百合悬浮细胞体系的建立及植株再生
摘要
为了建立良好的百合悬浮细胞培养体系,本试验首先研究了其愈伤组织诱导与再生体系,进而建立了3个基因型的5个悬浮细胞系。试验具体研究方法及取得的结果和结论如下:
(一)愈伤组织诱导与再生体系研究
(1)愈伤组织发生能力的测试:为了选择适宜的基因型和外植体类型,试验选用多个百合基因型的多种外植体(试管鳞茎、田间鳞茎、花器官或种子)为试材,利用激素picloram进行愈伤组织诱导。试验结果表明,愈伤组织发生能力为岷江百合、麝香百合>OT系百合、东方百合、亚洲百合>兰州百合,诱导效果为鳞片>花丝>种子>花柱>花药和子房。但具体到某一基因型或同一基因型的不同外植体其愈伤组织发生能力仍然存在差异。
(2)愈伤组织诱导与再生的研究:选用愈伤组织发生能力较强的岷江百合和麝香百合‘白狐’的试管鳞茎为试材。在愈伤组织诱导过程中,岷江百合研究了picloram浓度的影响,‘白狐’研究了光照、起始材料状态及激素种类(picloram和2,4-D)和浓度的影响;在愈伤组织再生过程中,两个基因型均研究了MS中大量元素含量和蔗糖浓度的影响。试验结果表明,岷江百合在picloram 2.0 mg?L-1条件下诱导效果最佳,诱导率约为80%;‘白狐’在picloram 1.0 mg?L-1条件下诱导效果最佳,诱导率约为100%,光照和起始材料状态对愈伤组织诱导的影响不显著;2个品种的最佳再生培养基均为?MS +蔗糖15 g?L-1。
(3)愈伤组织的再生:将生长良好的愈伤组织接种到培养基?MS+蔗糖15 g?L-1上进行植株再生,试验结果表明,基因型和愈伤组织类型在愈伤组织再生中起着重要作用。在基因型相同的条件下,愈伤组织的再生能力主要与愈伤组织类型有关。胚性愈伤组织的再生能力强,再生率基本为100%,非胚性愈伤组织的再生能力差,再生率偏低。
(二)悬浮细胞系建立与植株再生的研究
在(一)中选用生长良好且可成功再生的愈伤组织接种到培养液MS(NH4NO3含量减半)+2.0mg?L-1 picloram+30g?L-1蔗糖,置于25℃恒温摇床上,100 r·min-1、黑暗条件下进行悬浮培养。悬浮细胞在再生培养基?MS+15 g?L-1蔗糖上进行植株再生。试验结果表明,各悬浮细胞系的状态和再生情况与其起始愈伤组织的状态和再生情况相对应。结构松散的愈伤组织其悬浮细胞系的建立时间短,分散度、均一度和生长速率高,但由于为非胚性愈伤组织,其悬浮细胞系的再生率也偏低;颗粒状、结构紧实的愈伤组织其悬浮细胞系的建立时间长,分散度、均一度和生长速率均低于前者,但由于为胚性愈伤组织,其悬浮细胞系的再生能力强,再生率为100%。
To establish cell suspension cultures of Lilium spp., this research studied the callus induction and regeneration system firstly. Cell suspension cultures were established with the callus which was in good condition and could regenerate successfully. Five cell suspension cultures of three genotypes were established and different cell suspension cultures had different characters and regeneration ability. Specific research methods and the results and conclusions are as follows:
(一) Callus induction and regeneration system
(1) Callus formation examination: Callus formation were examined in 8 Lilium genotypes,Lilium regale wils., Lilium longiflorum 'White fox' and 'Snow Queen' , Lilium Oriental hybrids' Siberia' and 'Bernini', Lilium Oriental×Trumpet 'Manissa', Lilium davidii var. unicolor, Lilium Asiatic Hybrids 'Vermeer'. Seeds, bulb scales, and flower organs were placed on a medium containing 2.0 mg?L-1 picloram. The results showed that callus formation ability is Lilium regale wils., Lilium longiflorum> Lilium Oriental×Trumpet, Lilium Oriental hybrids, Lilium Asiatic Hybrids > Lilium davidii var. unicolor, respectively. Bulb scales and filament explants showed great ability to form calluses, whereas seeds had poor ability.
(2) Callus induction and regeneration: Bulb scales of Lilium regale wils. and Lilium longiflorum 'White fox' were used as explants to study callus induction and regeneration. In the study of callus induction, the effect of picloram concentrations on Lilium regale wils. and the effect of light, initial materials status and hormone types (picloram and 2,4-D) and oncentrations on Lilium longiflorum 'White fox' were analysed . During the regenerating, the effects of sucrose concentrations and mineral element content in MS basic medium on both genotypes were studied. The results showed that the optimum concentration for callus induction of Lilium regale wils. was picloram 2.0mg?L-1, and the induction rate was about 80%; the optimum concentration for callus induction of Lilium longiflorum 'White fox' was picloram 1.0mg?L-1, and the induction rate was about 100%; the optimum regenerating medium of both genotypes was 1/2MS supplemented with sucrose 15 g?L-1.
(3) Callus regeneration: Callus in good condition which were obtained from (1) were inoculated on solid 1/2MS medium with 15 g?L-1 sucrose. Genotypes and types of callus had a significant effect on callus regeneration. The genotypes mainly affected the patterns of regeneration and the types of callus had an effect on regeneration capacity. The regenerating rate of embryogenic callus with high regeneration ability was 100%. The non-embryonic callus with low regeneration ability showed lower regenerating rate.
(二) Establishment of cell suspension culture and plantlet regeneration: Callus in good condition obtained from (1) and regenerated in (3) were cultured in modified liquid MS medium (NH4NO3 with half-strength) supplemented with picloram 2.0mg?L-1 and sucrose 30g?L-1 , and shook at 100 rpm under dark condition at 25℃. Culture density were 2g(f.w.) per 40mL liquid medium, and cell suspension cultures were subcultured every 1~2w. Suspension cells regenerated plantlets on solid 1/2MS medium with 15 g?L-1 sucrose. The results showed that the state and regeneration ability of cell suspension cultures were related with the initial callus. Stably growing cell suspension cultures were established with loose callus in 10 to 20 days, which had high single-cells ,but low regenerating rate. The cell suspension cultures composed of cell groups,diameter 1~5mm and high regenerating rate,were established by the granular callus.
