匍匐翦股颖离体再生体系的建立及玻璃化超低温保存技术研究
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摘要
匍匐翦股颖(Agrostis stolonifera L.)是一种很重要的多年生冷地型草坪草,具有耐修剪、观赏性高等优点,常用于高尔夫球场果岭及高档草坪中。本研究探究了诱导匍匐翦股颖愈伤组织的最佳条件,建立了高频再生体系。并对匍匐翦股颖愈伤组织进行了玻璃化超低温保存,为匍匐翦股颖细胞工程和基因工程奠定了理论和实践基础,同时对匍匐翦股颖属种质资源长期保存提供了依据。主要研究结果如下:
1.建立了匍匐翦剪股颖再生体系:分别以A-4、A-1、帕特三个品种成熟种子为外植体进行愈伤组织诱导,其中品种A-4的愈伤组织诱导率最高,可达74.7%;2,4-D是影响愈伤组织诱导的最重要激素,以4 mg/L为宜,愈伤组织诱导率为73.2%;在MS+4mg/L2,4-D+0.3mg/L6-BA+蔗糖30g/L+琼脂7g/L中可显著提高愈伤组织诱导率,愈伤组织诱导率可达78.6%;继代培养条件:MS+2mg/L2,4-D+0.1mg/L6-BA +0.3mg/LABA+ 500 mg/L水解酪蛋白中对愈伤组织进行继代培养,胚性愈伤组织发生率可达36.8%;芽分化:在基本培养基中添加2mg/L 6-BA,芽的分化率在80%以上;根的诱导:在1/2MS培养基中添加0.5 mg/LNAA;
2.探索了玻璃化超低温保存体系:将继代两次的愈伤组织在4℃低温度下预培养5天,常温下用装载液(2mol/l甘油+0.4 mol/l蔗糖)过渡10min,再于0℃下用玻璃化溶液PVS2处理50min,换成新鲜的PVS2迅速投入液氮中保存一小时以上,取出后在30℃水浴中化冻,用MS+1.0mol/l蔗糖溶液洗涤3次,每次10min,细胞存活率可达85.6%。
3.超低温前后愈伤组织,过氧化物酶同工酶酶谱没有发生变化,染色体数目没有改变。该研究结果为匍匐翦股颖属植物种质资源的长期保存提供了基础数据。
Agrostis stolonifera L., a significant-grown cool season turf garss, with beautiful colour and the adventage of cut-resistance,Commonly used in the golf courses of the green and High-grade lawn. This study explores the best conditions of induction callus and establish high efficiency regeneration system. The procedure for cryopreservation by vitrification was preliminarily developed in Creeping bentgrass calli.It provide theoretical and practical basis for cell project and gene project of Creeping bentgrass. At the same time, it provided the basis for Long-term preservation of Agrostis stolonifera L. germplasm resources. The results showed that:
1. The regeneration system of Creeping Bentgrass have been estabilished. With A- 4、A-1and pate for the three varieties of explant on callus induction, A-4 have the highest rate of callus induction; 2,4-D is the the most important hormone affecting callus induction, It has shown that 4 mg/L 2,4-D was optimal for callus induction. It can significantly improve the induction rate, which show that MS medium appended with 4mg/L2,4-D , 0.3mg/L 6-BA, 30g/L sucrose and 7g/L agar can give highest rate of callus induction; Callus subculture: MS medium supplemented with 2,4-D2.0 mg/L, 6-BA0.1 mg/L,ABA 0.3mg/Land CH 500mg/L is the most effective subculture medium. The best differentiation medium is MS+6-BA2.0 mg/L,the inductivity can reach 80%.;Roots induction: The suggested medium is 1/2MS+0.5 mg/LNAA
2. The procedure for cryopreservation by vitrification:After subcultured twice, the callus was pre-cultured five days at 4℃,and then the excised calli were loaded with 2 mol/l glycerol and 0.4 mol/l sucrose.then the calli were exposed to PVS2 for 50 min at the zero temperature. after changing the solution with fresh PVS2, the calli were put into the liquid nitrogen at least one hour. the calli were thawed rapidly in a 30℃water bath. then they were washed with the MS medium with 1.0 mol/lsucrose for 3 times and each 10 minutes. The survival of cells can reached 85.6%.
3. Before and afte cryopreservation calli,The Peroxidase isozyme zymogram and chromosomes is not changed. The result provided a feasible proof of conservation Agrostis stolonifera L..through cryopreservation of in vitro shoot-tips by vitrification for long term
1.建立了匍匐翦剪股颖再生体系:分别以A-4、A-1、帕特三个品种成熟种子为外植体进行愈伤组织诱导,其中品种A-4的愈伤组织诱导率最高,可达74.7%;2,4-D是影响愈伤组织诱导的最重要激素,以4 mg/L为宜,愈伤组织诱导率为73.2%;在MS+4mg/L2,4-D+0.3mg/L6-BA+蔗糖30g/L+琼脂7g/L中可显著提高愈伤组织诱导率,愈伤组织诱导率可达78.6%;继代培养条件:MS+2mg/L2,4-D+0.1mg/L6-BA +0.3mg/LABA+ 500 mg/L水解酪蛋白中对愈伤组织进行继代培养,胚性愈伤组织发生率可达36.8%;芽分化:在基本培养基中添加2mg/L 6-BA,芽的分化率在80%以上;根的诱导:在1/2MS培养基中添加0.5 mg/LNAA;
2.探索了玻璃化超低温保存体系:将继代两次的愈伤组织在4℃低温度下预培养5天,常温下用装载液(2mol/l甘油+0.4 mol/l蔗糖)过渡10min,再于0℃下用玻璃化溶液PVS2处理50min,换成新鲜的PVS2迅速投入液氮中保存一小时以上,取出后在30℃水浴中化冻,用MS+1.0mol/l蔗糖溶液洗涤3次,每次10min,细胞存活率可达85.6%。
3.超低温前后愈伤组织,过氧化物酶同工酶酶谱没有发生变化,染色体数目没有改变。该研究结果为匍匐翦股颖属植物种质资源的长期保存提供了基础数据。
Agrostis stolonifera L., a significant-grown cool season turf garss, with beautiful colour and the adventage of cut-resistance,Commonly used in the golf courses of the green and High-grade lawn. This study explores the best conditions of induction callus and establish high efficiency regeneration system. The procedure for cryopreservation by vitrification was preliminarily developed in Creeping bentgrass calli.It provide theoretical and practical basis for cell project and gene project of Creeping bentgrass. At the same time, it provided the basis for Long-term preservation of Agrostis stolonifera L. germplasm resources. The results showed that:
1. The regeneration system of Creeping Bentgrass have been estabilished. With A- 4、A-1and pate for the three varieties of explant on callus induction, A-4 have the highest rate of callus induction; 2,4-D is the the most important hormone affecting callus induction, It has shown that 4 mg/L 2,4-D was optimal for callus induction. It can significantly improve the induction rate, which show that MS medium appended with 4mg/L2,4-D , 0.3mg/L 6-BA, 30g/L sucrose and 7g/L agar can give highest rate of callus induction; Callus subculture: MS medium supplemented with 2,4-D2.0 mg/L, 6-BA0.1 mg/L,ABA 0.3mg/Land CH 500mg/L is the most effective subculture medium. The best differentiation medium is MS+6-BA2.0 mg/L,the inductivity can reach 80%.;Roots induction: The suggested medium is 1/2MS+0.5 mg/LNAA
2. The procedure for cryopreservation by vitrification:After subcultured twice, the callus was pre-cultured five days at 4℃,and then the excised calli were loaded with 2 mol/l glycerol and 0.4 mol/l sucrose.then the calli were exposed to PVS2 for 50 min at the zero temperature. after changing the solution with fresh PVS2, the calli were put into the liquid nitrogen at least one hour. the calli were thawed rapidly in a 30℃water bath. then they were washed with the MS medium with 1.0 mol/lsucrose for 3 times and each 10 minutes. The survival of cells can reached 85.6%.
