Clonal propagation and cryogenic storage of virus-free grapevine (Vitis vinifera L.) via meristem culture

Authors : Mohamad SHATNAWI, Ghandi ANFOKA, Rida SHIBLI, Mohammed AL-MAZRA`AWI

Pages : 173-184

View : 19 | Download : 10

Publication Date : 2011-04-01

Article Type : Research Paper

Abstract :A protocol for production of virus-free Vitis vinifera using meristem culture was developed. Meristems insert ignore into journalissuearticles values(0.1-0.2 mm); of V. vinifera infected with grapevine fanleaf virus and grape leafroll associated viruses were excised from 1-year-old growing vines. Shoot tips were cultured on half-strength Murashige and Skoog insert ignore into journalissuearticles values(MS); medium, supplemented with 0.02 mg L-1 of benzylaminopurine insert ignore into journalissuearticles values(BAP); and 0.01 mg L-1 of naphthalene acetic acid insert ignore into journalissuearticles values(NAA);. BAP and kinetin resulted in differences in the number of new shoots per explant, shoot height, and number of new leaves per explant. BAP at 0.8 mg L-1 gave the highest in vitro multiplication rate, with 5.25 shoots per explant, whereas elongation was greatest in the presence of 0.2 mg L-1 of kinetin. Root initiation was tested on an MS medium supplemented with 0.0, 0.2, 0.4, 0.6, 0.8, or 1.0 mg L-1 of IBA insert ignore into journalissuearticles values(indole-3-butyric-acid);, IAA insert ignore into journalissuearticles values(indole-3-acetic-acid);, or NAA. Maximum root number was achieved using 0.6 mg L-1 of IBA. A survival rate of 95% was achieved when rooted explants were acclimatized ex vitro in a mixture of 1 soil: 1 perlite: 1 peat. Acclimatized plants grew in the greenhouse and were maintained as virus-free plants. Visual inspections as well as results of RT-PCR, using virus-specific oligonucleotide primers, showed that plants developed in vitro were free from grapevine fanleaf virus, grape leafroll associated virus-1, and grape leafroll associated virus-3 infections. Shoot tips from plantlets grown in vitro were cryopreserved by vitrification. Shoot tips were treated in a 2 mL cryotube filled with a solution of 5% insert ignore into journalissuearticles values(w/v); DMSO, 5% insert ignore into journalissuearticles values(w/v); glycerol, and 5% insert ignore into journalissuearticles values(w/v); sucrose at 25 °C for 20 min, then dehydrated with 1 mL of modified vitrification solution 2 insert ignore into journalissuearticles values(MPVS2); at 0 °C for 40 min. Maximum regrowth insert ignore into journalissuearticles values(55%); was obtained after cryogenic storage with cultures exposed to MPVS2 for 40 min at 0 °C. Cryopreserved shoot tips, after being warmed, resumed growth within 7 days and developed shoots directly without intermediate callus formation.
Keywords : Key words Clonal propagation, cryogenic storage, grapevine, in vitro root formation, meristem cultures, virus free grapevine, Vitis vinifera