fermentation pilot plant 41 3.0 BIOREACTORS FOR PLANT CELL TISSUE AND ORGAN CULTURES (by Shinsaku Takayama) 3.1 Background of the Technique--Historical Overview Haberlandt! first reported plant cell, tissue, and organ cultures in 1902. He separated plant tissues and attempted to grow them in a simple nutrient medium. He was able to maintain these cells in a culture medium for 20 to 27 days. Although these cells increased eleven-fold in the best case,no cell division was observed. Gautheret 2 was the first to succeed in multiply ing the cells from the culture in 1934. He used the cambial tissues of Acer pseudoplatanus, Salix capraea, Sambucus nigra. After 15 to 18 months in subculture, cell activity ceased. He reasoned that this inactiveness was due to the lack of essential substances for cell division. He suspected that auxin nay have been one of the deficient substances. This compound was first eported in 1928 and was isolated by Kogel in the 1930s. Addition of auxin to the medium prompted plant cell growth. This finding was reported almost simultaneously by gautheret! 3I and White!l in 1939. Plant cell tissue and organ culture techniques rapidly developed, and in the mid-1950s another important phytohormone, cytokinins, had been discovered(Miller, Skoog, Okumura, Von Saltza and Strong 1955). 15) By 1962 Murashige and Skoogl6) had reported a completely defined medium which allowed the culture of most plant cells. Their medium has now become the mostly widely used medium in laboratories around the world After these initial discoveries and some significant improvements in media, scientific research on the cultivation of plant cell, tissue, and organs shifted to the area of basic physiological research. Industrial applications were also sought in the production of secondary metabolites, clonal plants and the improvement of various plant tissues Plant cell, tissue, and organ culture can be performed by either solid or liquid culture methods, however, in order to scale up the culture to the level of industrial processes, the liquid culture method must be employed Recently, pilot bioreactors as large as 20 kl have been constructed in the research laboratories of japan tobacco and Salt Co. and in those ofNitto Denko Co. Solid culture methods were used in large scale pilot experiments for the production of tobacco cells, and liquid culture methods were used in the production of Panax ginseng cells. An outstanding example of cell suspension culture in a pilot scale bioreactor(750 D)was the production of shikonins by Mitui Petrochemical Industries. In all these examples, various technologies have been used to improve the productivity of the metabolites
Fermentation Pilot Plant 41 3.0 BIOREACTORS FOR PLANT CELL TISSUE AND ORGAN CULTURES fly Shinsaku Takayama) 3.1 Background of the Technique-Historical Overview HaberlandtL'] first reported plant cell, tissue, and organ cultures in 1902. He separated plant tissues and attempted to grow them in a simple nutrient medium. He was able to maintain these cells in a culture medium for 20 to 27 days. Although these cells increased eleven-fold in the best case, no cell division was observed. GautheretL2] was the first to succeed in multiplying the cells from the culture in 1934. He used the cambial tissues of her pseudoplatanus, Salix capraea, Sambucus nigra. After 15 to 18 months in subculture, cell activity ceased. He reasoned that this inactiveness was due to the lack of essential substances for cell division. He suspected that auxin may have been one of the deficient substances. This compound was first reported in 1928 and was isolated by Kogel in the 1930's. Addition of auxin to the medium prompted plant cell growth. This finding was reported almost simultaneously by Gauthered3] and in 1939. Plant cell tissue and organ culture techniques rapidly developed, and in the mid-1950's another important phytohormone, cytokinins, had been discovered (Miller, Skoog, Okumura, Von Saltza and Strong 1955).15] By 1962 Murashigeand Skoog[6] had reported a completely defined medium which allowed the culture of most plant cells. Their medium has now become the mostly widely