Plant Tissue Culture Media and Cell Culturing Notes | EduRev

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: Plant Tissue Culture Media and Cell Culturing Notes | EduRev

 Page 1


Chapter 4 
The Components of Plant Tissue Culture Media ll: 
 Organic Additions, Osmotic and pH Effects, 
 
1.  ORGANIC SUPPLEMENTS 
Growth and morphogenesis of plant tissue 
cultures can be improved by small amounts of some 
organic nutrients.  These are mainly vitamins 
(including some substances that are not strictly 
animal vitamins), amino acids and certain undefined 
supplements.  The amount of these substances 
required for successful culture varies with the species 
and genotype, and is probably a reflection of the 
synthetic capacity of the explant. 
1.1.  VITAMINS 
Vitamins are compounds required by animals in 
very small amounts as necessary ancillary food 
factors.  Absence from the diet leads to abnormal 
growth and development and an unhealthy condition.  
Many of the same substances are also needed by plant 
cells as essential intermediates or metabolic catalysts, 
but intact plants, unlike animals, are able to produce 
their own requirements.  Cultured plant cells and 
tissues can however become deficient in some 
factors; growth and survival is then improved by their 
addition to the culture medium. 
In early work, the requirements of tissue cultures 
for trace amounts of certain organic substances were 
satisfied by “undefined” supplements such as fruit 
juices, coconut milk, yeast or malt extracts and 
hydrolysed casein.  These supplements can contribute 
vitamins, amino acids and growth regulants to a 
culture medium.  The use of undefined supplements 
has declined as the need for specific organic 
compounds has been defined, and these have become 
listed in catalogues as pure chemicals. 
1.2.  THE DEVELOPMENT OF VITAMIN MIXTURES 
The vitamins most frequently used in plant tissue 
culture media are thiamine (Vit. B
1
), nicotinic acid 
(niacin) and pyridoxine (Vit. B
6
) and apart from these 
three compounds, and myo-inositol, there is little 
common agreement about which other vitamins are 
really essential. 
The advantage of adding thiamine was discovered 
almost simultaneously by Bonner (1937, 1938), 
Robbins and Bartley (1937) and White (1937).  
Nicotinic acid and pyridoxine appear, in addition to 
thiamine, in media published by Bonner (1940), 
Gautheret (1942) and White (1943b); this was 
following the findings of Bonner and Devirian (1939) 
that nicotinic acid improved the growth of isolated 
roots of tomato, pea and radish; and the papers of 
Robbins and Schmidt (1939a,b) which indicated that 
pyridoxine was also required for tomato root culture.  
These four vitamins; myo-inositol, thiamine, nicotinic 
acid, and pyridoxine are ingredients of Murashige 
and Skoog (1962) medium and have been used in 
varying proportions for the culture of tissues of many 
plant species (Chapter 3).  However, unless there has 
been research on the requirements of a particular 
plant tissue or organ, it is not possible to conclude 
that all the vitamins which have been used in a 
particular experiment were essential. 
The requirements of cells for added vitamins vary 
according to the nature of the plant and the type of 
culture. Welander (1977) found that Nitsch and 
Nitsch (1965) vitamins were not necessary, or were 
even inhibitory to direct shoot formation on petiole 
explants of Begonia x hiemalis.  Roest and 
Bokelmann (1975) on the other hand, obtained 
increased shoot formation on Chrysanthemum 
pedicels when MS vitamins were present. Callus of 
Pinus strobus grew best when the level of inositol in 
MS medium was reduced to 50 mg/l whereas that of 
P. echinata. proliferated most rapidly when no 
inositol was present (Kaul and Kochbar, 1985). 
Research workers often tend to adopt a ‘belt and 
braces’ attitude to minor media components, and add 
unusual supplements just to ensure that there is no 
missing factor which will limit the success of their 
experiment. Sometimes complex mixtures of as many 
as nine or ten vitamins have been employed. 
Experimentation often shows that some vitamins 
can be omitted from recommended media. Although 
four vitamins were used in MS medium, later work at 
Professor Skoog’s laboratory showed that the 
optimum rate of growth of tobacco callus tissue on 
MS salts required the addition of only myo-inositol 
and thiamine.  The level of thiamine was increased 
four-fold over that used by Murashige and Skoog 
(1962), but nicotinic acid, pyridoxine and glycine 
and Support Systems
115
E. F. George et al. (eds.), Plant Propagation by Tissue Culture 3rd Edition, 115–173. 
© 2008 Springer. 
Page 2


Chapter 4 
The Components of Plant Tissue Culture Media ll: 
 Organic Additions, Osmotic and pH Effects, 
 
