高山红景天颗粒状愈伤组织悬浮培养和红景天甙的诱导
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摘要
珍贵中药植物高山红景天（Rhodiola sachalinensis A.Bor）的根和根茎中含有以红景
天甙（salidroside）为主的次生代谢物质，具有抗疲劳、抗衰老、抗缺氧、抗微波辐射等
作用，是非常有开发前途的环境适应药物。本实验利用植物组织和细胞培养技术，以诱导
和建立较高红景天甙产量的高山红景天愈伤组织悬浮培养体系为目的，系统地对高山红景
天愈伤组织的诱导和状态调控、分化了的愈伤组织颗粒（CCA）悬浮培养体系的特点和
生长规律、红景天甙的积累规律、培养基优化和生产条件优化、利用诱导因子和添加前体
方法提高红景天甙产量、以及红景天甙生物合成途径几个方面做了调查。
利用高山红景天无菌苗的子叶、幼叶、幼茎和根成功诱导出了7种愈伤组织。以其
子叶最容易诱导产生愈伤组织，其次是幼叶、幼茎和根。对上述愈伤组织的状态进行调控，
得到红景天甙含量高、生长速度快、初步分化了的CCA型愈伤组织颗粒。
CCA悬浮培养的生长周期为25～30天。在继代后的0～15天左右为指数生长期，
15～20天以后生长速度减慢。最大红景天甙积累是在继代后的3～5天内。
外源8.27mg/L的BA与4.14mg/L的IBA的组合时，CCA悬浮培养的生长最好；
外源2,4-D抑制CCA悬浮培养的生长，但是对红景天甙积累的刺激作用最大。以MS基
本培养基的成分和含量为标准，总氮量不变、总糖量增至45g/L、pH值为5.7利于CCA
悬浮培养的生长，但抑制其红景天甙的积累；总氮量减少1/2、总糖量减少1/2、pH值为
4.2利于CCA悬浮培养细胞内红景天甙的积累，但抑制其生长。利于CCA悬浮培养生长
的培养条件为较低的温度（15℃）和较低的摇床转速（100rpm/min）；但利于红景天甙
积累的培养条件则为24℃和较高的摇床转速（150rpm/min）。
在继代培养后的第15天进行200W/m~2的254nm的紫外线照射20min对提高红景
天甙产量效果最好；牛皮杜鹃根的水提液的诱导效率大于高山凤毛菊根的水提液。前体
甙元酪醇（tyrosol）、苯丙氨酸（Phe）、水杨酸（SA）、色氨酸（Trp）和酪氨酸（Tyr）
对提高CCA细胞内红景天甙含量的效果依次减小。
建议生产上可以采用“两步法”培养来提高红景天甙产量。首先在利于CCA悬浮
培养生长的培养基上培养15～20天左右，使生物量积累达到最大，此时进行紫外线诱导
处理，或添加约4mg/L的高浓度2,4-D，或更换到生产培养基中培养3～5天再收获红景
天甙。CCA悬浮培养的生长培养基为总氮含量与基本MS培养基的含量相同、总糖含量
为45g/L、pH值为5.7并附加5mg/L的BA和2.5mg/L的IBA的外源激素；相应培养
条件为15～20℃、正常光照、摇床转速100rpm/min。红景天甙的生产培养基为总氮含
量减少1/2、总糖含量为15g/L、pH值为4.2并附加4mg/L的2,4-D；相应培养条件为
24℃、正常光照、摇床转速150rpm/min。
Rhodio/a sachalinensis A. Bor is a kind of precious herb of the traditional Chinese
medicine. Its root and rhizome contains salidroside, which has been shown to possess the
medical functions such as resisting anoxia, microwave radiation and fatigue. in order to
establish the differentiated callus suspension culture of Rh. Sachahnen.cis with high salidroside
production. the induction of callus of Rh. sachalinensis, the regulation of the morphas of the
callus, the establishment of the compact callus aggregates (CCA) suspension culture. the
characteristics of the growth of CCA suspension culture and the accumulation of salidroside.
the improvement of the culture media and culture conditions, the strategies of elicitation and
precursors feeding, and the biosynthesis pathway of salidroside were studied in this
experiments.
7 types of callus were induced from the cotyledon, the spire, the caulicle and the root
of Rh. Sachalinen.cis. The induction of callus from the cotyledon is easiest, from spire is easier
and from the root is very difficult. By regulating the morphas of the callus, the preIirninaril~
differentiated compact callus aggregates (CCA) with higher saLidroside content and higher
giowth rate was obtained.
