Development of in-vitro protocol for direct regeneration from thalamus ex-plant of Tagetes patula L. var. Pusa Deep

Abstract

The effect of growth hormones such as BAP, IAA and KINETIN in different treatment combinations on direct differentiation of shoots from thalamus ex-plants of French marigold (Tagetes patula L. var. "Pusa Deep") have been studied. A total of nine treatments was used to screen thalamus ex-plants to determine the capability for plant regeneration and subsequently to find out the optimum medium conditions for high potentiality of direct shoot formation. Thalamus ex-plants behaved differently in all over nine treatments and all the treatments are highly significant over the (T0) control-MS devoid of hormones. The best medium found for direct, shoot organogenesis from the thalamus of Pusa Deep was treatment (T7) MS + BAP 1.0 mg/l + KIN 0.5 mg/l + IAA 1.0 mg/. Pre-treatment with Carbendazim (0.2%) + Mancozeb (0.2%) + 8-HQC (200 mg/l) for 1.5hr resulted in minimum fungal (28.33%), bacterial (25.33%) contamination. Surface sterilization with 0.1% HgCl2 for 5 min reduced microbial contamination up to (44.33%) simultaneously increased the survival percentage upto (55.67%). This technique can be utilized in gene transfer technology, in vitro mutagenesis, production of lutein rich pharmaceutical compounds and secondary metabolites using tissue culture techniques. Introduction Marigold (Tagetes sp.) is a herbaceous ornamental plant grown all over the world as a loose and cut flower which belongs to the family Asteraceae. T. erecta (African marigold) and T. Patula (French marigold) are commonly cultivated species grown for decorative purpose while T. minuta L. (wild marigold) is cultivated for essential oil purposes. In India, it is one of the most commonly grown flowers and extensively used for decoration, social, religious, landscaping and carotenoid extraction purpose. To fulfil the increasing demand of quality planting material of marigold, micro-propagation is one of the viable approaches for large scale multiplication. There are very few published reports on the standardization of micropropagation protocols in marigold. Various research workers have used different types of ex-plants in the development of in-vitro protocols. (Unopened capitula) Kothari and Chandra 1984, 1986 [7, 8] and Ram and Mehta 1982 [15] , (leaves) Misra and Datta 1999, 2001 [9, 4] (hypocotyl and leaves) Belarmino et al. 1992 [1] and Venegas et al. 2002 [16] , (cotyledon and hypocotyl) Mohamed et al. 1998 [11] , Bespalhok and Hattori, 1998 [2] , (shoot apexes) Miranda-Ham et al. 2006, (hairy roots) Mukundan and Hjortso, 1991b [12] , (internode) Croes et al. 1989 [3] and (single-node stem segments) Pratibha et al. 2000, [14] (Anther) Kumar et al. 2018 [9] were used for successful in vitro establishment of cultures of Tagetes species. However, the regeneration through thalamus ex-plant have not yet reported in Tagetes for mass multiplication. The present study reports a highly efficient direct adventitious shoot regeneration system from thalamus ex-plants. This protocol will be helpful for genetic transformation, in-vitro pollination and fertilization, large scale production of disease free quality material, somaclonal variants, in-vitro mutation breeding, in-vitro induction of pigments and secondary metabolites and in-vitro selection for biotic and abiotic stresses.