草莓抗炭疽病突变体的诱变及筛选
|
摘要
本实验以江苏省农科院园艺研究所草莓资源圃保存的‘红颊’草莓品种的组培苗为外植体进行实验。‘红颊’是一种优质大果型草莓品种,其颜色鲜艳,着色一致,外形美观,香味口感极佳,但是炭疽病发病率高已经成为其产量增长的重要影响因素之一。本实验目的是通过EMS诱变筛选出定向突变的抗炭疽病新植株。
首先探讨激素,放置方式,暗培养时间,温度,不同部位等条件对其再生的影响,筛选出最佳组合以优化其再生体系,运用最小显著极差法处理实验数据,探讨不同植物生长调节剂组合(IBA, NAA, IAA)、暗培养时间(5,10,15,20d)、放置方式(正面朝上,反面朝上)、不同取材部位(叶柄,叶片)对其再生的影响。结果表明：MS+TDZ(2.0mg·L-1)+NAA(0.1mg·L-1)为最适宜的不定芽分化培养基,不定芽分化率最高可达到100.0%,一周(7天)是最佳的暗培养时间,叶片正面朝上放置较好。叶柄再生能力比叶片强,两者都是基部比顶部分化率高,诱导能力从强到弱可依次排列为叶柄基部>叶片基部>叶片顶部>叶柄顶部。最佳生根培养基为1/2MS+IBA (0.1mg-L-1),20天后100%的不定芽可长出3-4cm长的根,移栽成活率达到98.06%。由此建立起‘红颊’草莓品种的高效再生体系。
然后配置浓度分别为0.05%,0.10%,0.2%,0.4%,0.8%的5个梯度EMS诱变液,设空白对照。将诱变材料放入不同浓度诱变液1h探讨不同浓度对其影响。同时将诱变材料放入0.1%的诱变液处理时间分别为0.5,1.0,1.5,2.0h,以探讨不同诱变时间对其影响。还将诱变材料放入0.1%的诱变液处理1.0h,以探讨处理后其内部不同活性指标的变化。根据PPO、SOD等活性物质的变化,推测EMS较低浓度,较短时间处理下,细胞能自身修复,一旦超过临界点,则伤害无法得到修复。
0.8%浓度的EMS诱变液配以长时间处理,短时间内实验材料褐化死亡,而在0.2%浓度的EMS诱变液配以短时间处理,处理后的材料无褐化死亡现象或者褐化率过低。得出诱变组合为浓度为0.2%,处理时间为80min；浓度为0.4%,处理时间为60min；浓度为0.4%,处理时间为60min这三种组合均可。
将诱变得到的再生芽转接到Ms培养基培养中培养,待其长大后移入生根培养基。当根长到3-4cm长时移到穴盘内进行培养,待其长成后用用浓度为每毫升7×106个孢子的炭疽菌孢子悬浮液喷洒植株表面,初步筛选出表型为抗炭疽病的草莓植株5株。这些抗性植株的遗传基因需进一步进行分子学验证。
Strawberry cultivar' Benihoppe' is a high-quality type of strawberry cultivar with beautiful appearance and excellent fragrance taste. But anthracnose become one of the important factors affecting its production growth. The purpose of this study is to obtain new plants which resistant to anthracnose through the EMS mutagenesis screening.
The plantlets kept in vitro were used as test materials to study the influences of various combinations of plant growth regulators, culture durations under dark, positioning explant styles, and samplings on strawberry regeneration with the experimental data being analyzed by the shortest significant range method. The results showed that the optimal medium for adventitious bud regeneration was MS+TDZ (2.0mg·L-1)+NAA (0.1mg·L-1) with the highest differentiation rate of100.0%, for which7days of dark time and face up positioning were necessary. It was noticed that the adventitious bud regeneration capacity from petiole was better than that from leaf blade, and both of them could have better regeneration capacity at their bases. The best rooting medium was1/2MS+IBA (0.1mg·L-1), on which100%of the adventitious buds could grow3-4cm roots after20days of culture. After transplanting,98.06%of the rooted shoots could survived well in greenhouse.
The leaves used as mutagenic materials were treated with different concentrations of EMS solutions for different time to test the physiologically active substance and the degree of injury. The results indicated that the damage of the mutagenic materials was growing with concentration and time of EMS solutions increasing.0.8%concentration of EMS is too high. Most of all material was browning and died in short time under the0.8%concentration, on other side, browning of the materials was not heavy under0.2%of EMS.0.4%concentration of EMS for60min was the best combination of mutation.
According to the above results,many leaves used as mutagenic materials were treated with0.4%concentrations of EMS solutions for60min,then transferred the leaves into induction medium (MS+TDZ (2.0mg·L-1)+NAA (0.1mg·L-1)). to get regeneration bud. Take regeneration bud to the rooting medium. When the plantlets' root reach3-4cm length, transplant them in plug. Spraying plant with the7X106/ml concentrations of the anthrax spore suspension when the plants grew up, five plant showed relatively resistant to anthracnose. These resistant plants should be done further molecular genetic identification.
