Plant growth regulators for enhancing in vitro cormlet production in saffron(crocus sativus)

Abstract

The study entitled “Plant growth regulators for enhancing in vitro cormlet production in saffron (Crocus sativus L.)” was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani during 2023 2024. The objective of the study was to evaluate the effect of plant growth regulators in enhancing in vitro cormlet formation and differential expression of key genes involved in cormlet production. Experimental design followed was CRD with five treatments and three replications each. The explant, corms were collected from farmer’s fields in Pampore, Kashmir, and subjected to a rigorous three-step surface sterilization process using 0.02% bavistin (20min), 0.04% mancozeb (20min), 1% sodium hypochlorite bleach solutions (30min) and a final dip in 1.5% mercuric chloride solution. In vitro multiple shooting attempted using Murashige and Skoog (MS) medium supplemented with 2ppm Benzyl adenine (BA) showed sprouting, but did not grow further. Corms maintained under in vivo conditions of 16ºC temperature, 16/8hrs photoperiod and 60% humidity showed 100 per cent sprouting in 65 days. The sprouted corms were transferred to basal MS medium for elongation and shooting. Healthy multiple shoots were observed in 15 days with an average of 5 multiple shoots per culture and profuse roots were noticed in 2 days in all replications. Further, the in vitro multiple shoots were transferred to half-strength MS medium supplemented with 20 μM thidiazuron (TDZ), 10 μM indole acetic acid (IAA), 40% sucrose, and 0.25% of various anti-gibberellins viz., chlorocholine chloride (CCC), paclobutrazol (PBZ), chlormequat chloride (CMC) and mepiquat chloride (MPQ) along with control. Cultures maintained in full strength MS medium was used as absolute control. Cormlet initiation was first noticed in treatment using MPQ on 10th day followed by CCC (20 days), control (20 days) and absolute control (30 days). Cormlets were initiated in all 3 replications within 16 days in treatment using MPQ followed by CCC (36 days) and control (38 days). Total number of cormlets obtained was highest in treatment using MPQ (7 nos), followed by CCC (5 nos), control 64 (5nos), CMC (4nos) and absolute control (1no). The present study showed 0.25% MPQ as the best treatment with early initiation and maximum number of cormlet production. In vitro cormlets generated in treatment using 0.25% MPQ showed prominent size compared to control. In the treatment using 0.25% mepiquat chloride significant reduction was noticed in the expression level of GA3OX (0.08-fold) and upregulation of all the key genes involved in starch biosynthesis viz., SS (2.3-fold), SUSY (1.3-fold), PGM (1.1 fold). Similar results were noticed in treatment using 0.25% CCC, whereby significant reduction in the expression level of GA3OX (0.03-fold) and upregulation of SS (1.3-fold) and SUSY (1.8-fold) was found. Molecular studies could reveal a negative co-relation in the expression pattern of genes involved in biosynthesis pathway of gibberellin and starch metabolism during the early phase of cormlet initiation. To conclude, plant growth regulators viz., mepiquat chloride and chlorocholine chloride can be used effectively for early cormlet initiation and to enhance in vitro cormlet production in saffron. The inhibitory activity of MPQ & CCC against GA3oxidase noticed in the present study suggest the potential of these compounds in blocking the conversion of inactive GA to bioactive GA. The present study is the first report on differential expression analysis of key genes involved in in vitro cormlet formation in saffron and could reveal a low gibberellin mediated starch accumulation during early phase of cormlet production.