影响野葛幼叶悬浮细胞中葛根素等异黄酮类化合物产量的因素研究
|
摘要
野葛，Pueraria lobata(Willd.)Ohwi，富含葛根素、大豆苷、大豆苷元等异黄酮类化合物，在我国及其他东亚国家广泛用作医药业、食品业和轻工业原料。本研究以野葛幼叶愈伤组织建立悬浮细胞培养体系，研究了几种有机附加物、诱导子、有机溶剂等对野葛细胞生长和异黄酮类化合物生产的影响，为野葛细胞工程生产葛根素等异黄酮类化合物奠定了基础。
野葛未成熟种子萌发获得的无菌苗幼叶在含1mg／L NAA，2mg／L BA的固体培养基上诱导愈伤组织，愈伤组织在含0.5 mg／L 4-PU的MS固体培养基上继代形成结构疏松、生长迅速的淡黄色愈伤组织，接入含1mg／L 2,4-D，1mg／L NAA，0.5 mg／L KT，0.2％水解酪蛋白(CH)，20.g/L,蔗糖(简写为B_5+2,4-D_1+NAA_1+KT_(0.5)+0.2％CH)液体培养基中振荡培养建立了颗粒均一、分散良好、生长迅速的野葛幼叶细胞悬浮培养体系。悬浮培养条件为(25±2)℃、全天光照，光强5.34μmol·m~(-2)·s~(-1)，摇床转速110～130r／min。
野葛悬浮细胞的生物量在培养的第8d达最大值，为12.58mgDW／L，细胞产率为72.18％。悬浮细胞中葛根素的合成与细胞生长呈紧密偶联型，第8d时，细胞和培养液中的葛根素含量达最大值，分别为7.98mg／g DW和3.46mg／L，此时细胞和培养液中葛根素总产量为103.85mg／L。野葛悬浮细胞及培养液中总异黄酮类化合物积累的动力学曲线不同于葛根素积累的动力学曲线。野葛细胞接入新鲜培养液后，总异黄酮类化合物随细胞的生长而不断合成、积累，细胞进入稳定期后，总异黄酮类化合物的积累速度反而加快，第10d达最大值，为129.05mg／g DW，此时细胞和培养液中异黄酮类化合物的总产量为1.59g/L。野葛幼叶悬浮细胞中总异黄酮类化合物的最高含量超过野生葛根含量(68.7mg／gDW)，而葛根素最高含量则低于野生葛根含量(12.6 ms／g DW)。野葛幼叶悬浮细胞及培养液中葛根素的总含量占异黄酮类化合物总含量的9.22％。
过滤灭菌的椰乳和高压灭菌的椰乳的作用效应不同。10％(v／v)椰乳对野葛幼叶悬浮细胞的生长及次生代谢物的积累具有一定的抑制作用。0.2％的水解酪蛋白(CH)对野葛幼叶悬浮细胞的生长及葛根素和总异黄酮类化合物的积累和释放均有促进作用，可使葛根素和总异黄酮类化合物的产量分别提高34.05％和40.76％，因此野葛细胞悬浮培养宜用的培养基为B_5+2,4-D_1+NAA_1+KT_(0.5)+0.2％CH。
酵母提取物（YE）和脱乙酚几丁质（DC）对野葛幼叶悬浮细胞的生长均
无促进作用，较高浓度（0．5 g／L）的 DC处理较长时间6 d和 sd）抑制细胞的
生长。YE加到悬浮细胞培养液中后，引起细胞轻微褐化。YE不利于野葛悬浮
细胞中葛根素产量的提高。以 0刀 lgh DC处理 8 d对葛根素的增产效果最大（增
产28．40％人YE和DC对野葛幼叶悬浮细胞中异黄酮类化合物的积累及总产量
的提高均无促进作用。
SA、MJ和乙烯利对野葛幼叶悬浮细胞的生物量没有明显促进作用。SA和
MJ能引起细胞褐化，而乙烯利则能较好地保持细胞的活力。各种浓度的SA和
低浓度＊．ling／L和 lwt）的 MJ对葛根素总产量的影响不大，ling／L MJ
处理 8 d和 5 d略微促进葛根素的生产，而 0．ling／L乙烯利处理 3 d能显著提高
葛根素的总产量（增产27．37％）。SA、MJ和乙烯利对野葛悬浮细胞异黄酮类
化合物的生产没有促进作用。
浓度在1％－5％（v／V）范围内的DMSO即使处理5 d也不影响细胞的活力，浓
度在佃－5％（v／v）范围内的Tween－20和Titon X－100处理3 d内对细胞活力的影
响较小。三种有机溶剂均能促进细胞中葛根素及其它异黄酮类化合物的释放，但
均不利于葛根素的生产。5％（v／v）的Titon X－10o处理3d能够显著促进总异黄酮
类化合物产量的提高，增产率达40．56％。
Pueraria lobata (Willd.) Ohwi, rich in puerarin, daidzein, daidzine and other isoflavones, has been wildly used in China and other East Asia countries as resources in curatorial, food and light industries. In this study, the P. lobata cell suspension culture system was constructed from callus of seedling leaves. The effects of some organic appendices, elicitors and organic solutions on cell growth and isoflavones production were investigated. The purpose of the study is to provide technical support for P. lobata cell engineering to produce puerarin and other isoflavones.
Callus of P. lobata have been established on MS solid medium supplemented with 3% sucrose, 1.0 mg/L NAA, 2.0 mg/L BA using leaf explants of seedlings germinated from immature seeds. The callus grew fast and became yellowish and friable after subcultured on MS solid medium supplemented with 3% sucrose and 0.5 mg/L 4-PU. After these callus were inoculated and then subcultured for more than 8 times in B5 liquid medium containing 2% sucrose, 1.0 mg/L 2,4-D, 1.0 mg/L NAA, 0.5 mg/L KT and 0.2% CH, the cell cultures became homogeneous and grew fast. Hence the cell suspension culture system was established at (25?)癈, in 5.34 JJ mol ?m2 ?s"1 full day light, on gyratory shaker (110-130 r/min).