The growth c~ cle of the CCA suspension culture is about 25 ?--3(1 days. The period
from 0 to 1 5 days after subculture is the exponential stage of the gro~vth of the culture. From 15
to 20 days after subculture. the growth of the culture begins to decrease. The maximum of the
accumulation of salidroside appears on the day 3 ?? after subculture.
The basic MS liquid media with 5 nig/L BA and 2.5 mg/L IBA benefit the growth of
the culture. Exogenous 2,4-D inhibited the growth of the culture greatly but stimulated the
accumulation of salidroside significantly. If the basic MS medium is the standard. maintaining
the quantity of total nitrogen and pH value and increasing the quantity of total sugar to 45 g/L
improved the growth of the culture but inhibited the accumulation of salidroside. While.
decreasing quantity of both total nitrogen and total sugar to half level and decreasing pH ~ alue
to 4.2 separately improved the accumulation of salidroside but inhibited the growth of the
culture. Low temperature at 15?C and low shaking speed with 100 rprn/miii benefit the growth
of the culture, while temperature at 24~?C and high shaking speed with 150 rpm/rn in benefit the
accumulation of salidroside.
The ideal induction efficiency of UV irradiation (200 W/m2) on improvement of
salidroside production of the culture is carried out for 20 mm at day 15 after subculture. The
induction efficiency of the extract of the root of Rhododendron chrysanthum is higher than that
of Saussurea alpicola. The improvement efficiency of possible precursor of salidroside such as
Iv
I
/
tvrosol, phenylalanine, salicylic acid, tryptophan, and tyrosine become less.
~Two-step?culture method is suggested to improve the salidroside production in mass
CCA suspension culture of Rh. Sachalinensis. In order to gain the maximum biomass of the
culture first. the CCA is cultivated in the growth-medium for 15~?O days. Then the CCA is
transferred into production-medium to incubate for 3 5 days before cultivated. The
growth-medium has the same amount of total nitrogen, the same pH value and 1.5 folds of total
sugar as the basic MS medium with 5 mg/L BA and 2.5 mg
天甙（salidroside）为主的次生代谢物质，具有抗疲劳、抗衰老、抗缺氧、抗微波辐射等
作用，是非常有开发前途的环境适应药物。本实验利用植物组织和细胞培养技术，以诱导
和建立较高红景天甙产量的高山红景天愈伤组织悬浮培养体系为目的，系统地对高山红景
天愈伤组织的诱导和状态调控、分化了的愈伤组织颗粒（CCA）悬浮培养体系的特点和
生长规律、红景天甙的积累规律、培养基优化和生产条件优化、利用诱导因子和添加前体
方法提高红景天甙产量、以及红景天甙生物合成途径几个方面做了调查。
利用高山红景天无菌苗的子叶、幼叶、幼茎和根成功诱导出了7种愈伤组织。以其
子叶最容易诱导产生愈伤组织，其次是幼叶、幼茎和根。对上述愈伤组织的状态进行调控，
得到红景天甙含量高、生长速度快、初步分化了的CCA型愈伤组织颗粒。
CCA悬浮培养的生长周期为25～30天。在继代后的0～15天左右为指数生长期，
15～20天以后生长速度减慢。最大红景天甙积累是在继代后的3～5天内。
外源8.27mg/L的BA与4.14mg/L的IBA的组合时，CCA悬浮培养的生长最好；
外源2,4-D抑制CCA悬浮培养的生长，但是对红景天甙积累的刺激作用最大。以MS基
本培养基的成分和含量为标准，总氮量不变、总糖量增至45g/L、pH值为5.7利于CCA
悬浮培养的生长，但抑制其红景天甙的积累；总氮量减少1/2、总糖量减少1/2、pH值为
4.2利于CCA悬浮培养细胞内红景天甙的积累，但抑制其生长。利于CCA悬浮培养生长
的培养条件为较低的温度（15℃）和较低的摇床转速（100rpm/min）；但利于红景天甙
积累的培养条件则为24℃和较高的摇床转速（150rpm/min）。
在继代培养后的第15天进行200W/m~2的254nm的紫外线照射20min对提高红景
天甙产量效果最好；牛皮杜鹃根的水提液的诱导效率大于高山凤毛菊根的水提液。前体
甙元酪醇（tyrosol）、苯丙氨酸（Phe）、水杨酸（SA）、色氨酸（Trp）和酪氨酸（Tyr）
对提高CCA细胞内红景天甙含量的效果依次减小。
建议生产上可以采用“两步法”培养来提高红景天甙产量。首先在利于CCA悬浮
培养生长的培养基上培养15～20天左右，使生物量积累达到最大，此时进行紫外线诱导
