- Plant hormones or plant growth regulators are the organic compounds, other than nutrients which affect the morphological structure and/or physiological processes of plants in low concentrations.
- Phytohormones or plant hormones are naturally occurring growth regulators which in low concentrations control physiological processes in plants. More commonly, the term plant growth regulators is used, because it includes both the native (endogenous) and the synthetic (exogenous) substances; which modify the plant growth.
- As the native plant growth regulators, five major kinds of substances are reported, viz. auxins, gibberellins, cytokinins, abscisic acid and ethylene. All of these, except ethylene and abscisic acid, are multiple forms of endogenous plant growth regulators.
- In general, the plant growth regulators or substances regulate cell enlargement, cell division, cell differentiation, organogenesis, senescence and dormancy.
- They are employed in seed treatment to achieve earlier growth and root development, quality improvement like protein level and amino acid balance, etc.
- Plant growth regulators have given a real boost to plant tissue culture techniques by which now it is possible to culture almost any part of the plant in-vitro.
- In the domain of pharmacognosy, plant growth regulators established their status specifically in enhancing the production of secondary metabolites used as drug.
- Auxin is a general term used to indicate substances that promote elongation of coleoptiles tissues.
- Indole acetic acid (IAA) is an auxin that occurs naturally in plants.
- They are either natural auxins which are produced by plants themselves or synthetic auxins, which have the same action as natural auxins.
- IAA is the principal auxin and other natural auxins are indole-3-acetonitrile (IAN) 4-chloroindole 3-acetic acid and phenyl acetic acid.
- The synthetic auxins are indole-3-butyric acid (IBA), 2-napthyloxyacetic acid (NOA), 1-napthyl acetic acid (NAA), 1-napthyl acetamide (NAD), 2, 4-dichlorophenoxyacetic acid (2, 4-D), 2, 4, 5 trichlorophenoxy acetic acid and 5-carboxymethyl-N, N-dimethyl dithiocarbamate.
- Auxins are involved in different growth processes in plants like internodes’ elongation, leaf growth, initiation of vascular tissues, cambial activity, fruit setting in absence of pollination, fruit growth, apical dominance, inhibition of root growth, influencing physical and chemical properties in leaf abscission and inhibition of lateral buds.
- The proposed mechanism of action of IAA is its interaction with one or more components of biochemical systems involved in the synthesis of proteins. The other hypothesis suggested is the role of IAA to alter the active osmotic contents of cell vacuole during cell expansion or cell wall extension.
- IBA and NAA in combination are used in rooting of cuttings.
- NAA is used as a fruit setting spray 2, 4-D and 2, 4, 5-T are used both as plant growth regulators and in higher concentrations, as selective weed killers, especially for dicot plants.
- IBA has shown promising results to induce rooting in cuttings for cinchona, Pinus, papaya and coffee. The addition of different auxins like IAA, NAA and 2, 4-D in tissue cultures of ergot has led to increase in Indole alkaloids.
- Treatment of derivatives of NAA given to seedlings and young plants of Mentha piperita has shown about 40 percent increase in volatile oil content.
- They are a class of endogenous plant growth regulators and at present over 50 gibberellins are known.
- About 40 of these occur in green plants, while others are present in some fungi. They are present in different organs and tissues like roots, shoots, buds, leaves, floral apices, root nodules, fruits and callus tissues.
- The commercial formulations of gibberellins are used currently for promoting vegetative and fruit growth, breaking dormancy, flower initiation and induction of parthenocarpy.
- Kurosawa, a Japanese physiologist, is credited for initiating the discovery of gibberellins from fungus Gibberella fujikuroi (previously known as Fusarium heterospermum) grown on rice.
- According to Paleg, gibberellins are compounds having gibbane skeleton and biological activity in stimulating cell division or cell elongation, or both.
- The thorough research on gibberellins carried out in Japan, the United States of America and Great Britain has shown that gibberellin A — isolated in 1938 — is actually a mixture of at least 6 gibberellins referred to as GA1, GA2, GA3, GA4 GA7 and GA9. GA3 is termed as Gibberellic acid.
- All of these are the derivatives of gibbane ring skeleton. In addition to free gibberellins, they are also present in conjugated forms.
- GA has not yet been synthesized, but can be produced by large scale fermentation on commercial scale. The angiosperms, gymnosperms, ferns, algae, fungi and bacteria contain several forms of gibberellins, but no single plant contains all of them together.