The maximal biomass accumulation of P. lobata cell suspension cultures was obtained on day 8 for 12.58 mg DW/L. Cell productivity was 72.18%. The puerarin production was closely coupled with cell cultures' growth. It reached highest on day 8 both in cell cultures and medium with 7.98 mg/g DW and 3.46 mg/L, respectively. The total yield of puerarin reached peak (103.85 mg/L) on day 8. The time-courses of total isoflavones accumulation in P. lobata cell cultures and medium were different from those of puerarin. It coupled with cell cultures growth on the first 8 days and then became faster in cell stable growth phase. On day 10, it reached highest with 129.05 mg/g DW in cell cultures, and 1.59 g/L in cell cultures and medium, totaly. This content of total isoflavones in cell cultures surpassed the content in wild root of P. lobata that was 68.7 mg/g DW, while the content of
puerarin was lower than that in wild root of P. lobata that was 12.6 mg/g DW. In P. lobata cell cultures and medium, the total content of puerarin occupied 9.22% of total isoflavones.
The effect of coconut juice sterilized by autoclaving was different from that sterilized by filtration. 10% coconut juice restrained the growth of cell cultures and the accumulation of total isoflavones, while 0.2% casin acid hydrolysates (CH) promoted the growth of cell cultures and the accumulation and release of puerarin and total isoflavones. The total yield of puerarin and isoflavones were 34.05% and 40.76% highter than control, respectively. It was concluded that the optimizing medium for cell cultures in leaves of P. lobata seedlings was 85 liquid medium supplemented with 2% sucrose, 1.0 mg/L 2,4-D, 1.0 mg/L NAA, 0.5 mg/L KT and 0.2% CH.
Both yeast extract (YE) and deacetylated chitin (DC) had no promotion effects on the growth of P. lobata cell cultures. 0.5 g/L DC restrained the cell growth when treated for 5 d and 8 d. YE could cause the cell brown slightly, and didn't benefit the production of puerarin. 0.01 g/L DC improved the total yield of puerarin over 28.40% after treatment of 8 days. YE and DC had no effects on production of total isoflavones.
SA, MJ and ethrel had no promotion effects on the biomass accumulation of P. lobata cell cultures. SA and MJ both caused the cell brown, while ethylene had no negative effect on cell vitality. 0.1-5 mg/L SA and 0.1 mg/L MJ had not much effects on the production of puerarin. 1 mg/L MJ improved the yield of puerarin slightly after 8 d and 5 d of elicitation, while 0.1 mg/L ethrel had a better effect which improved 27.37% higher than control after treatment of 3 days. SAs MJ and ethrel had no effects on improving the yield of total isoflavones in cell cultures and medium.
DMSO at concentration 1%~5% had no effect on cell vitality even treated for 5 d
野葛未成熟种子萌发获得的无菌苗幼叶在含1mg／L NAA，2mg／L BA的固体培养基上诱导愈伤组织，愈伤组织在含0.5 mg／L 4-PU的MS固体培养基上继代形成结构疏松、生长迅速的淡黄色愈伤组织，接入含1mg／L 2,4-D，1mg／L NAA，0.5 mg／L KT，0.2％水解酪蛋白(CH)，20.g/L,蔗糖(简写为B_5+2,4-D_1+NAA_1+KT_(0.5)+0.2％CH)液体培养基中振荡培养建立了颗粒均一、分散良好、生长迅速的野葛幼叶细胞悬浮培养体系。悬浮培养条件为(25±2)℃、全天光照，光强5.34μmol·m~(-2)·s~(-1)，摇床转速110～130r／min。
野葛悬浮细胞的生物量在培养的第8d达最大值，为12.58mgDW／L，细胞产率为72.18％。悬浮细胞中葛根素的合成与细胞生长呈紧密偶联型，第8d时，细胞和培养液中的葛根素含量达最大值，分别为7.98mg／g DW和3.46mg／L，此时细胞和培养液中葛根素总产量为103.85mg／L。野葛悬浮细胞及培养液中总异黄酮类化合物积累的动力学曲线不同于葛根素积累的动力学曲线。野葛细胞接入新鲜培养液后，总异黄酮类化合物随细胞的生长而不断合成、积累，细胞进入稳定期后，总异黄酮类化合物的积累速度反而加快，第10d达最大值，为129.05mg／g DW，此时细胞和培养液中异黄酮类化合物的总产量为1.59g/L。野葛幼叶悬浮细胞中总异黄酮类化合物的最高含量超过野生葛根含量(68.7mg／gDW)，而葛根素最高含量则低于野生葛根含量(12.6 ms／g DW)。野葛幼叶悬浮细胞及培养液中葛根素的总含量占异黄酮类化合物总含量的9.22％。
过滤灭菌的椰乳和高压灭菌的椰乳的作用效应不同。10％(v／v)椰乳对野葛幼叶悬浮细胞的生长及次生代谢物的积累具有一定的抑制作用。0.2％的水解酪蛋白(CH)对野葛幼叶悬浮细胞的生长及葛根素和总异黄酮类化合物的积累和释放均有促进作用，可使葛根素和总异黄酮类化合物的产量分别提高34.05％和40.76％，因此野葛细胞悬浮培养宜用的培养基为B_5+2,4-D_1+NAA_1+KT_(0.5)+0.2％CH。
酵母提取物（YE）和脱乙酚几丁质（DC）对野葛幼叶悬浮细胞的生长均
无促进作用，较高浓度（0．5 g／L）的 DC处理较长时间6 d和 sd）抑制细胞的
生长。YE加到悬浮细胞培养液中后，引起细胞轻微褐化。YE不利于野葛悬浮
细胞中葛根素产量的提高。以 0刀 lgh DC处理 8 d对葛根素的增产效果最大（增
产28．40％人YE和DC对野葛幼叶悬浮细胞中异黄酮类化合物的积累及总产量
的提高均无促进作用。
SA、MJ和乙烯利对野葛幼叶悬浮细胞的生物量没有明显促进作用。SA和
MJ能引起细胞褐化，而乙烯利则能较好地保持细胞的活力。各种浓度的SA和
低浓度＊．ling／L和 lwt）的 MJ对葛根素总产量的影响不大，ling／L MJ
处理 8 d和 5 d略微促进葛根素的生产，而 0．ling／L乙烯利处理 3 d能显著提高
葛根素的总产量（增产27．37％）。SA、MJ和乙烯利对野葛悬浮细胞异黄酮类
化合物的生产没有促进作用。
浓度在1％－5％（v／V）范围内的DMSO即使处理5 d也不影响细胞的活力，浓
度在佃－5％（v／v）范围内的Tween－20和Titon X－100处理3 d内对细胞活力的影
响较小。三种有机溶剂均能促进细胞中葛根素及其它异黄酮类化合物的释放，但
均不利于葛根素的生产。5％（v／v）的Titon X－10o处理3d能够显著促进总异黄酮
类化合物产量的提高，增产率达40．56％。
Pueraria lobata (Willd.) Ohwi, rich in puerarin, daidzein, daidzine and other isoflavones, has been wildly used in China and other East Asia countries as resources in curatorial, food and light industries. In this study, the P. lobata cell suspension culture system was constructed from callus of seedling leaves. The effects of some organic appendices, elicitors and organic solutions on cell growth and isoflavones production were investigated. The purpose of the study is to provide technical support for P. lobata cell engineering to produce puerarin and other isoflavones.