处理，或添加约4mg/L的高浓度2,4-D，或更换到生产培养基中培养3～5天再收获红景
天甙。CCA悬浮培养的生长培养基为总氮含量与基本MS培养基的含量相同、总糖含量
为45g/L、pH值为5.7并附加5mg/L的BA和2.5mg/L的IBA的外源激素；相应培养
条件为15～20℃、正常光照、摇床转速100rpm/min。红景天甙的生产培养基为总氮含
量减少1/2、总糖含量为15g/L、pH值为4.2并附加4mg/L的2,4-D；相应培养条件为
24℃、正常光照、摇床转速150rpm/min。
Rhodio/a sachalinensis A. Bor is a kind of precious herb of the traditional Chinese
medicine. Its root and rhizome contains salidroside, which has been shown to possess the
medical functions such as resisting anoxia, microwave radiation and fatigue. in order to
establish the differentiated callus suspension culture of Rh. Sachahnen.cis with high salidroside
production. the induction of callus of Rh. sachalinensis, the regulation of the morphas of the
callus, the establishment of the compact callus aggregates (CCA) suspension culture. the
characteristics of the growth of CCA suspension culture and the accumulation of salidroside.
the improvement of the culture media and culture conditions, the strategies of elicitation and
precursors feeding, and the biosynthesis pathway of salidroside were studied in this
experiments.
7 types of callus were induced from the cotyledon, the spire, the caulicle and the root
of Rh. Sachalinen.cis. The induction of callus from the cotyledon is easiest, from spire is easier
and from the root is very difficult. By regulating the morphas of the callus, the preIirninaril~
differentiated compact callus aggregates (CCA) with higher saLidroside content and higher
giowth rate was obtained.
The growth c~ cle of the CCA suspension culture is about 25 ?--3(1 days. The period
from 0 to 1 5 days after subculture is the exponential stage of the gro~vth of the culture. From 15
to 20 days after subculture. the growth of the culture begins to decrease. The maximum of the
accumulation of salidroside appears on the day 3 ?? after subculture.
The basic MS liquid media with 5 nig/L BA and 2.5 mg/L IBA benefit the growth of
the culture. Exogenous 2,4-D inhibited the growth of the culture greatly but stimulated the
accumulation of salidroside significantly. If the basic MS medium is the standard. maintaining
the quantity of total nitrogen and pH value and increasing the quantity of total sugar to 45 g/L
improved the growth of the culture but inhibited the accumulation of salidroside. While.
decreasing quantity of both total nitrogen and total sugar to half level and decreasing pH ~ alue
to 4.2 separately improved the accumulation of salidroside but inhibited the growth of the
culture. Low temperature at 15?C and low shaking speed with 100 rprn/miii benefit the growth
of the culture, while temperature at 24~?C and high shaking speed with 150 rpm/rn in benefit the
accumulation of salidroside.
The ideal induction efficiency of UV irradiation (200 W/m2) on improvement of
salidroside production of the culture is carried out for 20 mm at day 15 after subculture. The
induction efficiency of the extract of the root of Rhododendron chrysanthum is higher than that
of Saussurea alpicola. The improvement efficiency of possible precursor of salidroside such as
Iv
I
/
tvrosol, phenylalanine, salicylic acid, tryptophan, and tyrosine become less.
