Micropropagation technique

micropropagation, a method in plant tissue culture.

1. Allie N U, MSc Biotechnology, MICROPROPAGATION

2. Introduction Micropropagation is a practice of rapidly multiplying stock plant material to produce a large number of progeny plants using modern PTC methods. Used to multiply novel plants, that are genetically modified or bred through conventional plant breeding methods. Also used to provide no. of plantlets from a stock plant that does not produce seeds, or does not respond well to vegetative propagation.

3. Clonal propagation Process of asexual reproduction by multiplication of genetically identical copies of individual plants. Clone refers to plant population derived from a single plant by asexual reproduction. Asexual reproduction through multiplication of vegetative parts is the method for invivo propagation of certain plants (as no viable seed is produced). Successfully applied for propagation of apple, potato, tuberous and several ornamental plants.

4. Invitro clonal propagation Invivo clonal propagation of plants is expensive and frequently unsuccessfull Invitro clonal propagation through tissue culture – micropropagation . Use of tissue culture technique for micropropagation - Morel(1960) for orchids. Used for rapid multiplication of plants.

5. Technique It’s a complicated process. Involves 3 stages (I,II and III). Sometimes two more stages are added (0 and IV). Stage 0:- Initial step in micropropagation . Involves the selection and growth of stock plants for about 3 months under controlled conditions. Stage I:- Initiation and establishment of culture in suitable medium. Commonly used explants are organs, shoot tips and axillary buds. Also explant is surface sterilized and washed before use.

6. Stage II:- Activity of micropropagation occurs in defined culture medium. Involves multiplication of shoots or rapid embryo formation from the explant . Stage III:- Involves the transfer of shoots to a medium for rapid development. Sometimes, shoots are directly planted in soil to develop roots. Invitro shooting is preferred while handling large number of species.

7. Stage IV:- Involves establishment of plantlets in soil. Done by transferring plantlets of stage III from lab to environment of green house. In some plants stage III is skipped and stage II shoots are planted in pots or in suitable compost mixture. These stages are useful for comparison between two or more plant systems, besides better understanding.

8. Approaches Two approaches: Multiplication by axillary bud/apical shoots. Multiplication by adventitious shoots. Besides these two approaches, plant regeneration processes namely ‘organogenesis’ and ‘somatic embryogenesis’ may also be used.

9. Multiplication by axillary bud and apical shoots Actively dividing meristems are present at axillary and apical shoots. Axillary buds located in axile of leaves are capable of developing into shoots. In the invitro state only limited no.of axillary meristem can form shoots. Possible to develop plants from meristem and shoot tip cultures , bud cultures

10. Meristem and shoot tip cultures Apical meristem - dome of tissue located at extreme tip of a shoot. Apical meristem along with young leaf primordia constitute the shoot apex. For disease free plants, meristem tips are cultured. Procedure :- Meristem or shoot tip is isolated from a stem by a V shaped cut. Size (0.2 to 0.5mm) of tip is critical for culture. For good results, explants should be taken from actively growing shoot tips, ideal timing is at end of plant dormancy period. Widely used media are MS media and White’s medium.

11. Stages of growth Stage I:- Culture of meristem is established. Addition of growth regulators, cytokinins (kinetin, BAP) and auxins (NAA, IBA) support growth and development. Stage II:- Shoot development along with axillary shoot proliferation occurs. High level of cytokinins are reqiured . Stage III:- Associated with rooting of shoots and further growth. Facilitated by low cytokinin and high auxin concentration. Thus both medium will be different in composition. Optimum temperature- 20- 28ºC and lower light intensity.

12. Bud cultures Plant bud possess active meristems depending on physiological state of plant. Two types of bud cultures- Single node culture:- natural method for vegetative propagation of plants. Bud found in axil of leaf is comparable to stem tips, for ablility in micropropagation . A bud along with piece of stem is isolated and cultured to develop into plantlet. Closed buds used to reduce infection. No cytokinin is added here.

13. 2. Axillary bud culture:- shoot tip along with axillary bud is isolated. Cultures carried out with high cytokinin concentration. Thus apical dominance stops and axillary bud develop Good axillary bud culture – cytokinin / auxin ratio is 10:1.

14. Multiplication by adventitious shoots Adventitious shoots include stems, bulbs, tubers and rhizomes. Useful for invivo and invitro clonal propagation.

15. Applications 1) High rate of plant propagation:- Large no.of plants can be grown from single piece of plant tissue within short period. Can be carried out throughout the year irrespective of seasonal variations. Small sized propagules obtained in micropropagation can be easily stored for many years. 2) Production of disease free plants:- Meristem tip cultures are generally employed to develop pathogen free plants Successfully used for production of virus free plants of sweet potato, cassava and yam

16. 3) Production of seeds in some crops:- Through axillary bud proliferation seed production can be done in some plants. Required in certain plants where limitation for seed production is high degree of genetic conservation. 4) Cost effective process:- Requires minimum growing space. Production cost is relatively low.

17. Automated micropropagation It is now possible to automate micropropagation at various stages. Bioreactors have been set up for large scale multiplication of shoots and bulbs. Some workers employ robots thus to reduce production cost. Commmercial exploitation of tissue culture technology is limited because of high cost of production. This can be reduced by devising systems. Bioreactors can be used for somatic embryogenesis.

18. Robotics Robotics can be used , for example, in media preparation, cutting microshoots and placing them back on fresh medium. First commercial automated tissue culture robot was introduced by Australian company for bio robotics in 1997. target species- eucalypts, acacia teak and pine. Miiwa 1991 – fully automated robotic multiplication system.

19. Types Total automation:- With use of image analysis system and a robot or other device Little or no human interventions, except to control the computer and repair parts Eg : Toshiba ptc robot 2) Semi automation:- Involvement of human operators contributing to any stage of process. Most commonly researched type of automation

20. Advantages Management – management of machine will be much easier. Contamination – reduction of contamination can be expected by using automation. Operation time – two or three shifts become possible.this increase the utilization of capacity and reduce the depreciation costs per unit Uniformity – machines act uniformly.

21. Disadvantages Flexibility – plant growth and shape are affected by uncontrollable environmental factors. Once production is started on non-uniform stock, it would be difficult to coorect it in machines. Correction ability- application of automation naturally decrease opportunity to detect contamination.