The top three plant growth promoters are: (1) Auxins (2) Gibberellins and (3) Cytokinins.
Plant Growth Promoter # 1. Auxins:
Auxins are the best known plant growth regulators. They promote growth of stem or coleoptile sections and decapitated (apex removed) coleoptiles, but in the same concentration are incapable of causing growth in intact plants.
Auxins are defined as “An organic compound characterized by its capacity in low concentration (below 10-3 M or 0.001 M) to induce elongation in shoot cells and inhibition of elongation of root cells. They resemble IAA (Indoleacetic acid) in physiological action. They may and generally do effect other processes besides elongation but elongation of shoot cells is considered as critical. Auxins are weak organic acids. The main naturally occurring auxin is Indole-3-acetic acid (IAA).”
Among the growth regulators, auxins were the first to be discovered and studied in detail.
Historical background of the work regarding the distribution, mode of action and chemical nature of auxins follows:
1. Julius Von Sachs was the first scientist to indicate the presence of organ-forming substances in plants.
2. Charles Darwin (1980-81):
The first indication of their existence came from the work of Charles Darwin (1880), who was studying the bending of the coleoptile of a grass (Phalaris sp.) towards light. He was able to establish that it was the tip of the coleoptile which was able to perceive the light stimulus. The latter was transmitted to the sub-apical region where the differential growth caused bending (Fig. 3.1). There was no phototropic response when the coleoptile tip was removed (Fig. 3.2), or covered by an opaque cap (Fig. 3.3)
3. Boysen-Jensen (1910-13):
Boysen-Jensen observed that when an excised coleoptile tip was replaced without (Fig. 3.4 AB-CD) or with gelatin (agar block; Fig. 3.4, AB-EF) inserted in between the two, phototropic curvature resulted as in normal coleoptile i.e., the tropic stimulus was transmitted through the incision and the agar block.
He further found that insertion of mica plate on the shaded side (Fig. 3.5) prevents curvature following unilateral illumination of the tip. When a mica sheet was inserted on the illuminated side (Fig. 3.6), a curvature was formed in the usual way. It was concluded that a substance migrates down the dark side promoting growth curvature towards light.
4. A. Paal (1914-1919):
He demonstrated that coleoptiles would bend even in the dark after certain treatments. He sliced off the Avena coleoptile tip and replaced it eccentrically (i.e., off centre) on one side of the coleoptile stump. He observed accelerated growth beneath the coleoptile tip, resulting in curvature (Fig. 3.7).
5. F.W. Went (1928):
He found that when an Avena coleoptile is decapitated its growth in length ceases. The addition of a plain agar block has no effect but growth is renewed by the addition of a block containing auxin extracted from the excised tip.
He further found that if we take tips and place them on an agar block for some time and then divide the agar block into smaller blocks which are now placed eccentrically on coleoptile stump, the plumule moves away from the side the agar block (+ growth substance) is kept and this is proportional to the amount of auxin in agar (Fig. 3.8).
He further found that when unilateral light falls upon an excised Avena coleoptile tip placed in contact with two agar blocks separated by a mica sheet, growth hormone is displaced towards the shaded side (65%) as opposed to illuminated side (35%) (Fig. 3.9).
These experiments suggested that the side of the coleoptile that received the growth substance elongated faster and caused the curvature towards the opposite side. These experiments further indicated that the substance is synthesized in the coloptile tip and is trans-located downward. He called this substance auxin (from the Greek wrord ‘auxein’ to grow).
It is interesting to note that the first auxin was isolated from human urine. Presently the term auxin is applied to indole-acetic acid (lAA) and to natural and synthetic compounds having similar structure and growth regulating properties.
On the basis of the work done so far, following conclusions are drawn:
(i) Growth regulating auxins are synthesized at the tips (apices) of the plant.
(ii) Auxins synthesis is inhibited on the illuminated side.
(iii) Auxins are transported from apex to the base (polar transport) on the dark or un-illuminated side.
(iv) Auxins are soluble in water.
(v) Light may inhibit the release of auxins or stimulate their degradation or even cause them to move laterally towards the dark or un-illuminated side of the stem.
Types of Auxins:
There are two major types of auxins:
(i) Natural auxins and
(ii) Synthetic auxins.
(i) Natural auxins:
These are naturally occurring auxins in plant parts and, therefore, regarded as phytohormones. The best known and universally present natural auxin is Indole-3-acetic acid. Other natural occurring auxins are hidole-3-pyruvic acid, Indole-3-ethanol, Indole-3-acetaldehyde, etc. The natural auxins may occur in plants in the form of free auxins and bound auxins.
