Elicitation mediated carotenoid prodouction and capsanthin capsorubin synthase gene expression in byadagi chilli (capsicum annuum L.)

Abstract

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.