In this article we will discuss about:- 1. Methods of Micro-Propagation 2. Stages of Micro-Propagation 3. Advantages 4. Commercial Uses.
Multiplication of genetically identical copies of a cultivar by asexual reproduction is called clonal propagation. The in vivo clonal propagation is often difficult, expensive and even unsuccessful. Tissue culture method offers an alternative way of clonal propagation which is popularly known as micro-propagation.
Here in this method a multiple number of miniatures of vegetative shoots are produced from a clone within a short time and space (Fig. 18.8). Use of tissue culture for micro-propagation was initiated by G. Morel (1960) who found this as the only commercially viable approach for orchid propagation.
The different techniques of single cell and protoplast culture also enable thousand plants to be derived within a short space and time. The products of these rapid vegetative propagation can also be regarded as clones when it is established that the cells they comprise are genetically identical.
Methods of Micro-Propagation:
(i) Multiplication by Meristematic Tissue of Axillary and Apical Shoots:
Axillary and apical shoots contain quiescent or active meristems depending on the physiological state of the plant. When these shoot tips are cultured on a basal medium containing no growth regulators, these typically develop into single seedling like shoots with strong apical dominance (Fig. 18.9A-B).
On the contrary, when the shoots of the same explant material are grown on culture media containing cytokinin, axillary shoots develop precociously which proliferate to form clusters of secondary and tertiary shoots. These clusters when subdivided and transferred onto fresh medium again these will form similar clusters.
This subdivision process may be continued indefinitely when provided with basic nutrients. About 5-10 multiplication rates on 4-8 weeks of micro-propagation cycle may ultimately lead to extremely impressive clonal propagation range of 0.1-3.0 x 106 within a year.
(ii) Multiplication by Adventitious Shoots:
Axillary buds are “preformed meristems” present at the leaf axils, whereas adventitious buds may arise from any plant structures and this regeneration is often dependent on the presence of organised plant tissue. The explants may be stems, internodes, leaf blades, cotyledons, root elongation zone, bulb, corms, tubers, rhizomes, etc.
If these explants are induced by using appropriate level of growth regulators in the medium they will form the meristematic zone which regenerates multiple shoots on a suitable culture medium. High number of adventitious shoots may arise only from a single epidermal cell.
Continuous propagation by adventitious shoot proliferation from bulbs and corms can be achieved by cultivating two vertically split piece of shoot bases. Clusters of shoots may develop from the abaxial surfaces of developing leave and scales. Trimming of the shoot apices is a good approach which has been found to ensure continuously productive cultures of different hybrid plants for indefinite period.
(iii) Multiplication by Adventitious Embryo Formation:
It is another useful approach followed for many important plant species. Adventitious embryos can arise directly from a group of cells within the original explants or from primary embryoids.
There are many plant species which develop embryos in vivo from diverse kind of explants e.g. the orchid leaf tips produce large number of embryoids whereas Citrus and Mangifera have shown to develop polyembryos from nucellar tissue.
These adventitious embryoids are diploid in nature and can be used as clonal material for micro-propagation. In in vitro similarly the adventitious embryos originated from different explants can be good materials for clonal propagation.
(iv) Multiplication through Callus Culture:
Direct plantlet formation from the explants in culture is more desirable in case of micro- propagation or clonal propagation. But shoot formation may occur through organogenesis or embryogenesis from the callus produced from the explant.
The limitation of this system is that the callus cells are not genetically stable so it cannot be called as a single clone and this process is more time consuming. Also the plant regeneration capacity may decline due to the genetic unstable condition.
In case of some plant species genetically stable calli have also been derived, in these types of calli slow growing meristematic zones are formed from the peripheral layer. The meristematic layers invariably comprise of diploid cells expressing totipotency. These types of calli can be subdivided into smaller parts and which may produce multiple shoots from each.
Stages of Micro-Propagation:
The process of micro-propagation or clonal propagation is a complicated process which can be subdivided into four prominent stages of operation:
Stage 0:
This is the initial step of micro-propagation in which stock plants has to be j grown under controlled condition before using for culture initiation.
Stage I:
The preparation of explants from stock plants is followed by its establishment j in a suitable culture medium.
The steps involved in this stage are:
(a) Explant isolation,
(b) Surface sterilisation,
(c) Washing,
(d) Establishment of explant on appropriate culture medium.
Stage II:
This stage involves the multiplication of shoots or rapid somatic embryo formation using a defined culture medium.
Various Approaches followed for Micro-Propagation Include:
(i) Multiplication through the growth and proliferations of meristems excised from apical and axillary shoot of the parent plant.
(ii) Induction and multiplication of adventitious meristems through processes of organogenesis or somatic embryogenesis directly on explants.