首先探讨激素,放置方式,暗培养时间,温度,不同部位等条件对其再生的影响,筛选出最佳组合以优化其再生体系,运用最小显著极差法处理实验数据,探讨不同植物生长调节剂组合(IBA, NAA, IAA)、暗培养时间(5,10,15,20d)、放置方式(正面朝上,反面朝上)、不同取材部位(叶柄,叶片)对其再生的影响。结果表明：MS+TDZ(2.0mg·L-1)+NAA(0.1mg·L-1)为最适宜的不定芽分化培养基,不定芽分化率最高可达到100.0%,一周(7天)是最佳的暗培养时间,叶片正面朝上放置较好。叶柄再生能力比叶片强,两者都是基部比顶部分化率高,诱导能力从强到弱可依次排列为叶柄基部>叶片基部>叶片顶部>叶柄顶部。最佳生根培养基为1/2MS+IBA (0.1mg-L-1),20天后100%的不定芽可长出3-4cm长的根,移栽成活率达到98.06%。由此建立起‘红颊’草莓品种的高效再生体系。
然后配置浓度分别为0.05%,0.10%,0.2%,0.4%,0.8%的5个梯度EMS诱变液,设空白对照。将诱变材料放入不同浓度诱变液1h探讨不同浓度对其影响。同时将诱变材料放入0.1%的诱变液处理时间分别为0.5,1.0,1.5,2.0h,以探讨不同诱变时间对其影响。还将诱变材料放入0.1%的诱变液处理1.0h,以探讨处理后其内部不同活性指标的变化。根据PPO、SOD等活性物质的变化,推测EMS较低浓度,较短时间处理下,细胞能自身修复,一旦超过临界点,则伤害无法得到修复。
0.8%浓度的EMS诱变液配以长时间处理,短时间内实验材料褐化死亡,而在0.2%浓度的EMS诱变液配以短时间处理,处理后的材料无褐化死亡现象或者褐化率过低。得出诱变组合为浓度为0.2%,处理时间为80min；浓度为0.4%,处理时间为60min；浓度为0.4%,处理时间为60min这三种组合均可。
将诱变得到的再生芽转接到Ms培养基培养中培养,待其长大后移入生根培养基。当根长到3-4cm长时移到穴盘内进行培养,待其长成后用用浓度为每毫升7×106个孢子的炭疽菌孢子悬浮液喷洒植株表面,初步筛选出表型为抗炭疽病的草莓植株5株。这些抗性植株的遗传基因需进一步进行分子学验证。
Strawberry cultivar' Benihoppe' is a high-quality type of strawberry cultivar with beautiful appearance and excellent fragrance taste. But anthracnose become one of the important factors affecting its production growth. The purpose of this study is to obtain new plants which resistant to anthracnose through the EMS mutagenesis screening.
The plantlets kept in vitro were used as test materials to study the influences of various combinations of plant growth regulators, culture durations under dark, positioning explant styles, and samplings on strawberry regeneration with the experimental data being analyzed by the shortest significant range method. The results showed that the optimal medium for adventitious bud regeneration was MS+TDZ (2.0mg·L-1)+NAA (0.1mg·L-1) with the highest differentiation rate of100.0%, for which7days of dark time and face up positioning were necessary. It was noticed that the adventitious bud regeneration capacity from petiole was better than that from leaf blade, and both of them could have better regeneration capacity at their bases. The best rooting medium was1/2MS+IBA (0.1mg·L-1), on which100%of the adventitious buds could grow3-4cm roots after20days of culture. After transplanting,98.06%of the rooted shoots could survived well in greenhouse.
The leaves used as mutagenic materials were treated with different concentrations of EMS solutions for different time to test the physiologically active substance and the degree of injury. The results indicated that the damage of the mutagenic materials was growing with concentration and time of EMS solutions increasing.0.8%concentration of EMS is too high. Most of all material was browning and died in short time under the0.8%concentration, on other side, browning of the materials was not heavy under0.2%of EMS.0.4%concentration of EMS for60min was the best combination of mutation.
According to the above results,many leaves used as mutagenic materials were treated with0.4%concentrations of EMS solutions for60min,then transferred the leaves into induction medium (MS+TDZ (2.0mg·L-1)+NAA (0.1mg·L-1)). to get regeneration bud. Take regeneration bud to the rooting medium. When the plantlets' root reach3-4cm length, transplant them in plug. Spraying plant with the7X106/ml concentrations of the anthrax spore suspension when the plants grew up, five plant showed relatively resistant to anthracnose. These resistant plants should be done further molecular genetic identification.
引文
[1]段辛嵋,赵亚夫.草莓品种与栽培[M].南京：江苏科技出版社,1993.
[2]赵密珍,王壮伟,钱亚明,袁骥,吴伟民,王静.草莓新品种‘宁玉’[J].园艺学报,2011,38(7)：1411-1412.
[3]Brooks AN. Anthracnose of strawberry caused by Colletotriehum fragariae, n.sp.[J] Phytopathology,1931,21:739-744
[4]叶正文,郑宏清,童尧明等.上海地区病原炭疽病病原种类及品种抗性研究初报[J].上海农业学报,1997,13(1)：75-80.
[5]Mcinnes TB, BlackLL, Gatt JM..Disease-free plants for management of strawberry anthracnose crown rot[J].Plant disease,1992,76:33,260-264.
[6]DenoyesB, and Baudry A. Species identification and Pathogenieity study of French Colletotrichum strains isolated from strawberry using morphologieal and cultural characteristics[J].Phytopathology,1995,85:53-57.
[7]Bosshard E.Why is Colletotrichum acutarum a quarantine organism, and C.gloeosporioides and C.fragariae are not [J].Acta Hort.(ISHS),1997,439:799-802.
[8]胡淼.草莓病害的初步调查.中国植物病理学会华东区分会第五次年会论文摘要汇编,江苏,1990：58-59
[9]邵小花.上海地区草莓新病害-炭疽病[J].上海农业学报,1992,8：86-87
[10]万瑶,马青,张敬泽.草莓炭疽病菌的鉴定、检测及附着胞形成过程中核相变化的研究[D].西北农林科技大学,2006.179907.
[11]钟霈霖.日本草莓育种及发展战略[J].种子.2005,24(12)：98-100.
[12]谢东,许仁林,魏书.草莓生物技术育种研究进展[J].果树科学,1996,13(3)：195-198,
[13]李卫东,周春江,葛会波.草莓现代生物技术育种研究进展[J].河北农业大学学报,2000,23(1)：32-36.
