Tissue culture in a pressure cooker

0 Comments Author(s): K N Shahjahan Oct 31, 1993 | From the print edition A new tissue culture technique that is claimed to be cheap and easy to understand is fast gaining popularity. "IF TISSUE culture companies can make profits, why can't villagers?" asks C R Raju, who has developed a tissue culture technique that he claims is cheap and easily transferable to the people -- a feat research laboratories are still to achieve. Raju developed the low-cost technique as part of voluntary work for the Centre of Science and Technology for Rural Development (COSTFORD), an NGO based in Mavelikara in Kerala. A senior Indian Council of Agricultural Research scientist working with the Central Plantation Crops Research Institute in Kayamkulam, Raju started work on the tissue culture technique in 1989, during weekend visits to his maternal house in a remote village near Mavelikara in Alappuzha district. The failure of initial trials, for nearly eight months, didn't discourage him. Careful detection and elimination of the sources of contamination readied the process for wider application. The next step was to scale down the cost of each and every equipment needed for the culture work -- a long, tedious process. Raju's tissue culture technique requires simple equipment such as a small balance, a hood, a pressure cooker, a heater, a tubelight and a small room that could be made airtight while culturing. Plants selected for tissue culture were those that could tolerate temperatures of 20-300C and expensive borosilicate glassware was replaced by cheap, colourless bottles. Chemicals of low purity levels were used. The cost reduction achieved was enormous. According to Raju, culture space of bottles worked out to less than 1 paise per sq cm as compared to Rs 250 per sq cm of culture space in the case of borosilicate glassware. Simple balance Most of the plant species tried for the tissue culture technique were found to tolerate considerable variations in the quantity of nutrients. Taking advantage of this tolerance, the costly chemical balance used in the conventional method of tissue culture was replaced by a simple balance worth Rs 15. Autoclaving (sterilisation under high pressure steam) of the tissue culture apparatus was done in the pressure cooker. Raju's technology is simple enough for anyone who can read English to understand. The total cost of setting up such a unit is Rs 10,000, of which Rs 9,000 goes towards capital costs. Using it, Raju was able to produce one lakh orchids, which he selected because they are easy to handle and multiply. An article by him in a local daily in 1990, in which

he described his technique, brought him an overwhelming response and prompted him to start a training programme for which he himself pays. Production of tissue cultured plants requires knowledge of suitable culture media, ways of maintaining an aseptic environment, techniques of judging developments in culture and methods of hardening in vitro plants. Except for the first step, all the other stages are included in the short training of 16 days spread over four months. Some 27 people from different parts of Kerala have already been trained by Raju. Of them, 12 have started their own laboratories and five have even started production. Santosh Srinivas, a literature graduate who has set up a tissue culture laboratory in Thiruvananthapuram after his training with Raju, says, "I am perfectly happy with the technique. There have been no complaints from the customers. And, because I use cheap equipment, I can sell at lower prices than others." He sells his cultured orchids to a mostly upper middle class clientele. M P Govindankutty, an economic botanist at the Central Plantation Crops Research Institute, says, "(Raju's work) demonstrates micropropagation for the common person through a simple technology that works. Media preparation and initiation of cultures is done by people who lack formal academic qualifications, but have grasped the idea behind aseptic manipulation." Raju is now experimenting with medicinal plants. His technique, with adequate financial and institutional support, can become a major source of employment. COSTFORD is contemplating charging fees from the trainees, especially those who can afford it. Says Raju, "Till now, I have spent Rs 3.5 lakh for developing the technique and giving free training. Can like-minded people sponsor my work?"

ow cost Tissue culture

Presented at NIRD Hyderabad.
NIRD Foundation day Seminar 2-3 January 2002 Rural Technology and Poverty Alleviation

Tissue culture an AGRICULTURE based rural technology: potential and problems in transfer
C.R. RAJU #, P. ANITHAKUMARI # * M.P. GOVINDANKUTTY , AND K.S.MANOJ
Biotechnology Unit Centre of Science and Technology for Rural Development Mavelikara 690106, Kerala, India ABSTRACT The tissue culture technique one of the modern tools in the area of biological

sciences- with enormous potential but veiled in a shroud of secrecy was unveiled through the consistent efforts of COSTFORD since early 90s. The basic reason for the benefits of S&T not being passing on to the lower strata of the society say for the rural areas is not the lack of technologies but the reluctance of the scientific community to come down to the level of the rural people. The jargons make the technology unacceptable to the rural people. The approach of the people towards tissue culture changed from that of a dream to a reality when it was rendered simple as any other systematic domestic activity. The transition was quite evident from the first person who was trained to the present state where the tissue culture is an art rather than a big scientific feat. They are also excited to see the modern technology is not only exciting to read in news but also work through their hands. The trend of the younger generation is to move to the urban areas hunting for pastures for a better tomorrow. However, the sustainable system can be created at the rural condition only through the intervention of S&T. In spite of rendering the tissue culture simple reaching the beneficiaries is the most difficult part and is where the effective interaction between the scientists and extension workers has to work hand in hand.

