Growth regulation aspects of propagation, embryogensis, seed and bud dormancy SUBMITTED BY VIKAS RAMTEKE PH.D. 1ST SEMESTER DEPT. OF FRUIT SCIENCE ACHF, NAU, NAVSARI Submitted to Dr. S.J. Patil I/C Professor and Head Dept. of Fruit Science ACHF, NAU, Navsari

Propagation

AUXINS Root initiation In contrast to stem, the higher concentration of auxin inhibits the elongation of roots. But the number of lateral roots is considerably increased i.e., higher concentration of auxin induces more lateral branch roots. Application of IAA in lanolin paste (lanolin is a soft fat prepared from wool and is good solvent for auxin) to the cut end of a young stem results in an early and extensive rooting. This fact is of great practical importance and has been widely utilized to promote root formation in economically useful plants which are propagated by cuttings.

Callus formation Besides cell elongation, auxin may also be active in cell division. In many tissue cultures, where the callus growth is quite normal, the continued growth of such callus takes place only after the addition of auxin. Commonly used synthetic auxins in tissue culture are 2,4-dichlorophenoxyacetic acid (2,4-D; often used for callus induction and suspension cultures), and 1-naphthaleneacetic acid (NAA; when organogenesis is required).

Plant tissue culture Within plant tissues, IAA and other naturally occurring auxins combine with small molecules (alcohols, amino acids, sugars) to produce ester, amide, or glycoside conjugates (Bandurski et al., 1995). This appears to be a mechanism for storing auxins in cells and stabilizing the level of free auxin by metabolizing the excess. Auxin in conjugated molecules is protected from oxidative breakdown and may be later enzymatically released when required.

Gibberellins Seed germination Certain light sensitive seeds eg. Lettuce and tobacco show poor germination in dark. Germination starts vigorously if these seeds are exposed to light or red light. This requirement of light is overcome if the seeds are treated with gibberellic acid in dark.

It has been suggested that there are related or common receptors for dormancy breaking agents within the plasma membrane of the responsive embryonic cells. When triggered, these receptors then initiate a signal transduction cascade, perhaps involving synthesis of or sensitization to germinationpromoting gibberellins that lead to the completion of germination. Changes in the phosphorylating activity of membrane- associated, Ca+2-dependent protein kinases that lead to dormancy or germination have been proposed as well.

Micropropagation When GAs are added to plant tissue culture media, they often diminish or prevent the formation of roots, shoots, or somatic embryos, although the opposite has also been seen. Some endogenous GAs may be necessary for normal callus growth (Lance et al., 1976), and inhibition of GA biosynthesis can influence development of cells in liquid cultures (Ziv and Ariel, 1991).

Some differences in the inhibition or promotion of adventitious root and shoot formation by GAs might be due to the fact that GAs inhibit meristemoid initiation (Thorpe and Murashige, 1970) but are required for assisting the further growth and development of preformed organs. GAs may also alter the availability of endogenous auxin. Growth of shoots in meristem and shoot cultures may be enhanced by addition of GA (Fry and Street, 1980).

CYTOKININS (Kinetin) In tissue culture, cytokinins play a crucial role as promoters of cell division and act in the induction and development of meristematic centers leading to the formation of organs, mainly shoots (Peeters et al., 1991).

Kinetin (KIN) has been applied for micropropagation of many ornamental plants (Jain and Ochatt, 2010). In vitro shoot proliferation and multiplication are largely based on media formulations containing cytokinins as a major plant growth regulators (Mamidala and Nanna, 2009; Hoque, 2010). Some reports indicated that the presence of cytokinin in the culture medium helped in the multiplication of shoots (Kumar et al., 2001).

Rooting can be initiated by transferring the regenerated shoots to a medium containing only auxins. The best explants for micropropagation of most plants, without any intervening callus phase, are shoot tips and axillary buds (Pati et al., 2005; Hashemabadi and Kaviani, 2010).

Seed and bud dormancy During the developmental cycle of the plant, at some phase or the other certain structures like buds, tubers, seeds, etc., go through a period of temporary suspension of growth activity.  Such a state is called dormancy. Generally plants or plant structures, in order to overcome or survive against hostile environmental conditions undergo a period of dormancy with suitable modifications. 