[14]洪燕萍,林顺权.草莓离体培养研究进展[J].福建农业学报,1999, 14(2)：37-44.
[15]郑永平,郭宝江,阮继红.茎尖组培与辐照对草莓遗传变异的影响[J].园艺学报,1988,15(4)：245-251.
[16]雷家军,吴禄平,代汉萍,等.草莓茎尖染色体加倍研究[J].园艺学报,1999,26(1)：13-18.
[17]赵密珍,王壮伟,钱亚明,袁骥,吴伟民,王静.草莓新品种‘宁玉’[J].园艺学报,2011,38(7)：1411-1412.
[18]施巾帼.理化诱变因素复合处理的诱变效应研究进展[A].王琳清.诱发突变与作物改良[e].北京：原子能出版社1995,271-277.
[19]梁国平,肖三元,管艳.钻60辐射红掌组培材料的实验初报[J].热带农业科技,2005,25(3)：24-26.
[20]陈少裕.膜脂过氧化对植物细胞的毒害.植物生理学通讯,1991(2)：84
[21]秦永华.丰香草莓高效再生的机理及遗传转化研究[D].浙江大学，2005.
[22]吴雪梅,汤浩茹.草莓叶片培养研究进展[J].果树学报,2004,21(6)：598-602.
[23]Toyoda H, Horikoshi K et al. Seleetion for Fusarium wilt disease resistance from regenerants derived from leaf callus of strawberry [J]. Plant Cell Reports,1991, 10:167～170.
[24]覃兰英,林翔鹰,吕斌.细胞工程选育草莓新品种[J].农业新技术,1992,3：12-16.
[25]NEHRA NS, STUSHNOFF C, KARTHA KK. Direct shoot regeneration from strawberry leaf disks [J]. Journal America HortScience,1989,114 (6):1014-1018.
[26]NEHRA NS, CHIBBAR RN, KARTHA KK. Agrobacterium mediated transformation of strawberry calli and recovery of transgenic plants[J]. Plant Cell Repors,1990,9:10-13.
[27]卢培秀,蔡汉松,王智发,郑继发.AT毒素胁迫体细胞诱导烟草抗赤星病的研究.见：董汉松主编[M].植物诱导抗病性原理和研究.北京：科学出版社,1995：348-407
[28]郑企成.离体诱变育种技术的发展和应用[A].王琳清.诱发突变与作物改良[C].北京：原子能出版社1995,385392.
[29]李周岐.高等植物体细胞突变体离体筛选技术及其在林木扛盐育种中的应用.陕西林业科技,1994,(4)：5055
[30]NEHAR NS, STASHNOFF C, KARRHA K K. Regeneartion of Plant from immature leaf-derived callus of Strawberry(Fragaria anannassa).Plant Cell ReP.1990, 66(1):119-126.
[31]NRHAR NS, STASHNOFF C. Direct shoot regeneration from strawberry leaf disks. J Amer Soc Hort Sci.1989,114(6):1014-1018.
[32]闫华晓,赵辉,崔德才.影响草莓叶片植株再生因素的研究[J].山东农业大学学报,2003,34(2)：177-180.
[33]冯利.草莓‘红颊’快速繁殖及遗传转化体系研究与应用[D].浙江大学,2011.
[34]孙崇波,谢鸣,蒋桂华,张慧琴,黄普乐.草莓主栽品种丰香高效再生体系的研究[J].浙江农业学报.2003,15(2)：69-72.
[35]KATI HANHINEVA, HARRI KOKKO, SIRPA KARENLAMPI1. Shoot regeneration from leaf explants of five strawberry (Fragaria×Ananassa) cultivars in temporary immersion bioreactor system [J]. Society for In Vitro Biology 41:826-831, 2005.
[36]Scala A, Bettini P, Buiatti M, Bogani P, Pellegrini G, TogoniF. In vitro analysis of the tomato-Fusarium oxysporum system and selection experiments. In:Novak F J, Havel L, Dolezel J.(Eds.). Plant tissue and cell culture applications to crop improvement,1985:361～362.
[37]Shahin E A, Spivey R. A single dominant gene for Fusarium wilt resistance in protoplast-derived tomato plants[J]. Theor. Appl. Genet.,1986,73(2):164～169
[38]Cassells A C, Farrell G, Coleoan M C.Somaclonal variation as a source of novel virus resistance in potato character improvement[J].Euphytica,1991,56(l):75～80
[39]Toyoda H, Shioizu K, Chatnai K, Kita N, Matsuda Y, Ouchis. Selection of bacterial wilt-resistant tomato through tissue culture[J].Plant Cell Rep.,1989, 8(6):317～320
[40]Gengenbach B G, Green C E. Selection of T-cytoplasm maize callus cultures resistant to Helminthosporim maydis race T pathotoxin[J]. Crop science,1975, 15(2):645～649
[41]Gengenbach B G, Green C E. Donovan C M. Inheritance of selected pathotoxin resistance in maize plants regenerated from cell cultures. Proc. Natl. Acad. Scl. USA,1997,74(11):5113～5117
[42]Brettell R I S, Thomas E, Ingram D S. Reversion to Texas male-sterile cytoplasm maizeinculture to give fertile, T-Toxin resistant plants[J]. Theor. Appl. Genet.1980, 57(4):55～58
[43]Behnke M. Selection of potato callus for resistance to culture filtrates of Phytophthora infestans and regeneration of resistant plants[J]. Theor. Appl. Genet., 1979,55(2):69～71
[44]Behnke M.General resistance to late blight of Solanum tuberosum plants regenerated from callus resistant to culture filtrates of Phytophthora infestans[J]. Thero. Appl. Genet,1980,56(4):151～152
[45]Behnke M. Selection of dihaploid Potato callus for resistance to culture filtrate of Fusarium oxysporum.Z.Pflanzenzuecht.,1980,85(1):254～58
[46]Sacristan MD. Resistance responses to Phoma lingamof plants regenerated from selected cell and embryogenic cultures of haploid Brassica napus[J]. Theor. Appl. Genet,1982,61(3):193～200
[47]Sacristan MD.Selection for disease resistnace in Brassica cultures[J]. Hereditas., 1985,3(Sup):57～63
[48]MacDonald M V, Ingram D S. In vitro selection for resistance to Alternaria brassicicola in Brassica napus ssp. Oleofera (winter oilseed rape) using partially purified culture filtrates[J]. Curc. Newslett,1985,10(2):97～100
[49]周春江.草莓悬浮细胞培养及原生质体技术研究[D].保定河北农业大学,1999.
