INTRODUCTION:

Hundreds of commercial micropropagation laboratories worldwide are currently multiplying large number of clones of desired varieties and local flora. Apart from the rapid propagation advantage, this technology is being used to generate disease-free planting material, and has been developed and applied to a wide range of crops, and forest and fruit trees. However, in many cases, the cost of micropropagule production precludes the adoption of the technology for large-scale commercial propagation.

 THE NEED FOR LOW COST TECHNOLOGY

Low-cost tissue culture technology is the adoption of practices and use of equipment to reduce the unit cost of micropropagule and plant production. In many developed countries, conventional tissue culture-based plant propagation is carried out in highly sophisticated facilities that may incorporate stainless steel surfaces, sterile airflow rooms, expensive autoclaves for sterilization of media and instruments, and equally expensive glasshouses with automated control of humidity, temperature and day-length to harden and grow plants. Many such facilities established at a high cost are high-energy users, and are run like a super-clean hospital.

The requirements to establish and operate such tissue culture facilities are expensive, and often are not available in the developing countries. For example, the cost of electricity in the developed countries is much lower, and its supply far better assured than in the developing countries. The same can be said of the supply of culture containers, media, chemicals, equipment and instruments used in micropropagation. Hence, alternatives to expensive inputs and infrastructure have been sought and developed to reduce the costs of plant micropropagation.  

ADOPTION OF LOW-COST OPTIONS Low cost options should lower the cost of production without compromising the quality of the micropropagules and plants. The primary application of micropropagation has been to produce high quality planting material, which in turn leads to increased productivity in agriculture. The generated plants must be vigorous and capable of being successfully transplanted in the field, and must have high field survival. In addition, they should be genetically uniform, free from diseases and viruses, and price competitive to the plants produced through conventional methods. Reducing the cost should not result in high contamination of cultures or give plants with poor field performance. 

 The foremost requirement of micropropagation is the aseptic culture and multiplication of plant material. Microbe-free conditions need to be maintained in culture containers, and during successive subcultures. In many cases, mistakes in concept or practice can introduce microbes in the culture containers from an external source or the plant material itself (endophytic contamination). As a result, the microbes overgrow the cultures, and wipe them out. Microbes may grow slowly under controlled low temperature, but they proliferate very fast under uncontrolled and high temperature. Thus the adoption of wrong low-cost options may make the production process prone to disasters. Low cost techniques will succeed only if the basic conditions for tissue culture are scrupulously adhered to maintain propagule quality.

 QUALITY OF MICROPROPAGULES

Low cost technology means an advanced generation technology, in which cost reduction is achieved by improving process efficiency, and better utilization of resources.  Presently, both the developing and the developed countries require low cost technology to progressively reduce the cost of propagule production. In many developing countries, the potential end-users of plants derived from tissue culture have been the resource-rich farmers. They know the benefits and potential of healthy planting material. Such growers are prepared to risk investment in the high productivity potential of the planting material.

For example, hybrid seeds of many vegetables, papaya, rice, and cotton cost 15-20 times more than the price of ordinary varieties. Yet there is a wide market for them. Hence, the production of low quality plants, just because they are less costly, is not going to be a sustainable approach for the application of micropropagation in agriculture.

Lowering of cost of production is possible only if the methods do not compromise the basic imperatives of tissue culture and quality of plants. Plant tissue culture techniques have a vast potential to produce plants of superior quality, but this potential has been not been fully exploited in the developing countries.

During in vitro growth, plants can also be primed for optimal performance after transfer to soil . In most cases, tissue-cultured plants out-perform those propagated conventionally. Thus in vitro culture has a unique role in sustainable and competitive agriculture and forestry, and has been successfully applied in plant breeding, and for the rapid introduction of improved plants.

Bringing new improved varieties to market can take several years if the multiplication rate is slow. For example, it may take a lily breeder 15-20 years to produce sufficient numbers of bulbs of a newly bred cultivars before it can be marketed. In vitro propagation can considerably speed up this process. Plant tissue culture has also become an integral part of plant breeding.

For example, the development of pest- and disease-resistant plants through biotechnology depends on a tissue culture based genetic transformation. The improved resistance to diseases and pests enables growers to reduce or eliminate the application of chemicals.  With this as the background if we move towards a design of a low cost technology TC unit the following parameters will weigh down in its implementation.

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A convenient location for a small laboratory can be a room or part of the basement of a house, a garage, a remodeled office or a room in the header house. The minimum area required for media preparation, transfer and primary growth shelves is about 14 m2. Walls should be installed to partition different areas. Before setting up a commercial micropropagation unit, it is essential to check out the area keeping in mind the climate, and access to water, electricity, transportation, and infrastructure for supplies.

A temperate climate is usually better suited to tissue culture ventures. This greatly reduces the cost of cooling required to maintain the temperature for optimum growth of the cultures.  The availability of electricity and water is of utmost importance, and should be taken into consideration while choosing the location of the facility.