- Many activities attributed to gibberellins are promotion of rapid expansion of plant cells, stimulation of seed germination, breaking dormancy of overwintering plants, induction of flowering under non- inductive conditions, marked increase in stem elongation, increase in the size of leaves and induction of parthenocarpy leading to seedless fruit sets.
- The effect of gibberellins in cell division is an increase in cell size similar to the effect of auxins. It is observed that gibberellins are more effective in intact plants, while major auxin effects are on excised organs.
- The applications of gibberellins are extended to various medicinal plants. The use of gibberellins in lower dose has shown increased yield of digitalis glycosides per shoot.
- The hormone tried with leaf and root culture of digitalis, showed higher production of digoxin. In case of Tinnevelley senna, GA shows a little positive effect on dry weight of shoot, but reduction in sennoside content of leaves.
- It is observed that GA treatment can cause an increase in height of castor plant up to five times, but does not show any change in fixed oi] content. The treatment significantly causes reduction in alkaloid content of vinca, datura, hyoscyamus, etc.
- The mechanism of action of Gibberellic acid appears mainly to induce activity of gluconeogenic enzymes during early stages of seed germination and this specificity ensures a rapid conversion of lipid to sucrose, which is further used in supporting growth and development of the embryonic axis to a competent root and shoot system.
- It is also found that gibberellins induce the synthesis of a-amylase and other hydrolytic enzymes during germination of monocot seeds. They are also involved in mobilizing seed storage reserves during germination and seedling emergence.
- These are either natural (zeatin) or synthetic (kinetin) compounds with significant growth regulating activity. Zeatin has effect on cell division and leaf senescence and synthetic cytokinins are useful in promoting lateral bud development and inhibition of senescence.
- Cytokinins influence a broad spectrum of physiological processes in plants like promotion of cell division. The other activities exerted are participation in orderly development of embryos during seed development, influencing the expansion of cells in leaf discs and cotyledons, delaying breakdown of chlorophyll and degradation of proteins in ageing leaves.
- Miller isolated the crystalline substance from autoclaved herring sperm DNA capable of inducing cell division in tobacco cultures and named it as kinetin, which was found to be 6-furfuryladenine.
- Further, some other adenine derivatives were also found having similar biological activity and were called ‘kinins’ collectively known as cytokinins. These substances are found in young and actively dividing tissues like embryos, seedlings and apical meristems.
- The naturally occurring cytokinins are zeatin, N° dimethylamino purine, and N6-Δ2-isopentenyl aminopurine. The synthetic cytokinins are kinetin, adenine, 6-benzy] adenine benzimidazole and N. N’-diphenyl urea.
- Cytokinins are reported to increase marginally sennoside content in Tinnevelley senna leaves and also enhance the dry weight of shoots. In opium, they cause formation of elongated capsule and reduce alkaloid content.
- In Duboisia hybrids, the cytokinins activity present in extract of seaweed shows marked increase in both leaf content and also hyoscine content.
- Kinetin is reported to play the role in nucleic acid metabolism and protein synthesis. In plant metabolism, it is proposed that some t-RNA contain cytokinins like activity. They have an action on some enzymes responsible for formation of certain amino acids.
- It is a simple organic molecule present in the form of volatile gas and shows profound physiological effects. It is present in ripening fruits, flowers, stems, roots, tubers and seeds. It is present in very small quantity in plant, say about 0.1 ppm — part per million — possibly, its quantity increases in local areas during the time of growth and development.
- Ethylene is produced by incomplete burning of carbon rich substances like natural gas, coal and petroleum. Denny (1924) showed the yellowing of lemons due to stove gas. Also, the plant damage was noticed after the introduction of illuminating gas.
- Gane (1935) found that a gas evolved from ripe apples can also effect the ripening of green apples and showed that it was ethylene gas which had a role in ripening of other fruits.
- Ethylene shows a broad array of growth responses in plants, which include fruit ripening, leaf abscission, stem swelling, leaf bending, flower petal discoloration, and inhibition of stem and root growth.
- It is commercially used for promotion of flowering and fruit ripening, induction of fruit abscission, breaking dormancy and stimulation of latex flow in rubber trees.
(E) ABSCISIC ACID (ABA)
- The physiological activities in plants like retaining or shedding of different organs such as leaves, stems, flowers and fruits have led to finding of natural growth inhibitor.
- A diffusible abscission-accelerating substance was found by Osborne (1955) in senescent leaves. Carns et al. isolated several abscission accelerating substances from cotton plants and named them as abscission I and abscission II.
