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, WESAM SHAHROUR, AREEJ AREBIAT

Abstract: A protocol for production of virus-free Vitis vinifera using meristem culture was developed. Meristems (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 (MS) medium, supplemented with 0.02 mg L^{-1} of benzylaminopurine (BAP) and 0.01 mg L^{-1} of naphthalene acetic acid (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 (indole-3-butyric-acid), IAA (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% (w/v) DMSO, 5% (w/v) glycerol, and 5% (w/v) sucrose at 25 °C for 20 min, then dehydrated with 1 mL of modified vitrification solution 2 (MPVS2) at 0 °C for 40 min. Maximum regrowth (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: Clonal propagation, cryogenic storage, grapevine, in vitro root formation, meristem cultures, virus-free grapevine, Vitis vinifera

Full Text: PDF