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Thesis written by Nivedita vaidya Research done by Nivedita vaidya ' INTRODUCTION  ''' The functioning of a multi- cellular organism  like a higher plant is controlled by many interacting regulatory systems.''' The regulatory systems of higher tiers are mechanisms that have grown through evolution out of lower-level regulatory systems and developed specific regulatory features, for example, the ability to coordinate the growth and development of organs in entire plants by means of set of phyto chromes, each exerting its influence in a defined way. Thus phytochromes are involved in the regulation of growth processes in the whole plant. Apart from phytochromes, a plant also contain their antagonists- endogenous growth inhibitors. It is the coordinated interaction of these two group of compounds that determines each normal growth process in plant. progress in the investigation of phytochromes been largly due to improvement of their purification and preparative isolation techniques. Since 1952 Chromatographic sepration of plant extracts has been used in study of    naturally occuring plant regulators combined with bio-assays. This technique helped detect auxins and other growth regulators including natural growth inhibitors in plant tissues. The discovery of natural growth inhibitors was undoubtedly a turning point as plant growth processes could not bev interpreted in terms of     balance between stimulatory and inhibitory factors. Relative concentrations of both growth promoters and inhibitors changes with phases of growth and development of plants  or organs. In present investigation it is proposed to study changes in amount of growth promoter ( auxin) and growth inhibitor ( phenolic compounds) with different development stages of leaf forced to grow from lignified shoots of    Salix babylonica L. stem cuttings kept under mist at controlled temperature. Auxin, mainly represented by indole-3-acetic acid (IAA) in plants, has common biosynthetic pathway with phenols ( i.e. Shikmic acid pathway). Phenolic compounds including wide range of plants substances, which possess in common an aromatic ring bearing one or more hydroxy/substituents like Benzoic acids: p- hydroxy benzoic acid, vanillic acid, cinnamic acids, Flavanols. Kaemferol quercetins, Antocyanidins, cyanidin, malvidin leicoantocyanidin, leucocyanidinic, While the function of some classes of phenolic compounds is well established (e.g the lignins and the  structural material of cellwall, the antocyanins  as flower pigments). Flavonols presence in Spinach chloroplast ( Octtmeir and Heapel,1971) has suggested that they may be involved in photosynthesis regulation The simple phenolic compounds have been implicated in regulation of plant growth and developement by modulating 1AA Oxidase activity. Rubery et al.(1972) reported the IAA effect over period of hours (12 to24),on the elongation of coleoptile and phenolics could affect coleoptile growth by inhibting the 1AA destruction and inhibiting the 1AA Oxidase where as ortho and para diphenolic acids such as caeffic acid inhibit its activity. Individual leaves have determinate growth pattern. Leaf primordia arise from apical meristems. Limited cell division may or may not occure after the establishment of leaf primordia. Cell enlargement and differentiation occurs and mature leaf acquires characteristic shapes and size of that species. Leaf passes through growing, maturing and senescent developmental stages during its short life span. These changes may be accompanied by changes in chemical constituents. Auxin content is considered to maximum in tissue undergoing cell enlargement and differentiation.Leaf( chloroplast) is also site of phenol carbonic acids and flavonols. Synthesis of phenol is also reported to be light dependent( Stenlid,1963). The maximum amount of growth inhibitors occurs in autumn buds .( Vietez and pena,1968) and growth promoter content in them is reported to be nil. Actively growing organs have high level of growth promoters with or without presence of growth inhibitors ( kefeli,1972). Leaf senesence is characterised by the presence of high level of growth inhibitors and low amount of growth promoters. Decline in IAA content with leaf age may be reflected in development of ethylene senesitivity. The choice of extractant for extracting plant constituents is guided by chemical nature of the compounds to be extracted. For more polar phenols alcohol i.e methanol or ethanol is most appropriate extractant Extraction with ether is also advantageous because this solvent extracts comparatively small group or compounds from tissue is distilled at alow temperature. Purification of extract for lipids and pigments is necessary for light growth test plant before estimation of growth inhibitors and growth promoters. Separation of substances on Chromatograms is known to be influenced by nature of solvent mixture used. There may be interconvertions or release from inactive conjugated complexes. Experimental work was carried out keeping in view the following objectives: 1. Extraction of indoles and phenols from mentioned test species at various stages of leaf development 2. Chromatographic sepration in acidic mixture. Detection of indoles and phenoles on chromatogram. Confirmation of auxin in elute by bioassay 3. Estimation of indoles and phenoles  REVIEW OF LITEATURE  Extraction of auxins from plant tissue is reported to be carried out with ether. acetone, ethanol or methanol. Alcohols and acetone provide more through extraction than ether, Since by direct ether extraction solid plant material may lose part of toatl auxins ( SRIVASTAVA,1963). reported that the alcohol, which terminated or inactivated most of the enzymes, as an excellent solvent for auxin extraction. Chesnut gall also contains a polar auxin, extractable into- n butanol  fraction. However, IAA was also isolated from this fraction indicating  that hydrolysis must have taken place during the purification ( yokata et al, 1974). IAA itself is chemically fragile compound which, when its solution is exposed to air and light produces colour materials and finally completely decomposes. In order to prevent such as sodium diethyl dithiocarbamate, santoquin ( Mann and jaworski, 1970),ascorbic acid (Nieder wieser and Giliberti. 