‘章姬’草莓茎段愈伤组织诱导及高频植株再生体系的建立
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摘要
该研究以‘章姬’脱毒苗带节匍匐茎段为外植体,以MS为基本培养基,进行单因素预实验,选取适合‘章姬’草莓生长的植物激素种类和质量浓度范围,进而通过L_9(3~4)正交实验研究不同植物激素种类及其质量浓度对愈伤组织诱导、丛芽发生及植株再生的影响。结果表明:采用的3种基本培养基中,B_5和1/2MS对抑制培养过程中的褐化现象明显优于MS,而附加20 g·L~(-1)Na_2S_2O_3在保证材料存活的前提下,大大降低了褐化率;去除褐化的材料在MS+0.1 mg·L~(-1)6-BA+0.05 mg·L~(-1)2,4-D+0.1 mg·L~(-1)NAA中可同步进行愈伤组织诱导和丛芽发生;丛芽增殖培养基为MS+0.1 mg·L~(-1)6-BA+0.1 mg·L~(-1)NAA,30 d后繁殖系数可达12.86;试管苗生根则在1/2MS+1.0 g·L~(-1)AC中进行,35 d后可获得生长健壮的再生植株,生根率92.50%。再生苗移栽成活率在95%以上。该研究建立了‘章姬’草莓体外高效快速繁殖体系,对短期内为‘章姬’草莓栽培提供大量种性稳定、质量优良的种苗具有重要意义,同时为其他草莓品种的体外快繁提供了技术参考。
In order to cultivate an improved variety with higher potential yield and establish an efficient in vitro regeneration system of strawberry‘Akihim’(Fragaria×ananassa),callus induction and plant regeneration protocol was designed for solve browning problem.A formal L_9(3~4)orthogonal experiment was designed to investigate the browning research in primary culture using explants of creeping stems excised from aseptic seedlings.Based on the improved medium above,singlefactor experiments were conducted to select effective plant growth regulators and appropriate concentrations on blank MS medium.A L_9(3~4)orthogonal experiment was designed to study effects of types and concentrations of plant growth regulators on callus induction,adventitious shoot formation,and plant regeneration.The results indicated that MS medium was inferior to B_5and 1/2MS in inhibiting browning condition.The browning rate was dramatically reduced while the callus still survived on the medium with the addition of 20 g·L~(-1)Na_2S_2O_3.The callus induction and shoot formation of the explants were observed on MS+0.1 mg·L~(-1)6-BA+0.05 mg·L~(-1)2,4-D+0.1 mg·L~(-1)NAA with effective inhibiting browning.The optimal medium protocol for multiple shoots proliferation was MS+0.1 mg·L~(-1)6-BA+0.1 mg·L~(-1)NAA where the proliferation coefficient was 12.86 after 30 d.Healthily regenerated plants were yielded on culture medium 1/2MS+1.0 g·L~(-1)AC after 35 d,with a rooting rate of 92.50%.More than 95%of plantlets survived after transplanting into field.The rapid propagation system is helpful to provide homogeneous progeny and high quality seedlings for cultivation of strawberry‘Akihime’as well as a technical reference for other strawberry species in vitro regeneration.
In order to cultivate an improved variety with higher potential yield and establish an efficient in vitro regeneration system of strawberry‘Akihim’(Fragaria×ananassa),callus induction and plant regeneration protocol was designed for solve browning problem.A formal L_9(3~4)orthogonal experiment was designed to investigate the browning research in primary culture using explants of creeping stems excised from aseptic seedlings.Based on the improved medium above,singlefactor experiments were conducted to select effective plant growth regulators and appropriate concentrations on blank MS medium.A L_9(3~4)orthogonal experiment was designed to study effects of types and concentrations of plant growth regulators on callus induction,adventitious shoot formation,and plant regeneration.The results indicated that MS medium was inferior to B_5and 1/2MS in inhibiting browning condition.The browning rate was dramatically reduced while the callus still survived on the medium with the addition of 20 g·L~(-1)Na_2S_2O_3.The callus induction and shoot formation of the explants were observed on MS+0.1 mg·L~(-1)6-BA+0.05 mg·L~(-1)2,4-D+0.1 mg·L~(-1)NAA with effective inhibiting browning.The optimal medium protocol for multiple shoots proliferation was MS+0.1 mg·L~(-1)6-BA+0.1 mg·L~(-1)NAA where the proliferation coefficient was 12.86 after 30 d.Healthily regenerated plants were yielded on culture medium 1/2MS+1.0 g·L~(-1)AC after 35 d,with a rooting rate of 92.50%.More than 95%of plantlets survived after transplanting into field.The rapid propagation system is helpful to provide homogeneous progeny and high quality seedlings for cultivation of strawberry‘Akihime’as well as a technical reference for other strawberry species in vitro regeneration.