(ii) Synthetic auxins:
These are the chemicals synthesized by chemists that cause various physiological actions similar to IAA. They are not treated as phytohormones but are considered as plant growth regulators. Some of the synthetic auxins are: Indole-3-butyric acid (IBA), Indole-3- propionic acid (IPA), α- and β-naphthalene acetic acid (NAA), 2, 4- dichlorophenoxy acetic acid (2, 4-D), benzoic acids, etc.
Various uses of synthetic auxins are given below in the table:
Distribution and Transport of Auxins in Plants:
Among the several kinds of auxins, indole-3-acetic acid is the most important and well studied natural auxin. They are synthesized continuously in the shoot apical meristems, cotyledons and young leaves (leaf primordia). In roots, these are produced in relatively very small amounts (Fig. 3.13). Elongating cells lose the ability to produce auxins. IAA has been found in all the plants studied so far and fungi.
It also occurs in the human urine, especially in persons suffering from pellagra, (niacin or nicotinic acid deficiency). The role of IAA in human beings is not known.
Observations clearly indicate that auxins move through the living cells (phloem parenchyma and parenchymatous cells) that surround the vascular bundles. The usual movement occurs from shoot apex downward to the base (i.e., basipetal) and from root upwards to the shoot (i.e., acropetal). Such a movement of auxins is known as polar transport.
Although movement of auxin occurs both basipetally and acropetally, the basipetal movement is comparatively predominant. The movement of auxin is slow taking place at 6.4 mm/hr. to 26 mm/hour. Importantly, synthetic auxins such as 2, 4-D, IBA, NAA, etc., move in all directions in plants.
IAA is present in very low concentration in the coleoptile tips (1 µg from 10,000 tips). Response of a cell to the auxins is affected by their concentration. Usually, a low concentration of auxin stimulates the growth while high concentration inhibits the growth of the same cell or organ.
Roots, in general, show growth stimulation at very low concentration (0.0001 ppm), while in stem the similar response occurs at a relatively high concentration (10 ppm) (Fig. 3.10) Thus, depending upon concentration, IAA induces both stimulation and inhibition of growth.
Isolation and Molecular Structure:
Kogl and Haagen Smit (1931) were the first to isolate a chemical from the human urine and named it auxin-a (auxentriolic acid). Later on, Kogl, Haagen Smit and Erxleben (1934) isolated a similar substance from corn germ oil (extracted from germinating corn seeds) and named it auxin-b (auxenolinic acid). Subsequently, they isolated another auxin from human urine and called it heteroauxin.
Later, it was identified as indole-3-acetic acid (IAA). Thimann (1935) isolated it from the culture filtrate in which the fungus Rhizopus suinus had been growing. Recently it has been isolated from coleoptile tips.
The molecular structure of some common auxins, both natural and synthetic are given in Fig. 3.11.
Antiauxins are the chemicals which inhibit the action of auxins. They do not interfere with the energy metabolism of the cells. Examples of the antiauxins are: 2, 3, 5 triiodobenzoic acid (TIBA); and naphthylthalamic acid (NPA).
Bioassay of Auxins:
Bioassay refers to the testing of a biological activity like growth response of a substance by employing a living material like plant or plant part. All such studies in which a living test material is used are known as bioassay. Auxin bioassary is a quantitative test as it measures concentration of auxin to produce the effect and the amount of effect.
1. Avena Coleoptile Curvature Test:
It is one of the most widely used bioassay for auxin activity. The test is based upon experiments of F.W. Went (1928).
This bioassay is based on two facts:
(i) There is rapid polar transport of auxins in the Avena coleoptile, and
(ii) There is always a correlation between the concentration of auxin and curvature produced.
The procedure for the Avena coleoptile curvature test involves following steps:
(1) When the oat seedlings have attained a height of 15 to 30 mm. approximately 1 mm tip of the coleoptile is removed. This apical part is the source of natural auxins.
(2) The tips are now placed on agar blocks for few hours (Figs. 3.12. A, B). During this period the auxins (which the tips are suspected to contain) diffuse out of these tips into the agar.
(3) The auxin containing agar block is now placed on one side of the decapitated stump of the Avena coleoptile (Fig. 3.12-C). The auxin (from the agar block) diffuses down through the coleoptile along the side to which the ‘auxin-agar block’ is placed. An agar block without auxin is placed on another decapitated coleoptile. This serves as control (3.12-F).
