紫苏离体再生体系的建立及其遗传转化初步研究
摘要
本研究以紫苏的子叶和下胚轴为外植体进行组织培养,建立了紫苏的离体再生体系,获得了正常的再生植株。本实验运用对比试验的方法,研究了影响紫苏再生的因素,同时对紫苏的遗传转化体系进行了初步探索。其实验结果表明:
1、紫苏子叶的最佳分化培养基为MS+BA 1.0mg/L+IAA 1.5 mg/L,再生率最高80.0%,平均每外植体长芽2.3个,下胚轴的最佳分化培养基为MS+BA 3.0mg/L+NAA 0.3 mg/L,再生率为70.9%,平均每外植体长芽2.5个。再生芽的生根培养基为1/2MS+1.0mg/L BA+0.3mg/L IAA,生根率达到100%,移栽成活率在85%以上。
2、以11天苗龄的紫苏苗作为实验材料,白砂糖添加30g/L,2,4-D预培养后转入最佳分化培养基,子叶暗培养12天,下胚轴暗培养8天后,给予每天14h光照处理,可以较大幅度提高紫苏外植体的再生率。培养基中添加4mg/L AgNO3的处理可使子叶外植体再生频率提高5.4%,下胚轴外植体的再生频率提高9.4%。
3、遗传转化研究表明,下胚轴抗性芽的筛选压力为40mg/L Kan,转化苗生根的筛选压力为35 mg/L Kan,研究发现培养基中添加15 mg/L Kan时,每外植体长芽7.6个,为对照组的3.3倍,较大地提高了每个外植体的再生芽数。农杆菌侵染后,选用羧苄青霉素作为抑菌剂,将外植体与农杆菌共培养2天,转接到附加羧苄青霉素400mg/L的培养基上进行抑菌处理,抑菌效果效果最好。
Perilla cotyledon and hypocotyl were used as explants for tissue culture in this research. This study established vitro regeneration system for perilla and got normal regenerative plants. Comparative experimental methods were used to study the factors affecting the regeneration for perilla, while the perilla genetic transformation system were studied as a preliminary exploration,The conclusions were obtained as follows:
1. Perilla cotyledons best differentiation medium was MS + BA 1.0mg/L + IAA 1.5 mg/L, the highest regeneration rate was 80.0%, every cotyledon germinated 2.3 buds. The perilla hypocotyl best differentiation medium was MS+BA 3.0mg/L + NAA 0.3 mg/L, the rate of regeneration was 70.9%, every cotyledon germinated 2.5 buds. Regeneration of shoots rooting medium was 1/2MS +1.0 mg/L BA +0.3 mg/L IAA, the rooting rate was up to 100% and the survival rate was more than 85%.
2. 11-days aged perilla was used as experimental material, added sugar 30g/L. The perilla was transferred after precultured in 2,4-D, cotyledons precultured for 12 days in darkness and hypocotyl precultured for 8 days in darknessthen cultured at the condition of 14h/d light. Cultured in the medium added 4mg/L AgNO3, cotyledons explant regeneration rates improve 5.4% and hypocotyl explant regeneration rates improve 9.4%.
3. Perilla hypocotyl selection pressure for regeneration was 40mg/L Kan, the rooting selective pressure was 35 mg/L Kan, The experment shows that every explant germinated 7.6 buds in the medium added Kan 15 mg/L. It is 3.3 times as control group, and greatly improve the number of regenerated shoots. After Agrobacterium infection, choose Carbenicillin as antibacterial agent, after two days co-culture, the explant transfered to the medium of 400 mg/L Car can get the best antimicrobial activity.
