MSc

The study entitled “In vitro selection for drought tolerance in black
pepper” was carried out in the Department of Plant Biotechnology, College of
Agriculture, Vellayani during 2014 to 2016.The objective was to develop drought
tolerance in black pepper (var. Panniyur-4) by in vitro selection using
polyethylene glycol (PEG) and to characterize the tolerance.
Callus was induced from different explants of black pepper namely leaf,
nodes and shoot tip. Murashige and Skoog’s (MS) medium containing different
combinations and concentrations of plant growth regulators (PGR) were used for
callus induction. Percentage of callus induction varied among the type of explants
used. In the present study leaf tissue was found to be most responsive to callus
induction than other explants. Among the fourteen treatments, 1.0 mg L-1 of 6-
benzyladenine (BA) recorded the highest callus induction of 75 % from leaves in
a period of 45 days. The same treatment produced 50 % callus induction from
nodes and 41.66 % from shoot tips.
In vitro selection for drought tolerance was done by growing the calli in
MS medium containing 1.0 mg L-1 of BA and different concentration of
polyethylene glycol ie., 5 g L-1 to 100 g L-1 for 30 days. It was observed that
increasing concentration of PEG resulted in reduction of growth and browning of
the calli. The calli were able to survive in PEG up to a maximum concentration of
10 g L-1 (83.3 %). None of them survived beyond that concentration of PEG. The
PEG tolerant calli were multiplied and maintained in MS supplemented with 1.0
mg L-1 of BA containing the respective concentration of PEG.
PEG tolerant callus were subjected to regeneration in MS medium
supplemented with different concentrations and combinations of PGRs, but they
failed to show regeneration.
Biochemical analysis of PEG tolerant calli showed significant difference in
proline, total free amino acids, soluble protein and ascorbic acid content. Calli
survived in 10 g L-1 of PEG showed the highest amount of proline content (0.87

ug g-1) than the one survived in 5 g L-1 of PEG. The amino acid (2.02 mg g-1),
soluble protein (1.71 mg g-1) and ascorbic acid (9.23 mg 100g-1) content were
significantly high in calli survived at 10 gL-1 of PEG. The increase in SOD content
in PEG tolerant calli was not significant.
The RNA isolated from PEG tolerant calli showed a maximum yield of
1600 ng/ul and purity value of 2.3. Amplification of cDNA using Actin gene
confirmed the quality of cDNA. Differential expression p5cs (Pyrroline-5-
carboxylate synthase) gene was observed in the PEG tolerant calli compared to
control. RT-PCR using degenerate primers designed for p5csgene amplified a
band of size approximately 120 bp and its expression was high in calli survived in
10 gL-1 of PEG.
In the present study, the PEG tolerant calli developed showed high levels of
proline, free amino acids, total soluble protein, SOD activity and ascorbic acid
content, indicating the development of water stress tolerance. Expression of p5cs
gene was also increased in PEG tolerant callus. Hence, this in vitro selection
system can be used for the selection of drought tolerance in black pepper.