1971) and carbon dioxide (Ray and Hutzinger, 1970) are used for stabilization of IAA during extraction and further purification steps. Careful experimentation in dim light is perferable. Mann and Jaworski(1970) estimated that considerable loss of IAA occurs through sublimation when an ether solution is evaporated to dryness by using a rotatory evaporator. This loss of IAA can be prevented by evaporating the ether at atmospheric pressure. The method of extraction and isolation of the phenols and phenolic acids are mainly based on the polarity, acidity and hydrogen-bonding capacity of the hydroxyl group attached to the aromatic ring( c.f. Harborne, 1989).The techniques used for the extraction of phenols have not changed although more schemes for the quantitative extraction of the compounds have been elaborated ( Van Sumere et al, 1985).Seikel ( 1964)stressed the importance of knowing the chemical composition of the biological source, before carrying out solvent extraction and chromatography. The same principles remain true when using newer chromatographic techniques such as gel filtration, thin layer chromatography ( METC),Centrifugal TLC,flash chromatography, dropllet counter current chromatography(DCCC) and high performance liquid chromatography (HPLC) ( Hostettman, 1985). Van Summere et al ( 1972)gave a variety of solvents except for phenolics bound to insoluble carbohydrates and protein. The majority of compounds can be extracted with methanol and / or ethanol. Alcohols are certainly the solvents of choice and methanol( lower boiling point)or mixtures of metanol and ethanol (1:1v/v)are often prefereed to ethanol. Aqueous alcoholic solutions( e.g 50% methanol)may be used in order to avoid too much contamination with lipid material or to avoid the partial esterification of phenolic acids. Thus,phenolic acids as well as their glucosides are normally extracted with alcohol-water mixtures.However alcoholysis is possible with some aromatic esters and therefore, care should be taken not to concentrate solution too much. Low PH values should also be avoided since hydrolysis of glycoside material may occure during vaccum concentration at 40degreeC ( Van Sumere et al,1985). Swain(1976)councluded that for the quantitative determinations, five successive extractions ( with intermittent homogenisation)under reflux in nitrogenare recommended before the extracts are bulked, concentrated and analysed. Dried plant tissues( roots, heartwood) can be extracted in similar way. pre extraction with light petroleum or hexane( to remove chlorophyll, carotenoids and sterols) should not be carried, if lipophillic phenols are to be quantified. if lipophilic phenols are known to absent or present in the tissue in question on a small scale, it is often useful to remove lipids from certain fat rich tissues ( e.g.seeds) with petrol before proceeding to extract the phenolics with aqueous alcohol. The pooled extracts should be concentracted under vaccum, at neutral PH  Values at 25-30 degree. These precautions are especially necessary with labile components and or when cinnamic acids are involved and acids have been used for extraction, because some phenolics might be lost under conditions of high vaccum. Danger and Basu (1990) reported IAA on TLC plates coated with Silica developed with isopropanol:ammonia :water (10:1:1) by volume. Yadava and Shrivastava (1992) reported the extraction, because some phenolics might be lost under conditions of high vaccum.Danger and Basu (1990) reported 1AA Separation with authentic IAA on TLC Plates coated with Silica developed with isopropanol : ammonia: Water(10:1:1)by volume. Yadava and Shrivastava(1992) reported the extraction of phenol acids from aerial parts of Christella dentata and Marislea minuta by ether. Bentley(1961) showed extraction of indoles from an ether solution by a water solution can cause spontaneous non- enzymatic conversation of certain indoles acetaldehyde to IAA. Extraction of IAA was found to be very easy with chloroform and ether and much worse with toluene and hexane. Glycoside containing phenols were found to be partially not extractable by these solvents. Selection of an extraction must be guided by the purpose of the investigation. Highly methylated phenols are extractable by benzene. Ethyl ether extract low molecular indoles, abscisic acid, phenol carbonic acids, some coumarins and chalcones( kefeli, 1970). if all indole and phenol products from plant tissues have to be extracted more or polar solvents like metanol of 70% ethanol must be used. Moriloshi et al (1980) had proposed indolepyrone fluorescence method for the estimation of nanogram quantities of IAA in plant extracts. Applied to shoots of maize seedlings it enables contents to estimate with ovewr 90% recovery in terms of IAA added at the beginning of the extraction. Law ( 1985) determined endogenous level of IAA and indole acetyl aspartic acid in small amounts of plant material using agueous methanolic extration. law(1985) determined endogeneous level IAA and indole acetyl aspartic acid in small amouts of plant material using aqueous methanolic extraction. In plant tissues free low molecular extraction. In plant tissues free low molecular indoles were found to easily undergo binding or to form amides, glycosides thioethers, protein complexes etc. Some of the bound compounds were found to be extractable by boiling methanol( Winter and Thimann,1966)or alkaline hydrolysis (zenk,1961). Phenolic growth inhibitors present in mature green plants can be maximally separated from endogenous indolic compounds during extraction and estimation by choosing appropriate chromatographic solvent mixture either acidic or alkaline. Alakline mixtures are practically inapplicable for phenol separation, because in such mixtures phenoles are oxidized and remain at the start or posses weak mobility (Kefeli and Turetskaya,1966).
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 * 1)  STUDY OF GROWTH REGULATORS DYNAMICS( INDOLES AND PHENOLS) IN FORCED SALIX SHOOT BUD WITH AGE<