- In an inhibitory way, ABA interacts with other plant growth substances. It inhibits the GA-induced synthesis of a-amylase and other hydrolytic enzymes.
- During maturation, ABA accumulates in many seeds and helps in seed dormancy. ABA concentrations are found to be enhanced in stress conditions, like mineral deficiency, injury, drought and flooding.
- ABA serves an important role as potential antitranspirant by closing the stomata, when applied to leaves.
- A number of other synthetic growth inhibitors and retardants reported are maleic hydrazide, daminozide, glyphosate, chlormequat chloride, S, S, S-tributyl phosphorothioate, ancymidol, chlorophonium chloride, piproctanyl bromide, etc.
- However, commercial use of these compounds is yet to be reported. A group of synthetic substances called morphactins is a potent inhibitor of auxin transport causing tropic responses, reduction of apical dominance and promoting lateral growth. Morphactins include chloroflurecol methyl, flurecol-butyl and TIBA (2, 3, 5-tri-iodobenzoic acid).
- Mutation is represented as variation in characters of the species. It is caused either due to environmental changes or changes in hereditary constitution.
- Normally, as a response to environmental changes, the variations are observed, but the original traits are restored when changes in environment are also withdrawn or disappeared.
- This type of change and restoring is not heritable and also not built into genotype. They are termed only as phenotypic variations and commonly called as modifications.
- It is evident that between organisms of similar genotype there are phenotypic variations. However, when a change occurs in the genome of an individual which is not caused due to the environment, it may make a permanent evolutionary change. This is termed as mutation and represents a sudden change in genotype causing qualitative or quantitative alterations of genetic material.
- Mutations are of two types namely chromosomal mutations and point mutations. The former type of mutation is also called chromosomal aberration, which in many cases leads to changes in amount or position of genetic material. On the other hand, the changes with a gene or cistron of DNA molecule cause point mutations and it is permanent and heritable.
- Mutation which occurs due to some unknown reason from nature is called as spontaneous mutation. This has been observed in some plants, bacteria, viruses, etc.
- Mutations can also be induced by artificial means with certain reagents called mutagens and are called induced mutations. The various mutagens used are exposure to UV rays, X-rays, ionizing radiations, certain chemicals, abnormal environment etc.
- The chemical mutagens used are nitrogen mustard, formaldehyde, nitrous acid, ethylethane sulphonate, 5-bromouracil, 2-aminopurine, manganese chloride etc.
- The changes caused due to mutations include morphological and anatomical changes, as well as, changes in the chemical composition of the plants.
- This is significant for the medicinal plants. In some cases, favourable changes and yields in active constituents of plants have been achieved.
- Mutations may cause building the resistance of a medicinal plant towards certain diseases. But, in all these cases, the plant may become susceptible to climatic conditions, certain other diseases, retardation in growth, etc. These undesirable effects are to be eliminated by breeding and selection.
- Polyploidy has exhibited various useful effects on medicinal plants like digitalis, mentha species, poppy, plants containing tropane alkaloids, lobelia, etc.
- The specific number of chromosomes is a character of each species and is called genome which is observed in all types of organisms.
- The term euploidy is a type of ploidy in which genome contains whole set of chromosomes and euploidy includes monoploidy, diploidy and polyploidy. When the organism contains more than two genomes, it is called polyploidy.
- The polyploidy occurs in a multiple series of 3, 4, 5, 6, 7, 8,etc. of the basic chromosome or genome number and then accordingly, it is called triploidy, tetraploidy, pentaploidy, hexaploidy, heptaploidy and octaploidy respectively.
- Polyploidy is caused through cell generation, physical agents like X-rays, centrifugation, temperature chocks and chemical agents, mainly colchicine, veratrine, sulphanilamide, hexachloro- cyclohexane, and mercuric chloride.
- The chemical agents cause disturbance to mitotic spindle of dividing diploid cell and cause non-segregation of already duplicated chromosome and thus convert diploids into tetraploid cells.
- The phenomenon of polyploidy is of greater significance to medicinal plants. It may cause formation of new species, adaptability to various habitats and mainly accumulation of vitamins.
- Perhaps, the best known chemical to cause polyploidy is colchicines, an alkaloid obtained from Colchicum species like Colchicum autumnale, C. luteum and C. speciosum.
- Colchicines prevents sister chromatids from separating into daughter nuclei at anaphase. These chromatids remain attached by their common centromere in C-metaphase.