Training the educated unemployeds and transfer of the technology was more difficult than actual development of the technology. Free training, training village women, training at community level were tried. Giving free training was in no way helping the actual transfer as the trainees did not feel the pinch of time spent and the expenditure but when the training was on actual charges people took the training serious and the response was excellent. Concerted effort from both the Scientific community as well as extension workers will be necessary for taking technologies to the rural people. In spite of rendering the tissue culture simple reaching the beneficiaries is the most difficult part and is where the effective interaction between the scientists and extension workers has to work hand in hand. Now it has been rendered more an art rather than a big science by itself. Training the educated unemployed and transfer of the technology was more difficult than actual development of the technology. Free training, training village women, training at community level were tried. TECHNOLOGY

Basic tissue culture technology is based on the fact that every cell has the potential to divide, differentiate and form a whole plant with the same genetic identity of the plant to which the original cell belonged. The second concept is that all the cells of a plant will be genetically identical as they are all formed through the same type of cell divisionmitosis. Apart from the usual concept of sophisticated equipment, highly qualified persons, extreme precautions for maintaining asepsis, complicated methods for providing light of specific colour temperature at specific photoperiodicity, keeping them at specified temperature and humidity controlled laboratories and humidity/temperature/ light controlled green houses for establishing the plants to the normal environment in the usual commercial laboratories tissue culture for large scale multiplication is just an aseptic technique of growing plants/tissues on nutrient media in transparent containers. Tissue culture simplified If one realises that tissue culture is just a technique, then it is possible to think about the alternatives for the sophisticated equipment to reduce the cost of establishment. It is known that in countries like Thailand and Singapore Tissue culture is practiced widely for the large-scale production of orchids and other valuable plants as a domestic industry. However, this is kept a trade secret and if one has to do it, it needs standardisation of the whole procedure. The first step was to identify the requirements. Tissue culture for propagation has two distinct parts: research for developing the methodology, which is done by the scientists yielding a series of simple steps to achieve the multiplication of the plant in question. These steps are like weighing chemicals, dissolving, mixing different solutions in specific proportions, melting in the flame or pressure cooker, taking plant parts, shaking in some solutions, washing them, cutting, placing on to the media etc. Once we divide the procedure into simple steps it becomes a simple mechanical activity of course requiring some precision of the hands, which on acquires with practice. The second part of multiplication is nothing but the repetition of the steps developed through research to get the same result. THE ART OF TISSUE CULTURE The technology once developed has to be repeated in the same manner given in the protocol to get the same level of propagation. This is purely mechanical activity and if

we look from the angle of the personal requirements they are just: Basic education to o o o o o o Identify chemicals Weigh them as given in the protocol Prepare stock solutions and to use at their convenience Prepare the final medium from stocks and to sterilise it Have an outlook on the general develop after training Logically handle the cultures as instructed

Steady hands and good eye sight o o o o Basic requirement as the explants may be small Dissection if tissues need precision and good sight The tissues put into and taken out of culture bottles require precision Good eyes only can perceive the subtle developments in culture early

Good sense of cleanliness o o o o Tissue culture is as aseptic technology The microbes which induce the contamination are too small The chances of contamination are from several sources Logical steps are to be taken when contamination is detected

Patience and persistent interest o o o o o The plants tissues grow only at a very slow pace One should patiently wait for the tissues to respond in culture There will be plant to plant variation in the response in some cases The percentage of explants which respond in culture is small Repeated trials may become inevitable I some cases

Love for plants o o Although tissue culture is a repeatable scientific procedure, plants grow better in caring hands A person used to keep and care plants alone can successfully grow them in culture as well

The plants as they are taken out of the aseptic condition are just like delicate babies and should be handled with care.

