Micropropagation

Plants are conventially propagated either by sexual (through generation of seeds) or by asexual (by multiplication of vegetative parts) means. Emergence of modern methods of plant tissue culture has provided a very useful alternative which allows rapid propagation of desired plant species in a limited space under strict control of growth conditions. These plants are microbiologically sterile and thus exempt from quarantine regulations that may apply to export/import of normal plants. In vitro production of plants is generally described as micropropagation. Micropropagation (micro = small area of glass vessels; propagation = to increase the number of propagules) can be defined as asexual multiplication of plants in a small area of glass vessel (in vitro) under controlled physio-chemical conditions.


Micropropagation can be achieved by any one of the following pathways:

1. Stimulated axillary bud proliferation.

2. De novo adventitious shoot bud differentiation.

3. Somatic embryogenesis and

4. Callus organogenesis.


Every method has its own advantages and disadvantages and the choice of a particular pathway will depend upon them.


1. Stimulated shoot bud proliferation:- This method of in vitro propagation utilizes the clue from the basic physiological phenomenon in plants called as "apical dominance". Vascular plants with intermediate mode of growth have in their leaf axils subsidiary meristem with the potential for the growing into shoot. Apical dominance is the phenomenon by which the presence of apical bud causes a complete or partial inhibition of pre-existing lateral buds. In this method of micropropagation nodal segments having pre-existing but inhibited axillary shoot buds are used to stimulate proliferation of shoots in the presence of cytokinins. Nodal segments can be taken from both sexually mature or seedling (cotyledonary nodes) materials. In this method of micropropagation, the shoot multiplication may be initially slower than the other methods but with each passage the number of shoot increases logarithmically. This method is currently the most popular approach for clonal propagation of crop plants because the cells of the shoot apex are uniformly diploid and are least susceptible to genotypic changes under culture conditions.


2. De novo adventitious shoot bud proliferation:- Adventitious shoot buds are those which develop on any part of the plant such buds are not pre-existing and differentiate de novo. Bryophylium is the best example in nature where adventitious shoots buds differentiate on the margin of fleshy leaves. Such shoot buds develop roots and serve as propagules. Adventitious shoot buds can also be induced in vitro on a variety of explants such as hypocotyl segments, cotyledons, leaf, stem segments etc. Differentiated shoot buds on their subsequent subculture to fresh medium can be multiplied to obtain a regular crop of shoots.


3. Somatic embryogenesis:- Development of an embryo in nature is the consequence of fertilization. It is enclosed in cotyledons and the seed coat. Embryo is unique in the sense that it is bipolar in nature. On germination, it develops into shoot and root simultaneously. Such embryos which develop from somatic cells are called as somatic embryos. Somatic embryos can develop in two different ways:

(a). Direct somatic embryogenesis
(b). Indirect somatic embryogenesis

Direct somatic embryogeny can be induced on any explant such as hypocotyls, cotyledons, leaf, stem segments etc. Somatic embryo development begins with stimulation of few cells on the explant which from a globular structure subsequently developing into heart shaped, torpedo and cotyledonary stage embryos.The somatic embryos are borne naked and can easily be distinguished from the adventitious shoot buds. Somatic embryos are discrete units and lack vascular connection with the mother explant while adventitious shoot buds have continuity of vascularization with the mother explants.
In case of indirect somatic embryogeny, the embryo develops adventitiously with an intervening callus phase. Callus can be induced from a variety of explants and subsequently the embryos can be induced on it.




4. Callus organogenesis:- Callus is an unorganized mass of cells which can be induced on any explant grown on a suitable medium. Callus formation is basically a result of dedifferentiation (an explant which is differentiated tissue gets converted into dedifferentiated mass of cells). Callus can be multiplied in culture for indefinite period even in absence of light in culture room environment. Callus can again be induced to develop shoots by the phenomenon called redifferentiation (callus organogenesis). By manipulating plant growth regulator in the medium, both dedifferentiation (auxin rich medium) and redifferentiation (cytokinin rich medium) can be easily controlled. Durig callus formation, the cell to cell contact is lost and the cells seem to be in the state of "chaos".This result into somaclonal variation on account of genetic changes (ex. change in ploidy level) that occur in culture.