(一)愈伤组织诱导与再生体系研究
(1)愈伤组织发生能力的测试:为了选择适宜的基因型和外植体类型,试验选用多个百合基因型的多种外植体(试管鳞茎、田间鳞茎、花器官或种子)为试材,利用激素picloram进行愈伤组织诱导。试验结果表明,愈伤组织发生能力为岷江百合、麝香百合>OT系百合、东方百合、亚洲百合>兰州百合,诱导效果为鳞片>花丝>种子>花柱>花药和子房。但具体到某一基因型或同一基因型的不同外植体其愈伤组织发生能力仍然存在差异。
(2)愈伤组织诱导与再生的研究:选用愈伤组织发生能力较强的岷江百合和麝香百合‘白狐’的试管鳞茎为试材。在愈伤组织诱导过程中,岷江百合研究了picloram浓度的影响,‘白狐’研究了光照、起始材料状态及激素种类(picloram和2,4-D)和浓度的影响;在愈伤组织再生过程中,两个基因型均研究了MS中大量元素含量和蔗糖浓度的影响。试验结果表明,岷江百合在picloram 2.0 mg?L-1条件下诱导效果最佳,诱导率约为80%;‘白狐’在picloram 1.0 mg?L-1条件下诱导效果最佳,诱导率约为100%,光照和起始材料状态对愈伤组织诱导的影响不显著;2个品种的最佳再生培养基均为?MS +蔗糖15 g?L-1。
(3)愈伤组织的再生:将生长良好的愈伤组织接种到培养基?MS+蔗糖15 g?L-1上进行植株再生,试验结果表明,基因型和愈伤组织类型在愈伤组织再生中起着重要作用。在基因型相同的条件下,愈伤组织的再生能力主要与愈伤组织类型有关。胚性愈伤组织的再生能力强,再生率基本为100%,非胚性愈伤组织的再生能力差,再生率偏低。
(二)悬浮细胞系建立与植株再生的研究
在(一)中选用生长良好且可成功再生的愈伤组织接种到培养液MS(NH4NO3含量减半)+2.0mg?L-1 picloram+30g?L-1蔗糖,置于25℃恒温摇床上,100 r·min-1、黑暗条件下进行悬浮培养。悬浮细胞在再生培养基?MS+15 g?L-1蔗糖上进行植株再生。试验结果表明,各悬浮细胞系的状态和再生情况与其起始愈伤组织的状态和再生情况相对应。结构松散的愈伤组织其悬浮细胞系的建立时间短,分散度、均一度和生长速率高,但由于为非胚性愈伤组织,其悬浮细胞系的再生率也偏低;颗粒状、结构紧实的愈伤组织其悬浮细胞系的建立时间长,分散度、均一度和生长速率均低于前者,但由于为胚性愈伤组织,其悬浮细胞系的再生能力强,再生率为100%。
To establish cell suspension cultures of Lilium spp., this research studied the callus induction and regeneration system firstly. Cell suspension cultures were established with the callus which was in good condition and could regenerate successfully. Five cell suspension cultures of three genotypes were established and different cell suspension cultures had different characters and regeneration ability. Specific research methods and the results and conclusions are as follows:
(一) Callus induction and regeneration system
(1) Callus formation examination: Callus formation were examined in 8 Lilium genotypes,Lilium regale wils., Lilium longiflorum 'White fox' and 'Snow Queen' , Lilium Oriental hybrids' Siberia' and 'Bernini', Lilium Oriental×Trumpet 'Manissa', Lilium davidii var. unicolor, Lilium Asiatic Hybrids 'Vermeer'. Seeds, bulb scales, and flower organs were placed on a medium containing 2.0 mg?L-1 picloram. The results showed that callus formation ability is Lilium regale wils., Lilium longiflorum> Lilium Oriental×Trumpet, Lilium Oriental hybrids, Lilium Asiatic Hybrids > Lilium davidii var. unicolor, respectively. Bulb scales and filament explants showed great ability to form calluses, whereas seeds had poor ability.
(2) Callus induction and regeneration: Bulb scales of Lilium regale wils. and Lilium longiflorum 'White fox' were used as explants to study callus induction and regeneration. In the study of callus induction, the effect of picloram concentrations on Lilium regale wils. and the effect of light, initial materials status and hormone types (picloram and 2,4-D) and oncentrations on Lilium longiflorum 'White fox' were analysed . During the regenerating, the effects of sucrose concentrations and mineral element content in MS basic medium on both genotypes were studied. The results showed that the optimum concentration for callus induction of Lilium regale wils. was picloram 2.0mg?L-1, and the induction rate was about 80%; the optimum concentration for callus induction of Lilium longiflorum 'White fox' was picloram 1.0mg?L-1, and the induction rate was about 100%; the optimum regenerating medium of both genotypes was 1/2MS supplemented with sucrose 15 g?L-1.
(3) Callus regeneration: Callus in good condition which were obtained from (1) were inoculated on solid 1/2MS medium with 15 g?L-1 sucrose. Genotypes and types of callus had a significant effect on callus regeneration. The genotypes mainly affected the patterns of regeneration and the types of callus had an effect on regeneration capacity. The regenerating rate of embryogenic callus with high regeneration ability was 100%. The non-embryonic callus with low regeneration ability showed lower regenerating rate.
(二) Establishment of cell suspension culture and plantlet regeneration: Callus in good condition obtained from (1) and regenerated in (3) were cultured in modified liquid MS medium (NH4NO3 with half-strength) supplemented with picloram 2.0mg?L-1 and sucrose 30g?L-1 , and shook at 100 rpm under dark condition at 25℃. Culture density were 2g(f.w.) per 40mL liquid medium, and cell suspension cultures were subcultured every 1~2w. Suspension cells regenerated plantlets on solid 1/2MS medium with 15 g?L-1 sucrose. The results showed that the state and regeneration ability of cell suspension cultures were related with the initial callus. Stably growing cell suspension cultures were established with loose callus in 10 to 20 days, which had high single-cells ,but low regenerating rate. The cell suspension cultures composed of cell groups,diameter 1~5mm and high regenerating rate,were established by the granular callus.
