General Procedure of Microprogation
In vitro micro propagation is a complicated process requiring many steps or stages Murashige (1978) , proposed four distinct stages that can be adopted for overall production technology of clones commercially. Stages I-III are followed under in vitro conditions. Where as stage IV is accomplished in greenhouse condition. Debergh and Maene (1981) suggested an additional stage O for various micro propagation systems. Establishment of a reproducible system with well characterised with well characterized stages is a pre-requisite for promotion of projection targets and schedule in the commercial of plants.
1. Stage O:
This is initial step of micro propagation in which stock plants used for culture initiation are grown for at least 3 months under carefully monitored conditions. Stock plants are grown at a relatively low humidity and watered either with irrigation tubes or by capillary sand beds or mats. This stock plant preconditioning stage also includes measures to be adopted for reduction of surface and systemic microbial contaminants.
2. Stage 1:
Murashige defined this stage as the initiation and establishment of aseptic cultures. The main steps involved are preparation of the explant followed by the establishment on a suitable culture medium. Cultures are initiated from explants several organs but shoot tips and auxillary buds are most often used for commercial micro propagation. Procedures to surface sterilise the explant and induce a healthy growth in the culture medium defined for each species may be devised. It may also be advisable to control microbial contaminantion within explant tissues in case such efforts at stage O were not successful. Stage I lasts 3 months to 2 years and requires atleast four passages of the subculture.
Usually explants carrying a performed vegetative bud are suitable for enhanced auxillary branching. When objective is to produce virus free plants from an infected individual, it becomes obligatory to use cultures derived from submillimetre shoot tips. If stock plants are tested virus-free, the most suitable explants are nodal cuttings. These are some advantages in using small sized explants for micro propagation. Small shoot-tip explants have low survival rate and show slow initial growth. Meristem- tip cultures may also result in the loss of certain horticultural traits exhibited by the presence of virus. Therefore sub-terminal or slightly older segments are desirable which can withstand the toxic effects of sterilization agents much better than the terminal cuttings. For rhizomatic plants, runner tips are commonly used.
3. Step II:
This stages takes up the bulk of micro propagation activity using a defined culture medium that stimulates maximum proliferation of regenerated shoots. Various approaches followed for micro propagation include:
a) Multiplication through the growth and proliferations of meristems excised from apical and axillary shoots of the parent plant.
b) Induction and multiplication of adventitious meristems through a process of organogenesis or somatic embryogenesis directly on explants.
c) Multiplication of calli derived from organs, tissues, cell or protoplasts and their subsequent expression of either organogenesis or somatic embryogenesis in serial subculture. Shoots obtained from these calli can be further multiplied following procedures a) and b).
Passage or harvest cycle generally requires 4 weeks. Shoots are harvested from the multiplying culture to either be sold as a Stage II product or carried onto Stage III. Generally stage II lasts to 10-36 months with large number of subcultures of similar age.
4. Stage III:
Shoots proliferated during stage II are transferred to a rooting medium. Sometimes shoots are directly established in the soil as micro cuttings to develop roots. Since such a possibility depends on the particular species and at present, a large number of species cannot be handled in this manner. The shoots are generally rooted in vitro. When the shoots or plantlets are prepared for soil, it may be necessary to evaluate the survival factors such as i) Dividing the shoots and rooting individually ii) Hardening the shoots to increase their resistances to moisture stress and diseases. iii) Rendering the plants capablr of autotrophic development in contrast to the heterotropic state induced by culture and iv) Fulfilling requirement of breaking dormancy, especially of bulb ceops. Stage III requires 1-6 weeks.
5. Stage IV:
Steps taken to ensure successful transfer of the plantlets of Stage III from the aseptic environment of the laboratory to the environment of greenhouse comprise stage IV. Unrooted stage II shoots are also acclimatised in Suitable compost mixture or soil in pots under control conditions of light, temperature and humidity inside the greenhouse. In such cases stage III is skipped. Supplying bottom head-aids to pots with plantlets or cuttings and maintenance of a dense fine- particle fog system, within the greenhouse enhances the rooting process. Complete plants can also be established in the artificial growing media such as soilless mixes, rockwood plugs or even sponges. It takes 4-16 weeks for the finished product to be ready for sale or shipment.