1.  ORGANIC SUPPLEMENTS 
Growth and morphogenesis of plant tissue 
cultures can be improved by small amounts of some 
organic nutrients.  These are mainly vitamins 
(including some substances that are not strictly 
animal vitamins), amino acids and certain undefined 
supplements.  The amount of these substances 
required for successful culture varies with the species 
and genotype, and is probably a reflection of the 
synthetic capacity of the explant. 
1.1.  VITAMINS 
Vitamins are compounds required by animals in 
very small amounts as necessary ancillary food 
factors.  Absence from the diet leads to abnormal 
growth and development and an unhealthy condition.  
Many of the same substances are also needed by plant 
cells as essential intermediates or metabolic catalysts, 
but intact plants, unlike animals, are able to produce 
their own requirements.  Cultured plant cells and 
tissues can however become deficient in some 
factors; growth and survival is then improved by their 
addition to the culture medium. 
In early work, the requirements of tissue cultures 
for trace amounts of certain organic substances were 
satisfied by “undefined” supplements such as fruit 
juices, coconut milk, yeast or malt extracts and 
hydrolysed casein.  These supplements can contribute 
vitamins, amino acids and growth regulants to a 
culture medium.  The use of undefined supplements 
has declined as the need for specific organic 
compounds has been defined, and these have become 
listed in catalogues as pure chemicals. 
1.2.  THE DEVELOPMENT OF VITAMIN MIXTURES 
The vitamins most frequently used in plant tissue 
culture media are thiamine (Vit. B
1
), nicotinic acid 
(niacin) and pyridoxine (Vit. B
6
) and apart from these 
three compounds, and myo-inositol, there is little 
common agreement about which other vitamins are 
really essential. 
The advantage of adding thiamine was discovered 
almost simultaneously by Bonner (1937, 1938), 
Robbins and Bartley (1937) and White (1937).  
Nicotinic acid and pyridoxine appear, in addition to 
thiamine, in media published by Bonner (1940), 
Gautheret (1942) and White (1943b); this was 
following the findings of Bonner and Devirian (1939) 
that nicotinic acid improved the growth of isolated 
roots of tomato, pea and radish; and the papers of 
Robbins and Schmidt (1939a,b) which indicated that 
pyridoxine was also required for tomato root culture.  
These four vitamins; myo-inositol, thiamine, nicotinic 
acid, and pyridoxine are ingredients of Murashige 
and Skoog (1962) medium and have been used in 
varying proportions for the culture of tissues of many 
plant species (Chapter 3).  However, unless there has 
been research on the requirements of a particular 
plant tissue or organ, it is not possible to conclude 
that all the vitamins which have been used in a 
particular experiment were essential. 
The requirements of cells for added vitamins vary 
according to the nature of the plant and the type of 
culture. Welander (1977) found that Nitsch and 
Nitsch (1965) vitamins were not necessary, or were 
even inhibitory to direct shoot formation on petiole 
explants of Begonia x hiemalis.  Roest and 
Bokelmann (1975) on the other hand, obtained 
increased shoot formation on Chrysanthemum 
pedicels when MS vitamins were present. Callus of 
Pinus strobus grew best when the level of inositol in 
MS medium was reduced to 50 mg/l whereas that of 
P. echinata. proliferated most rapidly when no 
inositol was present (Kaul and Kochbar, 1985). 
Research workers often tend to adopt a ‘belt and 
braces’ attitude to minor media components, and add 
unusual supplements just to ensure that there is no 
missing factor which will limit the success of their 
experiment. Sometimes complex mixtures of as many 
as nine or ten vitamins have been employed. 
Experimentation often shows that some vitamins 
can be omitted from recommended media. Although 
four vitamins were used in MS medium, later work at 
Professor Skoog’s laboratory showed that the 
optimum rate of growth of tobacco callus tissue on 
MS salts required the addition of only myo-inositol 
and thiamine.  The level of thiamine was increased 
four-fold over that used by Murashige and Skoog 
(1962), but nicotinic acid, pyridoxine and glycine 
and Support Systems
115
E. F. George et al. (eds.), Plant Propagation by Tissue Culture 3rd Edition, 115–173. 
© 2008 Springer. 
(amino acid) were unnecessary (Linsmaier and 
Skoog, 1965).  A similar simplification of the MS 
vitamins was made by Earle and Torrey (1965) for 
the culture of Convolvulus callus. 
Soczck and Hempel (1988) found that in the 
medium of Murashige et al. (1974) devised for the 
shoot culture of Gerbera jamesonii, thiamine, pyrid-
oxine and inositol could be omitted without any 
reduction in the rate of shoot multiplication of their 
local cultivars. Ishihara and Katano (1982) found that 
Malus shoot cultures could be grown on MS salts 
alone, and that inositol and thiamine were largely 
unnecessary. 
1.3.  SPECIFIC COMPOUNDS 
Myo-inositol.  Myo-inositol (also sometimes 
described as meso-inositol or i-inositol) is the only 
one of the nine theoretical stereoisomers of inositol 
which has significant biological importance. 
Medically it has been classed as a member of the 
Vitamin B complex and is required for the growth of 
yeast and many mammalian cells in tissue culture.  
Rats and mice require it for hair growth and can 
develop dermatitis when it is not in the diet.  Myo-
inositol has been classed as a plant ‘vitamin’, but note 
that some authors think that it should be regarded as a 
supplementary carbohydrate, although it does not 
contribute to carbohydrate utilization as an energy 
source or as an osmoticum. 
Historical use in tissue cultures.  Myo-inositol 
was first shown by Jacquiot (1951) to favour bud 
formation by elm cambial tissue when supplied at 20-
1000 mg/l. Necrosis was retarded, though the 
proliferation of the callus was not promoted.  Myo-
inositol at 100 mg/1 was also used by Morel and 
Wetmore (1951) in combination with six other 
vitamins for the culture of callus from the 
monocotyledon Amorphophallus rivieri (Araceae).  
Bud initials appeared on some cultures and both roots 
and buds on others according to the concentration of 
auxin employed. The vitamin was adopted by both 
Wood and Braun (1961) and Murashige and Skoog 
(1962) in combination with thiamine, nicotinic acid 
and pyridoxine in their preferred media fur the 
culture of Catharanthus roseus and Nicotiana 
tabacum respectively. Many other workers have since 
included it in culture media with favourable results 
on the rate of callus growth or the induction of 
morphogenesis. Letham (1966) found that myo-
inositol interacted with cytokinin to promote cell 
division in carrot phloem explants. 
Occurrence and biochemistry.  Part of the 
growth promoting property of coconut milk is due to 
its myo-inositol content (Pollard et al., 1961). 
Coconut milk also contains scyllo-inositol (Table 
4.1). This can also promote growth but to a smaller 
extent than the myo-isomer (Pollard et al., 1961). 
Inositol is a constituent of yeast extract (Steiner et al., 
1969; Steiner and Lester, 1972) and small quantities 
may also be contained in commercial agar (Wolter 
and Skoog, 1966).  Myo-inositol is a natural 
constituent of plants and much of it is often 
incorporated into phosphatidyl-inositol which may be 
an important factor in the functioning of membranes 
(Jung et al., 1972; Harran and Dickinson, 1978). The 
phosphatidylinositol cycle controls various cellular 
responses in animal cells and yeasts, but evidence of 
it playing a similar role in plants is only just being 
accumulated. Enzymes which are thought to be 
involved in the cycle have been observed to have 
activities in plants and lithium chloride (which 
inhibits myo-inositol-1-phosphatase and decreases the 
cycle) inhibits callus formation in Brassica oleracea 
(Bagga et al., 1987), and callus growth in 
Amaranthus paniculatus (Das et al., 1987). In both 
plants the inhibition is reversed by myo-inositol. 
As the myo-inositol molecule has six hydroxyl 
units, it can react with up to six acid molecules 
forming various esters. It appears that inositol 
phosphates act as second messengers to the primary 
action of auxin in plants: phytic acid (inositol hexa-
phosphate) is one of these. Added to culture media it 
can promote tissue growth if it can serve as a source 
of inositol (Watanabe et al., 1971). In some species, 
auxin can be stored and may be transported as IAA-
myo-inositol ester (Chapter 5). o-Methyl-inositol is 
present in quite large quantities in legumes; inositol 
methyl ethers are known to occur in plants of several 
other families, although their function is unknown 
(Phillips and Smith, 1974). 
The stimulatory effect of myo-inositol in plant 
cultures probably arises partly from the participation 
of the compound in biosynthetic pathways leading to 
the formation of the pectin and hemicelluloses needed 
in cell walls (Loewus et al., 1962; Loewus, 1974; 
Loewus and Loewus, 1980; Harran and Dickinson, 
1978; Verma and Dougall, 1979; Loewus and 
Loewus, 1980) and may have a role in the uptake and 
utilization of ions (Wood and Braun, 1961). In the 
experiments of Staudt (1984) mentioned below, when 
the P0
4
3–
 content of the medium was raised to 4.41 
mM, the rate of callus growth of cv. ‘Aris’ was 
The Components of Plant Tissue Culture Media II 116
Page 3