[50]李卫东,葛会波,周春江,等.草莓花药愈伤组织类型与状态调控研究[J].河北农业大学学报,2004,27(3)：59-63.
[51]GREENE AE, DAVIS TM. Regeneration of Fragaria vesca Plants from leaf tissue. Edited by Dale A and Luby J J.Portland, Oregon, USA. Timber Perss Inc. 1991,124-126.
[52]王翡,高志红,章镇,乔玉山,王萌,李国平,糜林.草莓高效离体叶片再生体系的建立[J].西北植物学报：2010,30(5)：1045-1049.
[53]祝丽英,池书敏,刘志增,等甲基磺酸乙醋(EMS)在创造玉米新种质中的应用[J].玉米科学,2000,8(1)：19-20.
[54]安学丽,蔡一林,王久光,等化学诱变及其在农作物育种上应用川.核农学报,2003,17(3)：239-242.
[55]赵永亮,宋同明.玉米化学诱变研究进展[J].华北农学报,1996,11(4)：24-28.
[56]祝丽英,池书敏,刘质增等.甲基磺酸乙醋(EMS)在创造玉米新品质中的应用.玉米科学,2001,9(3)：14-17
[57]郭利娟,胡启德,康绍兰等.诱发玉米抗小斑病突变体的研究,从玉米单倍体胚性细胞无性系筛选抗小斑病突变[J].遗传学报,1981,14(5)
[58]薛守旺,周洪生.利用花粉化学诱变剂创造玉米自交系研究[J].作物杂志,1998(6)：6-8、
[59]赵永亮等.利用花粉化学诱变快速创造特用玉米新种质[J].作物学报,1999,25(2)：157-160
[60]杜连恩,于秀普,魏玉昌.EMS处理小麦合子诱变筛选高蛋白突变体[J].河北农业大学学报,1990,13(4)：94-96
[61]王培英,王连铮.EMS诱发大豆脂肪酸组成优良突变的研究[J].核农学报,1939,7(2)：81-87
[62]朱保葛,路子显,耿玉轩等.烷化剂EMS诱发花生性状变异的效果及高产突变系的选育[J].中国农业科学,1997,30(6)：87-89
[63]朴铁夫,李国全.60Co-Y射线和EMS复合处理对大麦遗传效应的研究.核农学通报[J],1993,14(6)：263-266
[64]顾佳清,张智奇,周音等.EMS诱导水稻中花11突变体的筛选和鉴定[J].上海农业学报2005,21(1)：7-11
[65]Joong Ho Lee, Seung yeob Lee.Selection of stable mutants from cultured rice anthers Treated with ethyl methane sulfonic acid[J].Plant Cell, Tissue and Organ Culture 2002,71 (2):165～171
[66]F. Barro, J. Femnadez Escobar等.通过分离抱子的EMS处理诱变获得芥酸含量得到改良的埃塞俄比亚芥的加倍单倍体品系.国外作物育种,2002,21(1)：40-40
[67]Latado, R.R.Adames.Invitro mutation of chrysantheoum with EMS-in immature floral pedicels[J].Plant Cell Tissue&organ Culture.2004,77(4):103～107
f68]刘君绍,田时炳等.茄子抗黄萎病突变体离体筛选[J].西南农业学报,2003,16(4)：102-106
[69]徐德平,徐喜楼,徐惠瑛.葡萄试管化学诱变的研究[J].长江果树,2003(1)：9-11
[70]Mandal A K A, Chakrabraty D, Datta S K.. In vitro isolation of solid novel flower Colour mutants from induced chimeric ray florets of chrysanthemum[J]. Euphytica,2000,114(1):9～12
[71]Das A, Gosal S S, Sidhu J S, et al. Induction of mutations for heat tolerance in Potato by using in vitro culture and radiation[J]. Euphytica,2000,114(3):205～209
[72]Chen Q F, Wang C L, Lu Y M, etal. Anther culture in connection with induced mutations for rice ioprovement[J]. Euphytica,2001,120(3):401～408
[73]Khan A J, Hassan S, Tariq M, et al.Haploidy breeding and mutagenesis for drought tolerance in wheat[J].Euphytica,2001,120(3):409～414
[74]Lin M C, ShangK C, ChenW H. et al. Tissue and cell culture aids to sugarcane breeding. Aneuploid cells and plants induced by treatment of cell suspension cultures with colchicines[J]. Proc. hit. Soc.Sugarcane Technol.1977,16029～4
[75]Broer jes C & Harten A M Van.Applied mutation breeding for vegetatively propagated crops [M]. Elseciar, Amacretdam,1998
[76]Joong Ho Lee, Seung Yeob Lee.Selection of stable unjtants from cultured rice anthers treated with ethyl methane sulfonic acid [J]. Plant Cell, Tissue and Organ Culture 2002,71(2):165～171
[77]张秀省,张荣涛,曹岚等.EMS诱变的长春花细胞系突变研究[J].中草药,2004,35(11)：1293-1296