Callus of P. lobata have been established on MS solid medium supplemented with 3% sucrose, 1.0 mg/L NAA, 2.0 mg/L BA using leaf explants of seedlings germinated from immature seeds. The callus grew fast and became yellowish and friable after subcultured on MS solid medium supplemented with 3% sucrose and 0.5 mg/L 4-PU. After these callus were inoculated and then subcultured for more than 8 times in B5 liquid medium containing 2% sucrose, 1.0 mg/L 2,4-D, 1.0 mg/L NAA, 0.5 mg/L KT and 0.2% CH, the cell cultures became homogeneous and grew fast. Hence the cell suspension culture system was established at (25?)癈, in 5.34 JJ mol ?m2 ?s"1 full day light, on gyratory shaker (110-130 r/min).
The maximal biomass accumulation of P. lobata cell suspension cultures was obtained on day 8 for 12.58 mg DW/L. Cell productivity was 72.18%. The puerarin production was closely coupled with cell cultures' growth. It reached highest on day 8 both in cell cultures and medium with 7.98 mg/g DW and 3.46 mg/L, respectively. The total yield of puerarin reached peak (103.85 mg/L) on day 8. The time-courses of total isoflavones accumulation in P. lobata cell cultures and medium were different from those of puerarin. It coupled with cell cultures growth on the first 8 days and then became faster in cell stable growth phase. On day 10, it reached highest with 129.05 mg/g DW in cell cultures, and 1.59 g/L in cell cultures and medium, totaly. This content of total isoflavones in cell cultures surpassed the content in wild root of P. lobata that was 68.7 mg/g DW, while the content of
puerarin was lower than that in wild root of P. lobata that was 12.6 mg/g DW. In P. lobata cell cultures and medium, the total content of puerarin occupied 9.22% of total isoflavones.
The effect of coconut juice sterilized by autoclaving was different from that sterilized by filtration. 10% coconut juice restrained the growth of cell cultures and the accumulation of total isoflavones, while 0.2% casin acid hydrolysates (CH) promoted the growth of cell cultures and the accumulation and release of puerarin and total isoflavones. The total yield of puerarin and isoflavones were 34.05% and 40.76% highter than control, respectively. It was concluded that the optimizing medium for cell cultures in leaves of P. lobata seedlings was 85 liquid medium supplemented with 2% sucrose, 1.0 mg/L 2,4-D, 1.0 mg/L NAA, 0.5 mg/L KT and 0.2% CH.
Both yeast extract (YE) and deacetylated chitin (DC) had no promotion effects on the growth of P. lobata cell cultures. 0.5 g/L DC restrained the cell growth when treated for 5 d and 8 d. YE could cause the cell brown slightly, and didn't benefit the production of puerarin. 0.01 g/L DC improved the total yield of puerarin over 28.40% after treatment of 8 days. YE and DC had no effects on production of total isoflavones.
SA, MJ and ethrel had no promotion effects on the biomass accumulation of P. lobata cell cultures. SA and MJ both caused the cell brown, while ethylene had no negative effect on cell vitality. 0.1-5 mg/L SA and 0.1 mg/L MJ had not much effects on the production of puerarin. 1 mg/L MJ improved the yield of puerarin slightly after 8 d and 5 d of elicitation, while 0.1 mg/L ethrel had a better effect which improved 27.37% higher than control after treatment of 3 days. SAs MJ and ethrel had no effects on improving the yield of total isoflavones in cell cultures and medium.
