Personal collection and compilation of Dr Rajesh K Tiwari.

Available free of cost for students of AMITY Institute of Biotechnology

AMITY Institute of Biotechnology AMITY University Lucknow Campus ________Uttar Pradesh_______ Prepared by: Tiwari Dr Rajesh k

Plant Growth Hormones What is plant growth hormones / growth regulators ????:
A plant hormone is an organic compound synthesized in one part of a plant and translocated to another part where, in very low concentrations, it causes a physiological response. The response in the target organ need not be always promotive, because processes such as growth or differentiation are sometimes inhibited by hormones, especially abscisic acid. Because the hormone must be synthesized by the plants, such inorganic ions as _K+_ or Ca2+ that cause important responses are not hormones. Those Organic chemicals which regulates the growth e.g., 2,4-D, an auxin or synthesized in organisms other than plants can not be a hormone. It may be called growth regulators. The definition also states that a hormone must be translocated in plants from the site of synthesis to aite of action. Sucrose is not considered a hormone, even though it is synthesized and translocated by plants, because it causes growth only at relatively high concentrations .. Hormones are usually effective at internal concentrations of 1 uM or less, whereas sugars, amino acids, organic acids, and other metabolites necessary for growth and development (ex. eluding enzymes and most coenzymes) -are usually preset at concentaration of l to 50 mM.

The principle that plant development is influenced by special chemicals in plants is not new. About 100 years ago, the famous German botanist Julius von Sachs suggested that specific organ forming substances occur in plants; he supposed that one substance caused stem growth and others leaf, root, flower, or fruit growth; The first hormone identified in 1930 was Indoleacetic acid (IAA). Later gibberellins were discovered in the 1950s. As more hormones were identified and their effects and endogenous concentrations

studied, it became apparent that not only does each hormone affect the responses of many plant parts, those responses depend on the

concentrations and interactions of the various known hormones and possibly unknown' ones. But Von Sachs' concept that different tissues can respond differently to different chemicals is certainly valid.

Important points:
A plant growth hormones is an organic compound synthesized in one part of plant and translocated to another part, in very low concentration , it causes a physiological response. In addition to the nutrients, it is generally necessary to add one or more growth substances, such as auxins, cytokinins, and gibberellins, to support good growth of tissues and organs. However, the requirement for these substances varies considerably with the tissue, and it is believed that it depends on their endogenous levels. The growth regulators are required in very minute quantities (umoll values). There are many synthetic substances having growth regulatory activity, with differences in activity and species specificity.

It often requires testing of various types, concentrations and mixtures of growth substances during the development of a tissue culture protocol for a new plant species.

The Auxins
The term auxin was first used by Frits Went, who, as a graduate student in Holland in 1926, discovered that some unidentified compound probably caused curvature of oat coleoptiles. The curvature phenomenon is called phototropism.

Figure The demonstration by Went of auxin in the Avena coleoptile tip. Auxin is indicated by stippling. (a) The tip was removed and placed on a block of gelatin. (b) Another seedling was prepared by removing the tip, waiting a period of time, and removing the tip again (a new "physiological tip" sometimes forms). (c) The leaf inside the coleoptile was pulled out, and the gelatin block containing the auxin was placed against it. (d) Auxin moved into the coleoptile on one side, causing it to bend. (From Salisbury and Parke, 1964.)

The activity of auxin was demonstrated by curvature of coleoptiles caused by enhanced elongation on one side to which the the agar block was placed. Important salient features 1. In nature, the hormones of this group are involved with elongation of stem and internodes, apical dominance, abscission, rooting, formation of adventitious roots, inhibition of adventitious and axillary shoot formation. 2. In tissue cultures auxins have been used for cell division and root differentiation. 3. The auxins commonly used in tissue culture are: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthalene acetic acid (NAA), naphthoxyacetic acid (NOA), para-chlorophenoxyacetic acid (P-CPA), dichlorophenoxyacetic acid (2,4-D), and trichlorophenoxyacetic acid (2,4,5-T). 4. Of these, IBA and IAA are widely used for rooting and, in interaction with a cytokinin, for shoot proliferation.