Chapter 4 
The Components of Plant Tissue Culture Media ll: 
 Organic Additions, Osmotic and pH Effects, 
 
1.  ORGANIC SUPPLEMENTS 
Growth and morphogenesis of plant tissue 
cultures can be improved by small amounts of some 
organic nutrients.  These are mainly vitamins 
(including some substances that are not strictly 
animal vitamins), amino acids and certain undefined 
supplements.  The amount of these substances 
required for successful culture varies with the species 
and genotype, and is probably a reflection of the 
synthetic capacity of the explant. 
1.1.  VITAMINS 
Vitamins are compounds required by animals in 
very small amounts as necessary ancillary food 
factors.  Absence from the diet leads to abnormal 
growth and development and an unhealthy condition.  
Many of the same substances are also needed by plant 
cells as essential intermediates or metabolic catalysts, 
but intact plants, unlike animals, are able to produce 
their own requirements.  Cultured plant cells and 
tissues can however become deficient in some 
factors; growth and survival is then improved by their 
addition to the culture medium. 
In early work, the requirements of tissue cultures 
for trace amounts of certain organic substances were 
satisfied by “undefined” supplements such as fruit 
juices, coconut milk, yeast or malt extracts and 
hydrolysed casein.  These supplements can contribute 
vitamins, amino acids and growth regulants to a 
culture medium.  The use of undefined supplements 
has declined as the need for specific organic 
compounds has been defined, and these have become 
listed in catalogues as pure chemicals. 
1.2.  THE DEVELOPMENT OF VITAMIN MIXTURES 
The vitamins most frequently used in plant tissue 
culture media are thiamine (Vit. B
1
), nicotinic acid 
(niacin) and pyridoxine (Vit. B
6
) and apart from these 
three compounds, and myo-inositol, there is little 
common agreement about which other vitamins are 
really essential. 
The advantage of adding thiamine was discovered 
almost simultaneously by Bonner (1937, 1938), 
Robbins and Bartley (1937) and White (1937).  
Nicotinic acid and pyridoxine appear, in addition to 
thiamine, in media published by Bonner (1940), 
Gautheret (1942) and White (1943b); this was 
following the findings of Bonner and Devirian (1939) 
that nicotinic acid improved the growth of isolated 
roots of tomato, pea and radish; and the papers of 
Robbins and Schmidt (1939a,b) which indicated that 
pyridoxine was also required for tomato root culture.  
These four vitamins; myo-inositol, thiamine, nicotinic 
acid, and pyridoxine are ingredients of Murashige 
and Skoog (1962) medium and have been used in 
varying proportions for the culture of tissues of many 
plant species (Chapter 3).  However, unless there has 
been research on the requirements of a particular 
plant tissue or organ, it is not possible to conclude 
that all the vitamins which have been used in a 
particular experiment were essential. 
The requirements of cells for added vitamins vary 
according to the nature of the plant and the type of 
culture. Welander (1977) found that Nitsch and 
Nitsch (1965) vitamins were not necessary, or were 
even inhibitory to direct shoot formation on petiole 
explants of Begonia x hiemalis.  Roest and 
Bokelmann (1975) on the other hand, obtained 
increased shoot formation on Chrysanthemum 
pedicels when MS vitamins were present. Callus of 
Pinus strobus grew best when the level of inositol in 
MS medium was reduced to 50 mg/l whereas that of 
P. echinata. proliferated most rapidly when no 
inositol was present (Kaul and Kochbar, 1985). 
Research workers often tend to adopt a ‘belt and 
braces’ attitude to minor media components, and add 
unusual supplements just to ensure that there is no 
missing factor which will limit the success of their 
experiment. Sometimes complex mixtures of as many 
as nine or ten vitamins have been employed. 
Experimentation often shows that some vitamins 
can be omitted from recommended media. Although 
four vitamins were used in MS medium, later work at 
Professor Skoog’s laboratory showed that the 
optimum rate of growth of tobacco callus tissue on 
MS salts required the addition of only myo-inositol 
and thiamine.  The level of thiamine was increased 
four-fold over that used by Murashige and Skoog 
(1962), but nicotinic acid, pyridoxine and glycine 
and Support Systems
115
E. F. George et al. (eds.), Plant Propagation by Tissue Culture 3rd Edition, 115–173. 
© 2008 Springer. 
(amino acid) were unnecessary (Linsmaier and 
Skoog, 1965).  A similar simplification of the MS 
vitamins was made by Earle and Torrey (1965) for 
the culture of Convolvulus callus. 
Soczck and Hempel (1988) found that in the 
medium of Murashige et al. (1974) devised for the 
shoot culture of Gerbera jamesonii, thiamine, pyrid-
oxine and inositol could be omitted without any 
reduction in the rate of shoot multiplication of their 
local cultivars. Ishihara and Katano (1982) found that 
Malus shoot cultures could be grown on MS salts 
alone, and that inositol and thiamine were largely 
unnecessary. 
1.3.  SPECIFIC COMPOUNDS 
Myo-inositol.  Myo-inositol (also sometimes 
described as meso-inositol or i-inositol) is the only 
one of the nine theoretical stereoisomers of inositol 
which has significant biological importance. 
Medically it has been classed as a member of the 
Vitamin B complex and is required for the growth of 
yeast and many mammalian cells in tissue culture.  
Rats and mice require it for hair growth and can 
develop dermatitis when it is not in the diet.  Myo-
inositol has been classed as a plant ‘vitamin’, but note 
that some authors think that it should be regarded as a 
supplementary carbohydrate, although it does not 
contribute to carbohydrate utilization as an energy 
source or as an osmoticum. 
Historical use in tissue cultures.  Myo-inositol 
was first shown by Jacquiot (1951) to favour bud 
formation by elm cambial tissue when supplied at 20-
1000 mg/l. Necrosis was retarded, though the 
proliferation of the callus was not promoted.  Myo-
inositol at 100 mg/1 was also used by Morel and 