DMSO at concentration 1%~5% had no effect on cell vitality even treated for 5 d
引文
万平，宛晓春，杨晓春，陆翠珍，稽跃琴．葛根黄酮生物合成的研究．安徽农业大学学报，1997，24(2)：197～199
于树宏，李玲，刘传飞，潘瑞炽．发根农杆菌R1000对野葛的遗传转化及其影响因素．高技术通讯，2002，12(3)：34～37
于树宏，李玲．野葛的组织培养和植株再生．植物资源与环境，1999，8(1)：63～64
于树宏．野葛组织培养、遗传转化及毛状根中异黄酮的产生．华南师范大学硕士学位论文．广州，1999
于荣敏，赵鸿莲，张辉，姚新生．银杏细胞悬浮培养及其银杏内酯产生的研究．生物工程学报，1999，15(2)：207～210
王宁宁，王淑芳，王勇，田俊英，朱亮基．外源刺激物对长春花冠瘿细胞生长和吲哚生物碱含量的影响．生物工程学报，1996，12(增刊)：300～303
王红，叶和春，李国凤．诱导子的作用方式及其在植物组织培养中的应用．植物学通报，1999，16(1)：11～18
王琢成．2000年—2001年1季度化学药品产销回顾分析．中国药房，2001，12(7)：390～393
甘烦远，郑光植，彭丽萍．欧洲红豆杉细胞培养的研究．应用与环境生物学报，1996，2(3)：220～224
甘烦远，郑光植，彭丽萍，罗建平．云南红豆杉细胞的悬浮培养．植物生理学报，1997，23(1)：43～46
石瑛，杜昱光，白雪芳．寡糖素诱导的植物抗性信号转导．生命科学研究，2001，5(1)：29～32
刘传飞，于树宏，李玲，施和平，潘瑞炽．发根农杆菌对葛属药用植物的遗传转化，植物学报，2000a，42(9)：936～939
刘传飞，李玲，施和平，潘瑞炽，于树宏．野葛毛状根的诱导及离体培养．中国中药杂志，2000b，25(9)525～527
刘传飞，李玲，潘瑞炽，金乐红．发根农杆菌T-DNA对三种葛属植物形态和葛根素含量的影响。应用与环境生物学报，2001，7(2)：143～146
刘进平，郑成木，胡新文．体细胞无性系变异研究进展．华南热带农业大学学报，2001，7(2)：22～29
朱西儒，雷光富．药用植物细胞培养代谢全能性及产物检测技术的研究进展．广西科学院学报，1998，14(2)：1～6
刘松，钟建江，张以恒，孟宪丹．渗透处理三七细胞促使人参皂甙的有效释放．华东理工大学学报，1997，23(6)684～687
孙禄．葛的利用价值．特种经济动植物，2001，4(2)：24～25
刘慧丽．野葛细胞培养及其异黄酮类物质的产生．华南师范大学硕士学位论文．广州，2001
张长河，余龙江，梅兴国，李亚莉．Chitosan对红豆杉PAL及Taxol合成的影响．华中理工大学学报，1999，27(4)：104～106
余龙江，朱敏，刘幸福，周莹．茉莉酸甲酯对中国红豆杉胚性细胞紫杉醇生物合成的影响．武汉植物学研究，1999a，17(4)：371～374
余龙江，朱敏，周莹，柯铁，梅兴国．茉莉酸甲酯对紫杉醇生物合成的诱导作用．天然产物研究与开发，1999b，11(5)：1～7
余龙江，李为，梅兴国，吴元喜．代谢途径的调节对紫杉醇生物合成的促进作用．华中理工大学学报，1999，27(4)：100～103
李弘剑，张毅，郭勇，姚汝华．黄花蒿培养细胞中青蒿素合成代谢的体外调节．中
国生物化学与分子生物学报，1999，15(3)：479～483
张立莹，刘丽萍，贾景明，郝泗城，刘世强．东北红豆杉(Taxus cuspidata)细胞悬浮培养研究．沈阳农业大学学报，1997，28(3)：180～185
张学昆，唐章林，谌利，郭益红，陈云坪，李加纳．脱乙酰几丁质的乙酰化程度对诱导油菜抗性的影响．中国农业科学，2002，35(3)：287～290
李国婧，周燮．植物防御反应中水杨酸与茉莉酸的“对话”机制．细胞生物学杂志，2002，24(2)：101～105
应佩清，许建峰，苏志国．丹参冠瘿组织培养特性及培养动力学研究．应用与环境生物学报，1999，5(5)：478～482
何海金，陈西仓．葛藤的综合利用．特种经济动植物，2002，5(3)：30
苏湘鄂，梅兴国，龚伟，张舟宁．茉莉酸甲酯与真菌诱导物、水杨酸组合对红豆杉细胞中紫杉醇含量的影响．生物技术，2001，11(1)：10～13
张毅，李弘剑，郭勇，姚汝华．大蒜细胞悬浮培养中细胞生长与产酶的研究．华南理工大学学报(自然科学版)，1994，22(2)：67～73
杨宁．胡萝卜细胞培养生产胡萝卜素的研究．华南理工大学发酵工程专业工学硕士学位论文．1993
杨玉敏，元英进，胡宗定．植物细胞胞内产物释放行为研究进展．生物工程进展，1998，18(1)：17～21
欧阳藩，曹竹安．动植物细胞培养．化学进展，1995，7(3)：181～192
苗志奇，未作君，元英进．水杨酸在紫杉醇生物合成中诱导作用的研究．生物工程学报，2000a，16(4)：510～513
苗志奇，未作君，元英进．紫杉醇合成中甲基茉莉酮酸作用位点与配伍特性研究．生物物理学报，2000b，16(2)：204～211
周忠强，梅兴国．水杨酸对细胞培养生产紫杉烷的影响．天然产物研究与开发，2002，14(2)：11～13
周忠强，梅兴国，吴奇君．前体、诱导子及抑制剂对细胞培养生产紫杉醇的调节作用．天然产物研究与开发，2002，14(2)：19～21
赵心清，张冬艳，李宁，白凤武．植物细胞代谢产物的诱导释放．应用与环境生物学报，2000，6(3)：284～287
赵云强，方伊．甲壳素、壳聚糖的综合应用及其发展前景．贵州化工，2001，26 (1) 10～13
钦传光，李世杰，丁焰，林向东．发酵工程在医药研究和生产中的应用．湖北工学院学报，2000，15(1)：67～70
胡江琴，王利琳，余象煜．葛粉的化学成分分析．杭州师范学院学报，1999，16(3)：70～73
徐进，张卫明，马世宏，吴国荣．葛根在化妆品中的应用初探．中国野生植物资源，2000，19(3)：11～13
袁丽红，周名立，欧阳平凯．表面活性剂Tween20对紫草细胞生产紫草色素的影响．南京化工学院学报，1994，16(3)：16～19
袁丽红，欧阳平凯．产物释放促进剂对固定化紫草细胞生产紫草色素的影响．南京化工大学学报，1999，21(3)：44～47
袁丽红，欧阳平剀．提高西洋参细胞生长和皂苷生产的几种方法研究．南京化工学院学报，1994，16(1)：72～77
唐巍，吴绛云．人参悬浮细胞系的建立及其生长特性的研究．生物技术，1994，4(1)：26～29
梅乐和，姚善经，林东强．生化生产工艺学．北京：科学出版社．1999：233
梅兴国，张舟宁，苏湘鄂，龚伟．水杨酸对红豆杉细胞的诱导作用．生物技术，2000，10(6)：18～20
常钰，刘涤，胡之璧．植物细胞和器官大规模培养研究的进展．生物技术通报，2001，(1)：31～36
曾明，张汉明，郑水庆，苏中武．葛属植物根的异黄酮类成分分析．第二军医大学学报，1998，19(2)：?
