In vitro plant regeneration of Zelkova schneideriana, an endangered woody species in China, from leaf explants

Abstract

SummaryZelkova schneideriana Hand.-Mazz. is a high-value plant used for hardwood timber production in China. Because of over-harvesting and limited numbers of seedlings for plantations, Z. schneideriana has become an endangered species in China. To grow this plant sustainably, we generated a protocol to reproduce this species using tissue culture. Leaf explants were cultured on Wood Plant Medium (WPM), Murashige and Skoog (MS), 1/2MS (main elements), or B5 media variously supplemented with 6-benzylaminopurine (BA), naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2, 4-dichlorophenoxyacetic acid (2,4-D) to induce callus, adventitious shoots, and rooting. WPM was the most effective medium for callus induction and resulted in two types of calli from leaf explants. One was soft and straw-yellow coloured on WPM containing 0.45, 4.54, 9.08, or 13.62 µM 2,4-D or on WPM supplemented with 4.44 µM BA in combination with 0.45 or 4.54 µM 2,4-D. The other callus type was friable and green on WPM supplemented with 0.44, 4.44, 8.88, or 13.32 µM BA or on WPM supplemented with 4.44 µM BA in combination with 0.54 or 5.37 µM NAA. Only the latter callus type was observed to regenerate plantlets. WPM supplemented with 4.44 µM BA plus 2.68 µM NAA had a 64.5% shooting rate, with 6.75 shoots per callus. Rooting at 70.0%, with an average of 3.6 roots per shoot, was obtained on WPM supplemented with 2.46 µM IBA in combination with 2.0 g l−1 activated charcoal (AC). In total, 86% of regenerated Z. schneideriana plantlets survived after acclimation in a greenhouse at 24° ± 2°C under a 16 h photoperiod provided by cool-white fluorescent lights (PPFD = 65.5 µmol m−2 s−1), and 100% survived after transplanting to an experimental field plot.