If we look into the aspect of what all things they should know they are quite simple and are listed here: Basic training for o o o o o o Preparation of Chemical stocks Preparation of culture media and sterilisation Surface sterilisation and initiation of cultures Information about identifying the patterns of development How to follow the protocol and How to harden the plants

Protocol for the plant to be multiplied o o This is a serially arranged steps which can be follow easily after some exposure / training in tissue culture work. The basic difference between the protocols for multiplication will be just the changes in the composition of the media and other miner variations that has already been worked out. o There can be variations in the process of taking out the plants produced from the containers and getting them established in the ambient conditions. The details will be available in the protocol. The major requirements were identified to be: Infrastructure The infrastructure requirements vary considerably depending on the required output as well the number of individuals planning to do the multiplication together. The requirements of a small micropropagation unit are: A hood of wood or particle board UV lamp Plastic measuring cylinders Chemicals Good water source Bottles Test tubes with stopper Surgical knife & blade Pressure cooker Small balance with weights Pipettes pH paper Steel vessels Petri-plates or steel dishes A few flasks Spirit lamp

Forceps Paper for covering neck / cotton plug Rubber bands Cotton etc. All put together might work out to Rs 12,000/ Space This is an important aspect were due consideration is important. Space is needed for four basic activities: Medium preparation, aseptic culturing, incubation and hardening. o Medium preparation is basically mixing the stock solutions in specific proportions, adjusting pH, dispensing into culture containers and sterilisation in pressure cooker. All these can be done in the domestic kitchen itself with no additional expenditure. Culturing needs a small sterile space which can be a small room of size lot less than 5 x 6 feet with a table for placing the inoculation hood. This can be separated out from any part of a larger room or created in the veranda. o Incubation is just keeping the cultures in the appropriate conditions like light or dark at the temperatures tolerable to the plant under multiplication. Some cultures are to be kept in the dark and they can be kept in any place available in the house while the cultures that require light must be kept in such a way that they will get sufficient light for photo-differentiation and proper growth. The natural light at lower intensity as available in the shades of trees or at the veranda is sufficient for the growth of plants in culture bottles.

Hardening is just acclimatising the plants to the natural environment. At

this stage the plants taken out of the culture bottles are to be cleaned well in water, treated with some fungicides and maintain them in the high humidity environment which can be created in the shades of trees with polythene sheets which will be very cheap. The plants should be sprayed as per the schedule given in the protocol.

 Chemicals Usually in tissue culture research very high purity chemicals are routinely used but once the technology is developed, but for the actual multiplication the expensive high purity chemicals are not necessary. Several alternatives have been developed to maintain the cost of chemicals as low as possible.

Chemicals, facility to weigh chemicals, good water, equipment for sterilisation of media, a sterile environment, airy area with light for incubation of cultures and a shady area with high humidity for hardening plants. The requirements from the part of the person practicing tissue culture: they should have the basic sense of cleanliness, Basic education at least to matriculation, training in the various stages involved in the micropropagation, protocol for the plant to be propagated and finally a small culture facility costing anywhere from Rs 12,000/- to Rs 1,50,000/- (for a community based unit). When the resources are pooled then with a few equipments for ease of handling and to save time can also be purchased. POTENTIAL The major potential if immediate relevance is the income generation at the village level. Any young person of basic education, patience and aptitude to plants and a strong will can learn and adopt micropropagation for income generation. It has been proved to be a viable methodology by several people across the country up to Rajasthan and North eastern states. There are several horticultural plants of demand and those requiring conservation due to over exploitation but are not being attempted in a large scale as the tissue culture technology has the potential. Several of the plants are in great demand for horticultural purpose and also for medicinal value. Several of the native flora are getting eroded due to conversion of forest land for other purposes, and over exploitation of the native flora even before they produce the seeds to have the next generation. Micropropagation can be resorted to producing large quantities of planting material and to restore them in their natural habitats.

 APPROACHES IN TRANSFER OF TECHNOLOGY After partially standardising the methods for the multiplication with minimum resources two youngsters one girl (school drop out) and a boy (post matriculate) were taken for the training. With the continuous effort for about six months they were brought to the level of culturing orchid seeds with out laminar flow unit and other complicated equipment.. Simultaneously they were also taught the methods for initiate cultures from vegetative tissues of medicinal plants. By nine months both of them became proficient in culturing orchid seeds and culturing stem explants of the medicinal plant Tylophora asthmatica. With this first round of training two youngsters it became evident that the low cost appropriate tissue culture method would be a useful tool even at the grass root level for income generation. The next step was to make the training process simple so that one can learn it in less time with minimum effort. The experience of training the first two people helped in developing a simple alternative. Here the tissue culture is taught in the conventional method using laminar flow and when they were proficient in maintaining a satisfactory level of asepsis, hood was introduced in place of Laminar flow and thus the training period could be reduced to six months. Second culturing other plant multiplying unemployed Initially the training was imparted to graduates and postgraduates who were interested in the multiplication of orchids. There it was a heterogeneous group right from school drop outs to professionals who wanted to take up tissue culture as hobby. It was quite successful in which the experience proved that there is no need to tech all science behind tissue culture to enable a person to practice tissue culture. This lead to the cardinal change in the training process from the elaborate classes from the theory combined with practicals to purely mechanical training that is sufficient for the successful transfer of the technology. Training was given to groups to have a collective laboratory facility for multiplication. Two societies were formed by these trainees and started the propagation of orchids along with a nursery of ornamental so that the income ca be increased. The group effort is better than units started by an individual. Next stage planned is to give training to the dedicated activists sponsored by NGOs