used medium in laboratories around the world. After these initial discoveries and some significant improvements in media, scientific research on the cultivation of plant cell, tissue, and organs shifted to the area of basic physiological research. Industrial applications were also sought in the production of secondary metabolites, clonal plants, and the improvement of various plant tissues. Plant cell, tissue, and organ culture can be performed by either solid or liquid culture methods, however, in order to scale up the culture to the level of industrial processes, the liquid culture method must be employed. Recently, pilot bioreactors as large as 20 kl have been constructed in the research laboratories of Japan Tobacco and Salt Co. and in those ofNitto Denko Co. Solid culture methods were used in large scale pilot experiments for the production of tobacco cells, and liquid culture methods were used in the production of Panax ginseng cells. An outstanding example of cell suspension culture in a pilot scale bioreactor (750 1) was the production of shikonins by Mitui Petrochemical Industries. In all these examples, various technologies have been used to improve the productivity of the metabolites
42 Fermentation and Biochemical Engineering Handbook The technologies include: (i) selection of a high yielding cell strain, (ii) screening of the optimum culture condition for metabolite production, (iii) addition of precursor metabolites, (iv) immobilized cell culture, and (v) differentiated tissue and/or organ culture. The productivity of various metabolites such as ginsenoside, anthraquinones, rosmarinic acid, shikonins ubiquinones, glutathione, tripdiolide, etc., reached or exceeded the amount produced by intact plants. To date, the production costs remain very high which is why most of the metabolites are still not produced on an industrial orpilot plant scale. Development oflarge scaleindustrial culture systems and techniques for plant cell, tissue, and organs, and the selection of the target metabolites are the chief prerequisites for the establishment of the industrial production of plant metabolites Culture Collection Intact Plant Breeding Genetic Engineering Cell Culture Organ Culture Clonal Plant mobilized Isolation and Purification of the Cell Culture Metabolites Useful Plant Metabolites Figure 17. The area of plant cell, tissue and organ cultures
42 Fermentation and Biochemical Engineering Handbook The technologies include: (i) selection of a high yielding cell strain, (ii) screening of the optimum culture condition for metabolite production, (iii) addition of precursor metabolites, (iv) immobilized cell culture, and (v) differentiated tissue andor organ culture. The productivity of various metabolites such as ginsenoside, anthraquinones, rosmalinic acid, shikonins, ubiquinones, glutathione, tripdiolide, etc., reached or exceeded the amount produced by intact plants. To date, the production costs remain very high which is why most of the metabolites are still not produced on an industrial or pilot plant scale. Development oflarge scale industrial culture systems and techniques for plant cell, tissue, and organs, and the selection of the target metabolites are the chief prerequisites for the establishment of the industrial production of plant metabolites. I Culture Collection I I I Cell Culture Figure 17. The area of plant cell, tissue and organ cultures
Fermentation Pilot plant 3.2 Media Formulations The formulation of the medium for plant cell, tissue, and organ culture depend primarily on nutritional requirements. Intact plants grow photoau totrophically in the soil, (i.e, they use CO2 as the principal carbon source and synthesize sugars by photosynthesis). In the case of aseptic cultures however, establishment of an autotrophic culture is not achieved so that heterotrophic or mixotrophic growth becomes the distinguishing characteristic. Therefore such cultures require the addition of carbon as an energy source. Given this fact, the culture medium must be formulated as a chemically defined mixture of mineral salts(macro-and microelements)in combination with a carbon source(usually sucrose). In addition to these constituents, organic constitu- ents such as vitamins, amino acids, sugar alcohols, and plant growth regulators are usually added to the medium. Media commonly used are listed in Table 11 Table 11. Formulations of most frequently used plant tissue culture media Ingredients(mg (l) MS Heller NH4小2SO4 134 (NHAN 1650 NaNO KNO3 1900 2500 Ca(NO3)2 CaCl3 2H2O 440 150 MgSO4 7H,O 200 KH2PO4 170 125 NaH_PO4 H2O l50 16.5 KCI 750 FeSO, 7H,O 27.8 27.8 EDTA 37.3 37.3 Cont'd next page)
