycline 0.1 per cent for 10 minutes outside the laminar air flow chamber. Inside
the laminar air flow chamber, the explants were treated with Ethyl Alcohol 70 per cent
for 3 minutes followed by HgCl2 0.1 per cent for 5 minutes. This treatment showed the
highest survival percentage (91.67%) of cultures with minimum microbial
contamination (8.33%).
The medium WPM + 2-ip 1ppm + IAA 0.02ppm showed cent per cent shoot
induction in varieties CCRP 15 and Scavina; WPM + 2-ip 2ppm + IAA 0.04ppm in
varieties CCRP 2, CCRP 6 and Scavina; WPM + 2-ip 3ppm + IAA 0.06ppm in CCRP
8 and WPM + 2-ip 4ppm + IAA 0.08ppm in varieties CCRP 2 and Scavina. Period of
shoot induction for all the five varieties was significantly influenced by media, varieties
and their interactions. Among the media, the lowest period of shoot induction was
recorded in medium WPM + 2-ip 1ppm + IAA 0.02ppm (8.41). Among the varieties,
the lowest period of shoot induction was in CCRP 2 (7.10) and this was statistically on
par with variety Scavina (8.37). Among the interactions, the period of shoot induction
was lowest in variety CCRP 15 in medium WPM + 2-ip 1ppm + IAA 0.02ppm (6.33).
Culture establishment percentage in multiple shoot induction media varied
significantly among media. Highest culture establishment percentage (95%) was
recorded in media WPM + 2-ip 3ppm + IAA 0.06ppm + AgNO3 5ppm and WPM + 2-
ip 4ppm + IAA 0.08ppm + AgNO3 5ppm. Among the varieties, highest multiple shoot
induction percentage was recorded in Scavina (93.75%). Interaction effect of media
and varieties on number of multiple shoots per culture was significantly different and
the highest number of multiple shoots per culture (2.25) was observed in variety
Scavina when medium WPM + 2-ip 4ppm + IAA 0.08ppm + AgNO3 5ppm was used.
Rooting percentage was generally low under in vitro condition. But among the
media tried, 1⁄2 MS + IBA 4000 ppm (pulse treatment for 2-3 sec) and 1⁄2 MS + IBA
5000 ppm (pulse treatment for 2-3 sec) were found to be ideal. All the varieties showed
root induction in 1⁄2 MS + IBA 5000 ppm (pulse treatment for 2-3 sec) and only two
varieties, CCRP 15 and Scavina, had successful root induction in 1⁄2 MS + IBA 4000
ppm (pulse treatment for 2-3 sec). Hence in general, it can be concluded that, 1⁄2 MS +
IBA 5000 ppm (pulse treatment for 2-3 sec) was superior to all the media tried for root
induction.
Hardening of tissue culture cocoa plants were not successful in both sand:
vermicompost (1:1) and vermiculite: perlite: vermicompost (1:1:1). The vermiculite:
perlite: vermicompost (1:1:1) media showed a maximum survival of plantlets for one
week. However, later showed yellowing and finally the shoots were completely dried
off. The anatomical observation on root-shoot transition zone using rotary microtome
sectioning showed the presence of intervening callus between the vascular tissues of
root and shoot. This callus inhibits proper vascular connection and thus, the plants kept
for hardening were difficult to get established.