引文
ADAMS AN,1972.An improved medium for strawberry meristem culture[J].J Hortic Sci,48:263-264.
BOXUS P,1974.The production of strawberry plants by in vitro micropropagation[J].J Hortic Sci,49:209-210.
CHEN BY,HUANG JF,GAO ZH,et al,2012.Karyotype analysis of six wild strawberry species[J].Acta Bot BorealOccident Sin,32(8):1567-1572.[陈丙义,黄金凤,高志红,等,2012.6种野生草莓的核型分析[J].西北植物学报,32(8):1567-1572.]
DAI XM,SUN ZY,HAN L,2015a.Callus induction and inhibition effect of citric acid on browning of kentucky bluegrass callus[J].Acta Agric Nucl Sci,29(2):270-277.[代小梅,孙振元,韩蕾,2015a.草地早熟禾愈伤组织诱导及柠檬酸对其褐化的抑制效应[J].核农学报,29(2):270-277.]
DAI XM,SUN ZY,HAN L,et al,2015b.Callus induction and inhibition effect of citric acid on browning of perennial ryegrass callus[J].Acta Agric Nucl Sci,29(10):1893-1900.[代小梅,孙振元,韩蕾,等,2015b.多年生黑麦草愈伤组织诱导及柠檬酸对其褐化的抑制效应[J].核农学报,29(10):1893-1900.]
DUAN YX,GUO WW,2009.Study on callus browning in relation to polyphenol content and polyphenol oxidase activity among various Citrus embryogenic calli[J].Chin Agric Sci Bull,25(15):117-120.[段艳欣,郭文武,2009.多酚含量及多酚氧化酶活性与柑橘胚性愈伤组织褐化的关系[J].中国农学通报,25(15):117-120.]
FENG DD,WANG Y,CHEN JP,2015.Research progress of browning in the plant tissue culture[J].Acta Agric Zhejiang,27(6):1108-1116.[冯代弟,王燕,陈剑平,2015.植物组培褐化发生机制的研究进展[J].浙江农业学报,27(6):1108-1116.]
GUO ZY,QIAN SF,LUO Y,2009.Study on the browning control in tissue culture of Rubus suavissimus S.Lee[J].Anhui Agric Sci,37(19):8848-8850.[郭治友,钱绍方,罗应,2009.甜叶悬钩子植物组织培养褐化现象控制研究[J].安徽农业科学,37(19):8848-8850.]
HUANG HY,WANG MR,2014.Callus induction and regeneration system establishment of Ajugae nipponensis[J].Chin Trad Herb Drugs,45(9):1313-1318.[黄衡宇,王美蓉,2014.紫背金盘愈伤组织诱导与再生体系的建立[J].中草药,45(9):1313-1318.]
JIN MF,CAO Z,CAI JJ,et al,2017.Study on tissue culture and rapid propagation technique of red-flowered strawberry[J].Guihaia,37(4):1-12.[金美芳,曹智,蔡俊杰,等,2017.红花草莓的组织培养与快繁技术研究[J].广西植物,37(4):1-12.]
JIN Z,WANG KL,LIANG JH,et al,2015.Effects of exogenous hormones and medium on stem tip germination“Akihime”strawberry[J].J Agric,5(8):73-77.[金真,王康丽,梁佳惠,等,2015.培养基和外源激素对‘章姬’草莓茎尖培养的影响研究[J].农学学报,5(8):73-77.]
JOSUTTIS M,DIETRICH H,TREUTTER D,et al,2010.Solar UVB response of bioactives in strawberry(Fragaria×ananassa duch.l.):a comparison of protected and open-field cultivation[J].J Agric Food Chem,58(24):12692-12702.
KO CY,Al ABDULKARIM AM,Al JOWID SM,et al,2009.An effective disinfection protocol for plant regeneration from shoot tip cultures of strawberry[J].Afr J Biotechnol,8(11):2611-2615.
LEI Q,HUANG M,KANG J,et al,2011.Effect of ion beam irradiation on fresh-keeping of strawberry[J]Acta Agric Nucl Sci,25(3):510-513.[雷庆,黄敏,康菊,等,2011.电子束辐照草莓保鲜效果研究[J].核农学报,25(3):510-513.]
LI L,LONG H,GAO J,et al,2015.Efficient induction system of adventitious shoots and plant regeneration in Solanum pseudocapsicum[J].Chin Trad Herb Drugs,46(9):1360-1367.[李鹂,龙华,高洁,等,2015.珊瑚樱丛芽高效诱导体系及植株再生研究[J].中草药,46(9):1360-1367.]
