Studies on in Vitro Mutagenesis, Molecular Analysis And Selection in Plant Tissue Culture for Improvement In Carnation (Dianthus Caryophyllus L.)

Abstract

Carnation (Dianthus caryophyllus L.) is a popular floricultural crop grown worldwide for its attractive cut flowers, extraction of oil for medicinal and perfumery purpose. In present investigation an efficient sterilization, callusing and regeneration protocol was developed for in vitro response using various explants source for carnation cultivar Geolie. Effect of sodium nitroprusside (SNP) alone or with other PGRs was studied. Callus induction frequency from leaf explants was observed maximum (98%) with MS media supplemented with 5.0μM SNP + 2.5mg/l 2,4-D + 0.75 mg/l NAA and 20.0μM SNP + 2.5mg/l 2,4-D as compared to 90.47% CIF observed from media without SNP. Maximum shoot induction (88.09%) was produced by nodal segments maintained on MS medium supplemented with 12.0μM SNP + 2.0 mg/l TDZ + 0.25 mg/l NAA. Half strength MS basal medium supplemented with 15.0 μM SNP and 1.25 mg/l NAA, showed best rooting response (97.61%). In vitro mutagenesis was performed on carnation using various concentration combinations of ethyl methane sulphonate (EMS) and sodium azide (SA). Survival percent of plantlets in in vitro culture was highest treated with EMS concentration of 0.1% for 2 hours. The LD50 value was approximately 0.42% (v/v) for 4hours of EMS exposure. The LD50 value for 15minutes and 30minutes of sodium azide exposure was observed at 0.99mM and 0.62mM, respectively. Shoot induction was observed maximum (77.77%, 72.22% and 70.22%) in compare to control when nodal segments exposed with 0.10% EMS (2hrs) and 0.25 mM SA (15min), respectively. With an increase in dose and duration of EMS and SA, both the shoot and root growth was retarded. With increasing mutagen concentrations, all morphological parameters (plant height, inter-nodal length, and number of leaf and nodes) showed a linear reduction. The mutant lines took fewer days to bud appearance as compared to control. No abnormal effect of mutagen was observed on leaf shape and color. Molecular markers (SSR and ISSR) were used to observe the genetic variation among developed mutant lines of carnation under in vitro conditions. Molecular marker study showed that 0.10%, 0.75% EMS (2hrs) and 0.50mM SA (30 min) treated mutant lines were the most distinct from the control plants, with similarity values of 0.76, 0.84 and 0.91, respectively. Developed in vitro methodology from the present investigation may be valuable for commercial micropropagation of carnation.