1、紫苏子叶的最佳分化培养基为MS+BA 1.0mg/L+IAA 1.5 mg/L,再生率最高80.0%,平均每外植体长芽2.3个,下胚轴的最佳分化培养基为MS+BA 3.0mg/L+NAA 0.3 mg/L,再生率为70.9%,平均每外植体长芽2.5个。再生芽的生根培养基为1/2MS+1.0mg/L BA+0.3mg/L IAA,生根率达到100%,移栽成活率在85%以上。
2、以11天苗龄的紫苏苗作为实验材料,白砂糖添加30g/L,2,4-D预培养后转入最佳分化培养基,子叶暗培养12天,下胚轴暗培养8天后,给予每天14h光照处理,可以较大幅度提高紫苏外植体的再生率。培养基中添加4mg/L AgNO3的处理可使子叶外植体再生频率提高5.4%,下胚轴外植体的再生频率提高9.4%。
3、遗传转化研究表明,下胚轴抗性芽的筛选压力为40mg/L Kan,转化苗生根的筛选压力为35 mg/L Kan,研究发现培养基中添加15 mg/L Kan时,每外植体长芽7.6个,为对照组的3.3倍,较大地提高了每个外植体的再生芽数。农杆菌侵染后,选用羧苄青霉素作为抑菌剂,将外植体与农杆菌共培养2天,转接到附加羧苄青霉素400mg/L的培养基上进行抑菌处理,抑菌效果效果最好。
Perilla cotyledon and hypocotyl were used as explants for tissue culture in this research. This study established vitro regeneration system for perilla and got normal regenerative plants. Comparative experimental methods were used to study the factors affecting the regeneration for perilla, while the perilla genetic transformation system were studied as a preliminary exploration,The conclusions were obtained as follows:
1. Perilla cotyledons best differentiation medium was MS + BA 1.0mg/L + IAA 1.5 mg/L, the highest regeneration rate was 80.0%, every cotyledon germinated 2.3 buds. The perilla hypocotyl best differentiation medium was MS+BA 3.0mg/L + NAA 0.3 mg/L, the rate of regeneration was 70.9%, every cotyledon germinated 2.5 buds. Regeneration of shoots rooting medium was 1/2MS +1.0 mg/L BA +0.3 mg/L IAA, the rooting rate was up to 100% and the survival rate was more than 85%.
2. 11-days aged perilla was used as experimental material, added sugar 30g/L. The perilla was transferred after precultured in 2,4-D, cotyledons precultured for 12 days in darkness and hypocotyl precultured for 8 days in darknessthen cultured at the condition of 14h/d light. Cultured in the medium added 4mg/L AgNO3, cotyledons explant regeneration rates improve 5.4% and hypocotyl explant regeneration rates improve 9.4%.
3. Perilla hypocotyl selection pressure for regeneration was 40mg/L Kan, the rooting selective pressure was 35 mg/L Kan, The experment shows that every explant germinated 7.6 buds in the medium added Kan 15 mg/L. It is 3.3 times as control group, and greatly improve the number of regenerated shoots. After Agrobacterium infection, choose Carbenicillin as antibacterial agent, after two days co-culture, the explant transfered to the medium of 400 mg/L Car can get the best antimicrobial activity.
引文
[1]潘瑞炽.植物组织培养[M].广州:广东高等教育出版社, 2000. 1-3.
[2]赵国凡,王兴理.植物组织培养概论[M].大连:大连工学院出版社, 1988. 1-3.
[3]朱建华,彭士勇.植物组织培养实用技术[M].北京:中国计量出版社, 2002. 2-5.
[4] Atangana AR, Tchoundjeu Z, Asaah EK, et al. Domestication of Allanblackia floribunda: Amenability to vegetative propagation[J]. Forest Ecology and Management, 2006, (237): 246-251.
[5]盛玉婷.植物组织培养技术及应用进展[J].安徽农学通报, 2008, 14(9):45-47.
[6]胡孔峰,胡如善,张慎举等.植物组织培养技术及应用[M].郑州:河南科学技术出版社, 2006. 1-5.
[7]张国强,翟秋喜.我国植物组培技术的发展及展望[J].安徽农业科技, 2006, 34(16): 3893-3895.
[8]孙勇如,安锡培.植物原生质体培养[M].北京:科学出版社, 1991.