LI S,TIAN J,LU SW,et al,2014.Study on nursery effect of an elevated and layered seedling raising system for greenhouse strawberry[J].Northern Hortic,(14):41-45.[李邵,田婧,鲁少尉,等,2014.设施草莓高架分层育苗系统育苗效果研究[J].北方园艺,(14):41-45.]
LIAO YM,LEI NF,Chen JS,2006.Clonal integration of a stoloniferous plant(Fragaria vesca)in response to heterogeneous light[J].Guihaia,26(5):503-506.[廖咏梅,雷泞菲,陈劲松,2006.异质性光照条件下匍匐茎草本野草莓的克隆整合[J].广西植物,26(5):503-506.]
LIU Y,SU SC,LENG PS,et al,2007.Procedures of diminishing browning of in vitro explants in Pistacia vera[J].J Fruit Sci,24(3):393-396.[刘洋,苏淑钗,冷平生,等,2007.阿月浑子外植体褐变抑制方法[J].果树学报,24(3):393-396.]
LMP,WANG YZ,HUANG HY,2016.Callus induction and high efficiency plant regeneration system establishment of Pararuellia delavayana[J].Chin Bull Bot,51(1):89-97.[吕美萍,王元忠,黄衡宇,2016.地皮消愈伤组织诱导及植物高效再生体系的建立[J].植物学报,51(1):89-97.]
MA CJ,LIAO PY,2007.Tissue culture of petiole and leaf blade of Fragaria ananassa[J].Anhui Agric Sci,35(13):3841-3842.[马崇坚,廖佩颖,2007.草莓叶柄和叶片的组织培养[J].安徽农业科学,35(13):3841-3842.]
MA WQ,LI Q,LIU Y,2010.Studies on browning during the test-tube proliferation of Cymbidium hybridu[J].Chin Agric Sci Bull,26(7):186-190.[马文卿,李青,刘燕,2010.大花蕙兰试管苗增殖过程中的褐化研究[J].中国农学通报,26(7):186-190.]
NIU HY,WANG Q,XUE GB,et al,2004.Study on tissue culture of the stem of Fragaria ananassa Duch[J].J NEAgric Univ,35(5):542-545.[牛红云,王荃,薛贵彬,等,2004.草莓(Fragaria ananassa Duch)茎段组织培养的研究[J].东北农业大学学报,35(5):542-545.]
WANG F,GAO ZH,ZHANG Z,et al,2010.Establishment of regeneration system of strawberry[J].Acta Bot BorealOccident Sin,30(5):1045-1049.[王翡,高志红,章镇,等,2010.草莓高效离体叶片再生体系的建立[J].西北植物学报,30(5):1045-1049.]
WANG SY,Millner P,2009.Effect of different cultural systems on antioxidant capacity,phenolic content,and fruit quality of strawberries(Fragaria×aranassa Duch.)[J].J Agric Food Chem,57(20):9651-9657.
WU LL,KE BF,GONG C,et al,2015.Tissue culture and rapid propagation of Akebia trifoliate var.australis[J].Plant Physiol,51(6):903-908.[吴玲利,柯镔峰,龚春,等,2015.白木通组织培养及快速繁殖[J].植物生理学报,51(6):903-908.]
XIA YN,JIANG JX,YI ZL,et al,2014.Effect of three antioxidants on browning of explant and callus induction rate of Miscanthus lutarioriparius[J].Northern Hortic,(17):93-96.[夏亚男,蒋建雄,易自力,等,2014.三种抗褐化剂对南荻外植体褐变及愈伤诱导率的影响[J].北方园艺,(17):93-96.]
XIAO JZ,HUANG YH,JIANG FJ,et al,2011.Study on Virusfree rapid propagation of zhangji strawberry by anther tissue culture[J].Acta Agric Jiangxi,(10):49-50.[肖君泽,黄益鸿,姜放军,等,2011.章姬草莓花药组织培养脱毒好快速繁殖技术的研究[J].江西农业学报,(10):49-50.]
ZHOU LP,WANG SZ,RUAN SL,et al,2015.Optimization of extraction method for flow cytometry in strawberry[J].Acta Agric Zhejiang,27(11):2024-2028.[周历萍,王淑珍,阮松林,等,2015.草莓流式细胞检测提取方法的优化[J].浙江农业学报,27(11):2024-2028.]
ZHU HS,PAN DM,LIN YZ,et al,2007.In vitro efficient regeneration system of strawberry[J].Acta Bot BorealOccident Sin,27(5):859-863.[朱海生,潘东明,林义章,等,2007.草莓高频离体再生体系的研究[J].西北植物学报,27(5):859-863.]
