Tissue culture and induction of adventitious shoot regeneration from leaf explants of cold-hardy dwarfing apple rootstock ‘BP-176'

Abstract: 【Objective】Rootstocks play an important role in determining apple tree performance in the field. In practice, some clonal rootstocks with excellent horticultural trait are difficult to propagate with conventional methods. Tissue culture can be used for rapidly propagating new rootstocks realeased from breeding programs. Establishment of an efficient adventitious shoot regeneration protocol is an important prerequisite for genetic manipulation. The purposes of this study are to establish tissue culture and rapid micropropagation technological system and to establish an efficient shoot regeneration protocol from leaf explants of apple dwarfing rootstock‘BP-176'.【Methods】Semi-lignified shoots were selected as materials and single bud stem sectionswere used as explants. Stem section explants were first surface-disinfected by 70% ethanol for 1 min, followed by sterilization with sodium hypochloride containing 5% active chloride for 8 min. Then the stem section explants were rinsed five-times in sterile distilled water. Stem section explants were then inoculated into culture tubes (25 mm x 150 mm, one explant per tube) containing 20 mL axillary bud initiation medium of half-strength MS macroelements supplemented with 1.0 mg·L^-1 6-benzylaminopurine (BA) , 0.2 mg·L^-1 indole-3-butyric acid (IBA) , 30 g·L^-1 sucrose, and solidified with 6 g · L^-1 agar. Stem sections were cultured for 4 weeks under photoperiod of16 h light and 8 h dark before the axillary buds began to germinate, grow and develop into new aseptic shoots. In vitro shoots were obtained from the resultant aseptic shoot cultures. In vitro shoots were used for further shoot proliferation, shoot regeneration and rooting. For micropropagation. Te effects of basal medium composition and plant growth regulators on proliferation and rooting of in vitro shoots were investigated. For adventitious shoots induction, in vitro leaves were selected as explants, the effects of kinds and concentrations of cytokinin and carbon sources on shoot regeneration were examined.【Results】The germination rate of axillary buds was above 85%. When BA at lower concentration of 0.5 mg · L^-1, proliferation and elongation of in vitro shoots were not well. When BA at higher concentration of 1.0 mg · L^-1, proliferation and elongation were significantly improved. MS and QL basal media did nt make any differences in proliferation when BA was used at 1.0 mg · L^-1, although They did make obivous diffences in growth behavior.The color of the shoots was red the same as in the field when they wer cultured on QL, while it was green when they were cultured on MS. Both cytokinin and carbon source influenced shoot regeneration. From leaf explants When BA was used, the sucrose was more effective than the D-sorbitol, especially when BA was at a higher concentration of 3 mg·L^-1 or 4 mg·L^-1, shoot regeneration rate was significantly higher on the sucrose than that on the D-sorbitol. When TDZ was used, the D-sorbitol was more effective than the sucrose when TDZ was at 0.5 and 0.8 mg · L-1, but when TDZ was at a lower concentration of 0.3 mg · L^-1, shoot regeneration rate was higher on the sucrose than that on the D-sorbitol. The kinds of cytokinin influenced adventitious buds growth behavior.Adventitious buds induced with TDZ were unable to elongate and develop into shoots on regeneration medium, it was necessary to transfer the buds to elongation medium without TDZ to form shoots. Adventitious buds induced by BA could directly elongate to form shoots on regeneration medium without transfer. For rooting induction, basal medium1/2 MS was more effective than 1/4 MS, rooting rate was improved by IBA than by NAA. But rooting rate didn't show significant difference when the shoots were cultured on 1/2 MS with 0.3 mg · L^-1 NAA or 0.5 mg · L^-1 NAA and 1/4 MS with any auxin or any concentration. Root number was higher when the shoots were cultured on 1/2 MS with IBA than that on NAA, but it was higher when the shoots were cultured on 1/4 MS with NAA than that on IBA.【Conclusion】The proper proliferation medium of the shoots of apple rootstock‘BP-176'was QL supplemented with 1.0 mg · L^-1 BA and 0.1 mg · L^-1 IBA. The optimal shoot regeneration medium was NM supplemented with 3 mg · L^-1 BA, 0.3 mg · L-1 IBA and 30 g · L^-1 sucrose, regeneration rate was 71.6%. The optimal rooting medium was 1/2 MS supplemented with 0.3 mg·L^-1 IBA and 20 g·L^-1 sucrose, the highest rooting rate was 69.8%.The composition of basal medium influenced in vitro shoots growth behavior and the cytokinin and carbon source had a synergic effect on shoot regeneration.