5. 2,4-D and 2,4,5-T are very effective for the induction and growth of callus. 6. 2,4-D is also an important factor for the induction of somatic embryogenesis. Auxins are usually dissolved in either ethanol or dilute NaOH.

a: structure of some natural auxins b: Structure of some synthetic auxins

The Cytokinins.
About 1913, Gottlieb Haberlandt discovered in Austria that an unknown compound present in vascular tissues of various plants stimulated cell division that caused cork cambium formation and wound healing in cut Potato tubers. This was apparently the first demonstration that plants contain compounds, now called cytokinins, that stimulate cytokinesis.

In the 194Os, Johannes van Overbeek found that the milky endosperm from immature coconut is also rich in compounds that promote cytokinesis. In the early 1950s, Folke Skoog and his colleagues, who were then interested in auxin-stimulation of plants grown in tissue cultures, found that cells in pith sections from tobacco stems divided much more rapidly if a piece of vascular tissue was placed on the top of the pith, varifying Haberlandts results. They tried to identify the chemical factor from the vascular tissues, using growth of tobacco pith cells as a bioassay system. These cells were cultured on agar media containing known sugars, mineral salts, vitamins, amino acids, and IAA. IAA itself increased growth for a time by causing enormous cells to be formed, but these cells did not divide. Many of these cells were polyploids with several nuclei. In seeking substances that would promote cell division, they found an adenine-like compound in yeast extracts that was highly active. This led to investigations of the ability of DNA to promote cytokinesis (because DNA contains adenine) and, in 1954, to the discovery by Carlos Miller of a very active compound formed by partial breakdown of aged or autoclaved herring sperm DNA. They named this compound kinetin.

Although kinetin itself has not been found in plants and is not the active substance found by Haberlandt in phloem, related cytokinins are present in most plants. F. C. Steward, also using tissue culture techniques in the 1950s, found several cytokinins in coconut milk that enhance cell division in carrot root tissues. The most active of these were later shown by D. S. Letham (1974) to be compounds previously given the common name zeatin and zeatin riboside. Zeatin had first been identified by Letham in 1964 ard almost simultaneously by Carlos Miller, both of whom used the milky endosperm of Corn (Zea mays) as a source. Since then, other cytokinins with adenine-like

structures similar to kinetin and zeatin have been identified in numerous parts of seed plants. None of these cytokinins is present in DNA, or are they breakdown products of DNA, but some occur in transfer RNA (tRNA) and sometimes in ribosomal RNA molecules of seed plants, yeasts, bacteria, and even primates, and some exist as unbound free cytokinine.

Figure shows structure of the freebase form of the three most commonly detected end most physiologically active cytokinins in various .plants: zeatin, dihydrozeatin, and isopentenyl adenine (IPA). Also shown are kinetin and another synthetic cytokinin benzyladenine, which are highly active but which are probably not formed by plants. Benzyladenine might be present in some species, because its nucleoside derivative, 6-benzyladenine riboside, was recently found to occur in anise cells (Pimpinella anisum).

Site of Cytokinin synthesis and transport: Root represents an important cytokinin source for various plant parts but its present in rootless plant indicates that the aerial parts also synthesized some cytokinin they require. Evidence showed the root tip synthesized cytokinin and transport to various part through xylem.

Important salient features. 1. These hormones are concerned with cell division, shoot differentiation, stimulate axillary and adventitious shoot proliferation, etc. Cytokinin

promotes cell division in plant tissuesand able to produce a large mass of cell called callus. Hoever, If the cytokininto-auxin ratio is maintained high in culture media , certain cells become meristematic and develops shoots. But of the cytokininto-auxin ratio is maintained low , cells start forming roots. 2. Cytokinins promote lateral bud development in plant both in in vitro and field condition. If cytokinin is added in to nongrowing lateral buds dominated by shoot apex, the lateral buds begin to grow. It laso promot growth of dormant laternal buds in vitro. 3. Cytokinins delay senescence and increase nutrient sink activity in field condition. Cytokinins also increase cell expansion in dicot cotyledons and leaves. When seeds of certain dicots are germinated in dark, the cotyledons remain yellow and relatively small. If they are exposed to light, growth increases greatly. This is photomorphogenetic effect controlled by phytochrome. If these cotyledons are incubated with cytokinins, growth is enhanced by two fold. The growth is entily caused by water uptake that drives cell expansion, because the dry weight of the tissue does not increase. 4. In tissue culture media, cytokinins are incorporated mainly for cell division and differentiation of adventitious shoots from callus and organs. 5. These compounds are also used for shoot proliferation by the release of axillary buds from apical dominance. 6. More commonly used cytokinins are: benzylamino purine (BAP), isopentenyl-adenine (2-ip), furfurylamino purine (kinetin), thidiazuron (TDZ) and zeatin. 7. Compared to the other cytokinins, thidiazuron is generally used at very

low concentrations (0.1-5,ug 1-1). 8. Cytokinins are generally dissolved in dilute HCl or NaOH. For thidiazuron, DMSO may be used as the solvent.