[9]夏镇澳.植物组织培养与农业[J].植物生理学通讯, 1995, 31(1): 62-64.
[10]林蓉,谢春梅,谢世清.马铃薯茎尖脱毒培养关键因子分析[J].中国农学通报, 2005, 21(7): 338-340.
[11]郝爱平,詹亚光,尚杰.诱变技术在植物育种中的研究新进展[J].生物技术通报, 2004, (6): 30-33.
[12]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): 197-200.
[13]董诚明,苏秀红,王伟丽.碳源对冬凌草再生植株生长及次生代谢产物的影响[J].西北植物学报, 2009, 29(3): 494-498.
[14]杜勤,王金发.我国药用植物次生代谢产物功能基因研究概况[J].时珍国医国药, 2009, 20(8): 2019-2021.
[15] Verpoorte R, Memelink J. Engineering secondary metabolite production in plants[J]. Plant biotechnology, 2002, 31(2): 181-187.
[16]黄璐琦,郭兰萍.环境胁迫下次生代谢产物的积累及道地药材的形成[J].中国中药杂志, 2007, 32(4): 277-280.
[17] Ramachandra RS, Ravishankar GA. Plant cell cultures: Chemical factories of secondary metabolites[J]. Biotechnology Advances, 2002, 20(2): 101-153.
[18]吴雪梅,汤浩茹.包埋玻璃化法超低温保存植物种质的研究进展[J].植物学通报, 2005, 22(2): 238-245.
[19]徐刚标.植物种质资源离体保存研究进展[J].中南林学院学报, 2000, 20(4): 81-87.
[20]冯晓东,曹娟云,陈宗礼.水杨酸对枣树组织培养苗几种生理生化指标的影响[J].西北植物学报, 2003, 23(9): 1625-1627.
[21]谷兆萍,王昕,潘义宏.两种培养方式下砷对大叶井口边草生理生化的影响[J].农业环境科学学报, 2010, 29(7): 1254-1260.
[22]张玉红,曲伟娣,温慧.不同植物激素对黄檗叶片愈伤组织诱导效应[J].中国农学通报, 2010, 26(24): 179-182.
[23]金晶,刘小阳,蔡永萍.植物激素对砀山梨托病毒苗增殖生长的影响[J].激光生物学报, 2006, 15(4): 378-382.
[24]姚允聪.苹果三高栽培技术[M].北京市:中国农业大学出版社, 1997.
[25]刘琳,刘国杰.不同碳源对钙果组培苗的影响[J].北方园艺, 2005, (5): 68.
[26]陈俊伟,谢鸣,秦巧平等.植物糖信号与激素信号之间的联系[J].植物生理学通讯, 2005, 41(3): 279-285.
[27]庞红霞,祝长青,覃建兵.激素和外植体类型对新疆雪莲愈伤组织诱导的影响[J].华中农业大学学报, 2010, 29(2): 131-134.
[28] Vasil IK, Ahuja MR, Vasil V. Plant tissue cultures in Genetics and plant breeding[J]. Advances in Genetics, 1979, 20: 127-215.
[29]黄明发,郭莉,郑炯等.紫苏的研究进展[J].中国食品添加剂, 2007, (4): 85-89.
[30]于淑玲,王秀玲.药用紫苏的营养价值与综合利用的概述[J].食品科技, 2006, (8): 287-289.
[31] Yamazaki M, Nakajima J, Yamanashi M, et al. Metabolomics and differential gene expression in anthocyanin chemo-varietal forms of Perilla frutescens[J]. Phytochemistry, 2003, 62: 987-995.
[32]张麟,刘大川,李江平等.紫苏资源综合利用技术的中试生产研究[J].油脂工程, 2009, (8): 51-53.
[33]裴建文,毛学文.油料植物紫苏形态学的特性研究[J].天水师专学报, 1992, (2): 90-93.
[34]丁晨光,李芳.紫苏的医疗营养保健功效与开发研究进展[J].食用医药杂志, 2007, 24(10): 1253-1254.