(4) Within hours, the coleoptile with ‘auxin-agar block’ bends on the side opposite to which the agar block is placed (Fig. 3.12 D, E). The curvature occurs due to more rapid growth of the side on which the ‘auxin-agar block’ is placed. This curvature can be measured by exposing the curved coleoptile to a photographic plate. The amount of auxin in the agar block is directly proportional to the degree of curvature. 10° curvature is produced by auxin concentration of ISO μg/liter at 25°C and 90% relative humidity.
2. Root Growth Inhibition Test (Cress Root Inhibition Test):
Sterilized seeds of Cress are allowed to germinate on moist filter paper kept in a glass petri dish. As the roots reach a length of 1 cm or so, root lengths are measured. 50% of the seedlings are placed in a test solution while the remaining are allowed to grow over moist paper. Lengths of the roots are measured after 48 hours.
It is seen that the seedlings placed in test solution show very little root growth while root growth is normal in control seedlings. Here also the inhibition of root growth is proportional to the concentration of auxin in the test solution.
Physiological Roles of Auxins:
Auxins affect a number of physiological activities in plants. Some of these are described briefly below:
(i) Cell enlargement and longitudinal growth:
IAA promotes elongation and growth of shoots and enlargement of many fruits, by stimulating cell walls to stretch in more then one direction.
(ii) Cell Division in cambium:
It promotes cell division in vascular cambium. Thus, it id responsible for secondary growth
(iii) Reactivation of cambium:
The reactivation of cambium in the growing season is apparently triggered by IAA moving from the developing shoot buds.
(iv) Root formation:
IAA also stimulates cell division in the pericycle leading to the formation of lateral and adventitious roots. For this reason auxin particularly the Indole-butyric acid (IBA) is used by plant growers to induce root formation in cuttings. In brief it promotes root initiation. It may be noted that though the auxin stimulates formation of new roots, it inhibits the root growth.
(v) Tissue culture:
IAA promotes cell division and elongation leading to the development of callus (unorganized mass of parenchymatous cells) in tissue cultures.
(vi) Apical dominance:
In most plants it induces the phenomenon.of apical dominance, i.e. the terminal bud at the apex of a shoot suppresses the development of lateral buds into branches. Lateral buds start developing into branches when the apical bud is removed The process can be reversed if IAA is applied to the decapitated apex. Apical dominance is thus under the control of auxins.
(vii) Inhibition of Abscission:
Another inhibitory effect of auxin is abscission of leaves and fruits, which leads to leaf fall and fruit drop. It operates by the suppression to enzymatic degradation of cell wall components (pectins, celluloses and hemicelluloses) and thus prevents leaf or fruit fall. Thus, leaves and fruits must produce auxin continuously to prevent the formation of the abscission zone which cuts off their nutrient and water supply. Leaves are shed seasonally because they stop producing auxin.
IAA induces parthenocarpy i.e., the formation of seedless fruits without the act of fertilization.
(ix) Eradication of weeds:
Some of the synthetic auxins, especially 2, 4-D (2, 4-dichlorophenoxyacetic acid), act as herbicides and are used for the eradication of weeds. Spraying of this auxin in higher concentration induces the root cells to divide very rapidly. As a result the roots become distorted and sieve tubes get closed. Consequently the plant dies.Broad-leaved dicots (most weeds) are more sensitive to 2, 4-D. Therefore, the application of this synthetic auxin selectively kills the dicot weeds without effecting the monocots.
(x) Enzymatic activity.
While it stimulates the activity of certain enzymes (e.g. conjugates with aspartic acid), enzymatic activity of others (peroxidase isoenzymes) is lowered.
(xi) Flower initiation:
In some plants such as pineapple (Ananas sativus), flowering continues throughout the year. In such cases, spraying of dilute solution of the synthetic auxins (like 2, 4 – D; NAA, etc.) initiates simultaneous flowering in the whole crop. Spraying of high concentration of auxins is done to inhibit flowering in some plants such as lettuce. In lettuce, leaves are edible and hence inhibition of flowering increases its commercial value.
(xii) Increased metabolic activity:
It increases permeability of cells, phosphate utilization in biochemical reactions and active absorption of water by roots.
It stimulates respiration in many plants.
(xiv) Plant Growth Movements:
Differential distribution of auxin (IndoIe-3-acetic acid) regulates some of the important plant growth movements, viz. phototropism and geotropism.
Auxins induce early differentiation of xylem and phloem in tissue culture experiments.
a. Auxins + 2% of sucrose favour differentiation of xylem.
b. Auxins + 3% of sucrose favour differentiation of xylem and phloem.
c. Auxins + 4% sucrose favour differentiation of phloem.