(iii) Multiplication of calli derived from any kind of explant and subsequently shoots development either through organogenesis or embryogenesis. The harvest cycle generally takes 4-8 weeks. Either the shoots are marketed directly or carried over to the next stage for further development.
Stage III:
Shoots obtained from stage II are transferred to the next rooting or storage medium. These shoots are directly established in soil as micro-cuttings to develop roots. The shoot handling at this stage differs from species to species.
When the shoots are directly transferred to soil then the following procedures are to be maintained:
(i) Each shoot should be rooted individually,
(ii) Hardening of the shoots to increase their resistance,
(iii) Allowing the plants capable of autotrophic development rather than the heterotrophic development in culture,
(iv) Fulfilling the requirements for breaking dormancy.
Stage IV:
Transfer of plantlets to sterilized soil for hardening under greenhouse environment is achieved. This step is to ensure the successful transfer of the plantlets of stage III or un-rooted shoot apices of stage II into the suitable compost mixture or soil in pots under controlled condition of light, temperature, humidity.
For marketing sometimes these plat lets are established in an artificial growing medium such as soil-less mixes, Rockwood plugs or the sponges. It takes 4-16 weeks for marketing of the finished products.
Advantages of Micro-Propagation:
1. The technique of micro-propagation is an alternative approach to conventional methods of vegetative propagation, which has the enhanced rate of multiplication.
2. A million of shoot tips can be obtained from a small, microscopic piece of plant tissue within a short period of time and space.
3. The advantage in this type of propagation is that as shoot multiplication usually has a short cycle (2-6 weeks) and each cycle results in logarithmic increase in the number of shoots.
4. This method of propagation is more advantageous in case of bulb or corm producing plants, because mini-tubers or mini-corms for plant multiplication are available throughout the year irrespective of season.
5. Smaller size of propagules are advantageous for storing and transporting as it takes lesser space.
6. The propagules can be maintained in soil-free environment which facilitates their storage on a large scale.
7. Stocks of germplasm can be maintained for many years using this method of propagation.
8. This method is more applicable where disease free propagules are wanted. This in vitro technique helps to raise pathogen free plant and to maintain them.
9. Micro-propagation is very useful in case of dioecious plants, because there the seed progeny yield is 50% male and 50% female, but this technique helps to get the progeny according to the desired sex.
10. A major advantage of micro-propagation happens to be the minimum growing space required in commercial nurseries. Thousands to millions of plantlets can be maintained within the culture vials. This is specially useful for maintaining the horticultural species.
11. This method is more helpful in case of slow growing plants where the seeds are produced after a long term and the seeds are the only propagule. This method can overcome the difficulty to obtain the propagules.
12. Through seed production genetically uniform progeny is not possible always. But the micro-propagation method will help to maintain the genetic uniformity in the propagules.
13. Micro-propagation is one of the finest ways of plant multiplication by in vitro technique of plant tissue culture. The newer tissue material obtained through r DNA technology or haploid culture or somatic hybridization can be the source of tissue material for micro-propagation, as it is the easiest method for obtaining the multiple propagules.
Commercial Uses of Micro-Propagation:
Micro-propagation of orchids demonstrated profit all over the world, besides orchid about 600 species of other ornamental plants have been successfully cloned and some of them are commercially exploited (Chrysanthemum, Carnation, Gerbera and Anthurium), list of names is increasing day by day, the advantage is rapid cloning of selected colour individuals.
Except the horticultural flowering plants, a large number of forest trees, fruit trees, oil producing plants (Eucalyptus, Oil-palm), vegetables and many edible crops like peach, apple, pear, cherry, apricot and palm have been cloned and put into use commercially (Fig. 18.10).
Breeding and selection by sexual hybridization is a very slow procedure because of long generation time and they are difficult to propagate vegetatively. So the in vitro cloning of more than 100 woody species over a wide range of families has been successfully achieved.
Forest trees are important sources of our biodiversity and also they provide food, fuel, construction and industrial products for us.
These resources are diminishing at an unprecedented rate. Large scale, cost-effective and nursery-friendly in vitro technique like micro-propagation protocols have been developed for selective forest trees; about 70 forest tree species of Angiosperm and about 30 forest tree species of Gymnosperm.
The most important aspect of commercial micro-propagation is the economics involved and the unit cost of a plantlet. Research achievements in the field of micro-propagation are not always economically viable and not accepted by commercial houses. The investment in commercial tissue culture business will depend to a large extent on cost of the laboratory set up, type of plant to be propagated and the skill involved.
The commercial nursery man should start with those crop species for which published methods are available and also it is essential that the grower must have some training in tissue culture and plant husbandry. Another approach followed in micro- propagation is to automate it at its various stages.
In this connection the bioreactors are being used for large scale multiplication of somatic embryos, shoots and bulbs. Automatisation have also been used for subculture of shoots during stages III and IV of micro-propagation. This reduces the cost of labour component in micro-propagation.