- The chromatids eventually separate, but remain in the same nucleus. An interphase occurs, followed by a second C-metaphase, involving a doubled chromosome complement. Hence, the chromatid pairs are doubled in second
- C—metaphase. Likewise, the cell undergoes one, two or more than two rounds of DNA replication and causes polyploidy. The colchicines activity mentioned here is caused due to its interaction with disulphide bonds of spindle protein and by inhibition of conversion of globular proteins to fibrous proteins.
- The capacity of colchicines to induce polyploidy varies along with its different derivatives. Chemically induced mutations may lead to variations in biochemical composition of plant.
- Colchicines treatment given to medicinal plants has shown promising results in many cases. The observations in tropane alkaloids by way of polyploidy are more specific in stramonium, where yield of crop is enhanced by 60-150 percent in 4 n form.
- The plants like lobelia, cinchona, belladonna; acorus, squill, cannabis, and poppy also show increased yield of respective compounds in 4 n form.
- Polyploidy may cause a reduction of total glycosides of D. purpurea and D. lanata, but in the later case raises slightly the contents of lanatosides A and B.
- The increase in chemical contents may not be coincidental with phenotype of medicinal plant. There may be reduction in size also along with enhancement in content of active constituents. Some plants do not show any change in chemical contents as a response to polyploidy.
Chemodemes (Chemical Races)
- The knowledge about plant chemical races has surfaced, due to thorough chemical analysis of a huge number of different plants with the help of modern analytical instruments.
- Chemodemes are regarded as a group of plants of a species which have identical morphological characters, but differ in their chemical nature.
- Due to this, chemodemes are considered as chemically separate groups within species. The observation of chemodemes can be confirmed only by growing different plants of a species in identical conditions, preferably from the seeds and for many generations.
- By this way, it shows variations either in type or contents of certain constituents, like the secondary metabolites of medicinal importance. The chemical characters of chemodemes are hereditary.
- The artificial production of mutations in medicinal plants is an important milestone in the development of cultivation technology.
- The higher solasodine content is achieved by applying radiation and chemical mutagens in Solanum khasianum. It is also reported that chemical mutagens have a successful use in increasing morphine content of Papaver somniferum.
- The tuber yield and diosgenin content of Dioscorea bulbifera is increased by radiation. The economically important characters of Atropa belladonna have been enhanced by radiations and chemical mutagens.
- The agronomic performance and harvest index of Mentha arvensis var. piperascens (Japanese mint) have been improved by exposure to gamma radiations.
- The effects of artificial mutations have been extensively studied on different species of mentha. The capsicum seeds (Capsicum annuum) treated with sodium azide and ethyl methane sulphonate have led to plants giving higher contents of capsaicin.
- The process through which hybrids are produced is called hybridization. A hybrid is an organism which results from crossing of two species or varieties differing at least in one set of characters.
- The resultant hybrids are monohybrids (one pair of different characters), dihybrids (two pairs of different characters) or polyhybrid (more than tow pairs of different characters).
- Hybridization helps in inducing in a single variety, the favourable characters of other varieties or species, and sometimes producing new and favourable characters which are not present in both the parents.
- The hybridization of Withania somnifera Israeli chemotype II and W. somnifera South African chemotype has led to formation of a new hybrid which contains three new withanolides.
- The hybrids like Digitalis purpurea x D. lanata and D. purpurea x D. lutea contain principal glycoside as lanatoside A, along with lanatosides B and E, but devoid of lanatoside C or purpurea glycoside A.
- During the cross of Solanum incanum (1.8 per cent solasodine) and S. melongena (traces of solasodine), first-generation bears more fruits (berries) with solasodine up to 0.5 per cent. Second generation has proved to be a high yielding source for solasodine.
- A recent development in hybridization is through the medium of tissue culture. The protoplast cultures are employed for this purpose. Plant protoplasts are the cells without cell walls. Such protoplasts in cultures can be fused together (protoplast fusion or asexual hybridization). The fusion can be arranged in cells of same origin or between different species.
GENETIC ENGINEERING AND RECOMBINANT DNA TECHNOLOGY
In recent years, these techniques have taken greater strides in the field of drugs and pharmaceuticals. The method of artificial synthesis of new genes and their subsequent transplantation in the genome of an organism or the method of correcting defective genes of organism by molecular biological techniques form the discipline called genetic engineering.
Recombinant DNA technology involves gene splitting to change characters of plants and animals by implanting in them genes from other organisms and, in some cases, even from other species.
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