Cryoconservation of In Vitro Grown Shoot Tips of Cicer Microphyllum: A Crop Wild Relative of Chickpea

Abstract

BACKGROUND: Cicer microphyllum Benth. is a crop wild relative (CWR) of chickpea (C. arietinum L.), that possess useful genes for cold and drought tolerance. The species is being conserved in the In Vitro Active Genebank for short- to medium – term conservation. Cryopreservation would be a useful complementary approach for its long-term conservation. OBJECTIVE: The current work aimed to develop an efficient cryoconservation protocol for cryobanking of C. microphyllum shoot tips. MATERIALS AND METHODS: In vitro shoot tips excised from 4-month old shoot cultures grown on B5 + 0.5 mg L-1 KIN + 0.1 mg L -1 NAA + 10 mg L -1 AgNO 3medium were cryoconserved using a droplet-vitrification technique. Post-thaw regrowth was evaluated after: (i) preculture medium (B5 basal, B5 + 3, 4, 6 and 10% sucrose), (ii) preculture incubation temperature (25 ± 2, 10, 8 and 22/5°C), (iii) PVS2 duration (10, 20, 30. 40, 50 and 60 min) and (iv) regrowth medium (B5) supplemented with 0.5 mg L-1 KIN + 0.1 NAA mg L-1 ; 0.5 mg L-1 KIN + 0.1 mg L-1 NAA + 10 mg L-1 AgNO3; 0.2 mg L-1 BAP + 10 mg L-1 AgNO3 ; 0.2 mg L-1 BAP + 20 mg L-1 AgNO3 and 0.2 mg L-1 BAP + 30 mg L-1 AgNO3,. RESULTS: In vitro shoot tips grown on B5 + 0.5 mg L-1 KIN + 0.1 mg L-1 NAA + 10 mg L-1 AgNO3, precultured on B5 + 6% sucrose at 10°C for 3 days, followed by PVS2 treatment for 20 min, unloading solution for 60 min and regrowth on B5 + 0.2 mg L-1 BAP + 20 mg L-1 AgNO3 resulted in highest survival (57%) and regrowth (40%) after cryoconservation. CONCLUSION: The standardized protocol was successfully used for cryobanking of in vitro shoot tips of C. microphyllum in the In Vitro Base Genebank of ICAR-NBPGR, New Delhi.