Fermentation Pilot Plant 43 3.2 Media Formulations The formulation ofthe medium for plant cell, tissue, and organ culture depend primarily on nutritional requirements. Intact plants grow photoautotrophically in the soil, (i.e., they use CO, as the principal carbon source and synthesize sugars by photosynthesis). In the case ofaseptic cultures however, establishment of an autotrophic culture is not achieved so that heterotrophic or mixotrophic growth becomes the distinguishing characteristic. Therefore, such cultures require the addition of carbon as an energy source. Given this fact, the culture medium must be formulated as a chemically defined mixture of mineral salts (macro- and microelements) in combination with a carbon source (usually sucrose). In addition to these constituents, organic constituents such as vitamins, amino acids, sugar alcohols, and plant growth regulators are usually added to the medium. Media commonly used are listed in Table 11. Table 11. Formulations of most frequently used plant tissue culture media Ingredients (mg C1) MS B5 White Heller (NH4)2s04 W4W3 KNo3 NaNO, Ca(N03)2 CaCI,*2H20 MgS04*7H20 Na2SO4 =32po4 NaH,P O4.H2O KCI FeS O4.7H2O Na,,EDTA 134 1650 1900 2500 80 300 440 150 370 250 720 200 170 150 16.5 65 27.8 27.8 37.3 37.3 600 75 250 125 75 0 (Cont’d next page)
44 Fermentation and Biochemical Engineering Handbook Table 11.( Cont'd Formulations of most frequently used plant tissue culture media Ingredients(mg (.) MS White Heiler FeCl3 6H2O 1.0 Fe2(sO4) 3 MnSO4 4H2O 22.3 0.0l MnSO4 H2O ZnSO47H2O HaBO 6.2 0.83 075 Na,MoO 2H.O 0.25 CuSo→5H20 0.025 0.025 0.03 CoCl2 6H2O 0.025 NiCl2 6H,O AlCl3 0.03 100 Nicotinic acid 0.5 0.5 Pyridoxine HCI 0.5 0. Thiamine HCl Glycine 2.0 3.0 1.0 30,00020,00020,00020,000 Kinetin 0.04-100l 2.4D 0l-1.0 6.0 IAA pH 5.7-5.8 5.5 5.5
44 Fermentation and Biochemical Engineering Handbook Table 11. (Cont'd.) Formulations of most frequently used plant tissue culture media. Ingredients (mg 6-l) MS B5 White Heiler FeCl3*6H2O Mnso4-4H20 MnS04.H20 ZnS04.7H,0 K1 N%Mo04.2H20 CuS04.5H20 CoC12.6H20 NiC12*6H20 AlCI, Myo-inositol Nicotinic acid P yridoxine.HC1 Thiamine.HC1 Glycine Ca D-pantothenic acid Sucrose Kinetin Fe2(S04)3 H3BO3 2,4-D IAA PH 22.3 8.6 6.2 0.83 0.25 0,025 0.025 100 0.5 0.5 0.1 2.0 30,000 0.04- 10 1.0-30 5.7-5.8 10 2 3 0.75 0.25 0.025 0.025 100 1 .o 1 .o 10.0 20,000 0.1 0.1-1 .o 5.5 2.5 7 3 1.5 0.75 0.5 0.1 0.1 3 .O 1 .o 20,000 6.0 5.5 1 .o 0.01 1 1 0.01 0.03 0.03 0.03 1.0 20,000
Fermentation Pilot Plant 45 3.3 General applications The most important fields of research for industrial applications, plant cell tissue and organ cultures are clonal propagation and secondary metabo- lite production. Plants cultivated in vitro have great changes in their morphological features, from cell tissue to differentiated embryo, roots, shoots or plantlets Applications to Secondary Metabolite Production. Plant tissue culture is a potential method for producing secondary metabolites. Both shikonins(Fujita and Tabata 1987)]and ginseng saponins(Ushiyama etal 1986)[] have now been produced on a large scale by this method. However, the important secondary metabolites are usually produced by callus or cell suspension culture techniques. the amounts of some metabolites in the cell have exceeded the amounts of metabolites in the cells of the original plants grown in the soil. So it is expected that cell culturing may be applicable to industrial processes for the production of useful secondary metabolites. It is common knowledge that when a cell culture is initiated and then transferred, the productivity of the metabolite decreases(Kurz and Constabel, 1979).19) Once