苹果抗寒矮化砧木‘BP-176’的组织培养及其叶片不定梢诱导
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摘要
【目的】建立苹果抗寒矮化砧木‘BP-176’的组织培养快繁技术体系及其离体叶片不定梢再生技术体系,为工厂化生产优质苗木及利用生物技术手段改良品种奠定技术基础。【方法】以苹果抗寒矮化砧木‘BP-176’的半木质化新梢为试材,建立初代无菌试管苗。以试管苗为试材,研究了基本培养基、植物生长调节物质对试管苗继代生长及生根的影响。以离体叶片为外植体,研究了细胞分裂素种类和浓度及碳源对不定梢再生的影响。【结果】半木质化新梢在芽启动培养基上的腋芽萌发率达85%以上。在基本培养基MS和QL上,试管苗的增殖没有显著差异,但生长表现不同,QL培养基上的增殖苗表现出该品种田间生长的红色特征。当细胞分裂素为BA时,蔗糖比D-山梨醇易诱导出叶片不定梢;当细胞分裂素为TDZ并且浓度较高时,D-山梨醇比蔗糖易诱导出叶片不定梢。以添加3 mg·L-1BA和30 g·L-1蔗糖处理获得的不定梢再生率最高,为71.6%。生根诱导,基本培养基1/2MS比1/4MS有效,生长素IBA比NAA有利于提高生根率。【结论】苹果砧木‘BP-176’试管苗适宜的继代增殖培养基为添加1.0 mg·L-1BA和0.1 mg·L-1IBA的QL培养基;最佳不定梢再生培养基为:NM+3 mg·L-1BA+0.3 mg·L-1IBA+30 g·L-1蔗糖;最佳生根培养基为1/2MS+0.3 mg·L-1IBA+20 g·L-1蔗糖,最高生根率为69.8%。
【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-16-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.
【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-16-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.
引文
[1] WARSCHEFSKY E J,KLEIN L L,FRANK M H,CHITWOOD D H,LONDO J P,WETTBERG E J B,MILLER A J. Rootstocks:Diversity,domestication,and impacts on shoot phenotypes[J]. Trends in Plant Science,2016,21(5):418-437.
[2] BITE A,DRUDZEL I. Winter hardiness of apple cultivars and rootstocks[J]. Acta Horticulturae,2000,25:343-347.
[3] TOTH E N,SZABO L G Y,BOTZ L,OROSZ-KOVACS Z. Effect of rootstocks on floral nectar composition in apple cultivars[J]. Plant Systematics and Evolution,2003,238:43-55.
[4] DALAL M A,SHARMA A K,MIR M A,SOUNDUI A S. In vitro cloning of apple(Malus domestica Borkh.)employing forced shoot tip cultures of M9 rootstock.[J]. India Journal of Biotechnology,2006,6:543-550.
[5]孙清荣,孙洪雁,李林光,李芹,陶吉寒.苹果矮化砧GM256(Malus domestica Borkh)高效快繁技术体系的建立[J].中国农学通报,2014,30(7):95-99.SUN Qingrong,SUN Hongyan,LI Linguang,LI Qin,TAO Jihan. Establishment of high efficient proliferation technological system of apple dwarf rootstock‘GM256’(Malus domestica Borkh)[J]. Chinese Agricultural Science Bulletin,2014,30(7):95-99.
[6] SUN Q R,SUN H Y,BELL R L,LI L G,XIN L,TAO J H,LI Q. Optimisation of the media for in vitro shoot proliferation and root induction in three new cold-hardy and dwarfing or semidwarfing clonal apple rootstocks[J]. Journal of Horticultural Science&Biotechnology,2014,89(4):381-388.
[7] PREDIERI S,MALAVASI F F. High-frequency shoot regeneration from leaves of the apple rootstock M26(Malus pumila Mill.)[J]. Plant Cell,Tissue and Organ Culture,1989,17:133-142.
[8] HOHNLE M K,WEBR G. Efficient adventitious shoot formation of leaf segments of in vitro propagated shoots of the apple rootstock M.9/T337[J]. European Journal of Horticultural Science,2010,75(3):128-131.
[9]孙洪雁,孙清荣,李国田,张琼,李芹.苹果矮化砧木‘JM7’的组织培养及其离体叶片不定梢再生[J].植物生理学报,2014,50(6):779-784.SUN Hongyan,SUN Qingrong,LI Guotian,ZHANG Qiong,LI Qin. Tissue culture and shoot regeneration from in vitro leaf explants of apple dwarfing rootstock cultivar‘JM7’[J]. Plant Physiology Journal,2014,50(6):779-784.
[10] SUN Q R,SUN M J,SUN H Y,BELL R L,LI L G,ZHANG W,TAO J H. Comparative organogenic response of six clonal apple rootstock cultivars[J]. HortScience,2016,51(3):271-278.
[11]孙清荣,关秋竹,孙洪雁,李林光,陶吉寒,王海波,何平.苹果抗寒半矮化砧木‘54-118’的组织培养及其离体叶片不定梢再生[J].植物生理学报,2017,53(11):2007-2012.SUN Qingrong,GUAN Qiuzhu,SUN Hongyan,LI Linguang,TAO Jihan,WANG Haibo,HE Ping. Tissue culture and shoot regeneration from leaf explants of cold-hardy and semi-dwarf apple rootstock‘54-118’[J]. Plant Physiology Journal,2017,53(11):2007-2012.