引文
1.常立.岷江百合植株再生技术研究.[硕士论文].雅安:四川农业大学,2004.
2.陈发棣,蒋甲福,郭维明,房伟民,赵宏波.小菊悬浮细胞培养与植株再生研究.园艺学报,2006,33(5):1021~1026.
3.陈火英,张建华.番茄下胚轴离体诱导成株的激素调控.上海农业学报,1999,15(2):26~29.
4.陈巍,高文远,贾伟,等.人参属药用植物组织和细胞培养的研究进展.中草药,2005,36(4):616~620.
5.陈伟列.三倍体无籽西瓜快速繁殖.福建农业科技,1984,(3):23~26.
6.崔红,陈亮,沈明山,陈睦传.甘薯胚性悬浮系的建立及其细胞生长特性研究.厦门大学学报(自然科学版),2002,41(5):555-558.
7.董云洲,段胜军.谷子胚性悬浮细胞系植株再生体系的建立及转基因技术研究.应用基础与工程科学学报,1999,第7卷,第1期.
8.董志渊,郑思乡.百合的组织培养及在其育种中的应用.西部林业科学,2004,33(2):64-68.
9.付文奇,杜双田,陈建科,尹华.NAA和2 ,4-D对东方百合组织培养的影响.西北林学院学报,2008,23 (2):83~86.
10.方文娟,韩烈保,曾会明.植物细胞悬浮培养影响因子研究.生物技术通报,2005,5:11-14.
11.高桂珍,周吉源.喜树细胞悬浮培养中生理生化指标的测定.武汉植物学研究,2003,21(3):259-261.
12.高丽华,胡显文,胥照平.喂饲前体提高悬浮培养水母雪莲细胞的黄酮产量.高技术通讯,2007,17(4):418~423.
13.郝立新,王怀名.西瓜再生系统的建立.华北农学报,1995,23(3):112~115.
14.何钢,文亚峰,付杰,谢碧霞,李樊,韩文军,邢伟一.人心果细胞悬浮培养的初步研究.经济林研究,2004,22 (4) :43-46.
15.何勇,田志宏,郑用琏.马蹄金悬浮细胞系的建立与优化.华中农业大学学报,2005,24(4):401-405.
16.侯丙凯,陈际江.小麦不同类型愈伤组织及其悬浮系的原生质体培养和植株再生.植物学通报,1996,13 (2) :52～54.
17.胡博然,徐文彪,马峰旺.枸杞胚细胞悬浮培养系统建立的研究.西北农林科技大学学报(自然科学版),2003,31(1):99-100.
18.胡之璧.生物技术在传统药材中的应用前景.药学服务与研究,2002,2(1):1.
19.黄纯农,王军晖,颜秋生,张雪琴,严庆峰.大麦胚性悬浮细胞系的快速建立及其超低温保存.中国农业科学,1995,28(3):21~27.
20.蒋细旺,司怀军.百合的组织培养技术综述.湖北农业科学,2004,1:78-82.
21.赖钟雄,黄浅,林秀莲,林玉玲,陈义挺,赖呈纯,等.荔枝胚性悬浮细胞系的快速建立及其体胚植株的再生.农业生物技术科学,2007,第23卷,第1期.
22.雷鸣,李向辉,孙勇如.水稻原生质体的植株再生.科学通报,1986,22:1729~1731.
23.李东杰,魏景芳,刘淑清,等.药用植物细胞悬浮培养研究展.河北林业科技,2003,3(8):22~23.
24.李玉平,龚宁,李美玲,马威,毛健峰,张兴. 2,4-D和KT对大花金挖耳愈伤组织诱导的影响.西北农业学报,2006,15(4):147-152.
25.林拥军,邹玉兰,傅世耀.高效水稻细胞悬浮系建立的研究.江西农业大学学报,1995,17(4):410~413.
26.刘坤.岷江百合(Lilium regale Wils)悬浮细胞系的建立及耐盐筛选.[硕士论文].南京:南京林业大学,2008.
27.柳玉晶,龚束芳,樊金萍,王金刚,车代弟.百合愈伤组织的诱导及植株再生.东北农业大学学报,2007,38(3):352~355.
28.陆美莲,许新萍,周厚高,符红霓.均匀正交设计在百合组织培养中的应用.西南农业大学学报(自然科学版),2004,26(6).
29.孟新亚.大蒜胚性细胞悬浮系的建立.[硕士论文].郑州:河南农业大学,2002.
30.莫小路,黄学林.银杏悬浮培养细胞的生长、分化与萜内酯化合物的积累.生物工程学报,2004,20(3):445~449.
31.盛腊红,何光存,舒理慧.抗病资源疵粒野生稻(O.meyriana)悬浮培养与植株再生.华中农业大学学报,1996,22(增刊):82~85.
32.申茹,都晓伟,吴军凯.高人参皂苷产量的生物培养技术研究进展.医药学报,2007,35(4):57~59.
33.孙敬三,桂耀林.植物细胞工程实验技术.北京:科学出版社,1995.
34.孙君社,方晓华.植物激素对百合鳞片愈伤组织生长的影响.中国农业大学学报,2001,6(2):58-61.
35.孙世孟,赛吉灾,谢友菊.玉米获白莱的悬浮细胞系的建立及其再生植株的研究.作物学报,1994,20(2).
36.唐东芹,钱虹妹,黄丹枫,唐克轩.百合基因转化胚性愈伤组织受体系统的建立.浙江林学院学报,2003,20 (3):273~276.
37.王德强,王晓玲.银杏细胞的悬浮培养研究.食品与生物技术学报,2005,24(3):27~29,33.
38.王海波.细胞状态观点的提出及其生命科学意义.科技导报,2008,26(4)41.
39.王杰,刘国锋,包满珠,黄莉.麝香百合胚性愈伤组织状态的调整与植株再生.园艺学报,2008,35(12):1795~1802.
40.王俊丽,陈丕铃,杨庆仙.杜仲细胞的悬浮培养.河北大学学报(自然科学版),1997,17(3):78-80
41.王素芳,王志林,蒋琳兰,赵树进.植物细胞悬浮培养.中国生物制品学杂志,2002,15(6):381~383.
42.王旭英.杂交粳稻3优18胚性悬浮细胞系的建立.聊城大学学报(自然科学版),2005,第18卷,第4期.