productivity decreases, it becomes very difficult to arrest or reverse the decrease. In order to avoid this phenomenon, many cell strains were screened to select those which would maintain metabolite productivity. Some metabo- lites such as anthocyanins, shikonins, vinca alkaloids, and ubiquinones have been reported to have increased their productivity significantly. Deus Neumann and Zenk(1984) ol have checked the stability of the productivity of the selected cell strains reported in the literature and noted that the production of some metabolites such as anthraquinone Morinda citrifolia), rosmalinic acid (Colius blumei), visnagin (Ammi visnaga), diosgenin (Dioscorea deltoidea, etc were stable after several subcultures, but some metabolites such as nicotine(Nicotiana rustica), shikonin(Lithospermum erythrorhizon), ajmalicine(Catharanthus roseus), rotenoids( Derris elliptica) anthocyan(Daucus carota), etc, were shown to be unstable after several subcultures Clonal Plant Propagation. Plants are propagated clonally from vegetative tissue or organs via bypass sex. Conventional clonal propagation can be performed by leafor stem cutting and layering or dividing ofthe plants however the efficiency is very low. Recently, many plants were propagated efficiently through tissue culture. This technique was first reported in 1960 by G. Morell] for the propagation of orchids and since then, many plants have been propagated by tissue culture. Today there are many commercial
Fermentation Pilot Plant 45 3.3 General Applications The most important fields of research for industrial applications, plant cell tissue and organ cultures are clonal propagation and secondary metabolite production. Plants cultivated in vitro have great changes in their morphological features, from cell tissue to differentiated embryo, roots, shoots or plantlets. Applications to Secondary Metabolite Production. Plant tissue culture is a potential method for producing secondary metabolites. Both shikonins (Fujita and Tabata 1987)r'I and ginseng saponins (Ushiyama et al., 1986)[*] have now been produced on a large scale by this method. However, the important secondary metabolites are usually produced by callus or cell suspension culture techniques. The amounts of some metabolites in the cell have exceeded the amounts of metabolites in the cells of the original plants grown in the soil. So it is expected that cell culturing may be applicable to industrial processes for the production of useful secondary metabolites. It is common knowledge that when a cell culture is initiated and then transferred, the productivity of the metabolite decreases (Kurz and Constabel, 1979).['1 Once productivity decreases, it becomes very difficult to arrest or reverse the decrease. In order to avoid this phenomenon, many cell strains were screened to select those which would maintain metabolite productivity. Some metabolites such as anthocyanins, shikonins, vinca alkaloids, and ubiquinones have been reported to have increased their productivity significantly. DeusNeumann and Zenk ( 1984)[1°1 have checked the stability of the productivity of the selected cell strains reported in the literature and noted that the production of some metabolites such as anthraquinone (Uorinda citrofoliu), rosmalinic acid (Colius blumei), visnagin (Ammi visnaga), diosgenin (Dioscoreu deltoidea), etc., were stable after several subcultures, but some metabolites such as nicotine (Nicotiana rusticu), shikonin (Lithospermum erythrorhizon), ajmalicine (Catharanthus roseus), rotenoids (Derris eliptica), anthocyan (Duucus carota), etc., were shown to be unstable after several subcultures. Clonal Plant Propagation. Plants are propagated clonally from vegetative tissue or organs via bypass sex. Conventional clonal propagation can be performed by leaf or stem cutting and layering or dividing ofthe plants, however the efficiency is very low. Recently, many plants were propagated efficiently through tissue culture. This technique was first reported in 1960 by G. Morel["] for the propagation of orchids and since then, many plants have been propagated by tissue culture. Today there are many commercial