Gibberellins.
1. There are over 20 known gibberellins. Of these, generally, GA3 is used. 2. Compared to auxins and cytokinins, gibberellins are used very rarely. 3. They are reported to stimulate normal development of plantlets from in vitro formed adventive embryos or to break seed dormancy. 4. GA3 is readily soluble in cold water up to 1000 mg 1-L.

Ethylene. Volatile Hormone

The ability of certain gases to stimulate fruit ripening has been known for many years. Even the ancient Chinese knew that their picked fruits would ripen more quickly in a room with burning incense. It was also mentioned that oranges should not be stored with bananas on ships, because some emanation from the oranges caused the bananas to ripen prematurely. This was apparently the first suggestion that fruits release a gas that stimulates ripening, but it was not until 1934 that R Gane proved that ethylene is synthesized by plants and is responsible for faster ripening.

Another historical practice Implicating still a different role for ethylene was the building of bonfires by Puerto Rican 'pineapple growers and Philippine mango growers near their crops. These farmers apparently believed that the smoke helped to initiate and synchronize flowering. Ethylene causes these effects in both species, so ethylene is almost surely the most active smoke component. Stimulation of fruit ripening is a widespread phenomenon, while promoted flowering appears restricted to mangos and most bromeliad species, including the pineapple.

Still another effect of gases was reported as early 1864. Before the use of electric lights, trees were lighted with illuminating gas. Sometimes the gas pipes leaked, and in certain German cities this caused the leaves to fall off the shade trees. Ethylene causes senescence and abscission of leaves, so again it

presumably was responsible.

It was a Russian physiologist named Dimitry N Neljubow (1876-1926) who first established that-ethylene affects plant growth. He identified ethylene in illuminating gas and showed that it causes a triple response on pea seedlings: inhibited stern elongation, increase stem thickening and horizontal growth habit. Furthermore, leaf expansion is inhibited, and normal opening of the epicotyl hook is retarded. Ethylene Synthesis Ethylene production by various organisms. Only few

bacteria reportedly produce ethylene and no -algae are known to synthesize. Several fungal species produce it, including some that normally grow in soils. Essentially all parts of seed plants produce ethylene. In seedlings, the shoot apex is an important site production. It might result from the high amounts of IAA there; because auxins greatly stimulate ethylene formation

It was found that an unusual amino acid-like compound

1 amino

cyclopropane-1-carboxylic acid (ACC) is involved as a close precursor of ethylene. (Adams and Yang, 1979). ACC synthase is the enzyme which plays important role in production of ACC. Important salient features. 1. All kinds of plant tissue cultures produce ethylene, and the rate of production increases under stress conditions. 2. Produced in all cells of the plant and causes thickening of stems by inhibiting elongation. Root become thicker by radial expension. 3. Reduces adventitious shoot formation, causes epinasty of leaves by promoting elongation of cells of upper surface. 4. The induction of flowering in mango by ethylene was achieved. An ethylenereleasing commonly available. 5. In cultures, ethylene is also produced abiologically when the organic substance called ethephone and Ethrelis

constituents of the medium are subjected to heat, oxidation, sunlight or ionizing radiation. 6. Pure ethylene or chemical compounds which release ethylene during their decomposition, such as 2-chloroethylphosphonic acid (marketed under the trade names Ethrel, Ethaphon, Floridimex, Camposan), can be applied to study the effect of this gaseous growth regulator on plant tissue cultures. 7. Ethylene exerts various morphogenic influences on cultured tissues but its effects are not clear cut . 8. It may be promontory or inhibitory for the same process in different systems. For example, it promoted somatic embryogenesis in maize but the same process was inhibited in Hevea brasiliensis.

