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118 The Components of Plant Tissue Culture Media II Activity in tissue cultures. Cultured plant tissues vary in their capacity for myo-inositol biosynthesis. Intact shoots are usually able to produce their own requirements, but although many unorganised tissues are able to grow slowly without the vitamin being added to the medium (Murashige, 1974) the addition of a small quantity is frequently found to stimulate cell division. The compound has been discovered to be essential to some plants. In the opinion of Kaul and Sabharwal (1975) this includes all monocotyledons, the media for which, if they do not contain inositol, need to be complemented with coconut milk, or yeast extract. Fraxinus pennsylvanica callus had an absolute requirement for 10 mg/1 myo-inositol to achieve maximum growth; higher levels, up to 250 mg/l had no further effect on fresh or dry weight yields (Wolter and Skoog, 1966). The formation of shoot buds on callus of Haworthia spp was shown to be dependent on the availability of myo-inositol (Kaul and Sabharwal, 1972, 1975). In a revised Linsmaier and Skoog (1965) medium [Staudt (1984) containing 1.84 mM PO4 3– ], callus tissue of Vitis vinifera cv ‘Müller- Thurgau’ did not require myo-inositol for growth, but that of Vitis vinifera x V. riparia cv. ‘Aris’ was dependent on it and the rate of growth increased as the level of myo-inositol was increased up to 250 mg/l (Staudt, 1984). Gupta et al. (1988) found that it was essential to add 5 g/l myo-inositol to Gupta and Durzan (1985) DCR-1 medium to induce embryogenesis (embryonal suspensor masses) from female gametophyte tissue of Pseudotsuga menziesii and Pinus taeda. The concentration necessary seems insufficient to have acted as an osmotic stimulus (see section 3). myo- Inositol reduced the rate of proliferation in shoot cultures of Euphorbia fulgens (Zhang et al., 1986). Thiamine. Thiamine (Vit. B1, aneurine) in the form of thiamine pyrophosphate, is an essential cofactor in carbohydrate metabolism and is directly involved in the biosynthesis of some amino acids. It has been added to plant culture media more frequently than any other vitamin. Tissues of most plants seem to require it for growth, the need becoming more apparent with consecutive passages, but some cultured cells are self sufficient. The maize suspension cultures of Polikarpochkina et al. (1979) showed much less growth in passage 2, and died in the third passage when thiamine was omitted from the medium. MS medium contains 0.3 μM thiamine. That this may not be sufficient to obtain optimum results from some cultures is illustrated by the results of Barwale et al. (1986): increasing the concentration of thiamine-HCI in MS medium to 5 μM, increased the frequency with which zygotic embryos of Glycine max formed somatic embryos from 33% to 58%. Adding 30 μM nicotinic acid (normally 4 μM) improved the occurrence of embryogenesis even further to 76%. Thiamine was found to be essential for stimulating embryogenic callus induction in Zoysia japonica, a warm season turf grass from Japan (Asano et al., 1996). It has also been shown to stimulate adventitious rooting of Taxus spp. (Chée, 1995). There can be an interaction between thiamine and cytokinin growth regulators. Digby and Skoog (1966) discovered that normal callus cultures of tobacco produced an adequate level of thiamine to support growth providing a relatively high level of kinetin (ca. 1 mg/l) was added to the medium, but the tissue failed to grow when moved to a medium with less added kinetin unless thiamine was provided. Sometimes a change from a thiamine-requiring to a thiamine-sufficient state occurs during culture (see habituation – Chapter 7). In rice callus, thiamine influenced morphogenesis in a way that depended on which state the cells were in. Presence of the vitamin in a pre-culture (Stage I) medium caused thiaminesufficient callus to form root primordia on an induction (Stage II) medium, but suppressed the stimulating effect of kinetin on Stage II shoot formation in thiamine-requiring callus. It was essential to omit thiamine from the Stage I medium to induce thiamine-sufficient callus to produce shoots at Stage II (Inoue and Maeda, 1982). 1.4. OTHER VITAMINS Pantothenic acid. Pantothenic acid plays an important role in the growth of certain tissues. It favoured callus production by hawthorn stem fragments (Morel, 1946) and stimulated tissue proliferation in willow and black henbane (Telle and Gautheret, 1947; Gautheret, 1948). However, pantothenic acid showed no effects with carrot, vine and Virginia creeper tissues which synthesize it in significant amounts (ca. 1 μg/ml). Vitamin C. The effect of Vitamin C (L-ascorbic acid) as a component of culture media will be discussed in Chapter 12. The compound is also used during explant isolation and to prevent blackening.