43.王亚琴,朱媛.杜仲细胞悬浮培养生产桃叶珊瑚甙的研究.广西植物,2007,27(2):236~239.
44.王友生,李阳春,梁慧敏,刘自学,孙榕江.紫花苜蓿愈伤组织诱导及植株再生的研究.草原与草坪,2006,4:55-57.
45.魏岳荣,黄学林,李佳,黄霞,李哲,李筱菊.贡蕉胚性细胞悬浮系的建立和植株再生.生物工程学报,2005,第21卷,第1期.
46.吴晓玲.植物生长调节剂对银柴胡细胞生长特性的影响.植物研究,2005,25(2):173~176.
47.吴晓玲,邓光存,姜晓慧.黄芩细胞生长特性及次生代谢产物生产性能的研究.西北植物学报.2005,25(3):557~561.
48.吴永宏,张德颐,王丽蓉.植物细胞同步培养技术.植物生理学通讯,1993,29(2):108~110.
49.吴智鹏,冯俊涛,马志卿.除虫菊细胞悬浮体系营养条件优化研究.西北农业学报,2006,15(4):180~82.
50.许智宏,卫志明,刘贵云.用离体培养无性繁殖三倍体无籽西瓜.植物生理学报,1979,5(3):45~251.
51.杨柏云,杨慧琴,蔡奇英,管毕财,罗丽萍.龙牙百合体细胞胚的诱导及植株再生.南昌大学学报(理科版),2005,29(6).
52.杨剑波,吴家道,卫志明.粕型水稻原生质体再生植株.生物工程学报.1992,82(l):60~64.
53.杨远媛,贺窑青,郑彩霞.油松胚珠愈伤组织诱导和悬浮细胞系的建立.植物生理学通讯,2005,
41(5).
54.尹文兵,李伟,杜桂森,黄勤妮.西藏红景天组织培养研究.北方七省市植物学会年会暨学术讨论会,2004.
55.岳彩鹏,李品,黄象南.烟草愈伤组织的诱导及细胞悬浮培养体系的建立.烟草科技,2007,10:56~59.
56.张延龙,徐炎,李峰,罗佳,范铭.秦岭野百合鳞片植株再生体系的建立.西北植物学报,2004,24(7):1315~1318.
57.张艺萍,吴丽芳,吴学尉,崔光芬,丁丁,王继华.东方百合胚性愈伤组织诱导和植株a)再生研究.江西农业学报,2008,20(12):33~36.
58.张云,原雅玲,刘青林.百合品种改良与生物技术研究进展.北京林业大学学,2001,23(6): 56~59.
59.赵海涛.东方百合‘Siberia’试管鳞茎发育及休眠研究.[硕士论文].北京:中国农业科学院,2009.
60.赵鸿莲,于荣敏.诱导子在植物细胞培养中的应用研究进展.沈阳药科大学学报,2000,17(2):152~156.
61.赵珺,宋阳,雷家军.麝香百合子房离体培养的研究.辽宁农业科学,2006,(5):11~13.
62.赵军,徐莺,严钫,唐琳,陈放.红花愈伤组织的诱导与花柱愈伤组织的继代悬浮培养.四川大学学报(自然科学版),2001,38(3):421-424.
63.周春江,李卫东,葛会波.草莓悬浮细胞系的建立及某些影响因素.植物生理学通讯,2002,第38卷,第1期.
64.周晓罡,丁玉梅,孙茂林,陈亮,苏源,张绍松,等. 24个水稻抗稻瘟病单基因鉴别品系悬浮细胞系的建立.西南农业学报,2008,21(3).
65.朱宝成,吴爱民.掌叶半夏细胞悬浮培养及单细胞培养再生植株.作物学报,1996,22(2):197-200.
66.褚云霞,陈龙清,黄燕文,张永春.百合的花药培养研究.园艺学报,2001,28(5):472-474.
67.朱至清.植物细胞工程.北京:化学工业出版社,2003.
68. Aguettaz P.,Paffen A.,Delvallee I.,Van der Linde P.,& De Klerk G. J..The development of dormancy in bulblets of Lilium speciosum generated in vitro I. The effects of culture conditions.Plant Cell Tissue Organ Cult,1990,22:167~172.
69. Arzate-Fernández A. M.,Nakazaki T.,Okumoto Y.,& Tanisaka T. Efficient callus induction and plant regeneration from filaments with anther in lily (Lilium longiflorum Thunb.).Plant Cell Reports,1997,16:836~840.
70. Azevedo H.,Dias A.,& Tavares R. M..Establishment and characterization of Pinus pinaster suspension cell cultures.Plant Cell Tiss Organ Cult,2008,93:115~121.
71. Bui V. L.,Nhut D. T.,& Tran Thanh Van K..Plant production via shoot regeneration from thin cell layer pseudo-bulblet explants of Lilium longiflorum in vitro. C. R. Acad. Sci. Paris, Sciences de la vie/Life Sciences,1999,322:303~310.
72. Chin-wen Ho,Wei-ting Jian,& Hui-chun Lai. Plant regeneration via somatic embryogenesis from suspension cell cultures of Lilium×formolongi hort.using a bioreactor system.In Vitrp Cell.Dev.Biol.-Plant,2006,42:240~246.
73. Dong-Sheng Han,Yoshiji Niimi,& Masaru Nakano.Long term maintenance of an anther-derived haploid callus line of the Asiatic hybrid lily 'Connecticut King'.Plant Cell,Tissue and Organ Culture,2000,61:215~219.
74. Dong-Sheng Han,Yoshiji Niimi,& Masaru Nakano.Regeneration of haploid plants from anther cultures of the Asiatic hybrid lily 'Connecticut King'.Plant Cell,Tissue and Organ Culture,1997,47:153~158.
75. Enaksha R. M. Wickremesinhe,E. Jay Holcomb,& Richard N. Arteca.A pratical method for the production of flowering Easter lilies from callus cultures.Scientia Horticulturae,1994,60:143~152.
76. Famelaer I.,Bordas M.,Baliu E.,Ennik E.,Meijer H.,& Van Tuyl J. M. et al..The use of morphogenic suspension cultures for the development of a protoplast regeneration system in lily.Acta Hort,430,ISHS 1997.