BOXUS P,1974.The production of strawberry plants by in vitro micropropagation[J].J Hortic Sci,49:209-210.
CHEN BY,HUANG JF,GAO ZH,et al,2012.Karyotype analysis of six wild strawberry species[J].Acta Bot BorealOccident Sin,32(8):1567-1572.[陈丙义,黄金凤,高志红,等,2012.6种野生草莓的核型分析[J].西北植物学报,32(8):1567-1572.]
DAI XM,SUN ZY,HAN L,2015a.Callus induction and inhibition effect of citric acid on browning of kentucky bluegrass callus[J].Acta Agric Nucl Sci,29(2):270-277.[代小梅,孙振元,韩蕾,2015a.草地早熟禾愈伤组织诱导及柠檬酸对其褐化的抑制效应[J].核农学报,29(2):270-277.]
DAI XM,SUN ZY,HAN L,et al,2015b.Callus induction and inhibition effect of citric acid on browning of perennial ryegrass callus[J].Acta Agric Nucl Sci,29(10):1893-1900.[代小梅,孙振元,韩蕾,等,2015b.多年生黑麦草愈伤组织诱导及柠檬酸对其褐化的抑制效应[J].核农学报,29(10):1893-1900.]
DUAN YX,GUO WW,2009.Study on callus browning in relation to polyphenol content and polyphenol oxidase activity among various Citrus embryogenic calli[J].Chin Agric Sci Bull,25(15):117-120.[段艳欣,郭文武,2009.多酚含量及多酚氧化酶活性与柑橘胚性愈伤组织褐化的关系[J].中国农学通报,25(15):117-120.]
FENG DD,WANG Y,CHEN JP,2015.Research progress of browning in the plant tissue culture[J].Acta Agric Zhejiang,27(6):1108-1116.[冯代弟,王燕,陈剑平,2015.植物组培褐化发生机制的研究进展[J].浙江农业学报,27(6):1108-1116.]
GUO ZY,QIAN SF,LUO Y,2009.Study on the browning control in tissue culture of Rubus suavissimus S.Lee[J].Anhui Agric Sci,37(19):8848-8850.[郭治友,钱绍方,罗应,2009.甜叶悬钩子植物组织培养褐化现象控制研究[J].安徽农业科学,37(19):8848-8850.]
HUANG HY,WANG MR,2014.Callus induction and regeneration system establishment of Ajugae nipponensis[J].Chin Trad Herb Drugs,45(9):1313-1318.[黄衡宇,王美蓉,2014.紫背金盘愈伤组织诱导与再生体系的建立[J].中草药,45(9):1313-1318.]
JIN MF,CAO Z,CAI JJ,et al,2017.Study on tissue culture and rapid propagation technique of red-flowered strawberry[J].Guihaia,37(4):1-12.[金美芳,曹智,蔡俊杰,等,2017.红花草莓的组织培养与快繁技术研究[J].广西植物,37(4):1-12.]
JIN Z,WANG KL,LIANG JH,et al,2015.Effects of exogenous hormones and medium on stem tip germination“Akihime”strawberry[J].J Agric,5(8):73-77.[金真,王康丽,梁佳惠,等,2015.培养基和外源激素对‘章姬’草莓茎尖培养的影响研究[J].农学学报,5(8):73-77.]
JOSUTTIS M,DIETRICH H,TREUTTER D,et al,2010.Solar UVB response of bioactives in strawberry(Fragaria×ananassa duch.l.):a comparison of protected and open-field cultivation[J].J Agric Food Chem,58(24):12692-12702.
KO CY,Al ABDULKARIM AM,Al JOWID SM,et al,2009.An effective disinfection protocol for plant regeneration from shoot tip cultures of strawberry[J].Afr J Biotechnol,8(11):2611-2615.
LEI Q,HUANG M,KANG J,et al,2011.Effect of ion beam irradiation on fresh-keeping of strawberry[J]Acta Agric Nucl Sci,25(3):510-513.[雷庆,黄敏,康菊,等,2011.电子束辐照草莓保鲜效果研究[J].核农学报,25(3):510-513.]
LI L,LONG H,GAO J,et al,2015.Efficient induction system of adventitious shoots and plant regeneration in Solanum pseudocapsicum[J].Chin Trad Herb Drugs,46(9):1360-1367.[李鹂,龙华,高洁,等,2015.珊瑚樱丛芽高效诱导体系及植株再生研究[J].中草药,46(9):1360-1367.]