[35]陆洁静,任文彬.紫苏的研究概况[J].农产品加工, 2009, (6): 32-34.
[36]殷朝州.紫苏的开发利用[J].植物杂志, 1990, (4): 8.
[37]刘娟,雷焱霖,唐友红等.紫苏的化学成分与生物活性研究进展[J].时珍国医国药, 2010, 21(7): 1768-1769.
[38] Masumoto N, Korin M, Ito M. Geraniol and linalool synthases from wild species of perilla[J]. Phytochemistry, 2010, 71: 1068-1075.
[39]熊运海,王玫.化学计量学法对紫苏叶与紫苏子挥发油共有组分分析[J].食品科学, 2010, 31(2): 203-207.
[40]张洪,黄建韶,赵东海.紫苏营养成分的研究[J].食品与机械, 2006, 22(2): 41-43.
[41]蔡乾蓉,吴卫,郑有良等. HPLC法比较测定紫苏属植物种子维生素E含量差异[J].食品科学, 2010, 31(6): 243-247.
[42]彭小平,熊劲松.我国紫苏产业化研究现状与展望[J].安徽农业科学, 2010, 38(16): 8709-8711.
[43]赵贵红.营养紫苏果酒的研制[J].酿酒科技, 2008, (10): 86-88.
[44] Laureati M, Buratti S, Bassoli A, et al. Discrimination and characterisation of three cultivars of Perilla frutescens by means of sensory descriptors and electronic nose and tongue analysis[J]. Food research international, 2010, 43: 959-964.
[45]牛炳韬.紫苏再生体系的建立及体细胞无性系变异分析[D].兰州大学硕士学位论文, 2005, 34-41.
[46]于淑玲,石晓云.紫苏组织培养体系的研究[J].邢台学院学报. 2008, 23(2): 118-119.
[47] Zhang T, Wang XY, Cao ZY. Plant regeneration in vitro directly from cotyledon andhypocotyl explants of Perilla frutescens and their morphological aspects[J]. Biologia Plantarum, 2005, 49(3): 423-426.
[48]黄海泉,张蕾,黄美娟.紫苏组织培养及植株再生[J].亚热带植物科学, 2007, 36(4): 56.
[49] Kim KH, Lee YH. Agrobacterium-mediated genetic transformation of Perilla frutescens[J]. Plant Cell Rep, 2004, 23: 386-390.
[50] Lee BK, Yu SH. Agrobacterium-mediated transformation of Perilla[J]. Plant Cell, 2005, 83: 51-58.
[51] Venketeswaran S, Gandhi V. Mass propagation and genetic improvement of forest trees for biomass production by tissue culture[J]. Biomass, 1982, 2(1): 5-15.
[52]沈世华,张秀君,郭奕明.玉米基因转化的离体子房注射及其转基因植株的鉴定[J].植物学报, 2001, 43(10): 1055-1057.
[53]何俊平,陈发棣,陈素梅.石蒜凝集素基因转化切花菊及抗蚜性鉴定[J].西北植物学报, 2009, 29(11): 2318-2325.
[54] Almeida WAB, Filho FAM, Pino LE. Genetic transformation and plant recovery from mature tissues of Citrus sinensis L.Osbeck[J]. Plant Science, 2003, 164(2): 203-211.
[55]张玉翠,高海波.杜兰组培快繁技术的研究[J].中国农学通报, 2005, 21(8): 306-307.
[56]陈振东,高海筹,蔡坤秀.南方芦笋组培苗移栽技术研究[J].中国农学通报, 2006, 22(11): 282-284.
[57]张陈明,胡宗利,陈国平.根癌农杆菌介导转化马铃薯与抗病毒基因工程[J].生物技术通报, 2008, (6): 30-35.
[58]刘晓庆.油菜、烟草耐盐转基因的研究[D].扬州大学硕士学位论文, 2009, 43-44.