Wetmore (1951) in combination with six other 
vitamins for the culture of callus from the 
monocotyledon Amorphophallus rivieri (Araceae).  
Bud initials appeared on some cultures and both roots 
and buds on others according to the concentration of 
auxin employed. The vitamin was adopted by both 
Wood and Braun (1961) and Murashige and Skoog 
(1962) in combination with thiamine, nicotinic acid 
and pyridoxine in their preferred media fur the 
culture of Catharanthus roseus and Nicotiana 
tabacum respectively. Many other workers have since 
included it in culture media with favourable results 
on the rate of callus growth or the induction of 
morphogenesis. Letham (1966) found that myo-
inositol interacted with cytokinin to promote cell 
division in carrot phloem explants. 
Occurrence and biochemistry.  Part of the 
growth promoting property of coconut milk is due to 
its myo-inositol content (Pollard et al., 1961). 
Coconut milk also contains scyllo-inositol (Table 
4.1). This can also promote growth but to a smaller 
extent than the myo-isomer (Pollard et al., 1961). 
Inositol is a constituent of yeast extract (Steiner et al., 
1969; Steiner and Lester, 1972) and small quantities 
may also be contained in commercial agar (Wolter 
and Skoog, 1966).  Myo-inositol is a natural 
constituent of plants and much of it is often 
incorporated into phosphatidyl-inositol which may be 
an important factor in the functioning of membranes 
(Jung et al., 1972; Harran and Dickinson, 1978). The 
phosphatidylinositol cycle controls various cellular 
responses in animal cells and yeasts, but evidence of 
it playing a similar role in plants is only just being 
accumulated. Enzymes which are thought to be 
involved in the cycle have been observed to have 
activities in plants and lithium chloride (which 
inhibits myo-inositol-1-phosphatase and decreases the 
cycle) inhibits callus formation in Brassica oleracea 
(Bagga et al., 1987), and callus growth in 
Amaranthus paniculatus (Das et al., 1987). In both 
plants the inhibition is reversed by myo-inositol. 
As the myo-inositol molecule has six hydroxyl 
units, it can react with up to six acid molecules 
forming various esters. It appears that inositol 
phosphates act as second messengers to the primary 
action of auxin in plants: phytic acid (inositol hexa-
phosphate) is one of these. Added to culture media it 
can promote tissue growth if it can serve as a source 
of inositol (Watanabe et al., 1971). In some species, 
auxin can be stored and may be transported as IAA-
myo-inositol ester (Chapter 5). o-Methyl-inositol is 
present in quite large quantities in legumes; inositol 
methyl ethers are known to occur in plants of several 
other families, although their function is unknown 
(Phillips and Smith, 1974). 
The stimulatory effect of myo-inositol in plant 
cultures probably arises partly from the participation 
of the compound in biosynthetic pathways leading to 
the formation of the pectin and hemicelluloses needed 
in cell walls (Loewus et al., 1962; Loewus, 1974; 
Loewus and Loewus, 1980; Harran and Dickinson, 
1978; Verma and Dougall, 1979; Loewus and 
Loewus, 1980) and may have a role in the uptake and 
utilization of ions (Wood and Braun, 1961). In the 
experiments of Staudt (1984) mentioned below, when 
the P0
4
3–
 content of the medium was raised to 4.41 
mM, the rate of callus growth of cv. ‘Aris’ was 
The Components of Plant Tissue Culture Media II 116 Chapter 4
progressively enhanced as the myo- inositol in the 
medium was put up to 4000 mg/l. This result seems 
to stress the importance of inositol-containing 
phospholipids for growth. 
Table 4.1. Substances identified as components of coconut milk (water) from mature green fruits and market-purchased fruits. 
SUBSTANCE QUANTITY/REFERENCE SUBSTANCE QUANTITY/REFERENCE 
 Mature green 
fruits 
Mature fresh 
fruits 
 Mature 
green 
fruits 
Mature fresh 
fruits 
Amino acids (mg/l) Sugars (g/l) 
Alanine 127.3 (14) 312 (13), 177.1 
(14) 
Sucrose 9.2 (14) 8.9 (14) 
Arginine 25.6 (14) 133 (13), 16.8 
(14) 
Glucose 7.3 (14) 2.5 (14) 
Aspartic acid 35.9 (14) 65 (13), 5.4 (14) Fructose 5.3 (14) 2.5 (14) 
Asparagine 10.1 (14) ca.60 (13), 10.1 
(14) 
Sugar alcohols (g/l) 
?-Aminobutyric 
acid 
34.6 (14) 820 (13), 168.8 
(14) 
Mannitol  (1) 
Glutamine acid 70.8 (14) 240 (13), 78.7 
(14) 
Sorbitol  15.0 (12), (17) 
Glutamine 45.4 (14) ca.60 (13), 13.4 
(14) 
myo-Inositol  0.1 (12), (17) 
Glycine 9.7 (14) 13.9 (14) scyllo-Inositol  0.5 (12), (17) 
Histidine 6.3 (14) Trace (13,14) Vitamins (mg/l) 
Homoserine -- (14) 5.2 (14) Nicotinic acid  0.64 (4) 
Hydroxyproline  Trace (13,14) Pantolhenic acid  0.52 (4) 
Lysine 21.4 65.8 (14) Biotin, Riboflavin  0.02 (4) 
Methionine 16.9 (14) 8 (13), Trace (14) Riboflavin  0.01 (4) 
Phenylalanine -- (14) 12 (13), 10.2 (14) Folic acid  0.003 (4) 
Proline 31.9 97 (13), 21.6 (14) Thiamine, pyridoxine  Trace (4) 
Serine 45.3 (14)  Growth substances (mg/l) 
Typtophan  39 (13) Auxin  0.07 (7), (28) 
Threonine 16.2 (2) 44 (13), 26.3 (14) Gibberellin  Yes (10,28) 
Tyrosine 6.4 (14) 16 (13), 3.1 (14) 1,3-Diphenylurea  5.8 (8), (6,17) 
Valine 20.6 (14) 27 (13), 15.1 (14) Zeatin  (22,26) 
Other nitrogenous compounds Zeatin glucoside  (26) 
Ammonium  (19) Zeatin riboside  (20), (24), (25) 
Ethanolamine  (19) 6-Oxypurine growth 
promoter 
 (27) 
Dihydroxyphenyl
alanine 
 (19) Unknown cytokinin/s  6, (18) (22) 
Inorganic elements (mg/100g dry wt.) Other (mg/l) 
Potassium  312.0 (3) RNA-polymerase  (23) 
Sodium  105 (3) RNA-phosphorus 20.0 (14) 35.4 (14) 
Phosphorus  37.0 (3) DNA-phosphorus 0.1 (14) 3.5 (14) 
Magnesium  30.0 (3) Uracil, Adenine  21 
Organic acids (meq/ml) Leucoanthocyanins  (11) (15,17) 
Malic acid 34.3 (14) 12.0 (14) Phyllococosine  (16) 
Shikimic, Quinic 
and 2 unknowns 
0.6 (14) 0.41 (2) Acid Phosphatase  (5,9) 
Pyrrolidone 
carboxylic acid 
0.4 (14) 0.2 (14) Diastase  (2) 
Citric acid 0.4 (14) 0.3 (14) Dehydrogenase  (5) 
Succinic acid -- (14) 0.3 (14) Peroxidase  (5) 
   Catalase  (5) 
Numbered references (within brackets) in the above table are listed in Section 1.11 of this Chapter. 
117
Page 4