LI S,TIAN J,LU SW,et al,2014.Study on nursery effect of an elevated and layered seedling raising system for greenhouse strawberry[J].Northern Hortic,(14):41-45.[李邵,田婧,鲁少尉,等,2014.设施草莓高架分层育苗系统育苗效果研究[J].北方园艺,(14):41-45.]
LIAO YM,LEI NF,Chen JS,2006.Clonal integration of a stoloniferous plant(Fragaria vesca)in response to heterogeneous light[J].Guihaia,26(5):503-506.[廖咏梅,雷泞菲,陈劲松,2006.异质性光照条件下匍匐茎草本野草莓的克隆整合[J].广西植物,26(5):503-506.]
LIU Y,SU SC,LENG PS,et al,2007.Procedures of diminishing browning of in vitro explants in Pistacia vera[J].J Fruit Sci,24(3):393-396.[刘洋,苏淑钗,冷平生,等,2007.阿月浑子外植体褐变抑制方法[J].果树学报,24(3):393-396.]
LMP,WANG YZ,HUANG HY,2016.Callus induction and high efficiency plant regeneration system establishment of Pararuellia delavayana[J].Chin Bull Bot,51(1):89-97.[吕美萍,王元忠,黄衡宇,2016.地皮消愈伤组织诱导及植物高效再生体系的建立[J].植物学报,51(1):89-97.]
MA CJ,LIAO PY,2007.Tissue culture of petiole and leaf blade of Fragaria ananassa[J].Anhui Agric Sci,35(13):3841-3842.[马崇坚,廖佩颖,2007.草莓叶柄和叶片的组织培养[J].安徽农业科学,35(13):3841-3842.]
MA WQ,LI Q,LIU Y,2010.Studies on browning during the test-tube proliferation of Cymbidium hybridu[J].Chin Agric Sci Bull,26(7):186-190.[马文卿,李青,刘燕,2010.大花蕙兰试管苗增殖过程中的褐化研究[J].中国农学通报,26(7):186-190.]
NIU HY,WANG Q,XUE GB,et al,2004.Study on tissue culture of the stem of Fragaria ananassa Duch[J].J NEAgric Univ,35(5):542-545.[牛红云,王荃,薛贵彬,等,2004.草莓(Fragaria ananassa Duch)茎段组织培养的研究[J].东北农业大学学报,35(5):542-545.]
WANG F,GAO ZH,ZHANG Z,et al,2010.Establishment of regeneration system of strawberry[J].Acta Bot BorealOccident Sin,30(5):1045-1049.[王翡,高志红,章镇,等,2010.草莓高效离体叶片再生体系的建立[J].西北植物学报,30(5):1045-1049.]
WANG SY,Millner P,2009.Effect of different cultural systems on antioxidant capacity,phenolic content,and fruit quality of strawberries(Fragaria×aranassa Duch.)[J].J Agric Food Chem,57(20):9651-9657.
WU LL,KE BF,GONG C,et al,2015.Tissue culture and rapid propagation of Akebia trifoliate var.australis[J].Plant Physiol,51(6):903-908.[吴玲利,柯镔峰,龚春,等,2015.白木通组织培养及快速繁殖[J].植物生理学报,51(6):903-908.]
XIA YN,JIANG JX,YI ZL,et al,2014.Effect of three antioxidants on browning of explant and callus induction rate of Miscanthus lutarioriparius[J].Northern Hortic,(17):93-96.[夏亚男,蒋建雄,易自力,等,2014.三种抗褐化剂对南荻外植体褐变及愈伤诱导率的影响[J].北方园艺,(17):93-96.]
XIAO JZ,HUANG YH,JIANG FJ,et al,2011.Study on Virusfree rapid propagation of zhangji strawberry by anther tissue culture[J].Acta Agric Jiangxi,(10):49-50.[肖君泽,黄益鸿,姜放军,等,2011.章姬草莓花药组织培养脱毒好快速繁殖技术的研究[J].江西农业学报,(10):49-50.]
ZHOU LP,WANG SZ,RUAN SL,et al,2015.Optimization of extraction method for flow cytometry in strawberry[J].Acta Agric Zhejiang,27(11):2024-2028.[周历萍,王淑珍,阮松林,等,2015.草莓流式细胞检测提取方法的优化[J].浙江农业学报,27(11):2024-2028.]
ZHU HS,PAN DM,LIN YZ,et al,2007.In vitro efficient regeneration system of strawberry[J].Acta Bot BorealOccident Sin,27(5):859-863.[朱海生,潘东明,林义章,等,2007.草莓高频离体再生体系的研究[J].西北植物学报,27(5):859-863.]