[78]原亚平,朴铁夫,李国全等.EMS对蕙苡损伤效应的研究[J].吉林农业大学学报,1996,18(1)：18-20
[79]F. Barro, J. Femnadez Escobar等.通过分离孢子的EMS处理诱变获得芥酸含量得到改良的埃塞俄比亚芥的加倍单倍体品系.国外作物育种,2002,21(1)：40-40
[80]覃新程,王飞,王晓娟等.60Co-γ射线与EMS复合处理对山黧豆抗氧化酶活力及ODAP含量的影响[J].应用生态学报.2000,11(6)：957-958
[81]王幼平,徐晓霞,贾勇桐等.甲基磺酸乙酯及60Co-γ对海甘蓝萌发种子的生理效应[J].植物生理学通讯,1998,34(2)：101-103
[82]黄俊生等.EMS诱变菠萝愈伤组织选择抗性突变体的研究[J].热带作物学报,1995,16(12)：1-6
[83]Latado, R.R. Adames. In vitro mutation of chrysantheoum withEMS in immature floral pedicels[J]. Plant Cell Tissue&Organ Culture.2004,77(4):103—107
[84]刘君绍,田时炳等.茄子抗黄萎病突变体离体筛选[J].西南农业学报,2003,16(4)：102-106
[85]KuoashiroT. Selection for tenuazonic acid tolerant cells of tobacco and characteristics of the regenerates[J]. Jpn.J.Breeding,1983,33(Suppl.1):194～195
[86]李绍稳,金青.草莓超巨型突变育种初探[J].安徽农业科学,1996,24(1)：92-96.
[87]梁红艳.用EMS诱变“大五星”批把试管苗和愈伤组织的研究[D].四川农业大学,2004.
[88]沈守江.诱变因素研究利用的进展[A].王琳清.诱发突变与作物改良[C].北京：原子能出版社1995,187-190.
[89]夏英武.作物诱变育种[M].中国农业出版社,1993.
[90]张学月.诱变在作物育种中的应用[J].黑龙江农业科学,1994,(3)：43-44.
[91]王琳清.我国植物诱变育种进展剖析[J].核农学通报,1992,13(6)：282-295.
[92]王密恰.草莓叶片高效再生及细胞组织学观察[D].保定河北农业大学,2002.
[93]吴伟刚,刘桂茹,杨学举.诱变与组织培养相结合在植物育种中的应用[J].中国农学通报,2005,21(11)：197-201.
[94]郑企成.离体诱变育种技术的发展和应用[A].王琳清.诱发突变与作物改良[C].北京：原子能出版社1995,385-392.
[95]祝丽英,池书敏,刘志增,等.甲基磺酸乙醋(EMS)在创造玉米新种质中的应用[J].玉米科学,2000,8(1)：19-20.
[96]祝丽英,池书敏,刘志增,等.甲基磺酸乙醋(EMS)在创造玉米新种质中的应用[J].玉米科学,2000,8(1)：19-20.
[97]安学丽,蔡一林,王久光,等.化学诱变及其在农作物育种上应用[J].核农学报,2003,17(3)：239-242.
[98]Toyoda H, Horikshi K-Selection for Fusariuio wilt disease resistanee from regenerad from leaf callus of Strawberry [J]. Plant Cell Reports,1991(10):1
[99]郑永平,郭宝江,阮继红.茎尖组培与辐照对草莓遗传变异的影响[J].园艺学报,1988,15(4)：245-251
[100]洪燕萍,林顺权.草莓离体培养研究进展[J].福建农业学报,1999,14(2)：37-44
[101]张宪政.作物生理研究法[M].北京：农业出版社,1992.
[102]Heath R L, L Peaker. HotoPeroxidation in isolated ehloroPlasts I. Kineties and stoichiometry of fatted acid Peroeidation [J]. Arch Bioehem BioPhys,1986, 25:89～198.
[103]王宝山.生物自山基与植物膜伤害[J].植物生理学通讯,1988(2)：12-16.
[104]Sore K B. Peroxidase. Plant Mol Boil,1992,16:317～327
[105]张殿忠,汪沛洪,赵会贤.测定小麦叶片游离脯氨酸含量的方法[J].植物生理学通讯,1990(4)：62-65.
[106]汤章城.逆境条件下植物脯氨酸的累积及其可能的意义[J].植物生理学通讯,1984,1：15-21.
[107]闫华晓,赵辉,崔德才.影响草莓叶片植株再生因素的研究[J].山东农业 大学学报,2003,34(2)：177-180
[108]于冬梅,胡文玉,王关林,等.基因型和培养因子对诱导草莓叶片再生芽的影响[J].沈阳农业大学学报,1998，29(2)：138-143
[2]赵密珍,王壮伟,钱亚明,袁骥,吴伟民,王静.草莓新品种‘宁玉’[J].园艺学报,2011,38(7)：1411-1412.
[3]Brooks AN. Anthracnose of strawberry caused by Colletotriehum fragariae, n.sp.[J] Phytopathology,1931,21:739-744
[4]叶正文,郑宏清,童尧明等.上海地区病原炭疽病病原种类及品种抗性研究初报[J].上海农业学报,1997,13(1)：75-80.
[5]Mcinnes TB, BlackLL, Gatt JM..Disease-free plants for management of strawberry anthracnose crown rot[J].Plant disease,1992,76:33,260-264.
[6]DenoyesB, and Baudry A. Species identification and Pathogenieity study of French Colletotrichum strains isolated from strawberry using morphologieal and cultural characteristics[J].Phytopathology,1995,85:53-57.