Chapter 4 
The Components of Plant Tissue Culture Media ll: 
 Organic Additions, Osmotic and pH Effects, 
 
1.  ORGANIC SUPPLEMENTS 
Growth and morphogenesis of plant tissue 
cultures can be improved by small amounts of some 
organic nutrients.  These are mainly vitamins 
(including some substances that are not strictly 
animal vitamins), amino acids and certain undefined 
supplements.  The amount of these substances 
required for successful culture varies with the species 
and genotype, and is probably a reflection of the 
synthetic capacity of the explant. 
1.1.  VITAMINS 
Vitamins are compounds required by animals in 
very small amounts as necessary ancillary food 
factors.  Absence from the diet leads to abnormal 
growth and development and an unhealthy condition.  
Many of the same substances are also needed by plant 
cells as essential intermediates or metabolic catalysts, 
but intact plants, unlike animals, are able to produce 
their own requirements.  Cultured plant cells and 
tissues can however become deficient in some 
factors; growth and survival is then improved by their 
addition to the culture medium. 
In early work, the requirements of tissue cultures 
for trace amounts of certain organic substances were 
satisfied by “undefined” supplements such as fruit 
juices, coconut milk, yeast or malt extracts and 
hydrolysed casein.  These supplements can contribute 
vitamins, amino acids and growth regulants to a 
culture medium.  The use of undefined supplements 
has declined as the need for specific organic 
compounds has been defined, and these have become 
listed in catalogues as pure chemicals. 
1.2.  THE DEVELOPMENT OF VITAMIN MIXTURES 
The vitamins most frequently used in plant tissue 
culture media are thiamine (Vit. B
1
), nicotinic acid 
(niacin) and pyridoxine (Vit. B
6
) and apart from these 
three compounds, and myo-inositol, there is little 
common agreement about which other vitamins are 
really essential. 
The advantage of adding thiamine was discovered 
almost simultaneously by Bonner (1937, 1938), 
Robbins and Bartley (1937) and White (1937).  
Nicotinic acid and pyridoxine appear, in addition to 
thiamine, in media published by Bonner (1940), 
Gautheret (1942) and White (1943b); this was 
following the findings of Bonner and Devirian (1939) 
that nicotinic acid improved the growth of isolated 
roots of tomato, pea and radish; and the papers of 
Robbins and Schmidt (1939a,b) which indicated that 
pyridoxine was also required for tomato root culture.  
These four vitamins; myo-inositol, thiamine, nicotinic 
acid, and pyridoxine are ingredients of Murashige 
and Skoog (1962) medium and have been used in 
varying proportions for the culture of tissues of many 
plant species (Chapter 3).  However, unless there has 
been research on the requirements of a particular 
plant tissue or organ, it is not possible to conclude 
that all the vitamins which have been used in a 
particular experiment were essential. 
The requirements of cells for added vitamins vary 
according to the nature of the plant and the type of 
culture. Welander (1977) found that Nitsch and 
Nitsch (1965) vitamins were not necessary, or were 
even inhibitory to direct shoot formation on petiole 
explants of Begonia x hiemalis.  Roest and 
Bokelmann (1975) on the other hand, obtained 
increased shoot formation on Chrysanthemum 
pedicels when MS vitamins were present. Callus of 
Pinus strobus grew best when the level of inositol in 
MS medium was reduced to 50 mg/l whereas that of 
P. echinata. proliferated most rapidly when no 
inositol was present (Kaul and Kochbar, 1985). 
Research workers often tend to adopt a ‘belt and 
braces’ attitude to minor media components, and add 
unusual supplements just to ensure that there is no 
missing factor which will limit the success of their 
experiment. Sometimes complex mixtures of as many 
as nine or ten vitamins have been employed. 
Experimentation often shows that some vitamins 
can be omitted from recommended media. Although 
four vitamins were used in MS medium, later work at 
Professor Skoog’s laboratory showed that the 
optimum rate of growth of tobacco callus tissue on 
MS salts required the addition of only myo-inositol 
and thiamine.  The level of thiamine was increased 
four-fold over that used by Murashige and Skoog 
(1962), but nicotinic acid, pyridoxine and glycine 
and Support Systems
115
E. F. George et al. (eds.), Plant Propagation by Tissue Culture 3rd Edition, 115–173. 
© 2008 Springer. 
(amino acid) were unnecessary (Linsmaier and 
Skoog, 1965).  A similar simplification of the MS 
vitamins was made by Earle and Torrey (1965) for 
the culture of Convolvulus callus. 
Soczck and Hempel (1988) found that in the 
medium of Murashige et al. (1974) devised for the 
shoot culture of Gerbera jamesonii, thiamine, pyrid-
oxine and inositol could be omitted without any 
reduction in the rate of shoot multiplication of their 
local cultivars. Ishihara and Katano (1982) found that 
Malus shoot cultures could be grown on MS salts 
alone, and that inositol and thiamine were largely 
unnecessary. 
1.3.  SPECIFIC COMPOUNDS 
Myo-inositol.  Myo-inositol (also sometimes 
described as meso-inositol or i-inositol) is the only 
one of the nine theoretical stereoisomers of inositol 
which has significant biological importance. 
Medically it has been classed as a member of the 
Vitamin B complex and is required for the growth of 
yeast and many mammalian cells in tissue culture.  
Rats and mice require it for hair growth and can 
develop dermatitis when it is not in the diet.  Myo-
inositol has been classed as a plant ‘vitamin’, but note 
that some authors think that it should be regarded as a 
supplementary carbohydrate, although it does not 
contribute to carbohydrate utilization as an energy 
source or as an osmoticum. 
Historical use in tissue cultures.  Myo-inositol 
was first shown by Jacquiot (1951) to favour bud 
formation by elm cambial tissue when supplied at 20-
1000 mg/l. Necrosis was retarded, though the 
proliferation of the callus was not promoted.  Myo-
inositol at 100 mg/1 was also used by Morel and 