~Two-step?culture method is suggested to improve the salidroside production in mass
CCA suspension culture of Rh. Sachalinensis. In order to gain the maximum biomass of the
culture first. the CCA is cultivated in the growth-medium for 15~?O days. Then the CCA is
transferred into production-medium to incubate for 3 5 days before cultivated. The
growth-medium has the same amount of total nitrogen, the same pH value and 1.5 folds of total
sugar as the basic MS medium with 5 mg/L BA and 2.5 mg
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55． Hagendoom M.J.M., Poortinga A.M., Wong Fong Sang H.W., van der Plas L.H.W. and van Walraven H.S. Effect of elicitors on the plasmamembrane of Petunia hybrida cell suspensions. Plant Physiol. 1991, 96: 1261-1267
56． Hamill J.D., Parr A.J., Rhodes M. J.C., Robins R.J. and Walton N.J. New routes to plant secondary products. Biotechnol. 1987,5: 800-804
57． Harry K., Ann. Rev. Plant Physiol. 1987,38:467-486
58． Hartmeier W. Immobilisierte Biokatalysatoren-auf dem Weg zur zweirenGeneration. Naturwissenschaften. 1985,72:310-
59． Hartmeier W. Immobilisierte Biokatalysatoren-eine Einfuhrung. Springer. Berlin Heidelberg, New York 1986．
60． Ita Ananta, Doran P.M., Biotechnol. Bioeng. 1995,47: 541-549
61． Johnson T, Sundhaker, Ravishankar G.A. and Venkatamaran L.V. Elicitation of capsaicin production in freely suspended cells and immobilized cell cultures of Capsicum frutescens. Food Biotechnol. 1991, 5: 197-205
62． Kauss H., Jeblick W. and Domard A. The degree of polymerization and N-acetylation of chitosan determine its ability to elicit callose formation in suspension cells of Catharanthus roseus. Planta. 1989,178:385-392
63． Kingston D.G. I., Taxol and other anticancer agents from plants, presented at Int. Symp. New Drugs from Natural Sources, in London. June 13-14,1991．
64． Knuth M.E. and Sahai O.P., Flavor composition and method. US patent no. 5,057,424, Oct. 15, 1991
65． Kolodziejczyk P.P. and Fedec P., Recent progress in agricultural biotechnology and opportunities for contract research and development. In: Chemicals via Higher Plant Bioengineering. Shahidi F., kolodziejczyk P.P., Whitaker J.R., Munguia A.L. and Fuller G.(Ed.). Kluwer Academic/Plenum Publishers. New York, Boston. 1999, p: 5-20
66． Kolodziejczyk P.P. and Shahidi F., Novel chemicals from plants via bioengineering. In: Chemicals via Higher Plant Bioengineering. Shahidi F, kolodziejczyk P.P., Whitaker J.R., Munguia A.L. and Fuller G.(Ed.). Kluwer Academic/Plenum Publishers. New York, Boston. 1999, p: 1-5
67． Kurkin V.A., J. Pharm Chem. (Engl. Tromsl.). 1986,20:1231-1235
68． Lindsey K, Yeoman M.M., Planta. 1984,162:495-
69． Lindsey K., Yeoman M.M., Black G.M., and Mavituna F.A., A novel method for the immobilization and culture of plant cells. FEBS Lett. 1983,155: 143-149
70． Ma J.K. Generation and assembly of secretory antibodies in plants. Sciences. 1995,268: 716-719
71． Merchuk J., Ben-Zyi S., and Niranjan K. Why use bubble-column bioreactors? Tibtech. 1994． 12(2) : 501-511
72． Moldenhauer D. et al, Planta Medica. 1990,8:601-604?