77. Famelaer I.,Ennik E.,Van Tuyl J. M.,Meijer H.,& Creemers-Molenaar C..The establishment of suspension and meristem cultures for the development of a protoplast regeneration and fusion system in lily,Acta Hort,430,ISHS 1996.
78. Godo T.,Kobayashi K.,Tagami T.,Matsui K.,& Kida T..In vitro propagation utilizing suspension cultures of meristematic nodular cell clumps and chromosome stability of Lilium×formolongi hort.,Scientia Horticulturae,1998,72:193~202.
79. Godo T.,Matsui K.,Kida T.,& Mii M..Effect of sugar type on the efficiency of plant regeneration from protoplasts isolated from shoot tip-derived meristematic nodular cell clumps of Lilium×formolongi hort.Plant Cell Rep.,1996,15:401~404.
80. Godo T.,& Mii M..Regeneration of plants from protoplasts of Lilium×formolongi.In:Bajaj YPS(ed)Biotechnology in agriculture and forestry.Springer,Berlin Heidelberg New York,1996,38,79~89.
81. Gur'eva A. S.,Kintya P. K.,Mashchenko N. E.,& Shashkov A. S..Glycosides of the bulbs of Liliumregale.Chemistry of Natural Compounds,1996,Vol.32,No.3.
82. Horita M.,Morohashi H.,& Komai F..Regeneration of flowering plants from difficile lily protoplasts by means of a nurse culture.Planta,2002,215:880~884.
83. Horita M.,Morohashi H.,& Komai F..Production of fertile somatic hybrid plants between Oriental hybrid lily and Lilium×formolongi.Planta,2003,217:597~601.
84. Li L. Y.,Chen K..Towards a high and reproducible frequency of plant regeneration from protoplasts of riee(Oryzn sativn L).Genetic Manipulation in Crops Newsletter,1987,3:l~15.
85. Loretta Bacchetta,Patrizio C. Remotti,Claudia Bernardini,& Francesco Saccardo. Adventitious shoot regeneration from leaf explants and stem nodes of Lilium. Plant Cell,Tissue and Organ Culture,2003,74:37~44.
86. Komai F.,Morohashi H.,& Horita M..Application of nurse culture for plant regeneration from protoplasts of Lilium longiflorum Thunb. In Vitro Cell. Dev. Biol.-Plant,2006,42:252~255.
87. KyeungⅡP.,Jeong D. C.,Sun J. E..Effect of Sucrose and Plant Growth Regulator Concentrations on Callus Proliferation and Adventitious Bud Induction from Callus of Lilium Oriental Hybrid 'Casa Blanca'.J.Kor.Hort.Sci,2003,44(6):986-990.
88. Kyozlxka J.,Hayashi Y.,Shimamoto K..High frequeney plant regeneration from rice protoplasts by novel nurse culture methods.Mol.Gen.Genet.1987,206:408~413.
89. Mii M.,Yuzawa Y.,Suetomi H.,Motegi T.,& Godo T.. Fertile plant regeneration from protoplasts of a seed-propagated cultivar of Lilium×formolongi by utilizing meristematic nodular cell clumps.Plant Sci.,1994,100:221~226.
90. Mori S.,Adachi Y.,Horimoto S.,Suzuki S.,& Nakano M..Callus formation and plant regeneration in various Lilium species and cultivars.In Vitro Cell.Dev. Biol.-Plant,2005,41:783~788.
91. Nakano M.,Sakakibara T.,Suzuki S.,& Saito H.. Decrease in the regeneration potential of long-term cell suspension cultures of Lilium formosanum Wallace and its restoration by the auxin transport inhibitor,2,3,5-triiodobenzoic acid.Plant Sci.,2000,158:129~137.
92. Nhut D. T.,Jaime A.,Silva T. D.,& Aswath C. R.. The importance of the explant on regeneration in thin cell layer technology. In Vitro Cell. Dev. Biol.-Plant,2003,39:266~276.
93. Nhut D. T.,Bui V. L.,& Tran Thanh Van K.. Manipulation of the morphogenetic pathways of Lilium longiflorum transverse thin cell layer explants by auxin and cytokinin,In Vitro Cell. Dev. Biol.-Plant,2001,37:44~49.
94. Pelkonen V. P., Anneli K.. The effect of light and auxins on the regeneration of lily (Lilium Regale Wil.) cells by somatic embryogenesis and organogenesis.International Journal of Plant Sciences.1999,160(3):483-490.
95. Priyadarshi S., & Sen S.. A revised scheme for mass propagation of Easter Lily. Plant Cell Tissue Organ Cult,1992,30:193~197.
96. Ramsay J. L.,Galitz D. S.. Basal medium and sucrose concentration influence regeneration of Easter Lily in ovary culture. Hort Science,2003,38(3):404-406.
97. Simmonds J. A.,& Cumming B. G..Propagation of Lilium hybrids II. Production of plantlets from bulb-scale callus cultures for increased propagation rates. Scientia Hort.,1976,5,161~170.
98. Stimart D. P.,Ascher P. D.,& Zagorski J. S.. Plants from callus of the interspecific hybrid Lilium‘Black Beauty’. HortScience,1980,15,313~315.
99. Sugiura H.,Agong S. G.,Enami A.. Comparison between embryoid and shoot primordia methods of plantlet production for 'Asiatic Hybrid Lily' and 'Oriental Hybrid Lily'. African Crop Science Journal,2006,8(2): 117-127.
100. Tompson J. A.,R. Abdullan,E. C. Cooking.Protoplast culture of rice(Oryzn satvn.L)using media solidified with agarose.Plant Seienee,1986,47:123~133.
101. Tribulato A.,Remotti P. C.,L?ffler H. J. M.,& Van Tuyl J. M..Somatic embryogenesis and plant regeneration in Lilium longiflorum Thunb,Plant Cell Reports,1997,17,113~118.
102. Wang Dayuan,Miller P. D.,Sondahl M. R.. Plant regeration from protoplasts of Indica type rice and CMS rice.Plant Cell RePorts.1989,8:329~332.
103. Yong Qu,Machteld C. Mok,David W. S. Mok,& Jack R. Stang .Phenotypic and cytological variation among plants derived from anther cultures of Lilium longiflorum,In Vitro Cellular & Developmental Biology,1988,24(5).