Wetmore (1951) in combination with six other 
vitamins for the culture of callus from the 
monocotyledon Amorphophallus rivieri (Araceae).  
Bud initials appeared on some cultures and both roots 
and buds on others according to the concentration of 
auxin employed. The vitamin was adopted by both 
Wood and Braun (1961) and Murashige and Skoog 
(1962) in combination with thiamine, nicotinic acid 
and pyridoxine in their preferred media fur the 
culture of Catharanthus roseus and Nicotiana 
tabacum respectively. Many other workers have since 
included it in culture media with favourable results 
on the rate of callus growth or the induction of 
morphogenesis. Letham (1966) found that myo-
inositol interacted with cytokinin to promote cell 
division in carrot phloem explants. 
Occurrence and biochemistry.  Part of the 
growth promoting property of coconut milk is due to 
its myo-inositol content (Pollard et al., 1961). 
Coconut milk also contains scyllo-inositol (Table 
4.1). This can also promote growth but to a smaller 
extent than the myo-isomer (Pollard et al., 1961). 
Inositol is a constituent of yeast extract (Steiner et al., 
1969; Steiner and Lester, 1972) and small quantities 
may also be contained in commercial agar (Wolter 
and Skoog, 1966).  Myo-inositol is a natural 
constituent of plants and much of it is often 
incorporated into phosphatidyl-inositol which may be 
an important factor in the functioning of membranes 
(Jung et al., 1972; Harran and Dickinson, 1978). The 
phosphatidylinositol cycle controls various cellular 
responses in animal cells and yeasts, but evidence of 
it playing a similar role in plants is only just being 
accumulated. Enzymes which are thought to be 
involved in the cycle have been observed to have 
activities in plants and lithium chloride (which 
inhibits myo-inositol-1-phosphatase and decreases the 
cycle) inhibits callus formation in Brassica oleracea 
(Bagga et al., 1987), and callus growth in 
Amaranthus paniculatus (Das et al., 1987). In both 
plants the inhibition is reversed by myo-inositol. 
As the myo-inositol molecule has six hydroxyl 
units, it can react with up to six acid molecules 
forming various esters. It appears that inositol 
phosphates act as second messengers to the primary 
action of auxin in plants: phytic acid (inositol hexa-
phosphate) is one of these. Added to culture media it 
can promote tissue growth if it can serve as a source 
of inositol (Watanabe et al., 1971). In some species, 
auxin can be stored and may be transported as IAA-
myo-inositol ester (Chapter 5). o-Methyl-inositol is 
present in quite large quantities in legumes; inositol 
methyl ethers are known to occur in plants of several 
other families, although their function is unknown 
(Phillips and Smith, 1974). 
The stimulatory effect of myo-inositol in plant 
cultures probably arises partly from the participation 
of the compound in biosynthetic pathways leading to 
the formation of the pectin and hemicelluloses needed 
in cell walls (Loewus et al., 1962; Loewus, 1974; 
Loewus and Loewus, 1980; Harran and Dickinson, 
1978; Verma and Dougall, 1979; Loewus and 
Loewus, 1980) and may have a role in the uptake and 
utilization of ions (Wood and Braun, 1961). In the 
experiments of Staudt (1984) mentioned below, when 
the P0
4
3–
 content of the medium was raised to 4.41 
mM, the rate of callus growth of cv. ‘Aris’ was 
The Components of Plant Tissue Culture Media II 116 Chapter 4
progressively enhanced as the myo- inositol in the 
medium was put up to 4000 mg/l. This result seems 
to stress the importance of inositol-containing 
phospholipids for growth. 
Table 4.1. Substances identified as components of coconut milk (water) from mature green fruits and market-purchased fruits. 
SUBSTANCE QUANTITY/REFERENCE SUBSTANCE QUANTITY/REFERENCE 
 Mature green 
fruits 
Mature fresh 
fruits 
 Mature 
green 
fruits 
Mature fresh 
fruits 
Amino acids (mg/l) Sugars (g/l) 
Alanine 127.3 (14) 312 (13), 177.1 
(14) 
Sucrose 9.2 (14) 8.9 (14) 
Arginine 25.6 (14) 133 (13), 16.8 
(14) 
Glucose 7.3 (14) 2.5 (14) 
Aspartic acid 35.9 (14) 65 (13), 5.4 (14) Fructose 5.3 (14) 2.5 (14) 
Asparagine 10.1 (14) ca.60 (13), 10.1 
(14) 
Sugar alcohols (g/l) 
?-Aminobutyric 
acid 
34.6 (14) 820 (13), 168.8 
(14) 
Mannitol  (1) 
Glutamine acid 70.8 (14) 240 (13), 78.7 
(14) 
Sorbitol  15.0 (12), (17) 
Glutamine 45.4 (14) ca.60 (13), 13.4 
(14) 
myo-Inositol  0.1 (12), (17) 
Glycine 9.7 (14) 13.9 (14) scyllo-Inositol  0.5 (12), (17) 
Histidine 6.3 (14) Trace (13,14) Vitamins (mg/l) 
Homoserine -- (14) 5.2 (14) Nicotinic acid  0.64 (4) 
Hydroxyproline  Trace (13,14) Pantolhenic acid  0.52 (4) 
Lysine 21.4 65.8 (14) Biotin, Riboflavin  0.02 (4) 
Methionine 16.9 (14) 8 (13), Trace (14) Riboflavin  0.01 (4) 
Phenylalanine -- (14) 12 (13), 10.2 (14) Folic acid  0.003 (4) 
Proline 31.9 97 (13), 21.6 (14) Thiamine, pyridoxine  Trace (4) 
Serine 45.3 (14)  Growth substances (mg/l) 
Typtophan  39 (13) Auxin  0.07 (7), (28) 
Threonine 16.2 (2) 44 (13), 26.3 (14) Gibberellin  Yes (10,28) 
Tyrosine 6.4 (14) 16 (13), 3.1 (14) 1,3-Diphenylurea  5.8 (8), (6,17) 
Valine 20.6 (14) 27 (13), 15.1 (14) Zeatin  (22,26) 
Other nitrogenous compounds Zeatin glucoside  (26) 
Ammonium  (19) Zeatin riboside  (20), (24), (25) 
Ethanolamine  (19) 6-Oxypurine growth 
promoter 
 (27) 
Dihydroxyphenyl
alanine 
 (19) Unknown cytokinin/s  6, (18) (22) 
Inorganic elements (mg/100g dry wt.) Other (mg/l) 
Potassium  312.0 (3) RNA-polymerase  (23) 
Sodium  105 (3) RNA-phosphorus 20.0 (14) 35.4 (14) 
Phosphorus  37.0 (3) DNA-phosphorus 0.1 (14) 3.5 (14) 
Magnesium  30.0 (3) Uracil, Adenine  21 
Organic acids (meq/ml) Leucoanthocyanins  (11) (15,17) 
Malic acid 34.3 (14) 12.0 (14) Phyllococosine  (16) 
Shikimic, Quinic 
and 2 unknowns 
0.6 (14) 0.41 (2) Acid Phosphatase  (5,9) 
Pyrrolidone 
carboxylic acid 
0.4 (14) 0.2 (14) Diastase  (2) 
Citric acid 0.4 (14) 0.3 (14) Dehydrogenase  (5) 
Succinic acid -- (14) 0.3 (14) Peroxidase  (5) 
   Catalase  (5) 
Numbered references (within brackets) in the above table are listed in Section 1.11 of this Chapter. 
117
 