73． Morimoto H. and Murai F. The effect of gelling agents on plaunotol accumulation in callus cultures of Croton syblyratus. Plant cell Rep. 1989,8:210-213
74． Parr A., J. Secondary products from plant cell cultures. Adv Biotechnol. Proc. 1988,9:1-34
75． Pfeiffer N., FDA OKs Calgene's Flavr Savr?tomato for marketing in suspermarkets in the U.S.. Genetic Engineering News. 1994,14: 1． , 31
76． Richards J.H. et al. The biosynthesis of the storids terpenes and acetogenism. Benjaman W.A. INC. New York.1964．
77． Sauerwein M., Yamazaki T., and Koichiro S. Hernandulcin in hairy root cultures of Lippia dulcis. Plant Cell Rep. 1991,9:579-581
78． Schmauder H.P. and Doebel P., Plant cultivation as a biotechnological method. Acta Biotechnol. 1990,10: 501-516
79． Schripsema J, Fung S.Y. and Verpoorte R. Screening of plant cell cultures for new industrially interesting compounds. In "Plant cell culture secondary metabolism-toward industrial application". DiCosmo F. and Misawa M (Ed). CRC Press Inc., Tokyo. 1996． p: 1-10
80． Shahidi F., Kolodziejczyk P., Whitaker J.R., Munguia A.L. Chemicals via higher plant bioengineering. New York, Boston, Dordrecht, London, Moscow. Kluwer Academic/Plenum Publishers. 1999．
81． Sharp J.M., Crowley B.R., and Kwok K.H.(Ed). Plant cell culture Manual. 1995, p: 1-15
82． Staba E. J. (Ed), Plant Tissue Culture as a Source of Biochemicals. CRC Press, Boca Raton, Florida. 1979． p:1-10
83． Stafford A. The manufacture of food ingredients using plant cell and tissue cultures. Trends Food Sci. Technol.1991,2:116-122
84． Stafford A., Enzyme Microb. Technol. 1986,8． 578-586
85． Steward F.C., Mapes M.O., Keut A.E., Holsten R.D., Growth and development of cultured plant cells. Science. 1964,20:143-
86． Street H.E.(Ed.). Plant Tissue and Cell Culture. Blackwell Scientific Publications, Oxford. 1977
87． Tabata H et al. In: Colloques International CNRS No. 39, Lee Cultures De Tissue De Plantes. Centre National de la Recherche Scientitique 15． Quai Anatole-France, Paris-VII. 1970, p: 389-402
88． Tabata H., Fujita Y. Production of shikonin by plant cell cultures. In: Day P., Zaitlen X., Hollaender A. (Ed) Biotechnology in Plant Science: relevance toagriculture in the eighties. Acad. Press. Orlando 1985, p: 207
89． Takebe J., Labib G., and Melchers G., Regeneration of whole plants from isolated mesophyll protoplasts of tobacco. Naturwissenschaften. 1971,58:318-320
90． Tsoulpha P., Doran P.M., J. Biotechnol. 1991,19:99-110
91． Werrmann U. and Knorr D., Conversion of menthyl acetate or neomenthyl acetate to menthol or neomenthol by suspension cultures of Mentha canadensis and M. piperita. J. Agric. Food Chem. 1993,41: 517-520
92． West C:A. Fungal elicitors of the phytoalexin responses of high plants. Naturwissenschaften. 1981, 68． 447-457
93． White P.R., Potentially unlimited growth of excised tomato root tips in a liquid medium. Plant Physiol. 1934,9: 585-600
94． Williams D.H., Stone M. J, Hauck P.R., and Rahman S.K., Why are secondary metabolites (natural products) biosynthesized? J. Nat. Prod., 1989,52:1189-
95． Woerdenbag H.J., Lugt C.B., and Pras N. Artemisia annua L.: a sources of novel antimalarial drugs. Pharm. Weekbl. Sci. Ed., 1990． 12:169-
96． Wongsamuth R. and Doran P. M Foaming and cell flotation in suspended plant cell cultures and the effect of chemical antifoams. Biotechnology and bioengineering. 1994． 44:481-488
97． Xu J.F., Liu C.B., Han A.M. et al.. Strategies for the improvement of salidroside production in cell suspension cultures of Rhodiola sachalinensis. Plant Cell Reports. 1998a, 17:288-293
98． Xu J.F., Su Z.G., and Feng P.S. Activity of tyrosl glucosyltransferase and improved salidroside production through biotransformation of tyrosol in Rhodiola sachalinensis cell cultures. J. of Biotechnol. 1998c, 61: 69-73
