MSc

Byadagi chilli is well-known for its deep red colour and zero pungency.
The fruits of Byadagi chilli are brown to deep red at full maturity. The red colour
of chilli fruits are due to several carotenoid pigments. They are good source of
natural colourants used in food, feed, textile and cosmetic industries. The chilli
type is best suited for oleoresin extraction and exported as a substitute for paprika
oleoresin.
The present study ‘Elicitation mediated carotenoid production and
Capsanthin capsorubin synthase gene expression in Byadagi chilli (Capsicum
annuum L.)’ was undertaken at Centre for Plant Biotechnology and Molecular
Biology (CPBMB), College of Horticulture, Kerala Agricultural University,
Thrissur during 2016-2018. The study was carried out using two genetically
distinct chilli genotypes based on their colour at fully ripe fruit stage namely
Byadagi Dabbi and variety Anugraha.
Callus cultures were produced from tender leaves of both the genotypes
using MS media augmented with different plant growth regulators viz., Indole-3-
acetic acid (IAA), Kinetin (Kin) and Benzyl adenine (BA) at different
concentrations as reported by Kintzios et al. (1996), Kehie et al. (2012), Kabby et
al. (2015) and Santos et al. (2017). Though, callogeneses were obtained in all the
media tried, early callusing and higher callus index were achieved in MS medium
supplemented with 1 mgL-1 2,4-D and 1 mgL-1 Kin.
Elicitation of two month old calli was done with two different chemical
elicitors viz Salicylic acid and Methyl jasmonate at different concentrations like
20, 40 and 60 mgL-1 for 72h. The carotenoids were extracted and β-carotene was
quantified using HPLC. Elicitor treatment with salicylic acid increased β-carotene
content significantly. The highest β-carotene was recorded in Byadagi calli
elicited with salicylic acid 20 mgL-1 for 72 hours. In this treatment Byadagi Dabbi
recorded 6.75 times higher β-carotene (42.85 μgg-1 fresh weight) than the control
(6.34 μgg-1 fresh weight). In methyl jasmonate elicitation, both the genotypes
were found on par with respect to β-carotene production in the different
concentrations studied.
Expression of Capsanthin capsorubin synthase (Ccs) gene was studied in
the highest β-carotene yielding treatment viz elicitation with salicylic acid 20
mgL-1 for 72 hours using real time PCR. The total RNA extracted from elicited
calli were converted to cDNA. The Capsicum annuum L. β-tubulin gene was used
as the endogenous control. Dissociation curves were obtained as a single
dominant peak denoting that there was specific gene amplification for both
endogenous control and Ccs gene in different treatments. Relative quantification
of the Ccs gene expression was done using the Comparative CT method reported
by Livak and Schmittgen, (2001). The Ccs gene was found up-regulated 2.35 fold
in Byadagi Dabbi elicited calli with salicylic acid 20 mgL-1 for 72 hours. The
expression of Ccs gene in the variety Anugraha was down-regulated (0.906 fold)
when elicited with salicylic acid 20 mgL-1 for 72 hours.
The major outcome of the present investigations are the suitability of leaf
and calli induced from leaves for carotenoid production, scaling up of carotenoid
production through salicylic acid elicitation and upregulation of Ccsgene
expression in highest carotenoid yielding elicited calli.
Another significant finding is the response of calli to salicylic acid
elicitation which is a cheaper elictor as compared to Methyl jasmonate and the
highest content of β-carotene at lower concentration of elicitor which are plus
points as far as commercial exploitation of carotenoids is concerned.
However, much more elaborate studies are required on explant stage,
culture systems, age of cultures, culture conditions, duration and concentration of
elicitors and activity of regulatory enzymes and elicitor mediated expression of
genes involved in carotenoid metabolic pathway for commercial exploitation of
the system for carotenoid production. A more in-depth understanding of the
underlying biological mechanisms will enable to fully harness the potential of cell
cultures and to enhance carotenoid production on an industrial scale.