Mechanism of Induction of Dormancy in Buds: The onset of short day or long dark photoperiods in winter stimulates the synthesis of various growth inhibiting compounds of which Abscissin dominates. Abscissin is a well known growth inhibiting hormone.  By inhibiting the synthesis of proteins, RNA and other metabolic processes, ABA imposes dormancy on meristematic tissues of the plant body.

Breaking Bud Dormancy: Cytokinins are known to be synthesized in root tips but under cold conditions because of the snow fall, the root meristems are very inactive and they don’t synthesize sufficient quantities of cytokinin required for the buds to be active.  That is probably one of the reasons why buds remain dormant.  As soon as cytokinins are provided to dormant buds, mitotic activity is initiated and buds start sprouting. 

Gibberellins Another class of phytohormones, which overcomes the bud dormancy, is Gibberellins.  Now it is certain that Gibberellin synthesis takes place in plastids.  Moreover, the synthesis of GA and ABA starts from the same precursor called mevolonate.  Under short day conditions, the pathway from mevolonate is directed towards ABA synthesis and GA synthesis is inhibited, but during long day photoperiods it is directed towards GA synthesis and ABA synthesis is blocked. 

That is the reason why gibberellins under long day conditions or not light treatment, break bud dormancy and nullify the effect of ABA present in such dormant buds. Model for the regulation of dormancy and germination by ABA and GA in response to the environment; According to this model ambient environmental factors (e.g. temperature) affect the ABA/GA balance and the sensitivity to these hormones. ABA synthesis and signalling (GAcatabolism) dominates the dormant state, whereas, GA synthesis and signalling (ABA catabolism) dominates the transition to germination.   

Somatic embryogensis The effect of PGR is an important factor impacting SE and plant regeneration. In most cases, successful plant SE needs a mixture of the different concentration ratios of auxin and cytokinin (CTK), both of which are necessary for plant culture in vitro.

Auxins In Angiosperm monocots, primary embryogenesis was exclusively induced by auxin-supplemented media, there is a large variation of growth regulators used to induce somatic embryogenesis in dicot species. From a list of 65 dicot species reviewed by Raemakers et al. (1995), somatic embryogenesis was induced in 17 species on hormone-free media, in 29 species on auxin-containing media and in 25 species on cytokinin-supplemented media.

Among auxins, the most frequently used was 2,4-D (49%) followed by naphthalene acetic acid (27%), indole-3-acetic acid (IAA) (6%), indole-3-butyric acid (6%), Picloram (5%) and Dicamba (5%). However, not all plants requires 2,4-D for SE. IAA is necessary to induce SE of Begonia cathayana Hemsl. Picloram would induce the SE of banana rather than 2,4-D. The somatic embryo of Gladiolus could be induced in media containing NAA but not in media containing other PGR.

Gibberellins Despite the wide range of physiological effects of gibberellins, their effect when added to culture media, primarily as gibberellic acid (GA3), has only been minimal (Krikorian, 1995). Exogenous application of GA3 has been reported to inhibit somatic embryogenesis and somatic embryo development in several species; but it was also reported that this substance is required for germination of the mature somatic embryos if chilling is not applied provided (Takeno et al., 1983 and references therein).

Gibberellins Gibberellic acid (GA) and Abscisic acid (ABA) are not necessary for the induction of SE. GA has been shown to promote SE for Apple, Pear, Garnetberry, and Cherry and also plays an inhibition role in Citrus SE.

Cytokinins The reasonable ratio of CTK and auxin is one of the major factors to induce horticultural plant SE. The high efficiency of SE in poinsettia depends on the ratio of CTK to auxin. The high ratio of auxin to CTK would promote the SE of American chestnut. Whereas a high ratio of CTK to auxin would promote apple SE. It is a requirement of two types of auxin and CTK to induce direct SE for Chrysanthemum morifolium , But walnut and peach require two types of CTK and IAA to induce SE.

Absassic acid ABA has promoting or inhibitory effects depending on the specific plant. Wei et al. had shown that ABA inhibited the SE of banana embryogenic suspension callus (ESC), and the extent of callus increased with increasing ABA concentration in the media. Different plants have specific requirements for ABA concentration. For example, increased concentrations of ABA (100 mmol·L–1) was required for high quality somatic embryos of cherry to induce formation.However, the SE of Papaya would form in medium containing only ABA.

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