99． Xu J.F., Su Z.G., and Feng P.S. Suspension culture of compact callus aggregate of Rhodiola sachalinensis for improved salidroside production. Enzyme and Microbial Technology. 1998b, 23:20-27
100． Yamada et al., In: Proceedings of Chona-Japan Symposium on Plant Biotechnology, Shanghai. 1988, p: 14-21
101． Yeoman M.M., et al., In: Sala F. et al. (Ed) Proc. Int. Workship in Plant Cell Cultures: Resultes and Perspective. Italy. 1979． p: 61-71
102． Yoshikawa T, Plant Cell Rep. 1987,6:449-453
103． Zenk M.H., El-Shagi H., Arens E, Stockigt J., Weiler E., Deus B. Formation of the indole alkaloids serpentine and ajmalicine in cell suspension cultures of Catharanthus roseus. In: Barz W., Reinhard E., Zenk M.H. (Ed) Plant Tissue Culture and Its Biotechnological Application, Springer-Verlag, Berlin, Heidelbarg, New York. 1977． p: 27-43
105． Zenk M.H., El-Shagi H., Schulte U., Anthraquinone production by cell suspension cultures of Morinda citrifolia. Planta Med. Suppl. 1975． p: 97
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25．阎秀峰．王玉杰，王洋，孙海芹，祖元刚．长白山高山红景天的气体交换特性．植物研究．1999．19(2)：273-280
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30．张荫麟，宋经元，赵保华等．生物工程学报．1995，11(2)：150-152
31．周立刚，郑光植．生物工程进展．1991，11(1)：29-35
32．周重楚，李文亭，刘威等．高山红竟天化学成分与药理研究概况．特产研究．1993，(1)：25-27
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36. Blechert S. and Guenard D., Taxus alkaloids. In: The Alkaloids. Vol. 39. Brossi A. (Ed). Academic. San Diego. 1990. Chap. 6
37. Brodelius P. and Pedersen H. Increasing secondary metabolite production in plant-cell culture by redirecting transport. Tibtech. 1993.11(1): 30-36
38. Brodelius E, Funk C., H(?)ner A., Villegas M. A procedure for the determination of optimal chitosan concentrations for elicitation of cultured plants. Phytochemistry. 1986, 25:2803-2806
39. Curtin M.E., Harvesting profitable products from plant tissue culture. Biol. Tech. 1983.1:649-657
40. Curtis W. and Emery A. Plant cell suspension culture theology. Biotechnology and bioengineering. 1993.42: 520-526
41. Darvill A.G. and Albershein P. Phytoalexins and their elicitors-a defense against microbial infectionin
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42． Dicosmo F. and Misawa A, Eliciting secondary metabolism in plant cell cultures. Trends Biotechnol. 1985, 3(12) : 318-
43． DiCosmo F. and MisawaM. Plant cell culture secondary metabolism (toward industrial application). Boca Raton, New York, London, Tokyo. CRC Press. 1996
44． Dornenburg H. and Knorr D. Strategies for the improvement of secondary metabolite production in plant cell cultures. Enzyme and microbial technology. 1995,17:674-684．
45． Endress R. (Ed.). Plant Cell Biotechnology.. Springer-Verlag. Berlin Heidelberg, New York, Hong Kong, Barcelona. 1994． p: 121-242
46． Farnsworth N.R., The role of ethnopharmacology in drug development In: Bioactive Compounds from Plant, Ciba Foundation Symposium 154． John Wiley & Sons. Chichester. 1990, p: 2-
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48． Fujita Y, Hara Y, Suga C., Morimoto T, Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon of shikonin derivatives. Plant Cell Rep. 1981,1:61-64
49． Fuller K. W., Bartlett D.J., Plant-derived cell aggregates, plant granule, production and use thereof, European pattern application PCT/GB81/00186．
50． Funk C. and Brodelius P.E., Vanilla planifolia Andrews: In vitro biosynthesis of vanillin and other phenvpropanoid derivatives. In: Biotechnology in Agriculture and Forestry, vol. 26． Medicinal and Aromatic Plants VI (Bajaj Y.P.S. (Ed.)). Springer-Verlag, Berlin. 1994, p: 377-402
51． Furmanova M., Oledzka H., michalska M, Sokomicha I. and Radomska D. Rhodiola rosea L. (roseroot). In vitro regeneration and the biological activity of roots. In: Bajaj Y.P.S. (Ed) Biotechnology in Agriculture and Forestry,Vol.33: Medicinal and Aromatic Plants VIII. Springer-Verlag, Berlin, Heidelberg, New York. 1995, p:412-426．
