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The below mentioned article provides an overview on the organogenesis in plant tissue culture.
Introduction:
In culture, the explant develops into callus tissue in a medium containing either a particular concentration of auxin or a definite auxincytokinin ratio.
Such medium is known as callus inducing or initiation medium. Proliferation of callus mass in a relatively unorganised way will continue for a prolonged period, if the callus tissue is maintained in the same medium through a number of subcultures. But the main objective in plant tissue culture is to regenerate a plant or plant organ from the callus culture.
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The regeneration of plant or plant organ only takes place by the expression of cellular totipotency of the callus tissue. The callus tissue during its growth in callus inducing medium shows an extremely limited expression of totipotency, but in a certain number of plant species, this potentiality can be enhanced and extended by the adjustment of nutritional and hormonal conditions in culture.
Scattered areas of actively dividing cells, known as meristematic centres, develop as a result of differentiation and their further activity results in the production of root and shoot primordia. Skoog and his co-workers at Wisconsin, in their studies with tobacco stem pith culture, demonstrated that the initiation and the type of organ primordia formed from the resulting callus culture could be controlled by appropriate adjustment of the relative levels of the auxins and cytokinins.
With high auxin—low cytokinin roots develop, with low auxin—high cytokinin shoot buds develop; at intermediate levels undifferentiated callus tissue develops (Skoog and Miller, 1957). The expanded expression of totipotency of the callus tissue offers considerable potential for tissue culture technique as it is possible to grow the root or shoot or both. The production of adventitious roots and shoots from cells of tissue culture is called organogenesis.
Brief Past History:
F. Skoog (1944):
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The first indication that in vitro organogenesis could be chemically regulated to some extent was given by F. Skoog. He found that the addition of auxin to the culture medium served to stimulate root formation, whereas shoot initiation was inhibited. The latter effect on shoot production could be partially reversed by increasing the concentration of both sucrose and inorganic phosphate.
F. Skoog and C. Tsui (1948):
They found that adenine sulphate was active in promoting shoot initiation and this chemical reversed inhibitory effect of auxin.
F. Skoog and C. O, Miller (1957):
The studies of Skoog and his colleague led to the hypothesis that organogenesis is controlled by a balance between Cytokinin and auxin. A relatively high auxin—Cytokinin ratio induced root formation in tobacco callus whereas a low ratio of the same hormones favoured shoot production.
J. G. Torrey (1966):
He advanced the hypothesis that organogenesis in the callus tissue starts with the formation of clusters of meristematic cells (meristemoids).
K. Tran Thanh Van, H. Chlyah and H. Trinh (1978):
The precise regulation of organ formation such as floral buds, vegetative buds and roots has been demonstrated in thin cell layer explants (epidermal and sub epidermal explants) of several species by regulating auxin— cytokinin ratio, carbohydrate supply and environmental conditions.
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T. A. Thorpe (1980):
He advanced the hypothesis that the endogenous auxincytokinin balance is the key factor in the initiation of organogenesis.
N. Everett (1982):
Endogenous ethylene was identified as a factor in the induction of shoot buds from cultured tobacco cotyledons.
What is Organogenesis?
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Organogenesis means the development of adventitious organs or primordia from undifferentiated cell mass in tissue culture by the process of differentiation.
What is Caulogenesis?
Caulogenesis is a type of organogenesis by which only adventitious shoot bud initiation takes place in the callus tissue.
What is Rhizogenesis?
Rhizogenesis is a type of organogenesis by which only adventitious root formation takes place in the callus tissue.
What is Organoids?
In some cultured tissues, an error occurs in the development programming for organogenesis and an anomalous structure is formed. Such anomalous organ like structures is known as organoids. Although organoids contain the dermal, vascular and ground tissues present in plant organs, they differ from true organ in that the organoids are formed directly from the periphery of the callus tissue and not from organized meristemoids.
What is Meristemoids?
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Meristemoid is a localised group of meristematic cells that arise in the callus tissue and may give rise to shoots and/or roots.
General Account of Organogenesis:
In vitro organogenesis in the callus tissue derived from a small piece of plant tissue, isolated cells, isolated protoplasts, microspores etc. can be induced by transferring them to a suitable medium or a sequence of media that promote proliferation of shoot or root or both. The suitable medium is standardized by trial and error method.
The callus may remain in undifferentiated condition regardless of the hormones and nutrients to which they are exposed. Organ neo-formation generally follows cessation of unlimited proliferation. Individual cells or groups of cells of smaller dimensions may form small nests of tissue scattered throughout the callus tissue, the so-called meristemoids which become transformed into cyclic nodules from which shoot bud or root primordia may differentiate.
In most calli, initiation of shoot buds may precede rhizogenesis or vice-versa or the induced shoot bud may grow as rootless shoots. Shoot bud formation may decrease with age and subculture of the callus tissue, but the capacity of rooting may persist for longer period. In some calli, rooting occurs more often than other form of organogenesis.