[7]Bosshard E.Why is Colletotrichum acutarum a quarantine organism, and C.gloeosporioides and C.fragariae are not [J].Acta Hort.(ISHS),1997,439:799-802.
[8]胡淼.草莓病害的初步调查.中国植物病理学会华东区分会第五次年会论文摘要汇编,江苏,1990：58-59
[9]邵小花.上海地区草莓新病害-炭疽病[J].上海农业学报,1992,8：86-87
[10]万瑶,马青,张敬泽.草莓炭疽病菌的鉴定、检测及附着胞形成过程中核相变化的研究[D].西北农林科技大学,2006.179907.
[11]钟霈霖.日本草莓育种及发展战略[J].种子.2005,24(12)：98-100.
[12]谢东,许仁林,魏书.草莓生物技术育种研究进展[J].果树科学,1996,13(3)：195-198,
[13]李卫东,周春江,葛会波.草莓现代生物技术育种研究进展[J].河北农业大学学报,2000,23(1)：32-36.
[14]洪燕萍,林顺权.草莓离体培养研究进展[J].福建农业学报,1999, 14(2)：37-44.
[15]郑永平,郭宝江,阮继红.茎尖组培与辐照对草莓遗传变异的影响[J].园艺学报,1988,15(4)：245-251.
[16]雷家军,吴禄平,代汉萍,等.草莓茎尖染色体加倍研究[J].园艺学报,1999,26(1)：13-18.
[17]赵密珍,王壮伟,钱亚明,袁骥,吴伟民,王静.草莓新品种‘宁玉’[J].园艺学报,2011,38(7)：1411-1412.
[18]施巾帼.理化诱变因素复合处理的诱变效应研究进展[A].王琳清.诱发突变与作物改良[e].北京：原子能出版社1995,271-277.
[19]梁国平,肖三元,管艳.钻60辐射红掌组培材料的实验初报[J].热带农业科技,2005,25(3)：24-26.
[20]陈少裕.膜脂过氧化对植物细胞的毒害.植物生理学通讯,1991(2)：84
[21]秦永华.丰香草莓高效再生的机理及遗传转化研究[D].浙江大学，2005.
[22]吴雪梅,汤浩茹.草莓叶片培养研究进展[J].果树学报,2004,21(6)：598-602.
[23]Toyoda H, Horikoshi K et al. Seleetion for Fusarium wilt disease resistance from regenerants derived from leaf callus of strawberry [J]. Plant Cell Reports,1991, 10:167～170.
[24]覃兰英,林翔鹰,吕斌.细胞工程选育草莓新品种[J].农业新技术,1992,3：12-16.
[25]NEHRA NS, STUSHNOFF C, KARTHA KK. Direct shoot regeneration from strawberry leaf disks [J]. Journal America HortScience,1989,114 (6):1014-1018.
[26]NEHRA NS, CHIBBAR RN, KARTHA KK. Agrobacterium mediated transformation of strawberry calli and recovery of transgenic plants[J]. Plant Cell Repors,1990,9:10-13.
[27]卢培秀,蔡汉松,王智发,郑继发.AT毒素胁迫体细胞诱导烟草抗赤星病的研究.见：董汉松主编[M].植物诱导抗病性原理和研究.北京：科学出版社,1995：348-407
[28]郑企成.离体诱变育种技术的发展和应用[A].王琳清.诱发突变与作物改良[C].北京：原子能出版社1995,385392.
[29]李周岐.高等植物体细胞突变体离体筛选技术及其在林木扛盐育种中的应用.陕西林业科技,1994,(4)：5055
[30]NEHAR NS, STASHNOFF C, KARRHA K K. Regeneartion of Plant from immature leaf-derived callus of Strawberry(Fragaria anannassa).Plant Cell ReP.1990, 66(1):119-126.
[31]NRHAR NS, STASHNOFF C. Direct shoot regeneration from strawberry leaf disks. J Amer Soc Hort Sci.1989,114(6):1014-1018.
[32]闫华晓,赵辉,崔德才.影响草莓叶片植株再生因素的研究[J].山东农业大学学报,2003,34(2)：177-180.
[33]冯利.草莓‘红颊’快速繁殖及遗传转化体系研究与应用[D].浙江大学,2011.
[34]孙崇波,谢鸣,蒋桂华,张慧琴,黄普乐.草莓主栽品种丰香高效再生体系的研究[J].浙江农业学报.2003,15(2)：69-72.
[35]KATI HANHINEVA, HARRI KOKKO, SIRPA KARENLAMPI1. Shoot regeneration from leaf explants of five strawberry (Fragaria×Ananassa) cultivars in temporary immersion bioreactor system [J]. Society for In Vitro Biology 41:826-831, 2005.
[36]Scala A, Bettini P, Buiatti M, Bogani P, Pellegrini G, TogoniF. In vitro analysis of the tomato-Fusarium oxysporum system and selection experiments. In:Novak F J, Havel L, Dolezel J.(Eds.). Plant tissue and cell culture applications to crop improvement,1985:361～362.
