Culture of shoot tips from adventitious shoots can eradicate Apple stem pitting virus but fails in Apple stem grooving virus

Abstract

This study attempted to eradicate Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from ‘Gala’ apple using shoot tips from of adventitious shoots derived from leaf segments. Leaf segments were excised from in vitro 4-week-old stock shoots and cultured on shoot regeneration medium containing 9.1 µM thidiazuron and 0.25 µM indole-3-butyric acid (IBA) to induce adventitious shoot formation. Shoot tips of different size and different developmental stage were excised from adventitious shoots and cultured on shoot tip culture medium containing 1.1 µM 6-benzyladenine and 0.05 µM IBA. Results showed size and developmental stage of shoot tips excised from adventitious shoots did not influence survival rate, but significantly affected shoot regrowth rate and ASPV-free frequency. Shoot regrowth rates increased from 10 to 15 % in 0.3 mm shoot tips containing two leaf primordia (LP) excised after 2–3 weeks of shoot regeneration, to 53–55 % in those containing three LP excised after 3–4 weeks. The highest shoot regrowth rate (82 %) was obtained in shoot tips of 0.4 mm shoot tips containing four LP excised after 4 weeks. ASPV-free frequencies (95–100 %) were high in 0.2–0.4 mm shoot tips containing two to three LP excised after 2–4 weeks, but low (20 %) in 0.4 mm shoot tips containing four LP excised after 4 weeks. None of the shoots regenerated from the shoot tips were ASGV-free, regardless of the size and developmental stage at which shoot tips were excised. Histological studies and virus localization provided explanations to the varying frequencies of the virus eradication using different size of shoot tips that were excised from adventitious shoots at different developmental stages. The protocol described here was characteristic of high frequency ASPV eradication and high efficiency of production of shoot tips that were used for virus eradication, and thus may have potential applications to virus eradication in other plant species.