For example in India, of the 76 commercial tissue culture units, nearly 52 are located in and around the cities of Bangalore and Pune, where the climate is moderate. Hence, cooling is required only during certain periods of the year. However, in cities like Delhi, which have extremes of climate, tissue culture facilities require both heating and cooling. In a facility, which produces five million plants, the electricity cost per thousand plants is around US $0.30 in Bangalore and Pune; the same is about US $0.80 in Delhi. Disruption of power and water supply causes major breakdown in the smooth running of tissue culture units. Poor quality water adds to the cost of media.

Hence the optimum usage of electricity is a must in TC labs and here we use the technique of the direct sun instead of flurescent lamps for the growth chambers..

 

Bottle or containers for Tc work we use any and every one which is available. No constraints. See above picture..

The main parts of setting up a lab is a clean room for sterilisation and transfer to flasks and daily processing of cutting and cleaning and handling and replating work.

This clean room has mainly the Laminar flow units and the work tables including cutters, microscopes for work, TC chemicals, trays for handling and labware, shakers.

The location for autoclaving, cleaning of containers and strelisation can be hived of to an open area outside.Only sterlised containers can be brought in to the media room where the media can be dispensed into the culture vessels.

The decision to design an optimum work flow are depends on the workload per day and the design of handling of production loads.

The growth room where transferred and finished bottles are kept for growing should ideally be adjacent to the clean room or attached to it.

The clean room becomes the center and all others are placed in pheriphery to this facility. Cotamination hazards are best minimised by avoiding too many intrusions into the clean room and workforce movement. Timing is also very important function in the same. Some labes operate only for few hours in the morning of the clean room.

Hepa filters and air filtration in the room can be an ideal choice.

The design of the growth chambers where storage becomes the most important criterion and providing the right lux levels and day and night lengths are also critical. Lighting and design of shelving is of utmost importance. The design demands that the right choice of a plant flask can go a longway in the optimum space utilisation.

The last part of a good lab is the plant hardening room or the green house where babies from flasks are acclamatised for growing in the actual real environment. The time taken here may be a few weeks and care and consideration can help avoid losses due to fungal and bacterial attack.

The last journey of a plant Tc unit is the actual growing field and proepr care should be exercised in the first three months when the plant become adjusted to physical environs.

Here is a list that can be a starting point:

Plant tissue culture laboratories have specific design requirements. Careful initial planning is, therefore, a prerequisite for successful running of a facility. The location and design of the laboratories should take into account isolation from foot traffic, control of contamination from adjacent rooms, thermostatically controlled heating and cooling, water supply and drains for a sink, adequate electrical service, provisions for a fan and intake blower for ventilation, and good lighting. The heating system should be capable of maintaining room temperature at 20C during the coldest part of winter. A minimum of 2cm pipes should be used for water supply. Connection to a septic system or sanitary sewer should be provided. Air conditioning requirements should carefully estimated. Electrical service capacity for equipment, lights and future expansion should be calculated. For safety reasons, the electrical installation should be carried out professionally. Most electrical wiring will require 220 Volts, and autoclaves 230/250 Volts.

 

Cleanliness is the major consideration when designing a plant tissue culture laboratory to minimise contamination. A positive pressure module should be installed to circumvent air intake from outside. Routine cleaning and aseptic procedures can decrease contamination losses to less than 1%. An enclosed entrance should precede the laboratories, and sticky mats should be placed to collect dirt from shoes.

 

For a commercial tissue culture unit to be successful, it is essential to constantly find means to increase the efficiency of production, and bring down the cost of production. A number of low-cost alternatives can be used to simplify various operations and reduce costs in a tissue culture facility.

 

Instead of Petri dishes as stage for manipulation of culture, stainless steel plates, ceramic tiles and brown wrappingpaper can be used; all of these can be autoclaved. Ethanol for hand and workbench sterilization can be replaced by industrial spirit. The careless handling of inflammables used for sterilization can be hazardous. Glass bead sterilizers can be used to sterilize forceps and scalpels instead of the conventional flame sterilization using spirit lamps or gas cylinders. Commercial bleach has also been successfully used in several laboratories for bench and instrument sterilization to reduce cost, and to prevent fire hazard.

 

Most plants are seasonal in demand. To meet the requirements of extremely large number of plants, commercial production has to be backed by well defined working procedures and monitoring performance of the operation. Laboratories should produce a range of plants for different seasons to maximise the use of the facilities throughout the year. This lowers the unit cost of plant production.

 

Like wise I can make comments on each and every aspect of TC work..

 

A few pointers to include

 

Media preparation

 

Use of water that is ideal

 

laminar unit designs

 

Propagation facilities and mother stock plants

Growth room, Hardening room, shade houses

Documentation and Record maintanenece

Storage and sales areas.