3. Before and afte cryopreservation calli,The Peroxidase isozyme zymogram and chromosomes is not changed. The result provided a feasible proof of conservation Agrostis stolonifera L..through cryopreservation of in vitro shoot-tips by vitrification for long term
引文
[1]韩烈保,杨碚,邓菊芬.草坪全景:草坪草种及其品种[M].中国林业出版社, 1999, 112-118.
[2]李波.植物组织培养基本技术[M].哈尔滨:哈尔滨地图出版社,2002:1-4,11-21
[3]周维燕.植物细胞工程原理与技术[M].北京:中国农业出版社,2001:1-8,23-39.
[4]李浚明,朱登云.植物组织培养教程[M].第三版.北京:中国农业大学出版社,2005:1-64.
[5] Brent Tisserat and Carl E.Vandercook,Development of an automated Plant culture system[J].Plant Cell,Tissue and organ Culure.1985.1,5(2):107-117.German:Springer Veriag.
[6]朱至清.植物细胞工程[M].北京:化学工业出版社,2003:l-3.
[7]郭勇,崔堂兵,谢秀祯.植物细胞培养技术与应用附[M].北京:化学工业出版社,2003:7-14.
[8]吴桂容.植物组织培养的应用及存在问题[J].高教论坛.加2007.6,(3):119-121.南宁市:广西高等教育学会.
[9]王家麟.植物组织培养及其应用研究概况[J].黑龙江农业科学.2006,(3):86-89.黑龙江省哈尔滨市:黑龙江省农业科学院.
[10]周俊颜,植物体细胞在组织培养中的产生的胚体:植物体细胞的胚状体发生[J].植物生理学报, 1981, 7(4): 389-397.
[11]胡彦,赵艳.植物组织培养技术的应用以及在培养过程中存在的问题[J].陕西师范大学学报(自然科学版).2004.6,32(专辑):130-134.陕西省西安市:陕西师范大学.
[12]覃拥灵.植物组织培养技术及其应用[J].河池学院学报.2003.12,23(4):98-100.
[13]韩磊.汪旭东.昊先军等.植物组织培养技术及其应用研究进展[J].种子.2005.1,24(l):38-39.
[14]包英华.植物组织培养技术的应用[J].韶关学院学报.2002.12,23(l2):26-29.
[15]张红晓,经剑颖,张贯臣.组织培养技术在植物育种中的应用IJ].洛阳大学学报.2003.12, 18(4):43
[16]刘庆昌,吴国良.植物细胞组织培养[M].北京:中国农业大学出版社,2003:1-9,21-45.
[17]王秀丽.杨湿.徐平丽等.植物组织培养的应用及进展闭[J].山东农业科学.2005,(3):78-80.
[18]葛胜娟.植物组织培养中的快繁与脱毒技术及其应用切[J].中国农学通报.2005.5,21(5):104-107.
[19]李浚明,朱登云.植物组织培养教程[M].第三版.北京:中国农业大学出版社,2005:1-14.
[20]陈文武,彭兰华.药用植物的组织培养的应用[J].陕西农业科学.2006,(5):62-65.
[21] Tacicek R A,etal. Plant Cell Tissue Organ Culture, 1991(24):139-158.
[22]富桂荣,李勤贤,胡宗双.植物组织培养技术在农业生产中的应用[J].农业科技与信息.2007,(5):22-23.
[23]王雅茜,石轶松,阐建全.植物细胞和组织培养技术在食品添加剂生产上的应用及前景[J].食品工业科技.1999,20(3):66-67.
[24]王文静,袁道强,高松洁.植物组织培养的应用现状[J].河南师范大学学报(自然科学版).2000.8,28(3):137-139.
[25]孙敬三,陈维伦.植物生物技术和作物改良[M].北京.中国科学技术出版社,1990:55-59.
[26]伊华林.我国经济植物组织培养概况[ J].湖北农学院学报,2001, 21(3): 279-284.
[27]李永欣,王义强.植物组织培养的应用研究概述明.[J]江苏林业科技.2005.6,32(3):44-46.
[28] Rall WF et al. Nature, 1985, 313: 573.
[29] Helliot B et al. Plant Cell Rep, 2002, 20: 11-17
[30] Tumer S R,Senaratna T,Bunn E,et al. Cryopreservation of shoot tips from six endangered Australian species using a modified vitrification Protocol[J]. Annals of Botany.2001,87:371-378.
[31]刘峰,颜秋生.水稻胚性悬浮细胞玻璃化冻存中的超微结构变化[J1.中国水稻科学,1998,12(l):17-20.
[32]曾继吾,易干军,张秋明,等.番木瓜茎尖超低温保存过程中的细胞超微结构[J].园艺学报,2005,32(l):15-19.