Relation of Ethylene to Auxin Effects IAA stimulates ethylene formation several hundred fold. In these and other tissues auxins induce additional formation of ACC synthase, and the enhanced formation of ACC resulting from action of that enzyme that leads to increased ethylene production. Wounding also increases ethylene production by inducing formation of ACC synthase. The ability of IAA and all synthetic auxins to increase ethylene production raises the question of whether many auxin effects are really caused by ethylene. Indeed, ethylene appears to be responsible in many cases. Included are leaf epinasty, inhibition of stem, root, and leaf elongation, flower induction in bromeliads and mangos, inhibition' of epicotyl or hypocotyl hook opening in dicot seedlings, increased percentage of female flowers in dioecious plants, and perhaps apical dominance. Also, release of auxin by germinating pollen grains promotes ethylene production in the stigma, which contributes to senescence of the flower. Nevertheles, growth promotion, initial stages of adventitious root production, and many other effects of auxins appear to be independent of ethylene production. Only when the auxin concentration becomes relatively high, the ethylene production greatly influence and duplicate certain auxin effects.

Abscisic acid ( ABA)

1. Only one natural compound i.e ABA. 2. Promotes leaf abscission and seed dormancy. 3. Plays a dominant role in closing stomata in response to water stress. 4. Abscisic acid is most often required for normal growth and development of somatic embryos. 5. It is also known to promote morphogenesis in Begonia cultures.

Others
Polyamines - have a vital role in embryo development. Jasmonic acid - involved in plant wound responses.

Salicylic acid - Not universally acclaimed as plant hormones since they are usually needed at high concentrations.

Triazole - compounds have shown to influence in vitro growth and somatic embryogenesis in few fruit crops like Citrus. Paclobutrazol

during

the

acclimatization

stage

of

micropropagation to reduce hyperhydricity and regulate leaf growth and function in relation to control of water stress. Ancymidol : has been used to inhibit leaf formation and promote shoot formation in gladiolus

Preparation
To prepare a 1 mg/ml stock solution: Add 100 mg of the plant growth regulator to a 100 ml volumetric flask or other glass container. Add 2-5 ml of solvent to dissolve the powder. Once completely dissolved, bring to volume with double distilled water. Stirring the solution while adding water may be required to keep the material in solution. Store

Volume of Stock Solution =

Desired Hormone Conc. X Medium Volume Stock Solution Conc.

Table for Molar Equivalence and Solution Preparation

Auxins
Product Name p-Chlorophenoxyacetic acid (4-CPA) 2,4Dichlorophenoxyacetic acid 2,4Dichlorophenoxyacetic acid Sodium salt Molar Equivalence Mol. Wt. 186.6 M for 1mg/L 5.36 Solvent EtOH Diluent Solution Preparation Powder Storage RT Liquid Storage 2-8C SterilizWorking ation* Conc. (mg/L) CA 0.1-10.0

221

4.53

RT

2-8C

CA

0.01-6.0

243

4.12

Water EtOH/1N NaOH Water 0.5N NaOH EtOH/1N NaOH Water

RT

2-8C

CA

0.01-6.0

Indole-3-acetic acid Free 175.2 acid (IAA) Indole-3-acetic acid Sodium salt Indole-3-acetic acid methyl ester Indole-3-acetyl-Laspartic acid Indole-3-butyric acid (IBA) Indole-3-butyric acid Potassium salt (K-IBA) alphaNaphthaleneacetic acid Free acid (NAA) beta-Naphthoxyacetic acid Free acid (NOA) Phenylacetic acid (PAA) Picloram 2,4,5Trichlorophenoxyacetic acid (2,4,5-T) 2,3,5-Triiodobenzoic acid Free acid (TIBA) 197.2 189.2 290.3 203.2 241.3

5.71 5.07 5.29 3.45 4.90 4.14

Water Water Water Water

-0C 2-8C 2-8C -0C 2-8C 2-8C

-0C -0C 2-8C -0C -0C -0C

CA/F CA/F F CA/F CA/F

0.01-3.0 0.01-3.0 0.01-5.0 0.1-10.0 0.1-10.0

186.2

5.37

1N NaOH

Water

RT

2-8C

CA

0.1-10.0

202.2 136.2 241.5 255.5

4.95 7.34 4.14 3.91

1N NaOH EtOH DMSO EtOH

Water

RT RT RT RT

2-8C 2-8C 2-8C 2-8C

CA CA/F CA CA

0.1-10.0 0.1-50.0 0.01-10.0 0.01-5.0

499.8

2.00

1N NaOH

Water

-0C

-0C

0.05-5.0

Cytokinins
Product Name Product Number Mol. Wt. Molar Equivalence Solution Preparation