2.陈发棣,蒋甲福,郭维明,房伟民,赵宏波.小菊悬浮细胞培养与植株再生研究.园艺学报,2006,33(5):1021~1026.
3.陈火英,张建华.番茄下胚轴离体诱导成株的激素调控.上海农业学报,1999,15(2):26~29.
4.陈巍,高文远,贾伟,等.人参属药用植物组织和细胞培养的研究进展.中草药,2005,36(4):616~620.
5.陈伟列.三倍体无籽西瓜快速繁殖.福建农业科技,1984,(3):23~26.
6.崔红,陈亮,沈明山,陈睦传.甘薯胚性悬浮系的建立及其细胞生长特性研究.厦门大学学报(自然科学版),2002,41(5):555-558.
7.董云洲,段胜军.谷子胚性悬浮细胞系植株再生体系的建立及转基因技术研究.应用基础与工程科学学报,1999,第7卷,第1期.
8.董志渊,郑思乡.百合的组织培养及在其育种中的应用.西部林业科学,2004,33(2):64-68.
9.付文奇,杜双田,陈建科,尹华.NAA和2 ,4-D对东方百合组织培养的影响.西北林学院学报,2008,23 (2):83~86.
10.方文娟,韩烈保,曾会明.植物细胞悬浮培养影响因子研究.生物技术通报,2005,5:11-14.
11.高桂珍,周吉源.喜树细胞悬浮培养中生理生化指标的测定.武汉植物学研究,2003,21(3):259-261.
12.高丽华,胡显文,胥照平.喂饲前体提高悬浮培养水母雪莲细胞的黄酮产量.高技术通讯,2007,17(4):418~423.
13.郝立新,王怀名.西瓜再生系统的建立.华北农学报,1995,23(3):112~115.
14.何钢,文亚峰,付杰,谢碧霞,李樊,韩文军,邢伟一.人心果细胞悬浮培养的初步研究.经济林研究,2004,22 (4) :43-46.
15.何勇,田志宏,郑用琏.马蹄金悬浮细胞系的建立与优化.华中农业大学学报,2005,24(4):401-405.
16.侯丙凯,陈际江.小麦不同类型愈伤组织及其悬浮系的原生质体培养和植株再生.植物学通报,1996,13 (2) :52～54.
17.胡博然,徐文彪,马峰旺.枸杞胚细胞悬浮培养系统建立的研究.西北农林科技大学学报(自然科学版),2003,31(1):99-100.
18.胡之璧.生物技术在传统药材中的应用前景.药学服务与研究,2002,2(1):1.
19.黄纯农,王军晖,颜秋生,张雪琴,严庆峰.大麦胚性悬浮细胞系的快速建立及其超低温保存.中国农业科学,1995,28(3):21~27.
20.蒋细旺,司怀军.百合的组织培养技术综述.湖北农业科学,2004,1:78-82.
21.赖钟雄,黄浅,林秀莲,林玉玲,陈义挺,赖呈纯,等.荔枝胚性悬浮细胞系的快速建立及其体胚植株的再生.农业生物技术科学,2007,第23卷,第1期.
22.雷鸣,李向辉,孙勇如.水稻原生质体的植株再生.科学通报,1986,22:1729~1731.
23.李东杰,魏景芳,刘淑清,等.药用植物细胞悬浮培养研究展.河北林业科技,2003,3(8):22~23.
24.李玉平,龚宁,李美玲,马威,毛健峰,张兴. 2,4-D和KT对大花金挖耳愈伤组织诱导的影响.西北农业学报,2006,15(4):147-152.
25.林拥军,邹玉兰,傅世耀.高效水稻细胞悬浮系建立的研究.江西农业大学学报,1995,17(4):410~413.
26.刘坤.岷江百合(Lilium regale Wils)悬浮细胞系的建立及耐盐筛选.[硕士论文].南京:南京林业大学,2008.
27.柳玉晶,龚束芳,樊金萍,王金刚,车代弟.百合愈伤组织的诱导及植株再生.东北农业大学学报,2007,38(3):352~355.
28.陆美莲,许新萍,周厚高,符红霓.均匀正交设计在百合组织培养中的应用.西南农业大学学报(自然科学版),2004,26(6).
29.孟新亚.大蒜胚性细胞悬浮系的建立.[硕士论文].郑州:河南农业大学,2002.
30.莫小路,黄学林.银杏悬浮培养细胞的生长、分化与萜内酯化合物的积累.生物工程学报,2004,20(3):445~449.
31.盛腊红,何光存,舒理慧.抗病资源疵粒野生稻(O.meyriana)悬浮培养与植株再生.华中农业大学学报,1996,22(增刊):82~85.
32.申茹,都晓伟,吴军凯.高人参皂苷产量的生物培养技术研究进展.医药学报,2007,35(4):57~59.
33.孙敬三,桂耀林.植物细胞工程实验技术.北京:科学出版社,1995.
34.孙君社,方晓华.植物激素对百合鳞片愈伤组织生长的影响.中国农业大学学报,2001,6(2):58-61.
35.孙世孟,赛吉灾,谢友菊.玉米获白莱的悬浮细胞系的建立及其再生植株的研究.作物学报,1994,20(2).
36.唐东芹,钱虹妹,黄丹枫,唐克轩.百合基因转化胚性愈伤组织受体系统的建立.浙江林学院学报,2003,20 (3):273~276.
37.王德强,王晓玲.银杏细胞的悬浮培养研究.食品与生物技术学报,2005,24(3):27~29,33.
38.王海波.细胞状态观点的提出及其生命科学意义.科技导报,2008,26(4)41.
39.王杰,刘国锋,包满珠,黄莉.麝香百合胚性愈伤组织状态的调整与植株再生.园艺学报,2008,35(12):1795~1802.
40.王俊丽,陈丕铃,杨庆仙.杜仲细胞的悬浮培养.河北大学学报(自然科学版),1997,17(3):78-80
41.王素芳,王志林,蒋琳兰,赵树进.植物细胞悬浮培养.中国生物制品学杂志,2002,15(6):381~383.
42.王旭英.杂交粳稻3优18胚性悬浮细胞系的建立.聊城大学学报(自然科学版),2005,第18卷,第4期.