[33]吴黎明,曾继吾,彭抒昂,等.香蕉茎尖超低温保存过程中的细胞超微结构观察[J].分子细胞生物学,2006,39(6):563-567.
[34]卢新雄,陈晓玲.我国作物种质资源保存与研究进展.[J]中国农业科学,2003,36,(10): 1125~1132.
[35]刘燕,高荣孚,周晓阳.玻璃化超低温保存中拟南芥幼苗细胞膜ATPase的变化[J].北京林业大学学报,2001,23(5):1-5.
[36]王君辉,黄纯安.玻璃化法一园艺作物茎尖和分生组织超低温保存的新途径[J].园艺学报,1994,21(3):277-282.
[37]刘云国,王晓云.果树种质资源超低温保存研究进展.[J]生命科学研究,2001,5 3 : 227～232.
[38]间令成,吴素萱.植物抗寒性的细胞学研究—小麦越冬过程中的细胞结构形态的变化[J].植物学报,1965,(13):1-15.
[39] Withers L A.Long-term preservation of plant cells tissues and organs.[J]Oxford Surveys of Plant Molecular and Cell Biology,1987,4:221-272.
[40]刘贤旺,杜勤,罗光明等.杜仲愈伤组织超低温保存的研究.[J]生物学杂志,1996,4:21~24
[41] Cornejo MJ,Wong VL,Blechl AE.Cryopreserved callus:A source of protoplasts for rice transformation.Plant Cell Rep,1995,14(2):210~214.
[42]严庆丰,黄纯农.植物组织和细胞的玻璃化冻存研究.[J]细胞生物学杂志.1994,16(3):117-122.
[43] Pence V C.Desiccation and survival of Aesculus,Castanea and Quercus embryo axes through Cryopreservation[J].Cryobiology.1992,29:391-399.
[44] TAHTAMOUNI R W,SHIBLI R A. Preservation at low temperature and cryopreservation in wild pear( Pyrus syriaca) [J]. Adv Hortic Sci,1999(13): 156-160.
[45] Engelmann F,Danbire D,Ollitrault P.Cryopreservation of embryogenic cell suspensions and calluses of citrus using a simplified freezing process[J].Cryo-Letters,1994,l5(1):53-58.
[46] Steponkus P L,Langis R,Fujikawa S. Cryoprservation of plant tissue by vitrifieation.In: Advances Low- temperature Biology,edited by Steponkus P.L JAI Press, Ltd.London 1992,l:l-6
[47] Niino T,Tashiro K,Suzuki M et al.Cryopreservation of in vitro grown shoot tips of cherry and sweet cherry by one-step vitrification.Sci Hortic,1997,70:155~163.
[48] Shibli RA,Haagenson DM,Cunningham SM et al.Cryopreservation of alfalfa(Medicago sativa L.)cells by encapsulation-dehydration[J].Plant Cell Rep,2001,20:445-450.
[49] Uragami A,Sakai A,Nagai M et al,Survival of cultured cells and somatic embryos of Asparagus officinalis L.cryopreserved by vitrification,Plant cell Reports,1989,8:418-421.
[50] Finkle B J,Ulrieh J M,Cryoprotectants removal temperature as a factor in the survival of frozen rice and sugarcane cells[J].Cryobiology,1982,19:329-335.
[51] Li P H,Sakai A,Plant cold Hardiness and Freezing stress-Mechanisms and Crop Implieations,New York,San Francisco,london:Academic Press,1978:373-390.
[52]马锋旺,李嘉瑞.杏愈伤组织超低温保存的研究.[J]西北植物学报,1999,19(1):67~70.
[53]陈书安,王晓东,赵兵等.水母雪莲愈伤组织超低温保存条件的初探.[J]过程工程学报,2002,2(6):539~543.
[54] Lambardi M,Fabbri A..Caccavale A. Cryopreservation of white Poplar (Populusalba L.) by vitrification of invitro grown shoot tips[J].Plant Cell reports.2000,19:213-218.
[55]梁宏,王起华.植物种质的玻璃化超低温保存[J].细胞生物学杂志,2005,27: 43～45.
[56] Takagi H,Thinh N.T.,Islam O.M.,Senboku T.,Sakai A..Cryopreservation of in vitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure. Plant Cell Rep. 1997,16:594-599.
[57] Matsumoto T,Takahashi C,Sakai A,et al. Cryopreservation of invitro-grown apical meristems of hybrid statice by three different procedures.Scientia Horticture,1998,76:105-114.
[58]王子成,邓秀新.玻璃化法超低温保存柑桔茎尖及植株再生[J].园艺学报,2001,28(4):301-306.
[59] Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. Var. brasiliensis Tanaka) by vitrification. Plant Cell Rep. 9: 30-33
[60]陈品良.植物组织培养物的超低温保存[J].武汉植物研究,1989,7(4):390-398.
[61] Vandenbussche B, Weyens M, De Proft M.Cryopreservation of in vitro sugar beet (Beta vulgaris L)shoot tips by a vitrification technique[J].Plant Cell Reporters,2000,19: 1064～1068
[62] Touchell D H,Chiang V L, Tsai C J.Cryopreservation of embryogenic cultures of Picea mariana black spruce using vitrification[J].Plant cell Reporter,2002,21: 118～124.
[63] Ryynanen Leena.Aronen Tuija.Vitrification, a complementary cryopreservation method for Betula pendula Roth[J].Cryobiology,2005,51:208～219.
[64] Niino T, Tanaka D, Tantely R R, et al.cryopreservation of basal stembuds of in vitro -grownmat rush (Juncus spp.)by vitrification .CryoLetters,2007,28: 197～206.
[65]葛锋,王晓东,赵兵等.肉苁蓉愈伤组织的超低温保藏方法[J].过程工程学报,2006, 5 : 794～798.
[66] Towill L E.Cryopreservation of isolated mint shoot tips by vitrification [J].Plant Cell Reports.1990,9:178-1180.
[67] Yamada T, Sakai A, Matsumura T, Higuchi S Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification[J]. Plant Sci. 1991,78: 81-87.
[68] Reuff et al.Cryopreservation of Coleus blumei suspension and callus cultures[J].Plant physiol,1988,133:414～418.