M Working Powder Liquid Sterilizfor Solvent Diluent Conc. Storage Storage ation* 1mg/L (mg/L) RT RT RT RT RT -0C 2-8C -0C 2-8C 2-8C 2-8C 2-8C 2-8C -0C 2-8C -0C CA CA CA/F CA/F CA/F CA/F F CA/F 50-250 50-250 0.1-5.0 0.1-5.0 0.1-5.0 0.1-5.0 0.0011.0 1.0-30.0

Adenine Free base Adenine hemisulfate Hemisulfate salt 6-Benzylaminopurine (BA) 6-Benzylaminopurine Hydrochloride 6-Benzylaminopurine (BA)

A 5665 135.1 7.40 1.0 HCl Water A 2545 184.2 5.43 B 3408 225.3 4.44 B 5920 261.7 3.82 B 3274 225.3 4.44 Water 1N NaOH Water 1N NaOH EtOH DMSO 1N NaOH 1N NaOH DMSO 1N NaOH 1N NaOH 1N NaOH Water DMSO 1N NaOH 1N NaOH Water 1N NaOH Water Water Water

N-Benzyl-9-(2tetrahydropyranyl)adenine B 2275 309.4 3.23 (BPA) N-(2-Chloro-4-pyridyl)-N'C 2791 247.7 4.04 phenylurea (4-CPPU) 6-(gamma,gammaDimethylallylamino)purine D 7674 203.2 4.92 (2iP) 6-(gamma,gammaDimethylallylamino)purine D 5912 203.2 4.92 (2iP) 1,3-Diphenylurea (DPU) Kinetin Kinetin Kinetin Kinetin Hydrochloride 1-Phenyl-3-(1,2,3thiadiazol-5-yl)urea trans-Zeatin Free base Zeatin trans-Zeatin Hydrochloride trans-Zeatin riboside D 7535 212.3 4.71 K 0753 215.2 4.65 K 3378 215.2 4.65 K 3253 215.2 4.65 K 1885 251.7 3.97 P 6186 220.2 4.54 Z 0876 219.2 4.56 Z 0164 219.2 4.56 Z 2753 255.7 3.91 Z 3541 351.4 2.85

Water Water Water Water Water Water Water

-0C RT -0C -0C -0C -0C RT -0C -0C -0C -0C

-0C 2-8C -0C -0C -0C -0C 2-8C -0C -0C -0C -0C

CA/F F CA/F CA/F CA/F CA/F CA/F CA/F CA/F CA/F F

1.0-30.0 0.1-1.0 0.1-5.0 0.1-5.0 0.1-5.0 0.1-5.0 0.0010.05 0.01-5.0 0.01-5.0 0.01-5.0 0.01-5.0

Product Name

Molar Equivalence Mol. Wt. M for 1mg/L Solvent 1N NaOH DMSO Water

Solution Preparation Powder Liquid SterilizDiluent Storage Storage ation* Water Water -0C 2-8C RT 2-8C RT 2-8C -0C 2-8C RT RT 2-8C -0C -0C 2-8C 2-8C 2-8C -0C -0C -0C 2-8C 2-8C 2-8C CA/F CA/F F F CA/F CA/F F F CA/F F CA/F Working Conc. (mg/L) 0.1-10.0 1.0-10.0 up to 500 0.0110.0 0.01-5.0 0.01-5.0 0.01-5.0 0.01100.0 up to 162 0.1-10.0

()-cis,trans-Abscisic acid 264.3 3.78 (ABA) Ancymidol Chlorocholine chloride (CCC) 256.3 3.90 158.1 6.33

3,6-Dichloro-o-anisic acid 221.0 4.52 EtOH/Water (Dicamba) Gibberellic acid (GA3) 346.4 2.89 EtOH Water EtOH EtOH Water 1N NaOH Water Gibberellic acid Potassium 384.5 2.60 salt (K-GA3) Gibberellin A4 Free acid (GA4) ()-Jasmonic acid Phloroglucinol 332.4 3.01 210.3 4.76 126.1 7.93

N(Phosphonomethyl)glycine 169.1 5.91 (Glyphosate) Succinic acid 2,2dimethylhydrazide 160.2 6.24

*CA = coautoclavable with other media components. F = filter sterlize. CA/F = coautoclavable with other media components, however, some loss of activity may occur. This can be compensated for by increasing component concentration. Component may be filter sterilized.

