Micropropagation Techniques
1. Micropropagation by Axillary and Apical Buds:
Axillary and apical shoots contain quiescent or active meristems depending on the physiological state of the plant. Vascular plants with an interminate mode of growth have in their leaf axils subsidiary meristem with potential for growing into a shoot. However, only limited numbers of axillary meristems have the capacity to develop in vivo if the type of branching of a particular species display apical dominance. Shoot tips cultured on basal medium containing to growth regulators typically develop into single seedling like shoot with strong apical dominance. On the contrary , when the shoots of the same explant material are grown on culture media containing cytokinins, axillary shoots of the same explant material are grown on culture media containing cytokinins , axillary shoots develop precociously which proliferate to form clusters of secondary and tertiary shoots. These cultures can be further subdivided into smaller clumps of shoots or separate shoots, which in turn , will form similar clusters when subcultured on fresh medium. about 5-10 multiplication rates can be achieved on a regular 4-8 week micro propagation cycle, which may ultimately lead to extremely impressive rapid clonal propagation level in the range of 0.1 -3.0 X 10-6 within one year.
In general, the technique of proliferation by axillary shoots is applicable to any plant that produces regular axillary shoots and responds to cytokinins such as BAP, 2 ip and Zeatin many forest and orchid tree species are good candidates for in vitro clonal propagating using axillary shoots.
Apical shoots (1-5mm) are normally cultured on media containing mixture of auxin ( 0.01-0.1mg/l) and cytokinins ( 0.05-0.5 mg/l). The level of cytokinins is raised subsequent subcultures to induce an acceptable rate or proliferation without yellowing distoration of shoots. If the presence of cytokinins in the median inhibits root development culture material is often transferred to a rooting medium in stage III which contains either no or reduced level of cytokinins.
2. Micro Propagation by Adventitious Shoots:
Adventitious shoots are stem and leaf structures that arise naturally on plant tissues located in sites other than at normal leaf axil regions. These structures include stem, bulbs, corms, tubers and rhizomes. Almost every one of these organs can be used as cutting in conventional clonal propagation. Similar type of adventitious shoot development can be induced in cultures by using a suitable explant from preconditioned plant material and appropriate levels of growth regulators in the medium. Bulbs and corms grow from meristem at the base of leaves and scales where they join the basal plants. These meristematic regions regenerate multiple shoots on a suitable culture medium.
Continuous propagation by adventitious shoot proliferation from bulb and corms can be achieved by cultivating two vertically split piece of shoot bases. Clusters of shoots develop from around the abaxial surface of developing leaves and scales. Senescence and dormancy in such cultured material can be prevented in vitro by trimming of shoots within 2-3 mm of the basal plate. This method is found useful in cultures of Iris, Lilum and Tulipa hybrids.
Clonal propagation by adventive embryo formation is another useful approach following for many important plant species. Adventive embryos can directly arise from a group of cells within the explants or from primary embryoids. Orchids produce a large number of embryos at the tip of leaves in vitro, while cultivar of citrus and mangifera develop polyembryos from the nucellar tissues. Adventitious embryos obtained in vitro by inducing embryogenesis on explants are good material for clonal propagation.
3. Micropropagation:
Differentiation of plants from cultured cells via shoot-root formation or somatic embryogenesis, where applicable, can be the fastest method of shoot multiplication and cloning of plant species. However, cultures in which calli are produced tend to be of low value as a means of micro propagation. The most serious drawback in the use of callus cultures for shoot multiplication is the genetic instability of their cells, due to which the initial plant regeneration capacity of the tissue may decline with the passage of time. In vitro propagation via organogenic or embryogenic or embryogenic calli is unfavourable in some economically important species of cereals, forage legumes, citrus, and coffee. Forest and tropical palm trees. Even plant regeneration from protoplasts also requires passage through at least one callus stage. Production of many thousands of plantlets from calli either derived from cell suspension or isolated protoplasts constitutes unique cases of cloning. Such as ‘calliclones’ and ‘protoclones’. Such clones commonly exhibit Somaclonal variations.