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During organogenesis, if the roots are first formed, then it is very difficult to induce shoot bud formation from the same callus tissue. But if the shoots are first formed, it may form roots later on or may remain as rootless condition unless and until the shoots are transferred to another media or hormone less medium or conditions that induce root formation.
In certain cases, shoot and root formation may occur simultaneously. But the organic connection between two different organ primordia may or may not be established. Therefore, organic connection between shoot and root primordia is essential for the regeneration of complete plantlet from the same culture. Shoot formation followed by rooting is the general characteristic of organogenesis. The callus tissue may remain unchanged in colour during rhizogenesis or may develop yellow pigmentation. During shoot bud formation, the callus tissue generally develops green or pale green pigmentation.
The callus tissue in many cases shows a high potential for organogenesis when first initiated but gradually a decline sets in as subculture proceeds with eventual loss of organogenic response. The loss of potential for organogenesis may be due to either a genetic or a physiological change induced by either prolonged cultural conditions or the composition of culture media.
The genetic effects in a callus tissue are reflected in changes of chromosome structure or number such as aneuploidy, polyploidy, cryptic chromosomal rearrangements etc. Such chromosomal changes may lead to loss of totipotency of the cells. During prolonged culture, totipotent cells of the callus tissue are gradually replaced by non-totipotent cells. It is generally observed that shoot bud formation takes place from the diploid cells of the callus tissue.
At the early stage of culture, the callus tissue exhibits maximum number of diploid cells. Eventually at the later stage of culture, the cells of callus tissue become mixaploid due to alteration of chromosome number and organogenesis could not be induced in such callus tissue, Occasionally, rooting occurs but shoot bud does not develop.
But in some cases plant- lets could also be regenerated from old sub-cultured callus tissue and the potential for organogenesis or embryogenesis could be enhanced in the later part of culture. Again, an alteration in karyotype need not always result in organogenetic incapability as, for example, regeneration of extreme aneuploid plants from 20 years old tobacco tumour tissues has been observed.
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Therefore, it cannot be a generalized the concept that chromosomal changes are the main cause of organogenetic incapability of the callus tissue during prolonged culture. So an alternative physiological hypothesis has been put forward to explain the loss of organogenetic potential of the callus tissue during prolonged culture.
According to this hypothesis, subculture often leads to a loss of many endogenous factors or morphogens present at the critical stages of growth. Such factors present in the callus tissue at the initial stage may not be synthesized at all or synthesized only in insufficient quantity at later stages. As a result, callus tissue fails to exhibit the potential for organogenesis or embryogenesis.
However, if these factors are supplemented to the medium during subculture, then restoration of organogenetic potential should be regained. It has been reported that addition of kinetin could restore decline in regenerative response in long termed carrot culture, whereas, at the initial stages, no promotive effect of kinetin was observed. But the addition of kinetin or any other additives are not always conducive for the regeneration of plant in other plant species. Therefore, it is plausible that both genetic as well as physiological processes are involved in the decline and loss of organogenetic response during prolonged subculture.
The effect of chemical factors or organogenesis, especially those of phytohormones, has been studied in explant from a large number of species. The concept, as propounded by Skoog and Miller (1957), that induction of organogenesis would require, above all, the addition to culture medium of an appropriate balance of known phytohormones such as auxin and cytokinin has not proved to be so in many experimental materials.
In a few cultured tissue, the endogenous regulator complex can be adjusted to the required balance of phytohormones by an exogenous supply of auxin, cytokinin or gibberellin either separately or in combination. Generally high concentration of cytokinin brings about shoot bud initiation, whereas high levels of auxin favours rooting. Therefore, to obtain organogenesis, different permutation and combination of hormones as well as various concentrations of hormones are supplemented in the culture medium.
Certain phenolic compounds also induce shoot initiation in tobacco callus. Phenolic compounds actually inactivate the auxins and consequently rise in the physiologically effective level of cytokinins which untimately bring about the initiation of shoot buds. The use of auxin inhibitor or auxin antagonist via culture medium also causes the induction of shoot bud.
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Additions of adenine in the culture medium also induce shoot bud in the callus tissue. Shoot bud initiation takes place in haploid tobacco cultures in presence of chelating agent like 1, 3 diamino-2- hydoxypropane-N.N.N’.N’ tetra-acetic acid. Addition of abscisic acid in place of cytokinin also induces shoot bud formation in root tuber tissue of sweet potato and stem tuber tissue of potato.
Though the role of hormones and their quantitative interactions has been recognised, it is only recently that some efforts are initiated to gain some insight into the biochemistry of organ differentiation by hormonal interaction. It has tended to rather empirical. During last few years, some indirect studies have been made on organ forming tissues by estimating the level of structural and enzymatic proteins and the changes of isoenzyme pattern through gel electrophoresis during organogenesis.
Of different enzyme systems studied in plants, peroxidase is widely distributed among higher plants and has been investigated in relation to many different activities. One of the most important functions of peroxidase is involvement in the metabolism of auxin. Plant tissue cultures also require hormones like auxin and cytokinin for growth and differentiation in vitro.
