फसल सुधार के लि‍ए फसलों में सूक्ष्मप्रजनन 

Various methods of tissue culture and molecular biology have shown tremendous potential for creation, conservation and precise characterization of genetic variability for improvement of fruit, field, forest crops, medicinal and aromatic plants. Micropropagation of crops is now one of the best and most successful examples of the commercial application of tissue culture technology. Propagation of plants from very small plant tissues/ parts under in vitro conditions in the laboratory is called micropropagation.

Commercial application of tissue culture started in 1970 in USA with the micropropagation of orchids. Since then it has seen tremendous expansion globally both in terms of number of production units and the number of plants, as well. With the estimated global market of 15 billion US$ per annum for tissue cultured products, even with the exponential expansion in the industry, the demand far exceeds production, leaving enough scope for expansion.

Due to higher labour costs in the developed countries, micropropagation industry is now being expanded in the developing countries, with lower wage scales to multiply plants at much cheaper rates even for the international markets. Thus Indian micropropagation industry, though a late starter by almost a decade, has expanded exponentially from 5 million plants annual capacity in 1988 to around 200 million plants in 2003. There are now about 10 commercial tissue culture units in Punjab producing about 2 million plants of different plant species every year.

Advantages of Micropropagation:

1. It ensures true to type plants i.e. identical to mother plant.
2. Helps in rapid and large-scale multiplication of elite clones/varieties under aseptic and disease free conditions, which are otherwise difficult to mass multiply using conventional methods.
3. It helps in rejuvenating old clones or varieties.
4. It is independent of seasonal constraints. Therefore, starting from one small plant tissue, millions of plants can be produced in one year.
5. It helps in the exchange of germplasm both nationally and internationally by avoiding the risk of spreading the pathogens and insects.
6. Micropropagated field grown plants give high yield and exhibit better quality.

Starting from one plant, millions of plants can be produced in a year. Multiplication is generally in the form of shoot buds or shootswhich are further rooted to get complete plantlets. Micropropagation possesses special significance in the vegetatively propagated species where multiplication through vegetative means is very slow and pathogens keep on accumulating generation after generation, which cause huge losses in the quantity and quality ultimately causing decline of the clone/variety. Thus micropropagation a can play a significant role for crop improvement

Method of Micropropagation:

This technique includes four steps:

1. Establishment of aseptic cultures:

This is done by taking special care of selecting the mother plant, which should be disease free and healthy. The plant tissue is taken and disinfected by treating with any disinfectant like calcium hypochlorite, sodium hypochlorite, hydrogen peroxide, ethyl alcohol, silver nitrate, mercuric chloride or benzlalkonium chloride for a specific period of time depending upon the disinfectant used and the tissue being disinfected. The plant material is then cultured on synthetic medium, under aseptic laboratory conditions. The cultures are kept in the incubation room maintained at 25±2^oC, with relative humidity 60-80 % and light intensity of 5000 lux till new shoot buds/shoots appear (3-4 weeks).

2. Shoot bud/ shoot multiplication:

This is the real multiplication phase in which cultures from the previous cycle are cut or divided and then recultured aseptically on to fresh medium in separate culture vessels. During this shoot bud/shoot multiplication phase, there is about ten times multiplication per cycle (2 weeks) throughout. Thus 26 cycles can be completed in a year. The roots do not develop, hence multiplication is generally in the form of shoot buds or shoots.

3. Induction of rooting and hardening:

The shoots obtained from step 2 are transferred to rooting medium to get the root formation. Once root formation takes place, hardening of plants is done by taking plantlets out of the culture vessels and washing them under slow running tap water. The plantlets are then transferred to water moist cotton in enamel trays, with daily change of tray water kept in the incubation room for 7-10 days.

4. Transfer of plantlets to soil:

Hardened plantlets individually or in clumps are transferred to soil in polythene bags kept in the polyhouse for one and a half month before their delivery.

Uses of Micropropagation:

The micropropagation possesses special significance in the following areas:

1. Production of high quality, disease free, super elite planting material for commercial seed   production material for commercial seed production.
2. Rapid spread of new varieties of vegetatively propagated crops like sugarcane, potato, poplar,  mentha and other medicinal / aromatic plants for crop diversification.
3. Mass production of ornamental plants, which are otherwise difficult to multiply through conventional methods.
4. Rejuvenation of old varieties/clones of vegetatively propagated crops for improving their yield and quality. 
5. Mass cloning of rootstocks in horticultural plants.
6. Multiplication of male sterile lines for hybrid seed production in field, vegetable and floricultural crops.
7. Interstate/ international exchange of germplasm avoiding the risk of pathogens and insects.

Setting up of Micropropagation Unit:

Micropropagation unit requires the following items:

1. Building: About 4-5 rooms of 15'-20' each
2. Growth room: Air-conditioned room of size 20'-25', fitted with 3-4 air conditioners, humidifier and culture racks with florescent tube rods. This room is maintained at temperature 25±2⁰C, relative humidity 70-80 % and good illumination 5000 lux with 16 hours/8 hours (light/dark) regimes, throughout the year.
3. Polyhouse: 1-2 of size 30 meters (length) x 6 meters (width) x 3 meters (height) fitted with cooling pad/fan and overhead irrigation system.
4. Uninterrupted electricity supply: 30-40 KW connection.
5. Equipments: Laminar Air Flows, autoclave, water distillation, pH meter, electric balance, culture racks etc.
6. Man power: Technically trained 5-10 persons for handling various operations.       

This kind of unit will cost about Rs15-20 lacs with a capacity of producing 5-7 lac plants annually. The Department of Plant Breeding, Genetics and Biotechnology has developed micropropagation protocols for a number of plant species including sugarcane, potato, mentha, strawberry, citrus root stocks, eucalyptus, poplars, carnations, chrysanthemum, gladiolus, Aloe vera, brahmi and safed musli. Besides, this Department also provides on demand one month training to prospective entrepreneurs/farmers on various aspects relating to micropropagation of plants through tissue culture.

Authors:

Satish Kumar, Chandra Nath Mishra, Raj Pal Meena and Indu Sharma

Directorate of Wheat Research, Karnal

Haryana - 132 001

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