52． Furmanova M., Skopinska-Rozewska E., Rogala E. and Hartwich M. Rholidola rosea in vitro culture-phytochemical analysis and antioxidant action. Acta Societatis Botanicorum Poloniae 1998, 67(1) : 69-73
53． Gundlach H., Muller M.J., Kutchan T.M. and Zenk M.H. Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures, Proc. Natl. Acad. Sci. USA 1992,89:2389-2393
54． Haberlandt G., Culturversuche mit isolierten pflanzenzellen. Sitz Akad. Wiss. Wien., 1902, 111: 69-92
55． Hagendoom M.J.M., Poortinga A.M., Wong Fong Sang H.W., van der Plas L.H.W. and van Walraven H.S. Effect of elicitors on the plasmamembrane of Petunia hybrida cell suspensions. Plant Physiol. 1991, 96: 1261-1267
56． Hamill J.D., Parr A.J., Rhodes M. J.C., Robins R.J. and Walton N.J. New routes to plant secondary products. Biotechnol. 1987,5: 800-804
57． Harry K., Ann. Rev. Plant Physiol. 1987,38:467-486
58． Hartmeier W. Immobilisierte Biokatalysatoren-auf dem Weg zur zweirenGeneration. Naturwissenschaften. 1985,72:310-
59． Hartmeier W. Immobilisierte Biokatalysatoren-eine Einfuhrung. Springer. Berlin Heidelberg, New York 1986．
60． Ita Ananta, Doran P.M., Biotechnol. Bioeng. 1995,47: 541-549
61． Johnson T, Sundhaker, Ravishankar G.A. and Venkatamaran L.V. Elicitation of capsaicin production in freely suspended cells and immobilized cell cultures of Capsicum frutescens. Food Biotechnol. 1991, 5: 197-205
62． Kauss H., Jeblick W. and Domard A. The degree of polymerization and N-acetylation of chitosan determine its ability to elicit callose formation in suspension cells of Catharanthus roseus. Planta. 1989,178:385-392
63． Kingston D.G. I., Taxol and other anticancer agents from plants, presented at Int. Symp. New Drugs from Natural Sources, in London. June 13-14,1991．
64． Knuth M.E. and Sahai O.P., Flavor composition and method. US patent no. 5,057,424, Oct. 15, 1991
65． Kolodziejczyk P.P. and Fedec P., Recent progress in agricultural biotechnology and opportunities for contract research and development. In: Chemicals via Higher Plant Bioengineering. Shahidi F., kolodziejczyk P.P., Whitaker J.R., Munguia A.L. and Fuller G.(Ed.). Kluwer Academic/Plenum Publishers. New York, Boston. 1999, p: 5-20
66． Kolodziejczyk P.P. and Shahidi F., Novel chemicals from plants via bioengineering. In: Chemicals via Higher Plant Bioengineering. Shahidi F, kolodziejczyk P.P., Whitaker J.R., Munguia A.L. and Fuller G.(Ed.). Kluwer Academic/Plenum Publishers. New York, Boston. 1999, p: 1-5
67． Kurkin V.A., J. Pharm Chem. (Engl. Tromsl.). 1986,20:1231-1235
68． Lindsey K, Yeoman M.M., Planta. 1984,162:495-
69． Lindsey K., Yeoman M.M., Black G.M., and Mavituna F.A., A novel method for the immobilization and culture of plant cells. FEBS Lett. 1983,155: 143-149
70． Ma J.K. Generation and assembly of secretory antibodies in plants. Sciences. 1995,268: 716-719
71． Merchuk J., Ben-Zyi S., and Niranjan K. Why use bubble-column bioreactors? Tibtech. 1994． 12(2) : 501-511
72． Moldenhauer D. et al, Planta Medica. 1990,8:601-604?
73． Morimoto H. and Murai F. The effect of gelling agents on plaunotol accumulation in callus cultures of Croton syblyratus. Plant cell Rep. 1989,8:210-213
74． Parr A., J. Secondary products from plant cell cultures. Adv Biotechnol. Proc. 1988,9:1-34
75． Pfeiffer N., FDA OKs Calgene's Flavr Savr?tomato for marketing in suspermarkets in the U.S.. Genetic Engineering News. 1994,14: 1． , 31
76． Richards J.H. et al. The biosynthesis of the storids terpenes and acetogenism. Benjaman W.A. INC. New York.1964．
77． Sauerwein M., Yamazaki T., and Koichiro S. Hernandulcin in hairy root cultures of Lippia dulcis. Plant Cell Rep. 1991,9:579-581
78． Schmauder H.P. and Doebel P., Plant cultivation as a biotechnological method. Acta Biotechnol. 1990,10: 501-516
79． Schripsema J, Fung S.Y. and Verpoorte R. Screening of plant cell cultures for new industrially interesting compounds. In "Plant cell culture secondary metabolism-toward industrial application". DiCosmo F. and Misawa M (Ed). CRC Press Inc., Tokyo. 1996． p: 1-10