Effect of different explants on callus growth and survival

Number of Callus explants survived morphology (%) Mesocarp 6.0 90.3 Brown /compact Whitish Leaf 20.0 36.3 brown/loose Shoot -0.0 SE.m+ 1.06 2.02 CD (p=0.05) 2.61 4.95 * All the explants were inoculated on MS+BA(0.5)+NAA(0.1) ++++ High callusing, ++ Poor callusing Type of explants Days taken for callusing Callus frequency +++ ++ -

Table 2. Effect of growth regulators on callus morphology and frequency

Treatment
MS MS+2,4-D+BA

Concentration (mg/l)
0.0+0.0 0.5+0.1 1.0+0.1 5.0+0.1 O.1+0.2 0.1+0.05 0.5+0.1+150 0.5+0.1+250 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0

Colour of callus
wb wb wb wb wb wb wb wb C.Y. C.Y. C.Y. C.Y. gw gw gw gw

Texture of callus
L L L L L L L L C C C C C C C C C C C C C C C C

Frequency of callusing
*** *** *** *** *** *** **** *** *** *** *** *** **** *** *** ***

MS+BA+NAA+ADS MS+1,2,4Triazole

MS+1,2,4-1-H Triazole

MS+4L(1H-1,2,4Triazole-1Y) 1.0 2.0 3.0 4.0 MS+Paclobutrazole 1.0 2.0 3.0 4.0 Colour gw - Greenish white callus CY - Creamy white callus Wb - White brown callus

gw gw gw gw C.Y. C.Y. C.Y. C.Y. Texture of callus C Compact L Loose

*** *** *** *** *** *** *** **** Frequency **** High *** Moderate ** Low

Influence of different treatments on induction and growth of callus

Fresh callus weight(After 45 days) (mg)
2743 2615 2537 2182 2222 2134 3100 2674 2783 2229 2845 2394 2184 3061 1417 1845 1293 2106 1684 1813 2251 1777 1855 3140 168.9 454.05

Treatment
MS MS+ 2,4-D+BA

Concentration (mg/l)

Days taken for callusing

13.66 6.66 8.66 6.66 5.66 18.66 6.00 18.00 5.66 6.66 7.00 6.00 5.00 7.33 8.66 6.66 8.66 8.00 10.00 7.66 6.66 11.00 11.66 6.00 1.14 2.36

0.0+0.0 0.5+0.1 1.0+0.1 5.0+0.1 0.1+0.2 0.1+0.05 MS+BA+NAA+ADS 0.5+0.1+150 0.5+0.1+250 MS+1,2.4Triazole 1.0 2.0 3.0 4.0 MS+1,2.4-1-H Triazole 1.0 2.0 3.0 4.0 MS+4L(1H-1, 2,4Triazole-1-y) 1.0 2.0 3.0 4.0 MS+ Paclobutrazole 1.0 2.0 3.0 4.0 S.Em + CD (p=0.05)

Influence of different treatments on formation of somatic embryos

Treatment
MS MS+2,4-D+BA

Concentration( mg/l)
0.0+0.0 0.5+0.1 1.0+0.1 5.0+0.1 0.1+0.2 0.1+0.05 0.5+0.1+150 0.5+0.1+250 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0

Number of embryos
0.00 0.00 0.00 0.00 0.00 5.66 6.66 5.33 2.33 0.00 0.00 0.00 7.00 0.00 5.00 8.66 8.33 0.00 0.00 4.00 0.00 0.00 0.00 0.00 0.68 1.42

MS+BA+NAA+ADS MS+1,2,4Triazole

MS+1,2,4-1-H Triazole

MS+4L(1H-1.2.4Triazole-1-Y)

MS+Paclobutrozole

S.Em+ CD(p=0.05)