Intermediate cytokinins/low auxin – Callus formation.
Plant Growth Promoter # 2. Gibberellins:
Gibberellins, like auxins, are chemicals which have the capacity to influence both growth and development of plants. The first work on the gibberellins resulted from Japanese investigations into the Bakanae disease of rice. Bakanae was for the first time reported by Konishi in the year 1809. At that time it was thought that the giant rice plants were one of the varieties of rice.
The Japanese consider tall people foolish and hence they called those tall seedlings of rice ‘bakanae’ (foolish seedlings). Later it was found that it was not a new variety but the tallness was caused due to the infection of a fungus Gibberella fujikuroi.
It is a soil-borne fungus and its imperfect stage is called Fusarium moniliforme. One of the earliest symptoms of the infected plants is a marked elongation of the shoots and leaves. Gibberellins were first traced by Kurosawa (1926) who after application of fungus extracts to healthy and dwarf rice plants obtained symptoms seen in diseased plants. Once the presence of this active substance was known, other workers also became interested in this wonder chemical. Yabut and Hyashi (1934) were the first to obtain gibberellin A in pure form.
Takashi et al. (1955) obtained three qualitatively similar but quantitatively different forms of gibberellin. These were gibberellic acid (C19 H22 O5), gibberellin A1(C19 H24 O6) and gibberellin A2 (C19 H26 O5). Takashi (1957) isolated gibberellin A4 (C19 H24 O5) from cultures of Gibberella fujikuroi. MacMilan and Suter (1958) succeeded in isolating Gibberellin A1 from the seeds of Phaseolus multiflorus (runner bean).
Occurrence and Distribution:
Ever since the discovery of gibberellin numbers of individual Gibberellins (GAs) have been isolated from different plant sources. Of these, about 25 have been isolated from the fungus Gibberella fujikuroi. GA-like activity has been observed in almost all parts of higher plants. However, the reproductive organs, particularly immature seeds, possess more of GA than the vegetative parts.
Physiological Roles of Gibberellins:
(i) Effect on Shoot Elongation:
Gibberellins have marked effect on shoot elongation especially of the internode. When genetically dwarf plants of pea (Pisum sativum) are treated with GA, they elongate and become as tall as the genetically tall variety (see figure 3.14). Of course this chemically induced tallness is not carried to the next generation.
It is generally agreed that the internodes elongate not because of cell division but chiefly because of cell elongation. The cells elongate 3 to 4 times their original size. What physiological changes are exactly brought about by GA are not very well known.
(ii) Enhancement of apical dominance:
In a number of plants, enhancement of apical dominance as a result of GA treatment has been reported. For instance, in dwarf plants of French bean (Phaseolus vulgaris), where the growing point generally aborts, when treated with GA, shows activation and continuous growth of the apical point and suppression of laterals.
(iii) Breaking of dormancy:
Gibberellins are capable of breaking of dormancy in several economically important plants like potato. GA treatment on seed potatoes brings about simultaneous germination of ‘eye buds’. It is interesting to note that when GA is sprayed on the foliage of potato plants, tubers start sprouting even when attached to mother plant. Dormancy of lettuce, barley and the light sensitive seeds of kalanchoe has also been broken by GA treatment.
(iv) Increase in dry weight of plants:
Rice and other cereal plants when treated with GA show marked increase in the size of internodes, enhanced apical dominance and increased leaf growth. In other words GA increases the total area of plant exposed to light thereby increasing net photosynthesis. Increase in dry weight in pea, wheat, soybeans, grasses have been observed as a result of GA treatment.
(v) Breaking of dormancy of buds:
There are evidence that GA can replace long day treatment for breaking dormancy of buds. Breaking of dormancy of buds is controlled by critical light period, but dormancy of buds on the shoots of long-day plants is broken even when kept in short-day conditions as a result of GA treatment.
(vi) Replacement of Vernalisation:
It is well known that most of the plants require specific conditions of either temperature or light for flowering. Vernalisation (cold treatment) and photoperiodic treatments are the two processes used to bring about flowering in such plants. It has been postulated that as a result of vernalisation, a hormone known as Vernalin is produced in cold-requiring biennials.
Lang (1956) for the first time noted that GA treatment of plants of Hyoscyamus niger (a cold-requiring, long day biennial) could help in bringing about flowering in long day conditions without low temperature treatment. Thus it may be concluded that in H. niger, GA has the capacity to replace cold treatment required for stalk formation. Wittwer and Bukovac (1957) were able to induce flowering in carrot (a biennial, cold requiring, long day plant) under short day conditions as a result of prolonged treatment with high doses of GA.