[69] Kohmura H, Ikeda Y, Sakai A Cryopreservation of apical meristems of Japanese shallot (Allium wakegi A.) by vitrification and subsequent high plant regeneration[J]. Cryo Lett. 1994,15: 289-298.
[70]陈勇,陈娴婷,王君晖.区瓦柑愈伤组织的玻璃化法超低温保存研究[J].浙江大学学报(理学版),2004,31(2):197-201.
[71] Watanabe K and Steponkus P L.Vitrification of Oryza sativa L.cell suspensions[J],Cryo-letters.1995,16(5):255-262.
[72]间令成,孙德兰,孙龙华.甘蔗愈伤组织超低温保存中一些因素的研究[J]植物学报,1987,29(2):123-131.
[73]苗琦,谷运红,王卫东,等.植物组织培养物的超低温保存[J].植物生理学通讯,2005,41(3):350-354.
[74]艾鹏飞,卢利平.桔梗试管苗茎尖玻璃化法超低温保存及植株再生[J]中草药,2006, 37(9): 1409~1412.
[75]艾鹏飞,罗正荣.柿和君迁子试管苗茎尖玻璃化法超低温保存及再生植株遗传稳定性研究[J]中国农业科学,2004,37(12):2023-2027.
[76]刘云国.苹果种质资源玻璃化法超低温保存技术及其遗传变异分析[D].山东农业大学,博士论文.
[77]简令成.细胞组织培养物的超低温保存种质库的建立.植物生物技术和作物改良[M].中国科学技术出版社,1990,254-280.
[78] Moukadiri O,Deming J,O,Connor J E et al.Phenotypic characterization of the progenies of rice plants derived from cryopreserved calli[J].Plant Cell Rep,1999,18:625-632.
[79] Butenko R G,Popov A S,Volkova L A et al.Recovery of cell cultures and their biosynthetic capacity after storage of Dioscorea deltoidea and Panax ginseng in liquid nitrogen.Plant Sci Lett,1984,33:285-292..
[80] Withers L A.Long-term preservation of plant cells,tissues and organs[J].Oxford Surveys of Plant Molecular and Cell Biology,1987,4:221-272.
[81] Marin M I,Durnn-Vila N.Conservation of citrus germplasm in vitro[J].JAmet.Soc.Hort.Sci, 1991,116(4):740-746.
[82]郝玉金.柑桔和苹果等果树种质资源的立体保存及其遗传变异[D].武汉:华中农业大学园艺林学学院,2000
[83] Chaudhury R, ehandel K.Cryopreservation of embryonic axes of almond(Prumus amygcllus Batsch)seeds[J].Cryo-letlers,1995,16(1):51-56.
[84]刘月学,王家福,林顺权.超低温保存技术在果树种质资源中的应用[J].福建果树,2001,(3):25-27.
[85]袁立勇,李绍清,李阳生,冯双华,戴长柏,李达模.水稻悬浮细胞系的建立[J].云南大学学报,2003(4):373-376.
[86]付永彩,吴茂森,成卓敏.小麦不同品种的外植体的农杆菌转化方法的研究[J].农业生物技术学报,2002,20(1):25-28.
[87]周维燕.植物细胞工程原理与技术[M].北京:中国农业出版社,2001:1-8,23-39.
[88]林忠平,胡莺雷,朱先灿等.草坪草生物技术及应用[M].北京,化学工业出版社,2006:l-7,47-76.
[89]曹孜义.实用植物组织培养技术教程[M] .兰州:甘肃科学技术出版社,2001. 20～37.
[90]王忠.植物生理学[M] .北京:中国农业出版社,1999.323～328.
[91]王傲雪.李景富.转基因番茄的机理及现状[J].辽宁农业科学, 1998, 6: 33-36.
[92]郭光沁,夏光敏,李忠谊,等.小麦原生质体来源的体细胞胚形成和植株再生[J].中国科学编辑, 1990,9: 970~974.
[93]王海波,李向辉,孙勇如,等.小麦原生质体培养—细胞高频诱导和植株再生[J].中国科学编辑,1989,8: 828~836.
[94] Isserat B T,Murashige T,Effeets of ethePhon ethylene,and 2,4-D dichloro Phenoxyaeetic acid on asexual embryogenesis in vitro .Plant Physiol,1977,60:437-439.
[95] Bhaskaren S and Smith R H.Regeneration in cereal tissue culture:a review[J].Crop Sci, 1990 (30): 1328～1336.
[96]支月娥,何亚面,田龚.高羊茅组织培养研究初报[J].上海农学院学报,1998,16(1): 46-48.
[97]马忠华,张云芳,徐传详等.早熟禾的组织培养和基因枪介导的基因转化体系的初步建立[J].复旦学报(自然科学版), 1999, 38 (3): 540-543.
[98]潘瑞炽.植物组织培养[M].广州,广东高等教育出版社,2000,20-59.
[99] Nissen P. Stimulation of somatic embryogenesis in carrot by ethylene:effect of Spinach roots. J of the American Society for Hdrticultural[J]. Sciene ,2000,125(1):21-24.
[100]陈智勇,易自力,赵运林.匍匐翦股颖愈伤组织诱导及其分化的研究[J].园艺学报,2003.3(30):562-564.
[101] BAIY,QUR.Factors in fluencing tissue culture Responses of mature seeds and immature embryos in turf-type tall fescue[J].Plant Breeding,2001,1(20):239～42.
[102]于晓红,朱祯,付志明等.提高小麦愈伤组织分化频率的因素[J].植物生理学报,1999,25(4):388-394.
[103] Griffin J D,et al.High-frequency plant regeneration from seed-derived callus cultures of Kentuckybluegrass(Poa pratensis L.)[J].Plant Cell Reports,1995,14:721-724
[104] Chandrhury A,et al.Somatic embryogenesis and plant regeneration of turf-type bermugarass:effect of 6-benzyladenine in callus induction medium[J].Plant Cell tissueOrgan Culture,2000,60(2):113-120.
[105] Bai Y,et al.Factors influencing tissue culture responses of mature seeds and immature embryos in turf–type tall fescue[J].Plant Breeding,2001,120:239-242.
[106] Becher T, Haberland G,Koop H U. Callus formation and plant regeneration in standard and microexplants from seedlings of barley(Hordeum vulgare L) [J]. Plant cell Reports, 1992,11:39~43.