[59]盖钧镒.试验统计方法[M].北京:中国农业出版社, 2000. 227-236.
[60]赵普庆,於维维,汪俏梅.生长素与其他信号之间的相互作用[J].植物生理学通讯, 2004, 40(2): 246-249.
[61]黄超,李玲.植物激素信号间的相互作用[J].亚热带植物科学, 2005, 34(4): 66-70.
[62]袁晶,汪俏梅,张海峰.植物激素信号之间的相互作用[J].细胞生物学杂志, 2005,(27): 325-328.
[63] Zhang T, Wang XY, Cao ZY. Plant regeneration in vitro directly from cotyledon and hypocotyl explants of Perilla frutescens and their morphological aspects[J]. Biologia Plantarum, 2005, 49(3): 423-426.
[64]孙在红,刘荣堂,梁慧敏等.植物激素在草坪草组织培养及植株再生中的应用与进展[J].草原与草坪, 2004, (2): 13-16.
[65] Predieri S, Malavasi F. High-frequency shoot regeneration from leaves of the apple rootstock M26[J]. Plant Cell, 1989, 17: 133-142.
[66]张志兰.山茶花组织培养过程中的防外植体褐化试验[J].西部林业科学, 2007, 36(2): 118-121.
[67]杜勤,王振华,徐鸿华等.石牌广藿香试剂苗的研制[J].中国中药杂志, 2002, 27(3): 179-181.
[68]徐培洲,吴先军,胡保民等.油葵子叶外植体不定芽再生体系的建立[J].中国油料作物学报, 2004, 26(4): 16-19.
[69]杜启兰.不同培养基与碳源对丽佳秋海棠组培苗的影响[J].安徽农业科学, 2006, 34(11): 2404-2405.
[70]赵巧阳,赖钟雄.硝酸银在离体培养和转化中的作用及其机理[J].亚热带农业研究, 2008, 4(1): 62-66.
[71]刘娟,汤浩茹,刘玲梅. AgNO3对梨叶片不定梢再生过程中抗氧化酶活性的影响[J].果树学报, 2008, 25(4): 467-472.
[72]蒋明义,杨文英,徐江等.渗透胁迫下水稻幼苗中叶绿素降解的活性氧损伤作用[J].植物学报, 1994, 36(4): 289-295.
[73]韩雪梅.草莓试管苗分化培养基优化的研究[J].生物技术, 1997, 7(2): 27-29.
[74]孙清荣,孙洪雁.不同倍性苹果叶片植株再生研究[J].落叶果树, 2000, (2): 9-11.
[75]赵爽,雷建军,陈国菊等.卡那霉素对大豆生在转基因芥菜中的应用[J].遗传, 2008, 30(4): 501-507.
[76] Satyavathi VV, Prasad V, Gita Lakshmi B, et al. High efficiency transformation on protocol for three Indian cotton varieties via Agrobacterinm tumefaciens[J]. PlantScience, 2002, 162(2): 215-223.
[77]袁鹰,刘德璞,王玉民等.卡那霉素对大豆生长的抑制及筛选试验研究[J].大豆科学, 2003, 22(4): 261-263.
[78]张明洲,崔海瑞,舒庆尧等.抗生素对高粱愈伤组织诱导和生长的影响[J].浙江大学学报, 2004, 30(2): 221-225.
[79]周小梅,李君剑,赵军良等.抗生素对农杆菌的抑制和对油菜外植体分化的影响[J].西北植物学报, 2005, 25(1): 52-56.
[2]赵国凡,王兴理.植物组织培养概论[M].大连:大连工学院出版社, 1988. 1-3.
[3]朱建华,彭士勇.植物组织培养实用技术[M].北京:中国计量出版社, 2002. 2-5.
[4] Atangana AR, Tchoundjeu Z, Asaah EK, et al. Domestication of Allanblackia floribunda: Amenability to vegetative propagation[J]. Forest Ecology and Management, 2006, (237): 246-251.