80． Shahidi F., Kolodziejczyk P., Whitaker J.R., Munguia A.L. Chemicals via higher plant bioengineering. New York, Boston, Dordrecht, London, Moscow. Kluwer Academic/Plenum Publishers. 1999．
81． Sharp J.M., Crowley B.R., and Kwok K.H.(Ed). Plant cell culture Manual. 1995, p: 1-15
82． Staba E. J. (Ed), Plant Tissue Culture as a Source of Biochemicals. CRC Press, Boca Raton, Florida. 1979． p:1-10
83． Stafford A. The manufacture of food ingredients using plant cell and tissue cultures. Trends Food Sci. Technol.1991,2:116-122
84． Stafford A., Enzyme Microb. Technol. 1986,8． 578-586
85． Steward F.C., Mapes M.O., Keut A.E., Holsten R.D., Growth and development of cultured plant cells. Science. 1964,20:143-
86． Street H.E.(Ed.). Plant Tissue and Cell Culture. Blackwell Scientific Publications, Oxford. 1977
87． Tabata H et al. In: Colloques International CNRS No. 39, Lee Cultures De Tissue De Plantes. Centre National de la Recherche Scientitique 15． Quai Anatole-France, Paris-VII. 1970, p: 389-402
88． Tabata H., Fujita Y. Production of shikonin by plant cell cultures. In: Day P., Zaitlen X., Hollaender A. (Ed) Biotechnology in Plant Science: relevance toagriculture in the eighties. Acad. Press. Orlando 1985, p: 207
89． Takebe J., Labib G., and Melchers G., Regeneration of whole plants from isolated mesophyll protoplasts of tobacco. Naturwissenschaften. 1971,58:318-320
90． Tsoulpha P., Doran P.M., J. Biotechnol. 1991,19:99-110
91． Werrmann U. and Knorr D., Conversion of menthyl acetate or neomenthyl acetate to menthol or neomenthol by suspension cultures of Mentha canadensis and M. piperita. J. Agric. Food Chem. 1993,41: 517-520
92． West C:A. Fungal elicitors of the phytoalexin responses of high plants. Naturwissenschaften. 1981, 68． 447-457
93． White P.R., Potentially unlimited growth of excised tomato root tips in a liquid medium. Plant Physiol. 1934,9: 585-600
94． Williams D.H., Stone M. J, Hauck P.R., and Rahman S.K., Why are secondary metabolites (natural products) biosynthesized? J. Nat. Prod., 1989,52:1189-
95． Woerdenbag H.J., Lugt C.B., and Pras N. Artemisia annua L.: a sources of novel antimalarial drugs. Pharm. Weekbl. Sci. Ed., 1990． 12:169-
96． Wongsamuth R. and Doran P. M Foaming and cell flotation in suspended plant cell cultures and the effect of chemical antifoams. Biotechnology and bioengineering. 1994． 44:481-488
97． Xu J.F., Liu C.B., Han A.M. et al.. Strategies for the improvement of salidroside production in cell suspension cultures of Rhodiola sachalinensis. Plant Cell Reports. 1998a, 17:288-293
98． Xu J.F., Su Z.G., and Feng P.S. Activity of tyrosl glucosyltransferase and improved salidroside production through biotransformation of tyrosol in Rhodiola sachalinensis cell cultures. J. of Biotechnol. 1998c, 61: 69-73
99． Xu J.F., Su Z.G., and Feng P.S. Suspension culture of compact callus aggregate of Rhodiola sachalinensis for improved salidroside production. Enzyme and Microbial Technology. 1998b, 23:20-27
100． Yamada et al., In: Proceedings of Chona-Japan Symposium on Plant Biotechnology, Shanghai. 1988, p: 14-21
101． Yeoman M.M., et al., In: Sala F. et al. (Ed) Proc. Int. Workship in Plant Cell Cultures: Resultes and Perspective. Italy. 1979． p: 61-71
102． Yoshikawa T, Plant Cell Rep. 1987,6:449-453
103． Zenk M.H., El-Shagi H., Arens E, Stockigt J., Weiler E., Deus B. Formation of the indole alkaloids serpentine and ajmalicine in cell suspension cultures of Catharanthus roseus. In: Barz W., Reinhard E., Zenk M.H. (Ed) Plant Tissue Culture and Its Biotechnological Application, Springer-Verlag, Berlin, Heidelbarg, New York. 1977． p: 27-43
105． Zenk M.H., El-Shagi H., Schulte U., Anthraquinone production by cell suspension cultures of Morinda citrifolia. Planta Med. Suppl. 1975． p: 97