[107]米庆莉,何锶洁,曹守云,等.籼稻及粳稻对基因枪转化反应的差异及提高转化频率的研究[J].农业生物技术学报,1998,6(2):263~268.
[108] Cai, T. and Butler,L. Plant regeneration from embryogenesis callus initiated from immature inflorescences of several high-tannin sorghums[J].Plant Cell,Tissue and Organ Culture,1990,(20):101-110.
[109] Mcdonnel R E and Conger B V.Callus induction and plantlet formation from mature embryo explants of Kentucky bluegrass[J].Crop Sci,1984(24):573～578.
[110]余叔文.植物生理与分子生物学[M].北京:科学出版社,1992.37～52.
[111]罗士韦,唐惕.植物组织和细胞的超低温保存及种质库建立的研究现况[J].细胞生物学杂志,1983,5(1):3～9.
[112] Towill,L.E.and P.Mazur,1975:Studies on the reduction of 2,3,5- tirphenyl tetrazoliun chloride as a viability assay for plant tissue cultures.can.J.Bot,53:1097～1102.
[113]陈瑞阳,宋文芹,李秀兰等.植物有丝分裂染色体标本制作的新方法[J].植物学报,1979,21:297-298.
[114]刘贤旺,杜琴,罗光明等.杜仲愈伤组织超低温保存的研究[J].生物学杂志,1996(4):21～24.
[115] TAHTAMOUNI R W,SHIBLI R A.Preservation at low temperature and cryopreservation in wild pear(Pyrus syriaca) [J] Adv Hortic Sci, 1999(13 ): 156～160.
[116] TAKAGI H,TIENT N,ISLAM 0 M,et al. Cryopreservation of in vitri-grown shoot-tips of taro (Colocasia esculenta L. Schott) by vitrification[J]. Plant Cell Rep,1997(16):594～599.
[117]刘云国,王晓云.果树种质资源超低温保存研究进展[J].生命科学研究,2001,5(3):227-232.
[118] Withers L A,Davey M R.Protoplasma,1978,94:207～291.
[119] Sakai A,Yoshida S.Plant physiol,1967,42:1965～1701.
[120] Withers L A.In“Cryopreservation of plant cells and organs”, K.K.Kartha,eds. Cryopreservatio of cultured plant cells and protoplasts. Florida.CRC press inc.1985.243～267.
[121] Saklai A, Kobayashi S. Cryopreservation of Nuclear Cell of Navel Orange by Vitrification [J]. Plant Cell Report, 1990, 9(1):30-33.
[122] Brison M,de Boucaud M T,Pieronnet A,et al,Effect of cryopreservation On the sanitary state of A cv. Prunes rootstock experimentally Contaminated with Plum Pox Pot virus[J].Plant sci,1997,123:189-196. 55
[123] Niino T,Tashiro K,Suzuki M.Cryopreservation of in vitro grown shoot tips of cherry and sweet cherry by one-step verifications[J].Sci Hortic,1997.70:155-163.
[124]梁宏,王起华植物种质的玻璃化超低温保存[J].细胞生物学杂志,2005,27: 43～45.
[125]洪森荣,李明军.玻璃化法超低温保存怀山药种质的技术研究.中草药,2006,37(11) : 1715～1718.
[126] Ishikawa K,Harata R lvIii M,Sakai A,Yoshimatsu R Shim-omura K.Cryopreservation of Zygotic embryos of a Japariese terreslrial orchid (B1etilla striata)by vitrification[J].Plant Cell Reports,1997,16:754～757.
[127]周小梅,王国英,敖光明,赵云云.玉米组培材料的玻璃化法超低温保存及冻后植株再生[J]农业生物技术学报,2001,9(4):355～35.
[128]李嘉瑞,郭延平,王民柱.猕猴桃愈伤组织的超低温保存[J].果树科学,1996,13(2):88-91.
[129] Towill L E,Mazur P.Plant Physiol.1976.57:290～296.
[130] LANGIS R L,STEPONKUS P. Cryopreservation of rye protoplasts by Vitrification[J]. Plant Physiol,1990(92):666-67.
[131]陈辉,陈晓玲,陈龙清,卢欣熊.切花百合离体茎尖玻璃化法超低温保存研究[J].植物遗传资源学报,2007,8(2):170-173.
[2]李波.植物组织培养基本技术[M].哈尔滨:哈尔滨地图出版社,2002:1-4,11-21
[3]周维燕.植物细胞工程原理与技术[M].北京:中国农业出版社,2001:1-8,23-39.
[4]李浚明,朱登云.植物组织培养教程[M].第三版.北京:中国农业大学出版社,2005:1-64.
[5] Brent Tisserat and Carl E.Vandercook,Development of an automated Plant culture system[J].Plant Cell,Tissue and organ Culure.1985.1,5(2):107-117.German:Springer Veriag.
[6]朱至清.植物细胞工程[M].北京:化学工业出版社,2003:l-3.
[7]郭勇,崔堂兵,谢秀祯.植物细胞培养技术与应用附[M].北京:化学工业出版社,2003:7-14.
[8]吴桂容.植物组织培养的应用及存在问题[J].高教论坛.加2007.6,(3):119-121.南宁市:广西高等教育学会.
[9]王家麟.植物组织培养及其应用研究概况[J].黑龙江农业科学.2006,(3):86-89.黑龙江省哈尔滨市:黑龙江省农业科学院.
[10]周俊颜,植物体细胞在组织培养中的产生的胚体:植物体细胞的胚状体发生[J].植物生理学报, 1981, 7(4): 389-397.
[11]胡彦,赵艳.植物组织培养技术的应用以及在培养过程中存在的问题[J].陕西师范大学学报(自然科学版).2004.6,32(专辑):130-134.陕西省西安市:陕西师范大学.
[12]覃拥灵.植物组织培养技术及其应用[J].河池学院学报.2003.12,23(4):98-100.
[13]韩磊.汪旭东.昊先军等.植物组织培养技术及其应用研究进展[J].种子.2005.1,24(l):38-39.
[14]包英华.植物组织培养技术的应用[J].韶关学院学报.2002.12,23(l2):26-29.
[15]张红晓,经剑颖,张贯臣.组织培养技术在植物育种中的应用IJ].洛阳大学学报.2003.12, 18(4):43
[16]刘庆昌,吴国良.植物细胞组织培养[M].北京:中国农业大学出版社,2003:1-9,21-45.
