PhD.

ABSTRACT
Water deficit stress is considered as one of the most limiting factor for
production in cocoa. Preliminary efforts have been made in Kerala Agricultural
University to identify drought tolerant cocoa genotypes. The present study was an
attempt made to identify the cocoa genotypes and hybrids that can tolerate drought
condition.
The study was conducted at Department of Plantation crops and spices, College
of Agriculture (CoA) and Cocoa Research Centre and College of Agriculture,
Vellanikkara, Thrissur. Thirty exotic genotypes and six pre-released hybrids were
taken as materials for the study. They were budded on 6 month old rootstock of
seedling raised from polyclonal gardens. GIV 18.5 (progeny of pods from Nileshwar)
identified susceptible genotype was used as check in the experiment.
Five month old budded plants of thirty exotic cocoa genotypes and six hybrids
along with check variety were subjected to drought screening by following
gravimetric method. Control plants were maintained at fully irrigated condition under
100 per cent field capacity. Field capacity was maintained at 40 per cent for stress
condition. Based on the percentage of retained leaves genotypes and hybrids were
categorised as highly tolerant (more than 70% leaves retained), tolerant (40-70%
leaves retained), susceptible (10-40% leaves retained) and highly susceptible (less
than 10% leaves retained). Various physiological and biochemical parameters related
to drought were analyzed.
The physiological parameters, relative water content, chlorophyll stability
index, membrane stability, photosynthetic rate and chlorophyll content were recorded
high in tolerant and highly tolerant genotypes and hybrids whereas these parameters
were comparatively low in susceptible and check variety. The control plants at 100
per cent field capacity recorded high value for all these parameters. However in terms
of transpiration rate a reverse trend was observed among genotypes and hybrids. Low
transpiration rate was recorded in highly tolerant and tolerant hybrids whereas
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susceptible genotypes and hybrids had high transpiration rate under stress condition.
The control plants under fully irrigated condition of 100 per cent field capacity also
had high transpirational rate. This indicated the ability of tolerant genotypes and
hybrids to conserve water during stress. The leaf temperature did not shown any
significant variation among the genotypes and hybrids studied under both the field
capacity level.
In case of biochemical parameters, proline content, glycine betaine content and
superoxide dismutase activity were found to be high in tolerant and highly tolerant
genotypes and hybrids and was low in susceptible and check variety. The control
plants kept under 100 per cent filed capacity level shown less values for all these
parameters. However, all the plants showed reduced nitrate reductase activity under
drought stress. The control plants recorded high nitrate reductase activity at 100 per
cent field capacity level.
Correlation studies showed that all physiological and biochemical parameters
except transpiration rate and leaf temperature have positive correlation with
percentage of leaves retained. Effect of these parameters on percentage of leaves
retained was estimated using path analysis. Parameters having direct effect on leaf
retention were proline, nitrate reductase activity, SOD, glycine betaine, cell
membrane stability and relative water content. These characters were analyzed for
their genetic parameters and it was found that all these characters are having high
heritability and genetic gain. D
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statistics analysis revealed that the 30 genotypes and
6 hybrids along with check were grouped into 6 clusters indicating the presence of
diversity for different traits.
Four different isolation methods were compared in this experiment to identify
the best method that could produce high-quality total RNA free from contaminants
and genomic DNA. RNA obtained through the SDS- tertiary butanol method
(Gesteira et al., 2003) has high quantity and quality, followed by the modified SDSphenol method (Deepa et al., 2014) in both fresh and frozen leaves.
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The transcriptome of highly drought tolerant exotic genotype (T85/799) and
highly drought sensitive check variety CCRP 5 was analysed using mRNA
sequencing under control (100% FC) and drought stress condition (40% FC). Drought
has up-regulated 1540 and 1148 DEGs and downregulated 1502 and 1250 DEGs
respectively in tolerant and sensitive libraries. The PCA scatter plot shows that
replicates of the well watered samples (control) of highly drought tolerant genotype
and drought susceptible check variety CCRP 5 form distinct groups highlighting
differences between the two genotypes. Volcano plot depicted the significance of
differentially expressed genes in control and drought stress treated samples of highly
tolerant and susceptible genotype.
In both the tolerant and susceptible genotypes, the majority of the genes
within the biological process category were linked to GO terms that fall under the
subcategories of "metabolic process," "cellular process," and "single organism
process." “Enzyme catalytic activity”, “water channel activity”, “binding” and
“transporter activity” were the most abundant terms in molecular function category in
both the genotypes under stress. There were 17 pathways significantly enriched
(FDR-adjusted p-value < 0.05) in tolerant genotype for upregulated genes and the
most enriched pathways including metabolic pathways (178 genes) followed by
biosynthesis of secondary metabolites (121 genes), phenyl propanoid biosynthesis (29
genes), carbon metabolism (29 genes), glyoxylate and decarboxylate metabolism (16
genes), MAPK signalling pathway plant (14 genes) and starch and sucrose
metabolism etc.
Most of the Transcription Factors (TFs) identified families were enriched in,
B3, bHLH, WRKY, ARF, ARR B, FAR, LBD, MADS and ERF. These TFs could be
grouped into 10 families. The major upregulated genes families in tolerant genotype
includes protein kinase, protein phosphatase type 2c, zing finger proteins, laccase
gene, cytokinin dehydrogenase, cytochrome p450, galactinol synthase, NAC Domain
protein, peroxidase, protein ubiquitin E3 ligase, mannitol dehydrogenase, fatty acid
desaturase, UDP glycosyl transferase, Aquaporin PIP, Glutathione dependent genes,
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cellulose synthase and heat shock proteins etc.The major upregulated genes families
in susceptible genotype includes LEA protein, PSII protein, peroxisomal membrane
proteins, ABC transporter, ABA hydrolyse, phenyl alanine n-monoxygenase, tyrosine
carboxylase, plastocyanin, trehalose phosphate phosphatase and expansin etc.
RNA-Seq analysis showed that highly drought tolerant exotic genotype
(T85/799) activated more number of drought responsive genes than drought sensitive
check variety CCRP 5. Several drought-responsive genes were upregulated in the
tolerant genotype such as those encoding TFs, cytochrome 450, and membrane
transporters, and those associated with carbohydrate metabolism and flavonoid
biosynthesis. These genes might confer drought tolerance in this cocoa genotype at
the molecular level. Identified drought- responsive genes and metabolic pathways
were targets for future studies in order to understand the molecular mechanism of
drought tolerance in cocoa. Physiological and biochemical parameters also indicated
the better performance of the tolerant genotype over the susceptible genotype under
drought stress conditions. These results were a first step to understanding the
molecular mechanisms of drought tolerance in cocoa and lay a foundation for its
genetic improvement.