43.王亚琴,朱媛.杜仲细胞悬浮培养生产桃叶珊瑚甙的研究.广西植物,2007,27(2):236~239.
44.王友生,李阳春,梁慧敏,刘自学,孙榕江.紫花苜蓿愈伤组织诱导及植株再生的研究.草原与草坪,2006,4:55-57.
45.魏岳荣,黄学林,李佳,黄霞,李哲,李筱菊.贡蕉胚性细胞悬浮系的建立和植株再生.生物工程学报,2005,第21卷,第1期.
46.吴晓玲.植物生长调节剂对银柴胡细胞生长特性的影响.植物研究,2005,25(2):173~176.
47.吴晓玲,邓光存,姜晓慧.黄芩细胞生长特性及次生代谢产物生产性能的研究.西北植物学报.2005,25(3):557~561.
48.吴永宏,张德颐,王丽蓉.植物细胞同步培养技术.植物生理学通讯,1993,29(2):108~110.
49.吴智鹏,冯俊涛,马志卿.除虫菊细胞悬浮体系营养条件优化研究.西北农业学报,2006,15(4):180~82.
50.许智宏,卫志明,刘贵云.用离体培养无性繁殖三倍体无籽西瓜.植物生理学报,1979,5(3):45~251.
51.杨柏云,杨慧琴,蔡奇英,管毕财,罗丽萍.龙牙百合体细胞胚的诱导及植株再生.南昌大学学报(理科版),2005,29(6).
52.杨剑波,吴家道,卫志明.粕型水稻原生质体再生植株.生物工程学报.1992,82(l):60~64.
53.杨远媛,贺窑青,郑彩霞.油松胚珠愈伤组织诱导和悬浮细胞系的建立.植物生理学通讯,2005,
41(5).
54.尹文兵,李伟,杜桂森,黄勤妮.西藏红景天组织培养研究.北方七省市植物学会年会暨学术讨论会,2004.
55.岳彩鹏,李品,黄象南.烟草愈伤组织的诱导及细胞悬浮培养体系的建立.烟草科技,2007,10:56~59.
56.张延龙,徐炎,李峰,罗佳,范铭.秦岭野百合鳞片植株再生体系的建立.西北植物学报,2004,24(7):1315~1318.
57.张艺萍,吴丽芳,吴学尉,崔光芬,丁丁,王继华.东方百合胚性愈伤组织诱导和植株a)再生研究.江西农业学报,2008,20(12):33~36.
58.张云,原雅玲,刘青林.百合品种改良与生物技术研究进展.北京林业大学学,2001,23(6): 56~59.
59.赵海涛.东方百合‘Siberia’试管鳞茎发育及休眠研究.[硕士论文].北京:中国农业科学院,2009.
60.赵鸿莲,于荣敏.诱导子在植物细胞培养中的应用研究进展.沈阳药科大学学报,2000,17(2):152~156.
61.赵珺,宋阳,雷家军.麝香百合子房离体培养的研究.辽宁农业科学,2006,(5):11~13.
62.赵军,徐莺,严钫,唐琳,陈放.红花愈伤组织的诱导与花柱愈伤组织的继代悬浮培养.四川大学学报(自然科学版),2001,38(3):421-424.
63.周春江,李卫东,葛会波.草莓悬浮细胞系的建立及某些影响因素.植物生理学通讯,2002,第38卷,第1期.
64.周晓罡,丁玉梅,孙茂林,陈亮,苏源,张绍松,等. 24个水稻抗稻瘟病单基因鉴别品系悬浮细胞系的建立.西南农业学报,2008,21(3).
65.朱宝成,吴爱民.掌叶半夏细胞悬浮培养及单细胞培养再生植株.作物学报,1996,22(2):197-200.
66.褚云霞,陈龙清,黄燕文,张永春.百合的花药培养研究.园艺学报,2001,28(5):472-474.
67.朱至清.植物细胞工程.北京:化学工业出版社,2003.
68. Aguettaz P.,Paffen A.,Delvallee I.,Van der Linde P.,& De Klerk G. J..The development of dormancy in bulblets of Lilium speciosum generated in vitro I. The effects of culture conditions.Plant Cell Tissue Organ Cult,1990,22:167~172.
69. Arzate-Fernández A. M.,Nakazaki T.,Okumoto Y.,& Tanisaka T. Efficient callus induction and plant regeneration from filaments with anther in lily (Lilium longiflorum Thunb.).Plant Cell Reports,1997,16:836~840.
70. Azevedo H.,Dias A.,& Tavares R. M..Establishment and characterization of Pinus pinaster suspension cell cultures.Plant Cell Tiss Organ Cult,2008,93:115~121.
71. Bui V. L.,Nhut D. T.,& Tran Thanh Van K..Plant production via shoot regeneration from thin cell layer pseudo-bulblet explants of Lilium longiflorum in vitro. C. R. Acad. Sci. Paris, Sciences de la vie/Life Sciences,1999,322:303~310.
72. Chin-wen Ho,Wei-ting Jian,& Hui-chun Lai. Plant regeneration via somatic embryogenesis from suspension cell cultures of Lilium×formolongi hort.using a bioreactor system.In Vitrp Cell.Dev.Biol.-Plant,2006,42:240~246.
73. Dong-Sheng Han,Yoshiji Niimi,& Masaru Nakano.Long term maintenance of an anther-derived haploid callus line of the Asiatic hybrid lily 'Connecticut King'.Plant Cell,Tissue and Organ Culture,2000,61:215~219.
74. Dong-Sheng Han,Yoshiji Niimi,& Masaru Nakano.Regeneration of haploid plants from anther cultures of the Asiatic hybrid lily 'Connecticut King'.Plant Cell,Tissue and Organ Culture,1997,47:153~158.
75. Enaksha R. M. Wickremesinhe,E. Jay Holcomb,& Richard N. Arteca.A pratical method for the production of flowering Easter lilies from callus cultures.Scientia Horticulturae,1994,60:143~152.
76. Famelaer I.,Bordas M.,Baliu E.,Ennik E.,Meijer H.,& Van Tuyl J. M. et al..The use of morphogenic suspension cultures for the development of a protoplast regeneration system in lily.Acta Hort,430,ISHS 1997.