Hence the study of peroxidase level by estimating the activity and the changes of isoperoxidase patterns during organogenesis is very important. Increases in peroxidase activity in callus tissue have been demonstrated before the differentiation of both shoot as well as root. Distinctive changes in the isoperoxidase patterns have also been demonstrated during organ differentiation in cultured tissue.
Differences in isoperoxidase patterns associated with shoot and root differentiation have been elegantly demonstrated. Since cathodic isoperoxidases are considered to be involved in auxin catabolism and the last moving anodic bands have been associated with lignification, the changes in band patterns have been interpreted as creating situations .conducive to shoot or root formation.
It is also evident that certain isoperoxidase appeared several days prior to the actual emergence of root and shoot primodia from the tobacco callus. Later, these specific peroxidases were detected in the regenerated root and shoot respectively. Such isoperoxidases provide useful biochemical signals for morphogenetic events that follow.
The activities of some enzymes of the carbohydrate metabolism during organogenesis have been looked into. Starch accumulation, which has been known to be conspicuous feature in diverse morphogenetic processes in vitro, is also shown to occur prior to shoot differentiation from tobacco callus grown in vitro.
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Starch accumulation reflects high energy requirement for the organogenetic processes as strong correlation has been found between the starch content of the callus, its rate of respiration and shoot formation. Gibberellic acid, which represses starch accumulation by mobilising high amylase synthesis/activity, also inhibits shoot formation.
Comparison of malic dehydrogenase activity under root and shoot forming conditions revealed that this was more pronounced activity prior to shoot and root differentiation. Developmental patterns of the key Embden- Meyerhof-Parnas (EMP) and Pentose Phosphate (PP) Pathway enzymes namely phosphoglucose isomerase, aldolase, pyruvate kinase, glucose-6- phosphate dehydrogenase, 6-phosphogluconate dehydrogenase etc. were investigated in shoot forming and non-shoot forming sugarcane callus.
As compared with non-shoot forming callus, the shoot forming callus was characterised by higher activity level of these enzymes. Higher activity levels of the EMP and the PP pathway enzymes in the shoot forming sugarcane tissue are indicative of generation of energy molecules, reducing power and pentose sugars vital for energy- dependent reaction and the synthesis of nucleic acids during shoot differentiation.
Since differentiation took place by the synthesis of nucleic acids and proteins, many attempts have been made to correlate the two phenomena. It has been observed that shoot initiation in Cichorium intybus was associated with alterations in the pattern of RNA synthesis and nucleotides (Vasseur 1972). An increase in the ratio of RNA/DNA and histone/DNA was related to organogenesis in tobacco and to embryogenesis in carrot with DNA synthesis.
Protocol for Organogenesis in Tobacco Callus:
This is an experiment in which mature tobacco stem is initiated to give rise to callus tissue. Under the appropriate hormonal conditions callus is induced to form either root or shoot primordia.
The protocol is given below:
1. The upper part of the stem of 3-4ft tall tobacco plants are harvested and cut into 2 cm long internode segments.
2. Surface sterilization of the tissue is done by immersing the stem pieces in 70% v/v ethanol for 30 seconds, followed by a 15 minutes incubation in sodium hypochlorite (1.0% available chlorine). Then the tissue is washed in several changes of sterile distilled water.
3. The stem explants are taken in a sterilized petri dish and cut longitudinally into two equal pieces and inoculated onto Murashige and Skoog’s (1962) solid medium (MS) supplemented with 2mg/L indole acetic acid (IAA) and 0.2 mg/L kinetin. The cultures are then incubated at 25°C with an illumination of about 2,000 lux (16 hrs. photo period)
4. Callus tissue which is white/yellow in colour, begins to form in two weeks and after six weeks it should be sub cultured to fresh medium.
5. Organogenesis in callus culture can be stimulated by transferring tobacco callus onto MS medium with different auxin/cytokinin ratios. Shoot primordia develop within 3 weeks of transfer of callus to MS medium with IAA at 0.02 mg/L and kinetin at 1 mg/L (a high cytokinin/low auxin ratio). Root formation occurs within 2-3 weeks of transfer of callus to MS medium supplemented with 0.2 mg/L IAA and 0.02 mg/L kinetin (a high auxin/low cytokinin).
6. After 6 weeks, rootless shoots can be excised and placed onto the root inducing medium i.e. MS medium with 0.2 mg/L IAA and 0.02 mg/L.
7. It is possible to transplant the tobacco plantlets to soil. It should be noted that aseptic procedures are not required for the transplantation of plantlets. The plantlets are removed from the culture vessels and care should be taken not to damage root or shoot system. The plantlets are carefully washed with tap water to remove the residual agar medium.
Individual plantlets are separated out and transplanted into pot (75 mm) containing seedling compost. The soil is watered. The pot is covered with a small inverted polythene bag. This will reduce the amount of water lost by the plantlets due to transpiration.
After 7 days, several small holes are made in the polythene bag and gradually enlarged during next 2-3 weeks. At this stage, the tobacco plantlets should be sufficiently “hardened off” to allow the complete removal of plastic bag. They can be grown to maturity in a green house.