谢秋玲，郭勇．刺激剂(elicitor)在植物细胞培养次生代谢物生产中的应用．广西植物，1999，19(2)：146～149
崔堂兵，郭勇，林炜铁．提高植物细胞培养法生产次级代谢物产量的方法．植物生理学通讯，2001，37(5)：479～482
彭靖里，马敏象，安华轩，刘建中．论葛属植物的开发及综合利用前景．资源开发与市场，2000，16(2)：80～82
霍丹群，候长军．葛根保健食品的开发及利用．资源开发与市场，2000，16(1)：27～28
甄健存，陈志刚．葛根素注射液上市后产品质量比较．中国新药杂志，1999，8(12)：819～821
潘瑞炽．植物生理学．第四版．北京：高等教育出版社，2001a：123-141，182，295
潘瑞炽．植物组织培养．第二版．广州：广东高等教育出版社，2001b：23
Brodelius P. Appl Microbiol Biotechnol, 1988, 27:561～566
Brodelius P, Nilsson K. Permeabilization of immobilized plant cells, resulting in
release of intracelluarly stored products with preserved cell viability. Appl MicrolBiotechol, 1983, 17:275～280
Chen H, Chen F. Effect of yeast elicitor on the secondary metabolism of Ti-transformed Salvia miltiorrhiza cell suspension cultures. Plant Cell Reports, 2000,19 (7): 710～717
Chio JJ, Klosterman SJ, Hadwiger La. A comparison of the effects of DNA-damaging agents and biotic elicitors on the induction of plant defense genes, nuclear distortion, and cell death. Plant Physiology, 2001, 125 (2):752～762
Chiou SY; Su WW; Su YC. Optimizing production of polyunsaturated fatty acids in Marchantia polymorpha cell suspension culture. J Biotechnol, 2001, 85 (3),247～257
Cho GH, Kim DJ, Pedersen H, et al. Ethephon enhancement of secondary metabolite synthesis in plant cell culture. Biotechnol Prog, 1988, 4:184～188
Dai JG, Gong Z, Zhu DM, Guo HZ, Zhang JH, Guo DA. Biotransformation of gastrodin by cell suspension cultures of Catharanthus roseus. Acta Botanica Sinica, 2002, 44(3): 377～378
Felix H, Brodelius P, Mosbach K. Analytical Biochemistry, 1981, 116:462～470
Gamborg OL, Miller RA, Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res, 1968, 50:151～158
Hakamatsuka T, Mori K, Ishida S, Ebizuka Y, Sankawa U. Purification of 2-hydroxyisoflanone dehydratase from the cell cultures of Purearia lobata: In honour of Professor G. H. NEIL Towers 75th birthday. Phytochemistry, 1998, 49(2): 497-505
Hakamatsaka T, Noguchi H, Ebizuka Y, Sankawa U. Deoxychalcone synthase from cell suspension cultures ofPueraria lobata. Chem Pharm Bull, 1988, 36(10): 4225～4228
Hammerschmidt R. Induces disease resistance: how do induced plants stop
pathogens?. Physiol Mol Plant Pathol, 1999, 55: 77-84
Ketchum RE; Gibson DM; Croteau RB; Shuler ML. The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate. Biotechnol Bioeng, 1999, 62(1) ,: 97-105
Kim DI, Pedersen H, Chin CK. Cultivation of Thalictrum rugosum cell suspension in an improved airlift bioreactor: Stimulatory effect of carbon dioxide and ethylene on alkaloid production. Biotechnol Bioeng, 1991, 38: 331-339
Knorr D, Berlin J. Effects of immobilization and permeabilization procedures on growth of Chenopodium rubrum cells and amaranthin concentration. J Food Sci, 1987, 52(5) : 1397-1400
Kobayashi Y, Fukui H, Tabata M. Effect on carbon dioxide and ethylene on berberine production and cell browning in Thalictrum minus cell cultures. Plant Cell Report, 1991, 9: 496-499