Besides, its role as an antioxidant, ascorbic acid is involved in cell division and elongation, e.g., in tobacco cells (de Pinto et al., 1999). Ascorbic acid (4-8 x 10 –4 M) also enhanced shoot formation in both young and old tobacco callus. (Joy et al., 1988). It speeded up the shoot-forming process, and completely reversed the inhibition of shoot formation by gibberellic acid in young callus, but was less effective in old callus. Clearly its action here was not as a vitamin. Vitamin D. Some vitamins in the D group, notably vitamin D2 and D3 can have a growth regulatory effect on plant tissue cultures. Their effect is discussed in Chapter 7. Vitamin E. The antioxidant activity of vitamin E (α-tocopherol) will be discussed in Chapter 12. Other vitamins. Evidence has been obtained that folic acid slows tissue proliferation in the dark, while enhancing it in the light. This is probably because it is hydrolysed in the light to p-aminobenzoic acid (PAB). In the presence of auxin, PAB has been shown to have a weak growth-stimulatory effect on cultured plant tissues (de Capite, 1952a,b). Riboflavin which is a component of some vitamin mixtures, has been found to inhibit callus formation but it may improve the growth and quality of shoots (Drew and Smith, 1986). Suppression of callus growth can mean that the vitamin may either inhibit or stimulate root formation on cuttings. Riboflavin has been shown to stimulate adventitious rooting on shoots of Carica papaya (Drew et al., 1993), apple shoots (van der Krieken et al., 1992) and Eucalyptus globulus (Trindade and Pais, 1997). It also enhances embryogenic callus induction in Zoysia japonica in association with cytokinins and thiamine (Asano et al., 1996). Glycine is occasionally described as a vitamin in plant tissue cultures: its use has been described in the section on amino acids. Adenine. Adenine (or adenine sulphate) has been widely used in tissue culture media, but because it mainly gives rise to effects which are similar to those produced by cytokinins, it is considered in the chapter on cytokinins (Chapter 6). Stability. Some vitamins are heat-labile; see the section on medium preparation in Volume 2. 1.5. UNDEFINED SUPPLEMENTS Many undefined supplements were employed in early tissue culture media. Their use has slowly declined as the balance between inorganic salts has been improved, and as the effect of amino acids and Chapter 4 119 growth substances has become better understood. Nevertheless several supplements of uncertain and variable composition are still in common use. The first successful cultures of plant tissue involved the use of yeast extract (Robbins, 1922; White, 1934). Other undefined additions made to plant tissue culture media have been: • meat, malt and yeast extracts and fibrin digest; • juices, pulps and extracts from various fruits (Steward and Shantz, 1959; Ranga Swamy, 1963; Guha and Maheshwari, 1964, 1967), including those from bananas and tomatoes (La Rue, 1949); • the fluids which nourish immature zygotic embryos; • extracts of seedlings (Saalbach and Koblitz, 1978) or plant leaves (Borkird and Sink, 1983); • the extract of boiled potatoes and corn steep liquor (Fox and Miller, 1959); • plant sap or the extract of roots or rhizomes. Plant roots are thought to be the main site of cytokinin synthesis in plants (Chapter 6); • protein (usually casein) hydrolysates (containing a mixture of all the amino acids present in the original protein). Casein hydrolysates are sometimes termed casamino acids: they are discussed in Chapter 3). Many of these amendments can be a source of amino acids, peptides, fatty acids, carbohydrates, vitamins and plant growth substances in different concentrations. Those which have been most widely used are described below. 1.6. YEAST EXTRACT. Yeast extract (YE) is used less as an ingredient of plant media nowadays than in former times, when it was added as a source of amino acids and vitamins, especially inositol and thiamine (Vitamin B1) (Bonner and Addicott, l937; Robbins and Bartley, 1937). In a medium consisting only of macro- and micronutrients, the provision of yeast extract was often found to be essential for tissue growth (White, 1934; Robbins and Bartley, 1937). The vitamin content of yeast extract distinguishes it from casein hydrolysate (CH) so that in such media CH or amino acids alone, could not be substituted for YE (Straus and La Rue, 1954; Nickell and Maretzki, 1969). It was soon found that amino acids such as glycine, lysine and arginine, and vitamins such as thiamine and nicotinic acid, could serve as replacements for YE, for example in the growth of tomato roots (Skinner and Street, 1954), or sugar cane cell suspensions (Nickell and Maretzki, 1969).
Page 1 and 2: Chapter 4 The Components of Plant T
Page 3: progressively enhanced as the myo-
Page 7 and 8: 1.9. BANANA HOMOGENATE Homogenised
Page 9 and 10: Suboptimum stimulation and inhibiti
Page 11 and 12: 3.2 ALTERNATIVES TO SUCROSE 3.2.1.
Page 13 and 14: culture media it has been found to
Page 15 and 16: Street, 1972) the soluble invertase
Page 17 and 18: Chapman, 1975). Sugars apart from s
Page 19 and 20: ‘A’ and ‘B’, of different w
Page 21 and 22: derived in this way agree closely t
Page 23 and 24: contributory. The cells of many pla
Page 25 and 26: induced tobacco callus to form shoo
Page 27 and 28: salts and 2% sucrose (Ψs = ca. -0.
Page 29 and 30: Table 4.7 The relative humidity wit
Page 31 and 32: taken up in exchange for protons, m
Page 33 and 34: into media adjusted to pH 2.5-3.0 o
Page 35 and 36: It has been suggested that acidific
Page 37 and 38: Some other cultures may also be ben
Page 39 and 40: use of bioreactors often involves t
Page 41 and 42: contamination more easily detected
Page 43 and 44: mixture, the fresh mass of plantlet
Page 45 and 46: BORGES M., MENESES S., VAZQUEZ J.,
Page 47 and 48: DOUGALL D.K. 1980 Nutrition and Met
Page 49 and 50: HISAJIMA S., ARAI Y. & ITO T. 1978
Page 51 and 52: LANG A.R.G. 1967 Osmotic coefficien
Page 53 and 54: NESIUS K.K. & FLETCHER J.S. 1973 Ca
Page 55 and 56:
polar transport of auxin. pp. 50-60
Page 57 and 58:
STUART D.A., STRICKLAND S.G. & NICH
Page 59:
WHITE P.R. 1943b Nutrient deficienc
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