[17]王秀丽.杨湿.徐平丽等.植物组织培养的应用及进展闭[J].山东农业科学.2005,(3):78-80.
[18]葛胜娟.植物组织培养中的快繁与脱毒技术及其应用切[J].中国农学通报.2005.5,21(5):104-107.
[19]李浚明,朱登云.植物组织培养教程[M].第三版.北京:中国农业大学出版社,2005:1-14.
[20]陈文武,彭兰华.药用植物的组织培养的应用[J].陕西农业科学.2006,(5):62-65.
[21] Tacicek R A,etal. Plant Cell Tissue Organ Culture, 1991(24):139-158.
[22]富桂荣,李勤贤,胡宗双.植物组织培养技术在农业生产中的应用[J].农业科技与信息.2007,(5):22-23.
[23]王雅茜,石轶松,阐建全.植物细胞和组织培养技术在食品添加剂生产上的应用及前景[J].食品工业科技.1999,20(3):66-67.
[24]王文静,袁道强,高松洁.植物组织培养的应用现状[J].河南师范大学学报(自然科学版).2000.8,28(3):137-139.
[25]孙敬三,陈维伦.植物生物技术和作物改良[M].北京.中国科学技术出版社,1990:55-59.
[26]伊华林.我国经济植物组织培养概况[ J].湖北农学院学报,2001, 21(3): 279-284.
[27]李永欣,王义强.植物组织培养的应用研究概述明.[J]江苏林业科技.2005.6,32(3):44-46.
[28] Rall WF et al. Nature, 1985, 313: 573.
[29] Helliot B et al. Plant Cell Rep, 2002, 20: 11-17
[30] Tumer S R,Senaratna T,Bunn E,et al. Cryopreservation of shoot tips from six endangered Australian species using a modified vitrification Protocol[J]. Annals of Botany.2001,87:371-378.
[31]刘峰,颜秋生.水稻胚性悬浮细胞玻璃化冻存中的超微结构变化[J1.中国水稻科学,1998,12(l):17-20.
[32]曾继吾,易干军,张秋明,等.番木瓜茎尖超低温保存过程中的细胞超微结构[J].园艺学报,2005,32(l):15-19.
[33]吴黎明,曾继吾,彭抒昂,等.香蕉茎尖超低温保存过程中的细胞超微结构观察[J].分子细胞生物学,2006,39(6):563-567.
[34]卢新雄,陈晓玲.我国作物种质资源保存与研究进展.[J]中国农业科学,2003,36,(10): 1125~1132.
[35]刘燕,高荣孚,周晓阳.玻璃化超低温保存中拟南芥幼苗细胞膜ATPase的变化[J].北京林业大学学报,2001,23(5):1-5.
[36]王君辉,黄纯安.玻璃化法一园艺作物茎尖和分生组织超低温保存的新途径[J].园艺学报,1994,21(3):277-282.
[37]刘云国,王晓云.果树种质资源超低温保存研究进展.[J]生命科学研究,2001,5 3 : 227～232.
[38]间令成,吴素萱.植物抗寒性的细胞学研究—小麦越冬过程中的细胞结构形态的变化[J].植物学报,1965,(13):1-15.
[39] Withers L A.Long-term preservation of plant cells tissues and organs.[J]Oxford Surveys of Plant Molecular and Cell Biology,1987,4:221-272.
[40]刘贤旺,杜勤,罗光明等.杜仲愈伤组织超低温保存的研究.[J]生物学杂志,1996,4:21~24
[41] Cornejo MJ,Wong VL,Blechl AE.Cryopreserved callus:A source of protoplasts for rice transformation.Plant Cell Rep,1995,14(2):210~214.
[42]严庆丰,黄纯农.植物组织和细胞的玻璃化冻存研究.[J]细胞生物学杂志.1994,16(3):117-122.
[43] Pence V C.Desiccation and survival of Aesculus,Castanea and Quercus embryo axes through Cryopreservation[J].Cryobiology.1992,29:391-399.
[44] TAHTAMOUNI R W,SHIBLI R A. Preservation at low temperature and cryopreservation in wild pear( Pyrus syriaca) [J]. Adv Hortic Sci,1999(13): 156-160.
[45] Engelmann F,Danbire D,Ollitrault P.Cryopreservation of embryogenic cell suspensions and calluses of citrus using a simplified freezing process[J].Cryo-Letters,1994,l5(1):53-58.
[46] Steponkus P L,Langis R,Fujikawa S. Cryoprservation of plant tissue by vitrifieation.In: Advances Low- temperature Biology,edited by Steponkus P.L JAI Press, Ltd.London 1992,l:l-6
[47] Niino T,Tashiro K,Suzuki M et al.Cryopreservation of in vitro grown shoot tips of cherry and sweet cherry by one-step vitrification.Sci Hortic,1997,70:155~163.
[48] Shibli RA,Haagenson DM,Cunningham SM et al.Cryopreservation of alfalfa(Medicago sativa L.)cells by encapsulation-dehydration[J].Plant Cell Rep,2001,20:445-450.
[49] Uragami A,Sakai A,Nagai M et al,Survival of cultured cells and somatic embryos of Asparagus officinalis L.cryopreserved by vitrification,Plant cell Reports,1989,8:418-421.
[50] Finkle B J,Ulrieh J M,Cryoprotectants removal temperature as a factor in the survival of frozen rice and sugarcane cells[J].Cryobiology,1982,19:329-335.
[51] Li P H,Sakai A,Plant cold Hardiness and Freezing stress-Mechanisms and Crop Implieations,New York,San Francisco,london:Academic Press,1978:373-390.
[52]马锋旺,李嘉瑞.杏愈伤组织超低温保存的研究.[J]西北植物学报,1999,19(1):67~70.
[53]陈书安,王晓东,赵兵等.水母雪莲愈伤组织超低温保存条件的初探.[J]过程工程学报,2002,2(6):539~543.
[54] Lambardi M,Fabbri A..Caccavale A. Cryopreservation of white Poplar (Populusalba L.) by vitrification of invitro grown shoot tips[J].Plant Cell reports.2000,19:213-218.
[55]梁宏,王起华.植物种质的玻璃化超低温保存[J].细胞生物学杂志,2005,27: 43～45.