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 monocotyl-
edons, 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 PO
4
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. B
1
, aneurine) in the 
form of thiamine pyrophosphate, is an essential co-
factor 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
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 thiamine-
sufficient 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.  
118 The Components of Plant Tissue Culture Media II
 spp. (Chée, 
Page 5


Chapter 4 
The Components of Plant Tissue Culture Media ll: 
 Organic Additions, Osmotic and pH Effects, 
 
1.  ORGANIC SUPPLEMENTS 
Growth and morphogenesis of plant tissue 
cultures can be improved by small amounts of some 
organic nutrients.  These are mainly vitamins 
(including some substances that are not strictly 
animal vitamins), amino acids and certain undefined 
supplements.  The amount of these substances 
required for successful culture varies with the species 
and genotype, and is probably a reflection of the 
synthetic capacity of the explant. 
1.1.  VITAMINS 
Vitamins are compounds required by animals in 
very small amounts as necessary ancillary food 
factors.  Absence from the diet leads to abnormal 
growth and development and an unhealthy condition.  
Many of the same substances are also needed by plant 
cells as essential intermediates or metabolic catalysts, 
but intact plants, unlike animals, are able to produce 
their own requirements.  Cultured plant cells and 
tissues can however become deficient in some 
factors; growth and survival is then improved by their 
addition to the culture medium. 
In early work, the requirements of tissue cultures 
for trace amounts of certain organic substances were 
satisfied by “undefined” supplements such as fruit 
juices, coconut milk, yeast or malt extracts and 
hydrolysed casein.  These supplements can contribute 
vitamins, amino acids and growth regulants to a 
culture medium.  The use of undefined supplements 
has declined as the need for specific organic 
compounds has been defined, and these have become 
listed in catalogues as pure chemicals. 
1.2.  THE DEVELOPMENT OF VITAMIN MIXTURES 
The vitamins most frequently used in plant tissue 
culture media are thiamine (Vit. B
1
), nicotinic acid 
(niacin) and pyridoxine (Vit. B
6
) and apart from these 
three compounds, and myo-inositol, there is little 
common agreement about which other vitamins are 
really essential. 
The advantage of adding thiamine was discovered 
almost simultaneously by Bonner (1937, 1938), 
Robbins and Bartley (1937) and White (1937).  
Nicotinic acid and pyridoxine appear, in addition to 
thiamine, in media published by Bonner (1940), 
Gautheret (1942) and White (1943b); this was 
following the findings of Bonner and Devirian (1939) 
that nicotinic acid improved the growth of isolated 
roots of tomato, pea and radish; and the papers of 
Robbins and Schmidt (1939a,b) which indicated that 
pyridoxine was also required for tomato root culture.  
These four vitamins; myo-inositol, thiamine, nicotinic 
acid, and pyridoxine are ingredients of Murashige 
and Skoog (1962) medium and have been used in 
varying proportions for the culture of tissues of many 
plant species (Chapter 3).  However, unless there has 
been research on the requirements of a particular 
plant tissue or organ, it is not possible to conclude 
that all the vitamins which have been used in a 
particular experiment were essential. 
The requirements of cells for added vitamins vary 
according to the nature of the plant and the type of 
culture. Welander (1977) found that Nitsch and 
Nitsch (1965) vitamins were not necessary, or were 
even inhibitory to direct shoot formation on petiole 
explants of Begonia x hiemalis.  Roest and 
Bokelmann (1975) on the other hand, obtained 
increased shoot formation on Chrysanthemum 
pedicels when MS vitamins were present. Callus of 
Pinus strobus grew best when the level of inositol in 
MS medium was reduced to 50 mg/l whereas that of 
P. echinata. proliferated most rapidly when no 
inositol was present (Kaul and Kochbar, 1985). 
Research workers often tend to adopt a ‘belt and 
braces’ attitude to minor media components, and add 
unusual supplements just to ensure that there is no 
missing factor which will limit the success of their 
experiment. Sometimes complex mixtures of as many 
as nine or ten vitamins have been employed. 
Experimentation often shows that some vitamins 
can be omitted from recommended media. Although 
four vitamins were used in MS medium, later work at 
Professor Skoog’s laboratory showed that the 
optimum rate of growth of tobacco callus tissue on 
MS salts required the addition of only myo-inositol 
and thiamine.  The level of thiamine was increased 
four-fold over that used by Murashige and Skoog 
(1962), but nicotinic acid, pyridoxine and glycine 
and Support Systems
115
E. F. George et al. (eds.), Plant Propagation by Tissue Culture 3rd Edition, 115–173. 
© 2008 Springer. 
(amino acid) were unnecessary (Linsmaier and 
Skoog, 1965).  A similar simplification of the MS 
vitamins was made by Earle and Torrey (1965) for 
the culture of Convolvulus callus. 
Soczck and Hempel (1988) found that in the 
medium of Murashige et al. (1974) devised for the 
shoot culture of Gerbera jamesonii, thiamine, pyrid-
oxine and inositol could be omitted without any 
reduction in the rate of shoot multiplication of their 
local cultivars. Ishihara and Katano (1982) found that 
Malus shoot cultures could be grown on MS salts 
alone, and that inositol and thiamine were largely 
unnecessary. 
1.3.  SPECIFIC COMPOUNDS 
Myo-inositol.  Myo-inositol (also sometimes 
described as meso-inositol or i-inositol) is the only 
one of the nine theoretical stereoisomers of inositol 
which has significant biological importance. 
Medically it has been classed as a member of the 
Vitamin B complex and is required for the growth of 
yeast and many mammalian cells in tissue culture.  
Rats and mice require it for hair growth and can 
develop dermatitis when it is not in the diet.  Myo-
inositol has been classed as a plant ‘vitamin’, but note 
that some authors think that it should be regarded as a 
supplementary carbohydrate, although it does not 
contribute to carbohydrate utilization as an energy 
source or as an osmoticum. 
Historical use in tissue cultures.  Myo-inositol 
was first shown by Jacquiot (1951) to favour bud 
formation by elm cambial tissue when supplied at 20-
1000 mg/l. Necrosis was retarded, though the 
proliferation of the callus was not promoted.  Myo-
inositol at 100 mg/1 was also used by Morel and 
Wetmore (1951) in combination with six other 
vitamins for the culture of callus from the 
monocotyledon Amorphophallus rivieri (Araceae).  
Bud initials appeared on some cultures and both roots 
and buds on others according to the concentration of 