(vii) Sex Expression:
Gibberellins have been observed to induce maleness in plants.
(viii) Parthenocarpy (i.e., development of fruit without pollination and fertilization):
Gibberellins are more potent parthenocarpic agents than auxins.
(ix) Flowering and Gibberellins:
It is now known that there is some similarity between the action of light and gibberellin on flowering. GA can stimulate the wavelength of light responsible for flowering in long-day plants. GA also checks flowering of short-day plants as influenced by the action of light. In what way the action of GA resembles the light is not understood.
Plant Growth Promoter # 3. Cytokinins:
A third type of plant hormone, the cytokinins stimulate growth of cells in tissue culture or organ culture and have a marked increasing effect on cell division. The existence of specific substances which can control cell division in plants was suspected many years before such substances were finally discovered.
In the year 1956 Miller, for the first time, isolated a pure, highly active, cell division-inducing factor from autoclaved herring sperm DNA. This compound was identified as a 6-substituted amino purine 6-(furfurylamino) purine and was called Kinin or Kinetin. Kinetin was not a component of DNA, but was derived from breakdown and rearrangement during autoclaving.
Synthetic Kinetin was found to be a very potent promoter of cell division in tobacco pith. Later on it was discovered that substances division promoting activity were widely distributed in plants. Letham isolated it from immature seeds of maize and named it Zeatin.
The name “Kinetin” or “Kinins” was later on replaced with the name “cytokinin” because the former name conflicted with the same term used in animal biochemistry. A cytokinin is now defined as a compound which in the presence of optimal auxin, induces cell division. Cytokinins are compounds that are derived from the nitrogen-containing adenine.
Physiological Roles of Cytokinins:
Effects of cytokinins on plant growth and development:
Cytokinins not only promote cell division but also morphogenesis (i.e., differentiation of different tissues and organs). Cytokinins controlling the relative production of shoots and roots in tissue culture preparations suggests that they may perform a similar function in the intact plant.
1. High cytokinins/auxin ratio- Differentiation of shoot.
2. Low cytokinins/auxin ratio – Differentiation of root.
3. Intermediate Cytokinins/auxin ratio- Differentiation of both root and shoot.
Cytokinins and Cell Enlargement:
Cytokinins can bring about expansion of leaves and cotyledonary tissue by a process involving only cell enlargement. In combination with gibberellins, cytokinins are able to modify markedly the shapes of leaves on intact plants. It has been suggested that normal leaf development could be controlled by the gibberellin/cytokinin ratio.
Delay of senescence:
Cytokinins have a striking quality to retard senescence (i.e., process of ageing) in plants. External application of cytokinins delay the rate of chlorophyll disappearance and protein degradation which usually accompanies the process of senescence in leaves.
Localized application of cytokinins to yellowing leaves produces green areas in which the rate of senescence is markedly reduced. This is called Richmond-Land effect. Because of their senescence retarding quality, cytokinins are used commercially in the storage of green vegetables.
Cytokinins and Light:
It is interesting to note that cytokinins are capable of influencing not only the process of cell division, morphogenesis, cell enlargement and senescence in plants, they can also substitute for, or interact with, light in the control of a number of physiological and metabolic processes, such as seed germination, pigment synthesis and chloroplast development, in plants.
Breaking of dormancy of seeds:
Cytokinins can break dormancy of many seeds and also promote their germination.
External application of cytokinins promote growth of lateral buds by reducing apical dominance. Thus, cytokinins reverse the auxin-induced inhibition of lateral buds and counteracts the apical dominance.
Resistance to high and low temperatures:
Cytokinins provide resistance to plants against very high and very low temperatures, thus preventing temperature injuries.
Cytokinins are also known to enhance femaleness in some plants.
Cytokinins have been observed to induce parthenocarpy in some plants.
Site of cytokinin biosynthesis in plants:
There is sufficient circumstantial evidence available now to prove that cytokinins are synthesized in roots from which they move upwards in the xylem and pass into the leaves and fruits.
Applications of Cytokinins:
1. Prevention of Premature Senescence:
Cytokinins are used to prevent premature senescence in crop plants.
Cytokinins are applied to the marketed vegetables, cut soots and flowers to keep them fresh for several days. Cytokinins do it by delaying the senescence.
3. Tissue Culture:
Cytokinins are used in tissue culture because of their role in cell division and morphogenesis.