[37]Shahin E A, Spivey R. A single dominant gene for Fusarium wilt resistance in protoplast-derived tomato plants[J]. Theor. Appl. Genet.,1986,73(2):164～169
[38]Cassells A C, Farrell G, Coleoan M C.Somaclonal variation as a source of novel virus resistance in potato character improvement[J].Euphytica,1991,56(l):75～80
[39]Toyoda H, Shioizu K, Chatnai K, Kita N, Matsuda Y, Ouchis. Selection of bacterial wilt-resistant tomato through tissue culture[J].Plant Cell Rep.,1989, 8(6):317～320
[40]Gengenbach B G, Green C E. Selection of T-cytoplasm maize callus cultures resistant to Helminthosporim maydis race T pathotoxin[J]. Crop science,1975, 15(2):645～649
[41]Gengenbach B G, Green C E. Donovan C M. Inheritance of selected pathotoxin resistance in maize plants regenerated from cell cultures. Proc. Natl. Acad. Scl. USA,1997,74(11):5113～5117
[42]Brettell R I S, Thomas E, Ingram D S. Reversion to Texas male-sterile cytoplasm maizeinculture to give fertile, T-Toxin resistant plants[J]. Theor. Appl. Genet.1980, 57(4):55～58
[43]Behnke M. Selection of potato callus for resistance to culture filtrates of Phytophthora infestans and regeneration of resistant plants[J]. Theor. Appl. Genet., 1979,55(2):69～71
[44]Behnke M.General resistance to late blight of Solanum tuberosum plants regenerated from callus resistant to culture filtrates of Phytophthora infestans[J]. Thero. Appl. Genet,1980,56(4):151～152
[45]Behnke M. Selection of dihaploid Potato callus for resistance to culture filtrate of Fusarium oxysporum.Z.Pflanzenzuecht.,1980,85(1):254～58
[46]Sacristan MD. Resistance responses to Phoma lingamof plants regenerated from selected cell and embryogenic cultures of haploid Brassica napus[J]. Theor. Appl. Genet,1982,61(3):193～200
[47]Sacristan MD.Selection for disease resistnace in Brassica cultures[J]. Hereditas., 1985,3(Sup):57～63
[48]MacDonald M V, Ingram D S. In vitro selection for resistance to Alternaria brassicicola in Brassica napus ssp. Oleofera (winter oilseed rape) using partially purified culture filtrates[J]. Curc. Newslett,1985,10(2):97～100
[49]周春江.草莓悬浮细胞培养及原生质体技术研究[D].保定河北农业大学,1999.
[50]李卫东,葛会波,周春江,等.草莓花药愈伤组织类型与状态调控研究[J].河北农业大学学报,2004,27(3)：59-63.
[51]GREENE AE, DAVIS TM. Regeneration of Fragaria vesca Plants from leaf tissue. Edited by Dale A and Luby J J.Portland, Oregon, USA. Timber Perss Inc. 1991,124-126.
[52]王翡,高志红,章镇,乔玉山,王萌,李国平,糜林.草莓高效离体叶片再生体系的建立[J].西北植物学报：2010,30(5)：1045-1049.
[53]祝丽英,池书敏,刘志增,等甲基磺酸乙醋(EMS)在创造玉米新种质中的应用[J].玉米科学,2000,8(1)：19-20.
[54]安学丽,蔡一林,王久光,等化学诱变及其在农作物育种上应用川.核农学报,2003,17(3)：239-242.
[55]赵永亮,宋同明.玉米化学诱变研究进展[J].华北农学报,1996,11(4)：24-28.
[56]祝丽英,池书敏,刘质增等.甲基磺酸乙醋(EMS)在创造玉米新品质中的应用.玉米科学,2001,9(3)：14-17
[57]郭利娟,胡启德,康绍兰等.诱发玉米抗小斑病突变体的研究,从玉米单倍体胚性细胞无性系筛选抗小斑病突变[J].遗传学报,1981,14(5)
[58]薛守旺,周洪生.利用花粉化学诱变剂创造玉米自交系研究[J].作物杂志,1998(6)：6-8、
[59]赵永亮等.利用花粉化学诱变快速创造特用玉米新种质[J].作物学报,1999,25(2)：157-160
[60]杜连恩,于秀普,魏玉昌.EMS处理小麦合子诱变筛选高蛋白突变体[J].河北农业大学学报,1990,13(4)：94-96
[61]王培英,王连铮.EMS诱发大豆脂肪酸组成优良突变的研究[J].核农学报,1939,7(2)：81-87
[62]朱保葛,路子显,耿玉轩等.烷化剂EMS诱发花生性状变异的效果及高产突变系的选育[J].中国农业科学,1997,30(6)：87-89
[63]朴铁夫,李国全.60Co-Y射线和EMS复合处理对大麦遗传效应的研究.核农学通报[J],1993,14(6)：263-266
[64]顾佳清,张智奇,周音等.EMS诱导水稻中花11突变体的筛选和鉴定[J].上海农业学报2005,21(1)：7-11
[65]Joong Ho Lee, Seung yeob Lee.Selection of stable mutants from cultured rice anthers Treated with ethyl methane sulfonic acid[J].Plant Cell, Tissue and Organ Culture 2002,71 (2):165～171
[66]F. Barro, J. Femnadez Escobar等.通过分离抱子的EMS处理诱变获得芥酸含量得到改良的埃塞俄比亚芥的加倍单倍体品系.国外作物育种,2002,21(1)：40-40
[67]Latado, R.R.Adames.Invitro mutation of chrysantheoum with EMS-in immature floral pedicels[J].Plant Cell Tissue&organ Culture.2004,77(4):103～107