[12] ZHANG X,QIN Y,LIANG D,ZOU Y J,MA F W. Enhancement of in vitro shoot regeneration from leaf explants of apple rootstock G.41[J]. In Vitro Cellular Developmental Biology—Plant,2014,50:263-270.
[13]刘莹,徐继忠,邵建柱,高仪.苹果矮化砧木P59离体叶片高效再生体系的建立[J].河北农业大学学报,2006,29(3):10-12.LIU Ying,XU Jizhong,SHAO Jianzhu,GAO Yi. The establishment of a highly efficient regeneration system from leaves in vitro of apple dwarf stock P59[J]. Journal of Agricultural University of Hebei,2006,29(3):10-12.
[14] ZHU L H,HOLEFORS A,AHLMAN A,XUE Z T,WELANDER M. Transformation of the apple rootstock M.9:29 with the rol B gene and its influence on rooting and growth[J]. Plant Science,2001,160(3):433-439.
[2] BITE A,DRUDZEL I. Winter hardiness of apple cultivars and rootstocks[J]. Acta Horticulturae,2000,25:343-347.
[3] TOTH E N,SZABO L G Y,BOTZ L,OROSZ-KOVACS Z. Effect of rootstocks on floral nectar composition in apple cultivars[J]. Plant Systematics and Evolution,2003,238:43-55.
[4] DALAL M A,SHARMA A K,MIR M A,SOUNDUI A S. In vitro cloning of apple(Malus domestica Borkh.)employing forced shoot tip cultures of M9 rootstock.[J]. India Journal of Biotechnology,2006,6:543-550.
[5]孙清荣,孙洪雁,李林光,李芹,陶吉寒.苹果矮化砧GM256(Malus domestica Borkh)高效快繁技术体系的建立[J].中国农学通报,2014,30(7):95-99.SUN Qingrong,SUN Hongyan,LI Linguang,LI Qin,TAO Jihan. Establishment of high efficient proliferation technological system of apple dwarf rootstock‘GM256’(Malus domestica Borkh)[J]. Chinese Agricultural Science Bulletin,2014,30(7):95-99.
[6] SUN Q R,SUN H Y,BELL R L,LI L G,XIN L,TAO J H,LI Q. Optimisation of the media for in vitro shoot proliferation and root induction in three new cold-hardy and dwarfing or semidwarfing clonal apple rootstocks[J]. Journal of Horticultural Science&Biotechnology,2014,89(4):381-388.
[7] PREDIERI S,MALAVASI F F. High-frequency shoot regeneration from leaves of the apple rootstock M26(Malus pumila Mill.)[J]. Plant Cell,Tissue and Organ Culture,1989,17:133-142.
[8] HOHNLE M K,WEBR G. Efficient adventitious shoot formation of leaf segments of in vitro propagated shoots of the apple rootstock M.9/T337[J]. European Journal of Horticultural Science,2010,75(3):128-131.
[9]孙洪雁,孙清荣,李国田,张琼,李芹.苹果矮化砧木‘JM7’的组织培养及其离体叶片不定梢再生[J].植物生理学报,2014,50(6):779-784.SUN Hongyan,SUN Qingrong,LI Guotian,ZHANG Qiong,LI Qin. Tissue culture and shoot regeneration from in vitro leaf explants of apple dwarfing rootstock cultivar‘JM7’[J]. Plant Physiology Journal,2014,50(6):779-784.
[10] SUN Q R,SUN M J,SUN H Y,BELL R L,LI L G,ZHANG W,TAO J H. Comparative organogenic response of six clonal apple rootstock cultivars[J]. HortScience,2016,51(3):271-278.
[11]孙清荣,关秋竹,孙洪雁,李林光,陶吉寒,王海波,何平.苹果抗寒半矮化砧木‘54-118’的组织培养及其离体叶片不定梢再生[J].植物生理学报,2017,53(11):2007-2012.SUN Qingrong,GUAN Qiuzhu,SUN Hongyan,LI Linguang,TAO Jihan,WANG Haibo,HE Ping. Tissue culture and shoot regeneration from leaf explants of cold-hardy and semi-dwarf apple rootstock‘54-118’[J]. Plant Physiology Journal,2017,53(11):2007-2012.
[12] ZHANG X,QIN Y,LIANG D,ZOU Y J,MA F W. Enhancement of in vitro shoot regeneration from leaf explants of apple rootstock G.41[J]. In Vitro Cellular Developmental Biology—Plant,2014,50:263-270.
[13]刘莹,徐继忠,邵建柱,高仪.苹果矮化砧木P59离体叶片高效再生体系的建立[J].河北农业大学学报,2006,29(3):10-12.LIU Ying,XU Jizhong,SHAO Jianzhu,GAO Yi. The establishment of a highly efficient regeneration system from leaves in vitro of apple dwarf stock P59[J]. Journal of Agricultural University of Hebei,2006,29(3):10-12.
[14] ZHU L H,HOLEFORS A,AHLMAN A,XUE Z T,WELANDER M. Transformation of the apple rootstock M.9:29 with the rol B gene and its influence on rooting and growth[J]. Plant Science,2001,160(3):433-439.