77. Famelaer I.,Ennik E.,Van Tuyl J. M.,Meijer H.,& Creemers-Molenaar C..The establishment of suspension and meristem cultures for the development of a protoplast regeneration and fusion system in lily,Acta Hort,430,ISHS 1996.
78. Godo T.,Kobayashi K.,Tagami T.,Matsui K.,& Kida T..In vitro propagation utilizing suspension cultures of meristematic nodular cell clumps and chromosome stability of Lilium×formolongi hort.,Scientia Horticulturae,1998,72:193~202.
79. Godo T.,Matsui K.,Kida T.,& Mii M..Effect of sugar type on the efficiency of plant regeneration from protoplasts isolated from shoot tip-derived meristematic nodular cell clumps of Lilium×formolongi hort.Plant Cell Rep.,1996,15:401~404.
80. Godo T.,& Mii M..Regeneration of plants from protoplasts of Lilium×formolongi.In:Bajaj YPS(ed)Biotechnology in agriculture and forestry.Springer,Berlin Heidelberg New York,1996,38,79~89.
81. Gur'eva A. S.,Kintya P. K.,Mashchenko N. E.,& Shashkov A. S..Glycosides of the bulbs of Liliumregale.Chemistry of Natural Compounds,1996,Vol.32,No.3.
82. Horita M.,Morohashi H.,& Komai F..Regeneration of flowering plants from difficile lily protoplasts by means of a nurse culture.Planta,2002,215:880~884.
83. Horita M.,Morohashi H.,& Komai F..Production of fertile somatic hybrid plants between Oriental hybrid lily and Lilium×formolongi.Planta,2003,217:597~601.
84. Li L. Y.,Chen K..Towards a high and reproducible frequency of plant regeneration from protoplasts of riee(Oryzn sativn L).Genetic Manipulation in Crops Newsletter,1987,3:l~15.
85. Loretta Bacchetta,Patrizio C. Remotti,Claudia Bernardini,& Francesco Saccardo. Adventitious shoot regeneration from leaf explants and stem nodes of Lilium. Plant Cell,Tissue and Organ Culture,2003,74:37~44.
86. Komai F.,Morohashi H.,& Horita M..Application of nurse culture for plant regeneration from protoplasts of Lilium longiflorum Thunb. In Vitro Cell. Dev. Biol.-Plant,2006,42:252~255.
87. KyeungⅡP.,Jeong D. C.,Sun J. E..Effect of Sucrose and Plant Growth Regulator Concentrations on Callus Proliferation and Adventitious Bud Induction from Callus of Lilium Oriental Hybrid 'Casa Blanca'.J.Kor.Hort.Sci,2003,44(6):986-990.
88. Kyozlxka J.,Hayashi Y.,Shimamoto K..High frequeney plant regeneration from rice protoplasts by novel nurse culture methods.Mol.Gen.Genet.1987,206:408~413.
89. Mii M.,Yuzawa Y.,Suetomi H.,Motegi T.,& Godo T.. Fertile plant regeneration from protoplasts of a seed-propagated cultivar of Lilium×formolongi by utilizing meristematic nodular cell clumps.Plant Sci.,1994,100:221~226.
90. Mori S.,Adachi Y.,Horimoto S.,Suzuki S.,& Nakano M..Callus formation and plant regeneration in various Lilium species and cultivars.In Vitro Cell.Dev. Biol.-Plant,2005,41:783~788.
91. Nakano M.,Sakakibara T.,Suzuki S.,& Saito H.. Decrease in the regeneration potential of long-term cell suspension cultures of Lilium formosanum Wallace and its restoration by the auxin transport inhibitor,2,3,5-triiodobenzoic acid.Plant Sci.,2000,158:129~137.
92. Nhut D. T.,Jaime A.,Silva T. D.,& Aswath C. R.. The importance of the explant on regeneration in thin cell layer technology. In Vitro Cell. Dev. Biol.-Plant,2003,39:266~276.
93. Nhut D. T.,Bui V. L.,& Tran Thanh Van K.. Manipulation of the morphogenetic pathways of Lilium longiflorum transverse thin cell layer explants by auxin and cytokinin,In Vitro Cell. Dev. Biol.-Plant,2001,37:44~49.
94. Pelkonen V. P., Anneli K.. The effect of light and auxins on the regeneration of lily (Lilium Regale Wil.) cells by somatic embryogenesis and organogenesis.International Journal of Plant Sciences.1999,160(3):483-490.
95. Priyadarshi S., & Sen S.. A revised scheme for mass propagation of Easter Lily. Plant Cell Tissue Organ Cult,1992,30:193~197.
96. Ramsay J. L.,Galitz D. S.. Basal medium and sucrose concentration influence regeneration of Easter Lily in ovary culture. Hort Science,2003,38(3):404-406.
97. Simmonds J. A.,& Cumming B. G..Propagation of Lilium hybrids II. Production of plantlets from bulb-scale callus cultures for increased propagation rates. Scientia Hort.,1976,5,161~170.
98. Stimart D. P.,Ascher P. D.,& Zagorski J. S.. Plants from callus of the interspecific hybrid Lilium‘Black Beauty’. HortScience,1980,15,313~315.
99. Sugiura H.,Agong S. G.,Enami A.. Comparison between embryoid and shoot primordia methods of plantlet production for 'Asiatic Hybrid Lily' and 'Oriental Hybrid Lily'. African Crop Science Journal,2006,8(2): 117-127.
100. Tompson J. A.,R. Abdullan,E. C. Cooking.Protoplast culture of rice(Oryzn satvn.L)using media solidified with agarose.Plant Seienee,1986,47:123~133.
101. Tribulato A.,Remotti P. C.,L?ffler H. J. M.,& Van Tuyl J. M..Somatic embryogenesis and plant regeneration in Lilium longiflorum Thunb,Plant Cell Reports,1997,17,113~118.
102. Wang Dayuan,Miller P. D.,Sondahl M. R.. Plant regeration from protoplasts of Indica type rice and CMS rice.Plant Cell RePorts.1989,8:329~332.
103. Yong Qu,Machteld C. Mok,David W. S. Mok,& Jack R. Stang .Phenotypic and cytological variation among plants derived from anther cultures of Lilium longiflorum,In Vitro Cellular & Developmental Biology,1988,24(5).