[5]盛玉婷.植物组织培养技术及应用进展[J].安徽农学通报, 2008, 14(9):45-47.
[6]胡孔峰,胡如善,张慎举等.植物组织培养技术及应用[M].郑州:河南科学技术出版社, 2006. 1-5.
[7]张国强,翟秋喜.我国植物组培技术的发展及展望[J].安徽农业科技, 2006, 34(16): 3893-3895.
[8]孙勇如,安锡培.植物原生质体培养[M].北京:科学出版社, 1991.
[9]夏镇澳.植物组织培养与农业[J].植物生理学通讯, 1995, 31(1): 62-64.
[10]林蓉,谢春梅,谢世清.马铃薯茎尖脱毒培养关键因子分析[J].中国农学通报, 2005, 21(7): 338-340.
[11]郝爱平,詹亚光,尚杰.诱变技术在植物育种中的研究新进展[J].生物技术通报, 2004, (6): 30-33.
[12]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): 197-200.
[13]董诚明,苏秀红,王伟丽.碳源对冬凌草再生植株生长及次生代谢产物的影响[J].西北植物学报, 2009, 29(3): 494-498.
[14]杜勤,王金发.我国药用植物次生代谢产物功能基因研究概况[J].时珍国医国药, 2009, 20(8): 2019-2021.
[15] Verpoorte R, Memelink J. Engineering secondary metabolite production in plants[J]. Plant biotechnology, 2002, 31(2): 181-187.
[16]黄璐琦,郭兰萍.环境胁迫下次生代谢产物的积累及道地药材的形成[J].中国中药杂志, 2007, 32(4): 277-280.
[17] Ramachandra RS, Ravishankar GA. Plant cell cultures: Chemical factories of secondary metabolites[J]. Biotechnology Advances, 2002, 20(2): 101-153.
[18]吴雪梅,汤浩茹.包埋玻璃化法超低温保存植物种质的研究进展[J].植物学通报, 2005, 22(2): 238-245.
[19]徐刚标.植物种质资源离体保存研究进展[J].中南林学院学报, 2000, 20(4): 81-87.
[20]冯晓东,曹娟云,陈宗礼.水杨酸对枣树组织培养苗几种生理生化指标的影响[J].西北植物学报, 2003, 23(9): 1625-1627.
[21]谷兆萍,王昕,潘义宏.两种培养方式下砷对大叶井口边草生理生化的影响[J].农业环境科学学报, 2010, 29(7): 1254-1260.
[22]张玉红,曲伟娣,温慧.不同植物激素对黄檗叶片愈伤组织诱导效应[J].中国农学通报, 2010, 26(24): 179-182.
[23]金晶,刘小阳,蔡永萍.植物激素对砀山梨托病毒苗增殖生长的影响[J].激光生物学报, 2006, 15(4): 378-382.
[24]姚允聪.苹果三高栽培技术[M].北京市:中国农业大学出版社, 1997.
[25]刘琳,刘国杰.不同碳源对钙果组培苗的影响[J].北方园艺, 2005, (5): 68.
[26]陈俊伟,谢鸣,秦巧平等.植物糖信号与激素信号之间的联系[J].植物生理学通讯, 2005, 41(3): 279-285.
[27]庞红霞,祝长青,覃建兵.激素和外植体类型对新疆雪莲愈伤组织诱导的影响[J].华中农业大学学报, 2010, 29(2): 131-134.
[28] Vasil IK, Ahuja MR, Vasil V. Plant tissue cultures in Genetics and plant breeding[J]. Advances in Genetics, 1979, 20: 127-215.
[29]黄明发,郭莉,郑炯等.紫苏的研究进展[J].中国食品添加剂, 2007, (4): 85-89.
[30]于淑玲,王秀玲.药用紫苏的营养价值与综合利用的概述[J].食品科技, 2006, (8): 287-289.
