The most important challenge in a developing country is food for its growing population. The arable land area is fixed. Some means has to be there to increase the productivity.
Plant tissue culture technique offers an excellent opportunity for mass propagation of plants in laboratory test tubes, which are transferred to the field. Besides crop plants, the technique is also applied to regenerate saplings for plantation and regeneration of dwindled forests. Some rare and nearly extinct plant species can be rescued and propagated by this technique.
Embryos produced by incompatible crosses also are rescued, seed dormancy is overcome, life cycle is shortened and much more.
This technique is amalgamated with genetic engineering to regenerate plants with novel characters and combine two or more beneficial characters into a single plant. Things, once seemed impossible, have been made possible.
In this section, we shall discuss about four elementary applications of this technique, namely, (1) micro-propagation; (2) organogenesis; (3) somatic embryogenesis and (4) protoplast culture and fusion.
Basically, micro propagation is similar to rooting of plant cuttings and is, in a way, another method of vegetative propagation of plants. However, it differs from the conventional procedure in that it is carried out in an aseptic condition and requires a unique recipe i.e. an artificial nutrient medium.
It is used for forestry improvement and is an example of direct laboratory to land transfer of biotechnological benefits A small plant cutting or explant (usually an axillary bud) is surface sterilized and inoculated into a culture vessel containing a semi-solid nutrient medium. The inoculated culture vessel is incubated at room temperature.
In a day or two, a large number of shoots develop from the axillary bud in a process known as axillary bud proliferation. Each growing point is sub-cultured to give rise to shoot. This phenomenon is known as adventitious shoot formation. Each shoot is stimulated by an auxin to develop roots. The new plantlet is transferred to the field.
Organogenesis, in essence, refers to differentiation of organs, such as shoot and root from an undifferentiated mass of cells. The cells of an explant are highly differentiated. When an explant is placed in an artificially enriched nutrient medium, its differentiated cells first de-differentiate and form a mass of unorganized cells known as callus.
The cells of the callus then re-differentiate and produced the desired tissue and then an organ or organs under the influence of specific growth regulators (hormones). Single cells also can be cultured and made to develop shoot and root one followed by the other. Plant growth regulators (hormones) play an important role in this regard.
There are two important groups of plant hormones: cytokinins and auxins. Cytokinins such as kinetin and adenine promote shoot differentiation, while auxins, such as indole acetic acid (IAA) and naphthalene acetic acid (NAA) promote root differentiation.
It has been established that shoo root differentiation depends upon the ratio or quantitative interaction betwee cytokinins and auxins. For example, two molecules of kinetin or 15,000 molecule of adenine are required to neutralize one molecule of IAA. Thus, shoot /10 differentiations is a function of the quantitative interaction between cytokinin an auxin.
This principle is applied to plant cells or tissues cultured in vitro. Kinetin and IA are added to the in vitro culture in required amounts, one following the others- promote shoot and root differentiation.
In flowering plants, an embryo is the product of the zygote and the zygote is the product of the fusion of two gametes. The embryo undergoes a preprogrammed development and forms a plantlet. In a nutshell, under a normal circumstance, a embryo is the result of sexual reproduction.
Such embryos are known as zygoti embryos. However, plant tissue culture technique offers a method of producing; embryos from somatic cells bypassing sexual reproduction such embryos a known as somatic embryos. The process of formation of somatic embryos is known as somatic embryogenesis.
Somatic embryo formation starts with a mass of single cells or a tissue grown on a semirsolid nutrient medium. A cell repeatedly divides and forms a cell aggregate. The cell aggregate passes through different stages, such as globular, heart shaped and torpedo shaped stages.
The torpedo shaped stage is the mature stage. The culture is initially started on a semi-solid medium and the callus so formed is transferred to a liquid medium in an agitated and aerated bioreactor.
The cells breaking off from the callus develop into somatic embryos. Mature stages (torpedo shaped stages) are sorted out and grown to maturity on a semi-solid medium.
Dormancy is induced before these are processed for transfer to the field. There are four methods, by which the somatic embryos are transferred to the field.
1. These are germinated in the laboratory, transplanted in pots and then transferred to the field.
2. The dormant embryos are encapsulated in a gel containing an adequate nutrient for the embryo. These encapsulated embryos are known as artificial or synthetic seeds such seeds can be planted in the field.
3. This method involves the germination of the embryos under a controlled condition and then mixing of the seedlings is mixed with a gel like medium. The seedling- gel mix is sown in the field.
4. The embryos are germinated and then fluid drilled.
The major advantages of this process are: (1) rescue of zygotic embryos formed by: incompatible crosses and (2) overcoming seed sterility and dormancy.
Protoplast culture and fusion
Protoplasts are plant cells, whose cell wall is digested. The cell is bound by a plasma membrane. For the isolation and culture of protoplasts, see section isolated protoplasts from two different species of plants have been successfully fused to produce a single protoplast containing the genetic material and the cytoplasm of both the fusing protoplasts.
The process of fusion is not straightforward It is facilitated by some agents, known as fusogens. Fusogens are of two types: chemical and electrical. Poly ethylene glycol (PEG) is a chemical fusogen.
It is not used as a universal fusogen, since it is toxic to protoplasts of some plants; alternately, pulses of electric current (direct current) are applied to the fusing protoplasts. This method is known as electro-fusion. The depicts the sequential fusion of two protoplasts, resulting in a synkaryon.
There are three steps in this process. First, two protoplasts come in close proximity. Then the plasma membranes of the two fuse and then the two nuclei lie in the mingled cytoplasm. This stage is known as a heterokaryon. In the third stage, the two nuclei fuse forming a synkaryon.
The cell wall of the fused protoplast is regenerated and the cell is cultured in an artificially enriched nutrient medium. The following sequence of events is same as that in the callus culture. This process is also known as somatic hybridization and the products as somatic hybrids.
This method bypasses the fusion of gametes of two unrelated plant species. P. S. Carlson, H. H. Smith and R. D. Dearing (1972) obtained the first somatic hybrid by fusing the isolated protoplasts of Nicotiana glauca with N. langsdorfii. However, sometimes, due to cellular incompatibility, two nuclei can not co-exist. Consequently, one nucleus is eliminated and a protoplast containing the nucleus of one species and the cytoplasm of both results.
The ensuing hybrid is known as a cytoplasmic hybrid or cybrid. Somatic hybridization is attempted in plant species, which are sexually incompatible. The well known example of a somatic hybrid is ‘pomato’ obtained from the protoplasts of potato and tomato. However, this hybrid is of little commercial value.