f68]刘君绍,田时炳等.茄子抗黄萎病突变体离体筛选[J].西南农业学报,2003,16(4)：102-106
[69]徐德平,徐喜楼,徐惠瑛.葡萄试管化学诱变的研究[J].长江果树,2003(1)：9-11
[70]Mandal A K A, Chakrabraty D, Datta S K.. In vitro isolation of solid novel flower Colour mutants from induced chimeric ray florets of chrysanthemum[J]. Euphytica,2000,114(1):9～12
[71]Das A, Gosal S S, Sidhu J S, et al. Induction of mutations for heat tolerance in Potato by using in vitro culture and radiation[J]. Euphytica,2000,114(3):205～209
[72]Chen Q F, Wang C L, Lu Y M, etal. Anther culture in connection with induced mutations for rice ioprovement[J]. Euphytica,2001,120(3):401～408
[73]Khan A J, Hassan S, Tariq M, et al.Haploidy breeding and mutagenesis for drought tolerance in wheat[J].Euphytica,2001,120(3):409～414
[74]Lin M C, ShangK C, ChenW H. et al. Tissue and cell culture aids to sugarcane breeding. Aneuploid cells and plants induced by treatment of cell suspension cultures with colchicines[J]. Proc. hit. Soc.Sugarcane Technol.1977,16029～4
[75]Broer jes C & Harten A M Van.Applied mutation breeding for vegetatively propagated crops [M]. Elseciar, Amacretdam,1998
[76]Joong Ho Lee, Seung Yeob Lee.Selection of stable unjtants from cultured rice anthers treated with ethyl methane sulfonic acid [J]. Plant Cell, Tissue and Organ Culture 2002,71(2):165～171
[77]张秀省,张荣涛,曹岚等.EMS诱变的长春花细胞系突变研究[J].中草药,2004,35(11)：1293-1296
[78]原亚平,朴铁夫,李国全等.EMS对蕙苡损伤效应的研究[J].吉林农业大学学报,1996,18(1)：18-20
[79]F. Barro, J. Femnadez Escobar等.通过分离孢子的EMS处理诱变获得芥酸含量得到改良的埃塞俄比亚芥的加倍单倍体品系.国外作物育种,2002,21(1)：40-40
[80]覃新程,王飞,王晓娟等.60Co-γ射线与EMS复合处理对山黧豆抗氧化酶活力及ODAP含量的影响[J].应用生态学报.2000,11(6)：957-958
[81]王幼平,徐晓霞,贾勇桐等.甲基磺酸乙酯及60Co-γ对海甘蓝萌发种子的生理效应[J].植物生理学通讯,1998,34(2)：101-103
[82]黄俊生等.EMS诱变菠萝愈伤组织选择抗性突变体的研究[J].热带作物学报,1995,16(12)：1-6
[83]Latado, R.R. Adames. In vitro mutation of chrysantheoum withEMS in immature floral pedicels[J]. Plant Cell Tissue&Organ Culture.2004,77(4):103—107
[84]刘君绍,田时炳等.茄子抗黄萎病突变体离体筛选[J].西南农业学报,2003,16(4)：102-106
[85]KuoashiroT. Selection for tenuazonic acid tolerant cells of tobacco and characteristics of the regenerates[J]. Jpn.J.Breeding,1983,33(Suppl.1):194～195
[86]李绍稳,金青.草莓超巨型突变育种初探[J].安徽农业科学,1996,24(1)：92-96.
[87]梁红艳.用EMS诱变“大五星”批把试管苗和愈伤组织的研究[D].四川农业大学,2004.
[88]沈守江.诱变因素研究利用的进展[A].王琳清.诱发突变与作物改良[C].北京：原子能出版社1995,187-190.
[89]夏英武.作物诱变育种[M].中国农业出版社,1993.
[90]张学月.诱变在作物育种中的应用[J].黑龙江农业科学,1994,(3)：43-44.
[91]王琳清.我国植物诱变育种进展剖析[J].核农学通报,1992,13(6)：282-295.
[92]王密恰.草莓叶片高效再生及细胞组织学观察[D].保定河北农业大学,2002.
[93]吴伟刚,刘桂茹,杨学举.诱变与组织培养相结合在植物育种中的应用[J].中国农学通报,2005,21(11)：197-201.
[94]郑企成.离体诱变育种技术的发展和应用[A].王琳清.诱发突变与作物改良[C].北京：原子能出版社1995,385-392.
[95]祝丽英,池书敏,刘志增,等.甲基磺酸乙醋(EMS)在创造玉米新种质中的应用[J].玉米科学,2000,8(1)：19-20.
[96]祝丽英,池书敏,刘志增,等.甲基磺酸乙醋(EMS)在创造玉米新种质中的应用[J].玉米科学,2000,8(1)：19-20.
[97]安学丽,蔡一林,王久光,等.化学诱变及其在农作物育种上应用[J].核农学报,2003,17(3)：239-242.
[98]Toyoda H, Horikshi K-Selection for Fusariuio wilt disease resistanee from regenerad from leaf callus of Strawberry [J]. Plant Cell Reports,1991(10):1
[99]郑永平,郭宝江,阮继红.茎尖组培与辐照对草莓遗传变异的影响[J].园艺学报,1988,15(4)：245-251
[100]洪燕萍,林顺权.草莓离体培养研究进展[J].福建农业学报,1999,14(2)：37-44
[101]张宪政.作物生理研究法[M].北京：农业出版社,1992.
[102]Heath R L, L Peaker. HotoPeroxidation in isolated ehloroPlasts I. Kineties and stoichiometry of fatted acid Peroeidation [J]. Arch Bioehem BioPhys,1986, 25:89～198.
[103]王宝山.生物自山基与植物膜伤害[J].植物生理学通讯,1988(2)：12-16.
[104]Sore K B. Peroxidase. Plant Mol Boil,1992,16:317～327
[105]张殿忠,汪沛洪,赵会贤.测定小麦叶片游离脯氨酸含量的方法[J].植物生理学通讯,1990(4)：62-65.
[106]汤章城.逆境条件下植物脯氨酸的累积及其可能的意义[J].植物生理学通讯,1984,1：15-21.
[107]闫华晓,赵辉,崔德才.影响草莓叶片植株再生因素的研究[J].山东农业 大学学报,2003,34(2)：177-180
[108]于冬梅,胡文玉,王关林,等.基因型和培养因子对诱导草莓叶片再生芽的影响[J].沈阳农业大学学报,1998，29(2)：138-143