auxin employed. The vitamin was adopted by both 
Wood and Braun (1961) and Murashige and Skoog 
(1962) in combination with thiamine, nicotinic acid 
and pyridoxine in their preferred media fur the 
culture of Catharanthus roseus and Nicotiana 
tabacum respectively. Many other workers have since 
included it in culture media with favourable results 
on the rate of callus growth or the induction of 
morphogenesis. Letham (1966) found that myo-
inositol interacted with cytokinin to promote cell 
division in carrot phloem explants. 
Occurrence and biochemistry.  Part of the 
growth promoting property of coconut milk is due to 
its myo-inositol content (Pollard et al., 1961). 
Coconut milk also contains scyllo-inositol (Table 
4.1). This can also promote growth but to a smaller 
extent than the myo-isomer (Pollard et al., 1961). 
Inositol is a constituent of yeast extract (Steiner et al., 
1969; Steiner and Lester, 1972) and small quantities 
may also be contained in commercial agar (Wolter 
and Skoog, 1966).  Myo-inositol is a natural 
constituent of plants and much of it is often 
incorporated into phosphatidyl-inositol which may be 
an important factor in the functioning of membranes 
(Jung et al., 1972; Harran and Dickinson, 1978). The 
phosphatidylinositol cycle controls various cellular 
responses in animal cells and yeasts, but evidence of 
it playing a similar role in plants is only just being 
accumulated. Enzymes which are thought to be 
involved in the cycle have been observed to have 
activities in plants and lithium chloride (which 
inhibits myo-inositol-1-phosphatase and decreases the 
cycle) inhibits callus formation in Brassica oleracea 
(Bagga et al., 1987), and callus growth in 
Amaranthus paniculatus (Das et al., 1987). In both 
plants the inhibition is reversed by myo-inositol. 
As the myo-inositol molecule has six hydroxyl 
units, it can react with up to six acid molecules 
forming various esters. It appears that inositol 
phosphates act as second messengers to the primary 
action of auxin in plants: phytic acid (inositol hexa-
phosphate) is one of these. Added to culture media it 
can promote tissue growth if it can serve as a source 
of inositol (Watanabe et al., 1971). In some species, 
auxin can be stored and may be transported as IAA-
myo-inositol ester (Chapter 5). o-Methyl-inositol is 
present in quite large quantities in legumes; inositol 
methyl ethers are known to occur in plants of several 
other families, although their function is unknown 
(Phillips and Smith, 1974). 
The stimulatory effect of myo-inositol in plant 
cultures probably arises partly from the participation 
of the compound in biosynthetic pathways leading to 
the formation of the pectin and hemicelluloses needed 
in cell walls (Loewus et al., 1962; Loewus, 1974; 
Loewus and Loewus, 1980; Harran and Dickinson, 
1978; Verma and Dougall, 1979; Loewus and 
Loewus, 1980) and may have a role in the uptake and 
utilization of ions (Wood and Braun, 1961). In the 
experiments of Staudt (1984) mentioned below, when 
the P0
4
3–
 content of the medium was raised to 4.41 
mM, the rate of callus growth of cv. ‘Aris’ was 
The Components of Plant Tissue Culture Media II 116 Chapter 4
progressively enhanced as the myo- inositol in the 
medium was put up to 4000 mg/l. This result seems 
to stress the importance of inositol-containing 
phospholipids for growth. 
Table 4.1. Substances identified as components of coconut milk (water) from mature green fruits and market-purchased fruits. 
SUBSTANCE QUANTITY/REFERENCE SUBSTANCE QUANTITY/REFERENCE 
 Mature green 
fruits 
Mature fresh 
fruits 
 Mature 
green 
fruits 
Mature fresh 
fruits 
Amino acids (mg/l) Sugars (g/l) 
Alanine 127.3 (14) 312 (13), 177.1 
(14) 
Sucrose 9.2 (14) 8.9 (14) 
Arginine 25.6 (14) 133 (13), 16.8 
(14) 
Glucose 7.3 (14) 2.5 (14) 
Aspartic acid 35.9 (14) 65 (13), 5.4 (14) Fructose 5.3 (14) 2.5 (14) 
Asparagine 10.1 (14) ca.60 (13), 10.1 
(14) 
Sugar alcohols (g/l) 
?-Aminobutyric 
acid 
34.6 (14) 820 (13), 168.8 
(14) 
Mannitol  (1) 
Glutamine acid 70.8 (14) 240 (13), 78.7 
(14) 
Sorbitol  15.0 (12), (17) 
Glutamine 45.4 (14) ca.60 (13), 13.4 
(14) 
myo-Inositol  0.1 (12), (17) 
Glycine 9.7 (14) 13.9 (14) scyllo-Inositol  0.5 (12), (17) 
Histidine 6.3 (14) Trace (13,14) Vitamins (mg/l) 
Homoserine -- (14) 5.2 (14) Nicotinic acid  0.64 (4) 
Hydroxyproline  Trace (13,14) Pantolhenic acid  0.52 (4) 
Lysine 21.4 65.8 (14) Biotin, Riboflavin  0.02 (4) 
Methionine 16.9 (14) 8 (13), Trace (14) Riboflavin  0.01 (4) 
Phenylalanine -- (14) 12 (13), 10.2 (14) Folic acid  0.003 (4) 
Proline 31.9 97 (13), 21.6 (14) Thiamine, pyridoxine  Trace (4) 
Serine 45.3 (14)  Growth substances (mg/l) 
Typtophan  39 (13) Auxin  0.07 (7), (28) 
Threonine 16.2 (2) 44 (13), 26.3 (14) Gibberellin  Yes (10,28) 
Tyrosine 6.4 (14) 16 (13), 3.1 (14) 1,3-Diphenylurea  5.8 (8), (6,17) 
Valine 20.6 (14) 27 (13), 15.1 (14) Zeatin  (22,26) 
Other nitrogenous compounds Zeatin glucoside  (26) 
Ammonium  (19) Zeatin riboside  (20), (24), (25) 
Ethanolamine  (19) 6-Oxypurine growth 
promoter 
 (27) 
Dihydroxyphenyl
alanine 
 (19) Unknown cytokinin/s  6, (18) (22) 
Inorganic elements (mg/100g dry wt.) Other (mg/l) 
Potassium  312.0 (3) RNA-polymerase  (23) 
Sodium  105 (3) RNA-phosphorus 20.0 (14) 35.4 (14) 
Phosphorus  37.0 (3) DNA-phosphorus 0.1 (14) 3.5 (14) 
Magnesium  30.0 (3) Uracil, Adenine  21 
Organic acids (meq/ml) Leucoanthocyanins  (11) (15,17) 
Malic acid 34.3 (14) 12.0 (14) Phyllococosine  (16) 
Shikimic, Quinic 
and 2 unknowns 
0.6 (14) 0.41 (2) Acid Phosphatase  (5,9) 
Pyrrolidone 
carboxylic acid 
0.4 (14) 0.2 (14) Diastase  (2) 
Citric acid 0.4 (14) 0.3 (14) Dehydrogenase  (5) 
Succinic acid -- (14) 0.3 (14) Peroxidase  (5) 
   Catalase  (5) 
Numbered references (within brackets) in the above table are listed in Section 1.11 of this Chapter. 
117
 
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 monocotyl-
edons, 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 PO
4
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. B
1
, aneurine) in the 
form of thiamine pyrophosphate, is an essential co-
factor 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
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 thiamine-
sufficient 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.  
118 The Components of Plant Tissue Culture Media II
 spp. (Chée, 
 Chapter 4 
 
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 D
2
 and D
3
 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 
( a-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 
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 B
1
) (Bonner 
and Addicott, l937; Robbins and Bartley, 1937).  In a 
medium consisting only of macro- and micro-
nutrients, 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). 
119
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