Addition of growth regulators (Table3.4) to an otherwise complete medium, containing inor- ganic and organic nutrients and sucrose called Basal medium (BM), is invariably necessary to
trigger various types of growth and differentia- tion. Although the explants may have certain levels of endogenous growth hormones (natu- rally synthesized growth regulators), it is often necessary to supplement them exogenously to evoke certain responses. The growth regulators are generally required in very minute quantities (0.001–10 lM). The nature and quantity of growth regulators in the medium is varied according to the variety of plant, nature of the tissue and also the stage of culture (initiation of callus, induction of somatic embryogenesis, shoot differentiation or multiplication, rooting of shoots, etc.).
In order to develop a tissue culture protocol for a new plant species, various types and con- centrations of growth regulators in several per- mutation and combination need to be tested. The concentrations of growth regulators used are very often reported in mg L-1. However, for meaningful comparative studies of the responses induced by various growth regulators in cultures, it is desirable to express them in molar con- centrations, which is a reflection of the actual number of molecules of the growth regulator per unit volume of the medium.
(i) Auxins. Auxins are involved with many developmental processes in plants, including elongation of stem and internodes, tropisms, apical dominance, abscission and rooting. In tissue cultures, auxins have been used to induce cell division, cytodifferentiation and organogenic and embryogenic differentia- tion. Generally, auxin at low concentration favours root initiation, whereas at higher concentration induces callus formation. The auxins commonly used in tissue cultures are indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), a-naphthaleneacetic acid (NAA), 2,4-dicholorophenoxyacetic acid (2,4-D) and para-chlorophenoxyacetic acid (p-CPA). Whereas IAA and IBA are naturally occur- ring auxins, NAA, 2,4-D and p-CPA are synthetic. Some other synthetic auxins that have been used in tissue culture work are naphthoxyacetic acid (NOA), 2,4,5-trichol- orophenoxyacetic acid (2,4,5-T), 4-amino-3,
5,6-tricholoropyridinecarboxylic acid (piclo- ram) and 3,6-dichloro-o-anisic acid (dica- mba).
IBA, NAA and IAA are widely used for rooting and, in interaction with a cytokinin, for shoot proliferation. They have also been implicated in tracheidal differentiation in cell and callus cultures. 2,4-D and 2,4,5-T are very effective for the induction and growth of callus. For inducing somatic embryogen- esis, 2,4-D is an important factor. Dicamba has been used for monocots whereas piclo- ram has been effective for legumes.
Auxins are usually dissolved in a small amount of ethanol or dilute NaOH.
(ii) Cytokinins. This is another major group of plant hormones. Natural cytokinins are N6- substituted adenine derivatives and occur in plants as nucleosides and nucleotides. Roots are the possible sites of their synthesis. In nature, cytokinins are concerned with cell division, modification of apical dominance and shoot differentiation. Incorporation of these compounds in culture media is mainly
to trigger cell division, and to induce differ- entiation of adventitious shoots from callus and organs, and shoot proliferation by the release of axillary buds from apical domi- nance. The cytokinins commonly used in tissue cultures are kinetin (6-furfurylamino) purine, BAP [(benzylamino) purine], 2iP [(2- isopentenyl)-adenine or 6-(c,c-dimethylallyl amino) purine)] and zeatin [6-(4-hydroxy-3- methyl but-2-enyl amino)-purine)]. Thidi- azuron (TDZ), a dipheny1-substituted urea, has also been used as a cytokinin in tissue cultures. However, it is used at very low concentrations (0.05–0.1 lM) as at higher levels it promotes callusing. At times, adenine (2–120 mg L-1) has been added to tissue culture media as a weak cytokinin to promote shoot formation. The most efficient and commonly used cytokinin in plant tis- sue culture is BAP. It is used in the range of 1–10 lM.
Topolins, are a new class of highly active naturally occurring aromatic cytokinins. These cytokinins, particularly meta-topolin
Table 3.4 Some plant growth regulators used in plant tissue culture media, their molecular weights (M.W.) and solvents
Growth Regulator Abbreviation M.W. Solvent
Indole-3-acetic acid IAA 175.2 Ethanol/1 N NaOH
Indole-3-butyric acid IBA 203.2 Ethanol/1 N NaOH
2,4-Dichlorophenoxyacetic acid 2,4-D 221.04 Ethanol/1 N NaOH
a-Naphthalene acetic acid NAA 186.2 Ethanol/1 N NaOH
Naphthoxyacetic acid NOA 202.2 1 N NaOH
2,4,5-Trichlorophenoxyacetic acid 2,4,5-T 255.5 Ethanol
para-Chlorophenoxyacetic acid p-CPA 100.59 Ethanol
4-Amino-3,5,6-tricholoro pyridinecarboxylic acid Picloram 241.5 Acetone
3,6-Dichloro-o-anisic acid Dicamba 221 Ethanol/Acetone
6-(Furfurylamino)-purine (kinetin) KIN 215.2 dil HCl/1 N NaOH
6-(Benzylamino)-purine BAP 225.3 1 N NaOH
2-(Isopentinyl)-adenine 2iP 203.2 1 N NaOH
6-(4-Hydroxy-3-methylbut-2-enylamino)-purine (Zeatin) ZEA 219.2 dil HCl/1 NaOH 1-Phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (thiadiazuron) TDZ 220.3 DMSO
6-(3-Hydroxybenzylamino)-purine mT 243.26 1 N NaOH
Gibberellic acid GA3 346.4 Water
Abscisic acid ABA 264.31 1 N NaOH
Tri-iodo benzoic acid TIBA 499.81 1 N NaOH
Phloroglucinol PG 126.11 Ethanol/water
or mT [6-(3-hydroxybenzylamino)-purine], are becoming increasingly popular with plant tissue culturists because of their positive effects on several parameters of tissue cul- ture, such as high rate of shoot multiplica- tion, reduced physiological abnormalities, and better rooting and acclimatization. Cytokinins are generally dissolved in small amounts of dilute HCl or NaOH. For thidi- azuron, DMSO (dimethylsulphoxide) is used as the solvent.
(iii) Gibberellins. These are less commonly used in plant tissue culture. There are over 20 known gibberellins, of which GA3 is
used most often. They are reported to stimulate elongation of internodes, meri- stem growth for some species and more importantly to attain normal development of plantlets from in vitro formed adventive embryos.
GA3is readily soluble in cold water (up to
1000 mg L-1). Being heat sensitive (90 % of the biological activity is lost after auto- claving), GA3is filter sterilized and added
to autoclaved medium after it has cooled. (iv) Ethylene and Abscisic acid. Ethylene (eth-
ane, C2H4) is an unusual, gaseous plant
hormone. It is produced by ageing and stressed tissues. In plant tissue cultures, ethylene is also produced by the organic constituents of the medium on exposure to heat, oxidation, sunlight or ionizing radia- tion. Ethylene appears to influence various morphogenic processes, such as embryo- genesis and organogenesis, but its effects are not clear. It may be promotory or inhibitory for the same process in different plant systems. For example, ethylene pro- motes somatic embryogenesis in maize but inhibits it in rubber tree tissue cultures. Ethrel or ethapon (2-chloroethane phos- phonic acid), which releases ethylene upon decomposition, is used to study the effect of this gaseous growth regulator on plant tis- sue cultures. It generally inhibits growth and differentiation, but in some cases, it promoted somatic embryogenesis.
Abscisic acid, a naturally occurring growth inhibitor, is often required for normal growth, development and maturation of somatic embryos.