[31] Yamazaki M, Nakajima J, Yamanashi M, et al. Metabolomics and differential gene expression in anthocyanin chemo-varietal forms of Perilla frutescens[J]. Phytochemistry, 2003, 62: 987-995.
[32]张麟,刘大川,李江平等.紫苏资源综合利用技术的中试生产研究[J].油脂工程, 2009, (8): 51-53.
[33]裴建文,毛学文.油料植物紫苏形态学的特性研究[J].天水师专学报, 1992, (2): 90-93.
[34]丁晨光,李芳.紫苏的医疗营养保健功效与开发研究进展[J].食用医药杂志, 2007, 24(10): 1253-1254.
[35]陆洁静,任文彬.紫苏的研究概况[J].农产品加工, 2009, (6): 32-34.
[36]殷朝州.紫苏的开发利用[J].植物杂志, 1990, (4): 8.
[37]刘娟,雷焱霖,唐友红等.紫苏的化学成分与生物活性研究进展[J].时珍国医国药, 2010, 21(7): 1768-1769.
[38] Masumoto N, Korin M, Ito M. Geraniol and linalool synthases from wild species of perilla[J]. Phytochemistry, 2010, 71: 1068-1075.
[39]熊运海,王玫.化学计量学法对紫苏叶与紫苏子挥发油共有组分分析[J].食品科学, 2010, 31(2): 203-207.
[40]张洪,黄建韶,赵东海.紫苏营养成分的研究[J].食品与机械, 2006, 22(2): 41-43.
[41]蔡乾蓉,吴卫,郑有良等. HPLC法比较测定紫苏属植物种子维生素E含量差异[J].食品科学, 2010, 31(6): 243-247.
[42]彭小平,熊劲松.我国紫苏产业化研究现状与展望[J].安徽农业科学, 2010, 38(16): 8709-8711.
[43]赵贵红.营养紫苏果酒的研制[J].酿酒科技, 2008, (10): 86-88.
[44] Laureati M, Buratti S, Bassoli A, et al. Discrimination and characterisation of three cultivars of Perilla frutescens by means of sensory descriptors and electronic nose and tongue analysis[J]. Food research international, 2010, 43: 959-964.
[45]牛炳韬.紫苏再生体系的建立及体细胞无性系变异分析[D].兰州大学硕士学位论文, 2005, 34-41.
[46]于淑玲,石晓云.紫苏组织培养体系的研究[J].邢台学院学报. 2008, 23(2): 118-119.
[47] Zhang T, Wang XY, Cao ZY. Plant regeneration in vitro directly from cotyledon andhypocotyl explants of Perilla frutescens and their morphological aspects[J]. Biologia Plantarum, 2005, 49(3): 423-426.
[48]黄海泉,张蕾,黄美娟.紫苏组织培养及植株再生[J].亚热带植物科学, 2007, 36(4): 56.
[49] Kim KH, Lee YH. Agrobacterium-mediated genetic transformation of Perilla frutescens[J]. Plant Cell Rep, 2004, 23: 386-390.
[50] Lee BK, Yu SH. Agrobacterium-mediated transformation of Perilla[J]. Plant Cell, 2005, 83: 51-58.
[51] Venketeswaran S, Gandhi V. Mass propagation and genetic improvement of forest trees for biomass production by tissue culture[J]. Biomass, 1982, 2(1): 5-15.
[52]沈世华,张秀君,郭奕明.玉米基因转化的离体子房注射及其转基因植株的鉴定[J].植物学报, 2001, 43(10): 1055-1057.
[53]何俊平,陈发棣,陈素梅.石蒜凝集素基因转化切花菊及抗蚜性鉴定[J].西北植物学报, 2009, 29(11): 2318-2325.
[54] Almeida WAB, Filho FAM, Pino LE. Genetic transformation and plant recovery from mature tissues of Citrus sinensis L.Osbeck[J]. Plant Science, 2003, 164(2): 203-211.
[55]张玉翠,高海波.杜兰组培快繁技术的研究[J].中国农学通报, 2005, 21(8): 306-307.
