PhD

The genetic analysis of inbreds, inbred crosses and hybrid was carried out at College of
Horticulture and Cocoa Research Centre (CRC), Vellanikkara, Thrissur. A total of 113 inbreds was
evaluated for qualitative and quantitative characters. Cocoa is predominantly out breeding with highly
complex genetic structure. The cross pollinating nature coupled with existence of self/cross
incompatibility, poses much difficulty to the cocoa breeders. The hybrid progeny from the same cross
exhibit high level of variability due to heterogeneous nature of the parents. This can be overcome to
certain extent by using fully homozygous inbreds of diverse genotypes. The CRC succeeded in producing
first ever fifth generation inbred and it was proved self incompatible. In this context, the present study
was formulated to quantify the magnitude of inbreeding depression in yield and yield attributes in various
self-generations and to establish a physiological relationship between the vigour of inbreds, inbred crosses
and hybrids in the early stages of plant growth. The different pollination techniques were tested to
overcome the self incompatibility and the extraction of proteins from self incompatible plants was
attempted.
Morphological characterization of 113 inbreds were carried out by recording qualitative
characters such as pod shape, ridge colour, pod apex, pod basal constriction, pod rugosity and bean
colour. High variability was observed for all qualitative characters except ridge colour. The pod and bean
characterization for 21 characters expressed wide variation among the inbreds and within the inbreds of
same genotype. Characterization of inbreds based on the biochemical parameters such as fat and phenol
expressed wide variability.
The inbreeding depression was estimated for 21 characters in 12 genotypes over generations. In
S1 generation of M 18.7, the husk furrow thickness and number of flat beans expressed negative
inbreeding depression, whereas the average yield per tree per year has shown a very meager inbreeding
depression over the preceding generation. In S2 generation of M 18.7, pod value, conversion index and
dry matter recovery have shown an inbreeding depression between 5 to 10 percent. Negative inbreeding
depression was observed for husk ridge thickness, number of flat bean per pod, pod index, efficiency
index and fat content. In S3 generation of M 18.7, maximum inbreeding depression was observed for wet
bean weight per pod followed by pod length. In S4 generation of M 18.7, the highest inbreeding
depression of 43.58 per cent was observed for number of pods/tree/year. In S1 generation of G II 7.4,
positive inbreeding depression was observed for 10 characters out of 21. In S2 generation of G II 7.4,
positive inbreeding depression was observed for 15 characters. Only seven characters expressed positive
inbreeding depression in S3 generation of G II 7.4 genotype. In S5 generation inbred, the maximum
inbreeding depression (63.95%) was observed for husk furrow thickness, followed by husk ridge
thickness (49.02%), In S1 and S2 generation of H1 1.2, positive inbreeding was observed in 16 characters.
In general, inbreeding depression was less for economic characters confirming that most of the characters
are controlled by additive gene action and lethal gene canceled in heterozygous condition is less.
In the comparative evaluation of inbreds, inbred crosses and hybrids, the inbreds were found
superior over inbred crosses and hybrids for morphological characters such as plant height, collar girth,
plant spread and leaf area. The inbreds also had maximum chlorophyll content, leaf nutrient status and
relative water content. The superiority of inbreds was mainly attributed to the growing environmental
condition with more of openness in the inbred plantation, which was confirmed with spherical
densiometer, an instrument for measuring plantation overstory density.
A total of nine techniques to overcome self incompatibility barriers such as bud pollination,
surgical technique, intra-ovarian technique, salt spray (1% and 3%), high humidity, high temperature,
Naphthalene Acetic Acid (100 and 200 ppm), gamma irradiation and flower organ extract were attempted
from September 2016 to March 2017 and September 2017 to March 2018. In all the methods, the fruit set
was not obtained except in surgical technique. The majority of self incompatible S3 and S5 inbreds set in
to pods through surgical techniques. In the fluorescent microscopic study, the clear disintegration of
pollen tube was observed in self incompatible flower in the style.
Flowers from fifth generation cocoa inbreds were used for extraction of protein. All the available
protein extraction methods were tried and the protein was quantified. In all the methods, the protein
content was below 5μg per ml in the cocoa flower. The protein from cocoa leaf was extracted and
quantified. In lysis buffer extraction with some modification, a protein content of 7 μg per ml was
obtained. Faint bands were observed in SDS PAGE. The flower and leaf protein were subjected for LC Q
ToF. The protein content obtained from the flower was insufficient to run LC Q ToF. A total of 87
proteins were found in cocoa leaf sample, of which, 85 proteins were similar to cocoa and two proteins
were similar to forest coconut.
From the above, it can be concluded that the genotypes in S5/S6 can be crossed to get highly
heterotic hybrids. For extraction of protein from cocoa flowers, fine tuning of the available methods under
ideal laboratory conditions must be employed. Molecular basis of self-incompatibility has to be studied in
detail.