Mirjalili N, Linden JC. Methyl jasmonate induced production of taxol in suspension cultures of Taxus cuspidata: ethylene interaction and induction models. Biotechnol Prog, 1996, 12: 110-118
Mori T, Sakurai M, Sakyta M. Changes of PAL, CHS, DAHP synthase (DS-Co and DS-Mn) activity during anthocyanin synthesis in suspension culture of Fragaria ananassa. Plant Cell, Tissue and Organ Culture, 2000, 62: 135-139
Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Planta, 1962,15: 473-497
Nakajima O, Akiyama T, Hakamatsuka T, ShibuyaM, Noguchi H, Ebizuka Y, Sankawa U. Isolation, sequence and bacterial expression of a cDNA for chalcone synthase from the cultured cells of Pueraria lobata. Chem Pharm Bull (Tokyo), 1991, 39(7) : 1911-1913
Park CH, Martinez BC. Enhaneed release of rosmarinic acid from Coleus blumei permeabilized by dimethy sufoxide (DMSO) while preserving cell viability and growth. Biotech Bioeng, 1992, 40: 459-464
Park HH, Hakamatsuka T, Sankawa U, Ebizuka Y. Rapid metabolism of isoflavonoids in elicitor-treated cell suspension cultures of Pueraria lobata. Phytochemistry, 1995, 38(2) : 373-380
Schlatmann JE, Fonck E, ten Hoopen HJG, Heijnen JJ. The negligible role of carbon dioxide and ethylene in ajmalicine production by Catharanthus roseus cell suspensions. Plant Cell Report, 1994, 14(2-3) : 157-160
Smith HB. Signal transduction in systemic acquired resistance. Plant Cell, 2000, 12(2) : 179-180
Susko DJ, Mueller JP, Spears JF. An evaluation of methods for breaking seed dormancy in kudzu (Pueraria lobata). Canadian Journal of Botany, 2001, 79(2) : 197-203
Szabo E, Thelen A, Petersen M. Fungal elicitor preparations and methyl jasmonate enhance rosmarinnic acid accumulation in suspension cultures of Coleus blumei. Plant Cell Reports, 1999, 18: 485-489
Takeya K, Itokawa H. Isoflavoids and the other constituents in callus tissue of Pueraria lobata,Chem Pharm Bull, 1982, 30(4) : 1496-1499
Ton J, van Pelt JA, van Loon LC, Pieterse CMJ. Differential effectiveness of salicylate-dependent and jasmonate/ethylene-dependent induced resistance in Arabidopsis. Molecular Plant-Microbe Interactions, 2002, 15 (1) : 27-34
Undberg PI, Linsefors L, Vogel HJ et al. Plant Cell Reports, 1986, 5: 13-15
van der Plas LHW, Eijkelboom C, Hagendoorn MJM. Relation between primary and secondary metabolism in plant cell suspensions. Competition between secondary metabolite production and growth in a model system (Morinda citrifolia). Plant Cell, Tissue and Organ Culture, 1995, 43: 111-116
Verpoorte R, van der Heijden R, the Hoopen HJG, Memelink J. Metablic engineering of plant secondary metabolite pathways for the production of fine chemicals. Biotechnology Letters, 1999, 21: 467-479
Yang Y, Shah J, Klessing DF. Signal perception and transduction in plant defense responses. Genes Dev, 1997, 11: 1621-1639
Yeoman MM, Yeoman CL. Manipulating secondary metabolism in cultured plant cells. New Phytol, 1996, 134: 553-569