[56]陈振东,高海筹,蔡坤秀.南方芦笋组培苗移栽技术研究[J].中国农学通报, 2006, 22(11): 282-284.
[57]张陈明,胡宗利,陈国平.根癌农杆菌介导转化马铃薯与抗病毒基因工程[J].生物技术通报, 2008, (6): 30-35.
[58]刘晓庆.油菜、烟草耐盐转基因的研究[D].扬州大学硕士学位论文, 2009, 43-44.
[59]盖钧镒.试验统计方法[M].北京:中国农业出版社, 2000. 227-236.
[60]赵普庆,於维维,汪俏梅.生长素与其他信号之间的相互作用[J].植物生理学通讯, 2004, 40(2): 246-249.
[61]黄超,李玲.植物激素信号间的相互作用[J].亚热带植物科学, 2005, 34(4): 66-70.
[62]袁晶,汪俏梅,张海峰.植物激素信号之间的相互作用[J].细胞生物学杂志, 2005,(27): 325-328.
[63] Zhang T, Wang XY, Cao ZY. Plant regeneration in vitro directly from cotyledon and hypocotyl explants of Perilla frutescens and their morphological aspects[J]. Biologia Plantarum, 2005, 49(3): 423-426.
[64]孙在红,刘荣堂,梁慧敏等.植物激素在草坪草组织培养及植株再生中的应用与进展[J].草原与草坪, 2004, (2): 13-16.
[65] Predieri S, Malavasi F. High-frequency shoot regeneration from leaves of the apple rootstock M26[J]. Plant Cell, 1989, 17: 133-142.
[66]张志兰.山茶花组织培养过程中的防外植体褐化试验[J].西部林业科学, 2007, 36(2): 118-121.
[67]杜勤,王振华,徐鸿华等.石牌广藿香试剂苗的研制[J].中国中药杂志, 2002, 27(3): 179-181.
[68]徐培洲,吴先军,胡保民等.油葵子叶外植体不定芽再生体系的建立[J].中国油料作物学报, 2004, 26(4): 16-19.
[69]杜启兰.不同培养基与碳源对丽佳秋海棠组培苗的影响[J].安徽农业科学, 2006, 34(11): 2404-2405.
[70]赵巧阳,赖钟雄.硝酸银在离体培养和转化中的作用及其机理[J].亚热带农业研究, 2008, 4(1): 62-66.
[71]刘娟,汤浩茹,刘玲梅. AgNO3对梨叶片不定梢再生过程中抗氧化酶活性的影响[J].果树学报, 2008, 25(4): 467-472.
[72]蒋明义,杨文英,徐江等.渗透胁迫下水稻幼苗中叶绿素降解的活性氧损伤作用[J].植物学报, 1994, 36(4): 289-295.
[73]韩雪梅.草莓试管苗分化培养基优化的研究[J].生物技术, 1997, 7(2): 27-29.
[74]孙清荣,孙洪雁.不同倍性苹果叶片植株再生研究[J].落叶果树, 2000, (2): 9-11.
[75]赵爽,雷建军,陈国菊等.卡那霉素对大豆生在转基因芥菜中的应用[J].遗传, 2008, 30(4): 501-507.
[76] Satyavathi VV, Prasad V, Gita Lakshmi B, et al. High efficiency transformation on protocol for three Indian cotton varieties via Agrobacterinm tumefaciens[J]. PlantScience, 2002, 162(2): 215-223.
[77]袁鹰,刘德璞,王玉民等.卡那霉素对大豆生长的抑制及筛选试验研究[J].大豆科学, 2003, 22(4): 261-263.
[78]张明洲,崔海瑞,舒庆尧等.抗生素对高粱愈伤组织诱导和生长的影响[J].浙江大学学报, 2004, 30(2): 221-225.
[79]周小梅,李君剑,赵军良等.抗生素对农杆菌的抑制和对油菜外植体分化的影响[J].西北植物学报, 2005, 25(1): 52-56.