across the country and then they in turn can give training to the rural masses in their local dialect. The spade work for this has already been established and the first batch of trainers will be given the training soon. This approach alone can take the technology to the grass root level across the country where ever this can be exploited for income generation or environment restoration. PROBLEMS AT TRANSFER LEVEL The low cost tissue culture technology of quite sound and has been proved handy with several people after training. But the whole difficulty is after the transfer of technology stage or at the implementation stage. There is a dearth of adequately trained persons for the mushrooming tissue culture laboratories in the country. After the training most of them are absorbed the large companies with quite attractive salaries so that the whole effort that was put in the training is exploited by the private agencies while the training is intended for the trainees to develop their own micro propagation units. The training when given free the beneficiaries do not take the training seriously. Often the people mainly housewives come for the training to avoid the monotony at home after the days routine work is over. However, after the training they seldom show any interest in taking it as an avenue for income generation. This category of people are not much concerned about the potential income that can be derived. They will continue to come for the training till they are told the training is over. Another category of women who come for the free training are those are having the spare time. These women often like to have good backing to carryout the and tissue culture programme for the finance as well as morel support from the family. The self help groups would be one of the possible ways in encouraging the people to go in for such innovative technologies. However the proper monitoring till it becomes self sustained is a necessity. Another problem prevails is the difficulty to find out a source of input. For the set up of laboratory facility, a considerable sum is required. But by adopting low cost method the initial investment can bring down to a manageable level. But still it will remain as considerable amount to rural layman (for setting up a lab, by adopting low cost methods

Rs 12,000/- is required.). The possible solution to this is, manage to rise funds by a group of individuals together. Progressive lack of care in the successive stages of development of protocorm can be taken as a third problem at the level of transfer. There is a time lag in realising the returns from a propagation unit. But many a time it has been observed that, adequate care required is not given after the initiation. Since micropropagation requires a persistent effort and patience, in many causes the interest seemed to be lost in the later stage. If the cultured materials do not get adequate care in the subsequent stages it will result in a peril of ruin of the entire culture. Lack of adequate marketing facilities for the produce also play a key role in lost of interest in the micropropagation by the trainees in later stage. In rural areas inaccessibility to transportation facilities always prevails. That in turn may result in perishing produce, if it couldnt be marketed timely. CONCLUSION The concept of the scientists who develop the technology is quite different from the way the layman the end user thinks. In the Indian context almost all the commercial firms are going in for either imported technology or resort to their own technologies due to the lack of confidence. Literature shows enormous technologies but the scientists are not willing to come to the level of the end users so that the expenditure incurred goes waste. It would be possible to find areas of S & T which can have of immense use in the rural sector but the need of the hour is to find techniques that can be translated to the rural areas and to demonstrate the feasibility for income generation. Creating awareness among the scientific community to make the technologies simple will be the first step. It is then the handing over of these technologies to the extension agencies through training and finally the extension persons who work with the rural people should be entrusted the transfer of technology. In terms of poverty alleviation any effort to increase the income generation capacity of the rural people will be of immense application. Tissue culture as a modern tool will help in the rural sector in several ways: for income generation, production of planting material for gardening, forestry and to multiply as well as to maintain the genetic diversity. The National Institutes like NIRD should identify this type of technologies and

should be the nodal agency for the transfer of these technologies on the national level giving due credits to the Institutions developing the technologies. NIRD should also take a lead role in identifying the needs of the rural people and pass on the information to the agencies involved in the R&D activities so that they in turn will develop the technologies, test them ion a pilot scale and transfer to the needy. The contacts of the NGOs engaged in R&D with the public will be limited. This emphasises the need for the intervention of National Institutes to be a forum for such discussions as well as reaching out the needy.