Yukimune Y, Tabata H, Higashi Y, Kara Y. Methyl jasmonate-induced over production of paclitaxel and baccatin in Taxus cell suspension cultures. Nature Biotechnology, 1996, 14(9) : 1129-1132
于树宏，李玲，刘传飞，潘瑞炽．发根农杆菌R1000对野葛的遗传转化及其影响因素．高技术通讯，2002，12(3)：34～37
于树宏，李玲．野葛的组织培养和植株再生．植物资源与环境，1999，8(1)：63～64
于树宏．野葛组织培养、遗传转化及毛状根中异黄酮的产生．华南师范大学硕士学位论文．广州，1999
于荣敏，赵鸿莲，张辉，姚新生．银杏细胞悬浮培养及其银杏内酯产生的研究．生物工程学报，1999，15(2)：207～210
王宁宁，王淑芳，王勇，田俊英，朱亮基．外源刺激物对长春花冠瘿细胞生长和吲哚生物碱含量的影响．生物工程学报，1996，12(增刊)：300～303
王红，叶和春，李国凤．诱导子的作用方式及其在植物组织培养中的应用．植物学通报，1999，16(1)：11～18
王琢成．2000年—2001年1季度化学药品产销回顾分析．中国药房，2001，12(7)：390～393
甘烦远，郑光植，彭丽萍．欧洲红豆杉细胞培养的研究．应用与环境生物学报，1996，2(3)：220～224
甘烦远，郑光植，彭丽萍，罗建平．云南红豆杉细胞的悬浮培养．植物生理学报，1997，23(1)：43～46
石瑛，杜昱光，白雪芳．寡糖素诱导的植物抗性信号转导．生命科学研究，2001，5(1)：29～32
刘传飞，于树宏，李玲，施和平，潘瑞炽．发根农杆菌对葛属药用植物的遗传转化，植物学报，2000a，42(9)：936～939
刘传飞，李玲，施和平，潘瑞炽，于树宏．野葛毛状根的诱导及离体培养．中国中药杂志，2000b，25(9)525～527
刘传飞，李玲，潘瑞炽，金乐红．发根农杆菌T-DNA对三种葛属植物形态和葛根素含量的影响。应用与环境生物学报，2001，7(2)：143～146
刘进平，郑成木，胡新文．体细胞无性系变异研究进展．华南热带农业大学学报，2001，7(2)：22～29
朱西儒，雷光富．药用植物细胞培养代谢全能性及产物检测技术的研究进展．广西科学院学报，1998，14(2)：1～6
刘松，钟建江，张以恒，孟宪丹．渗透处理三七细胞促使人参皂甙的有效释放．华东理工大学学报，1997，23(6)684～687
孙禄．葛的利用价值．特种经济动植物，2001，4(2)：24～25
刘慧丽．野葛细胞培养及其异黄酮类物质的产生．华南师范大学硕士学位论文．广州，2001
张长河，余龙江，梅兴国，李亚莉．Chitosan对红豆杉PAL及Taxol合成的影响．华中理工大学学报，1999，27(4)：104～106
余龙江，朱敏，刘幸福，周莹．茉莉酸甲酯对中国红豆杉胚性细胞紫杉醇生物合成的影响．武汉植物学研究，1999a，17(4)：371～374
余龙江，朱敏，周莹，柯铁，梅兴国．茉莉酸甲酯对紫杉醇生物合成的诱导作用．天然产物研究与开发，1999b，11(5)：1～7
余龙江，李为，梅兴国，吴元喜．代谢途径的调节对紫杉醇生物合成的促进作用．华中理工大学学报，1999，27(4)：100～103
李弘剑，张毅，郭勇，姚汝华．黄花蒿培养细胞中青蒿素合成代谢的体外调节．中
国生物化学与分子生物学报，1999，15(3)：479～483
张立莹，刘丽萍，贾景明，郝泗城，刘世强．东北红豆杉(Taxus cuspidata)细胞悬浮培养研究．沈阳农业大学学报，1997，28(3)：180～185
张学昆，唐章林，谌利，郭益红，陈云坪，李加纳．脱乙酰几丁质的乙酰化程度对诱导油菜抗性的影响．中国农业科学，2002，35(3)：287～290
李国婧，周燮．植物防御反应中水杨酸与茉莉酸的“对话”机制．细胞生物学杂志，2002，24(2)：101～105
应佩清，许建峰，苏志国．丹参冠瘿组织培养特性及培养动力学研究．应用与环境生物学报，1999，5(5)：478～482
何海金，陈西仓．葛藤的综合利用．特种经济动植物，2002，5(3)：30
苏湘鄂，梅兴国，龚伟，张舟宁．茉莉酸甲酯与真菌诱导物、水杨酸组合对红豆杉细胞中紫杉醇含量的影响．生物技术，2001，11(1)：10～13
张毅，李弘剑，郭勇，姚汝华．大蒜细胞悬浮培养中细胞生长与产酶的研究．华南理工大学学报(自然科学版)，1994，22(2)：67～73
杨宁．胡萝卜细胞培养生产胡萝卜素的研究．华南理工大学发酵工程专业工学硕士学位论文．1993
杨玉敏，元英进，胡宗定．植物细胞胞内产物释放行为研究进展．生物工程进展，1998，18(1)：17～21
欧阳藩，曹竹安．动植物细胞培养．化学进展，1995，7(3)：181～192
苗志奇，未作君，元英进．水杨酸在紫杉醇生物合成中诱导作用的研究．生物工程学报，2000a，16(4)：510～513
苗志奇，未作君，元英进．紫杉醇合成中甲基茉莉酮酸作用位点与配伍特性研究．生物物理学报，2000b，16(2)：204～211
周忠强，梅兴国．水杨酸对细胞培养生产紫杉烷的影响．天然产物研究与开发，2002，14(2)：11～13
周忠强，梅兴国，吴奇君．前体、诱导子及抑制剂对细胞培养生产紫杉醇的调节作用．天然产物研究与开发，2002，14(2)：19～21
赵心清，张冬艳，李宁，白凤武．植物细胞代谢产物的诱导释放．应用与环境生物学报，2000，6(3)：284～287
赵云强，方伊．甲壳素、壳聚糖的综合应用及其发展前景．贵州化工，2001，26 (1) 10～13
钦传光，李世杰，丁焰，林向东．发酵工程在医药研究和生产中的应用．湖北工学院学报，2000，15(1)：67～70
胡江琴，王利琳，余象煜．葛粉的化学成分分析．杭州师范学院学报，1999，16(3)：70～73
徐进，张卫明，马世宏，吴国荣．葛根在化妆品中的应用初探．中国野生植物资源，2000，19(3)：11～13
袁丽红，周名立，欧阳平凯．表面活性剂Tween20对紫草细胞生产紫草色素的影响．南京化工学院学报，1994，16(3)：16～19
袁丽红，欧阳平凯．产物释放促进剂对固定化紫草细胞生产紫草色素的影响．南京化工大学学报，1999，21(3)：44～47
袁丽红，欧阳平剀．提高西洋参细胞生长和皂苷生产的几种方法研究．南京化工学院学报，1994，16(1)：72～77
唐巍，吴绛云．人参悬浮细胞系的建立及其生长特性的研究．生物技术，1994，4(1)：26～29
梅乐和，姚善经，林东强．生化生产工艺学．北京：科学出版社．1999：233
梅兴国，张舟宁，苏湘鄂，龚伟．水杨酸对红豆杉细胞的诱导作用．生物技术，2000，10(6)：18～20
常钰，刘涤，胡之璧．植物细胞和器官大规模培养研究的进展．生物技术通报，2001，(1)：31～36
曾明，张汉明，郑水庆，苏中武．葛属植物根的异黄酮类成分分析．第二军医大学学报，1998，19(2)：?