[56] Takagi H,Thinh N.T.,Islam O.M.,Senboku T.,Sakai A..Cryopreservation of in vitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure. Plant Cell Rep. 1997,16:594-599.
[57] Matsumoto T,Takahashi C,Sakai A,et al. Cryopreservation of invitro-grown apical meristems of hybrid statice by three different procedures.Scientia Horticture,1998,76:105-114.
[58]王子成,邓秀新.玻璃化法超低温保存柑桔茎尖及植株再生[J].园艺学报,2001,28(4):301-306.
[59] Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. Var. brasiliensis Tanaka) by vitrification. Plant Cell Rep. 9: 30-33
[60]陈品良.植物组织培养物的超低温保存[J].武汉植物研究,1989,7(4):390-398.
[61] Vandenbussche B, Weyens M, De Proft M.Cryopreservation of in vitro sugar beet (Beta vulgaris L)shoot tips by a vitrification technique[J].Plant Cell Reporters,2000,19: 1064～1068
[62] Touchell D H,Chiang V L, Tsai C J.Cryopreservation of embryogenic cultures of Picea mariana black spruce using vitrification[J].Plant cell Reporter,2002,21: 118～124.
[63] Ryynanen Leena.Aronen Tuija.Vitrification, a complementary cryopreservation method for Betula pendula Roth[J].Cryobiology,2005,51:208～219.
[64] Niino T, Tanaka D, Tantely R R, et al.cryopreservation of basal stembuds of in vitro -grownmat rush (Juncus spp.)by vitrification .CryoLetters,2007,28: 197～206.
[65]葛锋,王晓东,赵兵等.肉苁蓉愈伤组织的超低温保藏方法[J].过程工程学报,2006, 5 : 794～798.
[66] Towill L E.Cryopreservation of isolated mint shoot tips by vitrification [J].Plant Cell Reports.1990,9:178-1180.
[67] Yamada T, Sakai A, Matsumura T, Higuchi S Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification[J]. Plant Sci. 1991,78: 81-87.
[68] Reuff et al.Cryopreservation of Coleus blumei suspension and callus cultures[J].Plant physiol,1988,133:414～418.
[69] Kohmura H, Ikeda Y, Sakai A Cryopreservation of apical meristems of Japanese shallot (Allium wakegi A.) by vitrification and subsequent high plant regeneration[J]. Cryo Lett. 1994,15: 289-298.
[70]陈勇,陈娴婷,王君晖.区瓦柑愈伤组织的玻璃化法超低温保存研究[J].浙江大学学报(理学版),2004,31(2):197-201.
[71] Watanabe K and Steponkus P L.Vitrification of Oryza sativa L.cell suspensions[J],Cryo-letters.1995,16(5):255-262.
[72]间令成,孙德兰,孙龙华.甘蔗愈伤组织超低温保存中一些因素的研究[J]植物学报,1987,29(2):123-131.
[73]苗琦,谷运红,王卫东,等.植物组织培养物的超低温保存[J].植物生理学通讯,2005,41(3):350-354.
[74]艾鹏飞,卢利平.桔梗试管苗茎尖玻璃化法超低温保存及植株再生[J]中草药,2006, 37(9): 1409~1412.
[75]艾鹏飞,罗正荣.柿和君迁子试管苗茎尖玻璃化法超低温保存及再生植株遗传稳定性研究[J]中国农业科学,2004,37(12):2023-2027.
[76]刘云国.苹果种质资源玻璃化法超低温保存技术及其遗传变异分析[D].山东农业大学,博士论文.
[77]简令成.细胞组织培养物的超低温保存种质库的建立.植物生物技术和作物改良[M].中国科学技术出版社,1990,254-280.
[78] Moukadiri O,Deming J,O,Connor J E et al.Phenotypic characterization of the progenies of rice plants derived from cryopreserved calli[J].Plant Cell Rep,1999,18:625-632.
[79] Butenko R G,Popov A S,Volkova L A et al.Recovery of cell cultures and their biosynthetic capacity after storage of Dioscorea deltoidea and Panax ginseng in liquid nitrogen.Plant Sci Lett,1984,33:285-292..
[80] Withers L A.Long-term preservation of plant cells,tissues and organs[J].Oxford Surveys of Plant Molecular and Cell Biology,1987,4:221-272.
[81] Marin M I,Durnn-Vila N.Conservation of citrus germplasm in vitro[J].JAmet.Soc.Hort.Sci, 1991,116(4):740-746.
[82]郝玉金.柑桔和苹果等果树种质资源的立体保存及其遗传变异[D].武汉:华中农业大学园艺林学学院,2000
[83] Chaudhury R, ehandel K.Cryopreservation of embryonic axes of almond(Prumus amygcllus Batsch)seeds[J].Cryo-letlers,1995,16(1):51-56.
[84]刘月学,王家福,林顺权.超低温保存技术在果树种质资源中的应用[J].福建果树,2001,(3):25-27.
[85]袁立勇,李绍清,李阳生,冯双华,戴长柏,李达模.水稻悬浮细胞系的建立[J].云南大学学报,2003(4):373-376.
[86]付永彩,吴茂森,成卓敏.小麦不同品种的外植体的农杆菌转化方法的研究[J].农业生物技术学报,2002,20(1):25-28.
[87]周维燕.植物细胞工程原理与技术[M].北京:中国农业出版社,2001:1-8,23-39.
[88]林忠平,胡莺雷,朱先灿等.草坪草生物技术及应用[M].北京,化学工业出版社,2006:l-7,47-76.
[89]曹孜义.实用植物组织培养技术教程[M] .兰州:甘肃科学技术出版社,2001. 20～37.
[90]王忠.植物生理学[M] .北京:中国农业出版社,1999.323～328.
[91]王傲雪.李景富.转基因番茄的机理及现状[J].辽宁农业科学, 1998, 6: 33-36.
[92]郭光沁,夏光敏,李忠谊,等.小麦原生质体来源的体细胞胚形成和植株再生[J].中国科学编辑, 1990,9: 970~974.
[93]王海波,李向辉,孙勇如,等.小麦原生质体培养—细胞高频诱导和植株再生[J].中国科学编辑,1989,8: 828~836.
[94] Isserat B T,Murashige T,Effeets of ethePhon ethylene,and 2,4-D dichloro Phenoxyaeetic acid on asexual embryogenesis in vitro .Plant Physiol,1977,60:437-439.