谢秋玲，郭勇．刺激剂(elicitor)在植物细胞培养次生代谢物生产中的应用．广西植物，1999，19(2)：146～149
崔堂兵，郭勇，林炜铁．提高植物细胞培养法生产次级代谢物产量的方法．植物生理学通讯，2001，37(5)：479～482
彭靖里，马敏象，安华轩，刘建中．论葛属植物的开发及综合利用前景．资源开发与市场，2000，16(2)：80～82
霍丹群，候长军．葛根保健食品的开发及利用．资源开发与市场，2000，16(1)：27～28
甄健存，陈志刚．葛根素注射液上市后产品质量比较．中国新药杂志，1999，8(12)：819～821
潘瑞炽．植物生理学．第四版．北京：高等教育出版社，2001a：123-141，182，295
潘瑞炽．植物组织培养．第二版．广州：广东高等教育出版社，2001b：23
Brodelius P. Appl Microbiol Biotechnol, 1988, 27:561～566
Brodelius P, Nilsson K. Permeabilization of immobilized plant cells, resulting in
release of intracelluarly stored products with preserved cell viability. Appl MicrolBiotechol, 1983, 17:275～280
Chen H, Chen F. Effect of yeast elicitor on the secondary metabolism of Ti-transformed Salvia miltiorrhiza cell suspension cultures. Plant Cell Reports, 2000,19 (7): 710～717
Chio JJ, Klosterman SJ, Hadwiger La. A comparison of the effects of DNA-damaging agents and biotic elicitors on the induction of plant defense genes, nuclear distortion, and cell death. Plant Physiology, 2001, 125 (2):752～762
Chiou SY; Su WW; Su YC. Optimizing production of polyunsaturated fatty acids in Marchantia polymorpha cell suspension culture. J Biotechnol, 2001, 85 (3),247～257
Cho GH, Kim DJ, Pedersen H, et al. Ethephon enhancement of secondary metabolite synthesis in plant cell culture. Biotechnol Prog, 1988, 4:184～188
Dai JG, Gong Z, Zhu DM, Guo HZ, Zhang JH, Guo DA. Biotransformation of gastrodin by cell suspension cultures of Catharanthus roseus. Acta Botanica Sinica, 2002, 44(3): 377～378
Felix H, Brodelius P, Mosbach K. Analytical Biochemistry, 1981, 116:462～470
Gamborg OL, Miller RA, Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res, 1968, 50:151～158
Hakamatsuka T, Mori K, Ishida S, Ebizuka Y, Sankawa U. Purification of 2-hydroxyisoflanone dehydratase from the cell cultures of Purearia lobata: In honour of Professor G. H. NEIL Towers 75th birthday. Phytochemistry, 1998, 49(2): 497-505
Hakamatsaka T, Noguchi H, Ebizuka Y, Sankawa U. Deoxychalcone synthase from cell suspension cultures ofPueraria lobata. Chem Pharm Bull, 1988, 36(10): 4225～4228
Hammerschmidt R. Induces disease resistance: how do induced plants stop
pathogens?. Physiol Mol Plant Pathol, 1999, 55: 77-84
Ketchum RE; Gibson DM; Croteau RB; Shuler ML. The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate. Biotechnol Bioeng, 1999, 62(1) ,: 97-105
Kim DI, Pedersen H, Chin CK. Cultivation of Thalictrum rugosum cell suspension in an improved airlift bioreactor: Stimulatory effect of carbon dioxide and ethylene on alkaloid production. Biotechnol Bioeng, 1991, 38: 331-339
Knorr D, Berlin J. Effects of immobilization and permeabilization procedures on growth of Chenopodium rubrum cells and amaranthin concentration. J Food Sci, 1987, 52(5) : 1397-1400
Kobayashi Y, Fukui H, Tabata M. Effect on carbon dioxide and ethylene on berberine production and cell browning in Thalictrum minus cell cultures. Plant Cell Report, 1991, 9: 496-499
Mirjalili N, Linden JC. Methyl jasmonate induced production of taxol in suspension cultures of Taxus cuspidata: ethylene interaction and induction models. Biotechnol Prog, 1996, 12: 110-118
Mori T, Sakurai M, Sakyta M. Changes of PAL, CHS, DAHP synthase (DS-Co and DS-Mn) activity during anthocyanin synthesis in suspension culture of Fragaria ananassa. Plant Cell, Tissue and Organ Culture, 2000, 62: 135-139
Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Planta, 1962,15: 473-497
Nakajima O, Akiyama T, Hakamatsuka T, ShibuyaM, Noguchi H, Ebizuka Y, Sankawa U. Isolation, sequence and bacterial expression of a cDNA for chalcone synthase from the cultured cells of Pueraria lobata. Chem Pharm Bull (Tokyo), 1991, 39(7) : 1911-1913
Park CH, Martinez BC. Enhaneed release of rosmarinic acid from Coleus blumei permeabilized by dimethy sufoxide (DMSO) while preserving cell viability and growth. Biotech Bioeng, 1992, 40: 459-464
Park HH, Hakamatsuka T, Sankawa U, Ebizuka Y. Rapid metabolism of isoflavonoids in elicitor-treated cell suspension cultures of Pueraria lobata. Phytochemistry, 1995, 38(2) : 373-380
Schlatmann JE, Fonck E, ten Hoopen HJG, Heijnen JJ. The negligible role of carbon dioxide and ethylene in ajmalicine production by Catharanthus roseus cell suspensions. Plant Cell Report, 1994, 14(2-3) : 157-160
Smith HB. Signal transduction in systemic acquired resistance. Plant Cell, 2000, 12(2) : 179-180
Susko DJ, Mueller JP, Spears JF. An evaluation of methods for breaking seed dormancy in kudzu (Pueraria lobata). Canadian Journal of Botany, 2001, 79(2) : 197-203
Szabo E, Thelen A, Petersen M. Fungal elicitor preparations and methyl jasmonate enhance rosmarinnic acid accumulation in suspension cultures of Coleus blumei. Plant Cell Reports, 1999, 18: 485-489
Takeya K, Itokawa H. Isoflavoids and the other constituents in callus tissue of Pueraria lobata,Chem Pharm Bull, 1982, 30(4) : 1496-1499
Ton J, van Pelt JA, van Loon LC, Pieterse CMJ. Differential effectiveness of salicylate-dependent and jasmonate/ethylene-dependent induced resistance in Arabidopsis. Molecular Plant-Microbe Interactions, 2002, 15 (1) : 27-34
Undberg PI, Linsefors L, Vogel HJ et al. Plant Cell Reports, 1986, 5: 13-15
van der Plas LHW, Eijkelboom C, Hagendoorn MJM. Relation between primary and secondary metabolism in plant cell suspensions. Competition between secondary metabolite production and growth in a model system (Morinda citrifolia). Plant Cell, Tissue and Organ Culture, 1995, 43: 111-116
Verpoorte R, van der Heijden R, the Hoopen HJG, Memelink J. Metablic engineering of plant secondary metabolite pathways for the production of fine chemicals. Biotechnology Letters, 1999, 21: 467-479
Yang Y, Shah J, Klessing DF. Signal perception and transduction in plant defense responses. Genes Dev, 1997, 11: 1621-1639
Yeoman MM, Yeoman CL. Manipulating secondary metabolism in cultured plant cells. New Phytol, 1996, 134: 553-569
Yukimune Y, Tabata H, Higashi Y, Kara Y. Methyl jasmonate-induced over production of paclitaxel and baccatin in Taxus cell suspension cultures. Nature Biotechnology, 1996, 14(9) : 1129-1132