[95] Bhaskaren S and Smith R H.Regeneration in cereal tissue culture:a review[J].Crop Sci, 1990 (30): 1328～1336.
[96]支月娥,何亚面,田龚.高羊茅组织培养研究初报[J].上海农学院学报,1998,16(1): 46-48.
[97]马忠华,张云芳,徐传详等.早熟禾的组织培养和基因枪介导的基因转化体系的初步建立[J].复旦学报(自然科学版), 1999, 38 (3): 540-543.
[98]潘瑞炽.植物组织培养[M].广州,广东高等教育出版社,2000,20-59.
[99] Nissen P. Stimulation of somatic embryogenesis in carrot by ethylene:effect of Spinach roots. J of the American Society for Hdrticultural[J]. Sciene ,2000,125(1):21-24.
[100]陈智勇,易自力,赵运林.匍匐翦股颖愈伤组织诱导及其分化的研究[J].园艺学报,2003.3(30):562-564.
[101] BAIY,QUR.Factors in fluencing tissue culture Responses of mature seeds and immature embryos in turf-type tall fescue[J].Plant Breeding,2001,1(20):239～42.
[102]于晓红,朱祯,付志明等.提高小麦愈伤组织分化频率的因素[J].植物生理学报,1999,25(4):388-394.
[103] Griffin J D,et al.High-frequency plant regeneration from seed-derived callus cultures of Kentuckybluegrass(Poa pratensis L.)[J].Plant Cell Reports,1995,14:721-724
[104] Chandrhury A,et al.Somatic embryogenesis and plant regeneration of turf-type bermugarass:effect of 6-benzyladenine in callus induction medium[J].Plant Cell tissueOrgan Culture,2000,60(2):113-120.
[105] Bai Y,et al.Factors influencing tissue culture responses of mature seeds and immature embryos in turf–type tall fescue[J].Plant Breeding,2001,120:239-242.
[106] Becher T, Haberland G,Koop H U. Callus formation and plant regeneration in standard and microexplants from seedlings of barley(Hordeum vulgare L) [J]. Plant cell Reports, 1992,11:39~43.
[107]米庆莉,何锶洁,曹守云,等.籼稻及粳稻对基因枪转化反应的差异及提高转化频率的研究[J].农业生物技术学报,1998,6(2):263~268.
[108] Cai, T. and Butler,L. Plant regeneration from embryogenesis callus initiated from immature inflorescences of several high-tannin sorghums[J].Plant Cell,Tissue and Organ Culture,1990,(20):101-110.
[109] Mcdonnel R E and Conger B V.Callus induction and plantlet formation from mature embryo explants of Kentucky bluegrass[J].Crop Sci,1984(24):573～578.
[110]余叔文.植物生理与分子生物学[M].北京:科学出版社,1992.37～52.
[111]罗士韦,唐惕.植物组织和细胞的超低温保存及种质库建立的研究现况[J].细胞生物学杂志,1983,5(1):3～9.
[112] Towill,L.E.and P.Mazur,1975:Studies on the reduction of 2,3,5- tirphenyl tetrazoliun chloride as a viability assay for plant tissue cultures.can.J.Bot,53:1097～1102.
[113]陈瑞阳,宋文芹,李秀兰等.植物有丝分裂染色体标本制作的新方法[J].植物学报,1979,21:297-298.
[114]刘贤旺,杜琴,罗光明等.杜仲愈伤组织超低温保存的研究[J].生物学杂志,1996(4):21～24.
[115] TAHTAMOUNI R W,SHIBLI R A.Preservation at low temperature and cryopreservation in wild pear(Pyrus syriaca) [J] Adv Hortic Sci, 1999(13 ): 156～160.
[116] TAKAGI H,TIENT N,ISLAM 0 M,et al. Cryopreservation of in vitri-grown shoot-tips of taro (Colocasia esculenta L. Schott) by vitrification[J]. Plant Cell Rep,1997(16):594～599.
[117]刘云国,王晓云.果树种质资源超低温保存研究进展[J].生命科学研究,2001,5(3):227-232.
[118] Withers L A,Davey M R.Protoplasma,1978,94:207～291.
[119] Sakai A,Yoshida S.Plant physiol,1967,42:1965～1701.
[120] Withers L A.In“Cryopreservation of plant cells and organs”, K.K.Kartha,eds. Cryopreservatio of cultured plant cells and protoplasts. Florida.CRC press inc.1985.243～267.
[121] Saklai A, Kobayashi S. Cryopreservation of Nuclear Cell of Navel Orange by Vitrification [J]. Plant Cell Report, 1990, 9(1):30-33.
[122] Brison M,de Boucaud M T,Pieronnet A,et al,Effect of cryopreservation On the sanitary state of A cv. Prunes rootstock experimentally Contaminated with Plum Pox Pot virus[J].Plant sci,1997,123:189-196. 55
[123] Niino T,Tashiro K,Suzuki M.Cryopreservation of in vitro grown shoot tips of cherry and sweet cherry by one-step verifications[J].Sci Hortic,1997.70:155-163.
[124]梁宏,王起华植物种质的玻璃化超低温保存[J].细胞生物学杂志,2005,27: 43～45.
[125]洪森荣,李明军.玻璃化法超低温保存怀山药种质的技术研究.中草药,2006,37(11) : 1715～1718.
[126] Ishikawa K,Harata R lvIii M,Sakai A,Yoshimatsu R Shim-omura K.Cryopreservation of Zygotic embryos of a Japariese terreslrial orchid (B1etilla striata)by vitrification[J].Plant Cell Reports,1997,16:754～757.
[127]周小梅,王国英,敖光明,赵云云.玉米组培材料的玻璃化法超低温保存及冻后植株再生[J]农业生物技术学报,2001,9(4):355～35.
[128]李嘉瑞,郭延平,王民柱.猕猴桃愈伤组织的超低温保存[J].果树科学,1996,13(2):88-91.
[129] Towill L E,Mazur P.Plant Physiol.1976.57:290～296.
[130] LANGIS R L,STEPONKUS P. Cryopreservation of rye protoplasts by Vitrification[J]. Plant Physiol,1990(92):666-67.
[131]陈辉,陈晓玲,陈龙清,卢欣熊.切花百合离体茎尖玻璃化法超低温保存研究[J].植物遗传资源学报,2007,8(2):170-173.