Micro Prorogation - Tissue Culture

Introduction:-

Tissue culture, an important area of biotechnology can be use to improve the productivity of planting material through enhanced availability of identified planting stock with desired traits. The idea of cell and tissue culture were put forth by a German Scientist Haberlandt in 1902. All attempts to culture the plant cells and tissue were unsuccessful till the 1930’s. Around 1939 the possibility of culturing plant tissues for definite periods was independently reported by white, Nobecourt and Gautheret.

Micro propagation is one of the important contribution of Plant Tissue Culture to commercial plant propagation and has vast significance. The name micro propagation derives Item the miniature shoots/plantlets initially produced horn this method of plant propagation. Micro propagation is the true to type propagation of selected genotype using in vitro culture technique. This technique provides a rapid reliable system for a production of large number of genetically uniform disease free plantlets The pioneering work in this area of research was reported by Indian scientists. Today almost all the prime universities and institutes like BARC, Bombay, NCL, Pune, ISC Bangalore etc. in India are carrying out research in tissue culture.

Commercial tissue culture born in India in 1987 when NV. Thomas & Go. in kerala established their commercial unit for large scale production of cardarnurn. This was based on the bench scale protocol developed by Dr. R. S. Nadgauda and Dr. A. F. Mascarenhas at N.C.L. Pune, India. These pioneering efforts of AVI, Kerala, were followed by servile other commercial companies who entered in this area. The second unit was put by Indo-American Hybrid Seed Company at Bangalore in 1988. Now there are several companies in this field viz.


Hindustan Lever, Tata Tea, Unicorn Biotech, Nath seeds, RPG Enterprises, Indian tobacco, and Hindustan Agri Genetics limited etc. are the major companies in the field or micro propagation in the country.

There has been an increasing awareness about Bio-technology in India in recent years. The discussions are not only confined to workshops, seminars, conferences, symposium but people from various section of the society are curious F. know and use this technology in various fields. Modem bio technology offers unlimited opportunities for enhancing genetic potential of crops, management of biotic and biotic stresses, waste management by organic recycling. and also faster multiplication of plaiting material. the technique of bio technology like tissue culture has attracted the attention of farmers in the Country.

Tissue culture has helped to develop new strain of food crops, cereals, vegetables flowers, oilseeds and plantation crops such as spices, coffee, tea and rubber. Maharashtra is in forefront in the use of bio -technology in the country with tissue culture technology the production of quality planting material is gaining immense popularity for micro propagation of various cash crops flowering shrub and trees. Since the farmers are attracted immensely towards this technology, Govt. of Maharashtra would like to decide the slate policy regarding use of bio technology in agriculture. In order to take a stock of present scenario of various aspects pertaining to bio technology Govt. of Maharashtra has appointed a committee under the chairmanship of Principal Secretary, Agriculture of Govt. of Maharashtra. One Sub committee is also appointed under the chairmanship of Director of Horticulture with the experts to evaluate the present situation arm suggest appropriate interventions to boost this technology in the sin ~ Members of this committee are listed as

appendix-I

Such committee is to look into the following issues:-
(A)  To collect and consolidate the information regarding present scenario of micro propagation technology and study the utility of this technology.
(B)  To study the potential for increasing agriculture production with the help of tissue culture and decide strategy for next 5 years for use of this technology.
(C)  To decide strategy for establishment of tissue culture laboratories along with criteria regarding financial assistance.
(D)  To suggest extension plan for use of micro propagated plants by the farmers.
(E)  To suggest quality control measures for micro propagated plants.
(F)  Decide future direction for research and development in the field of tissue culture considering needs of the state.

Accordingly the sub committee members met and discussed various issues, four meetings were conducted on 10/6(99, 5/7199, 1911199 — and 30/7/99. Abstract of discussions is included the report.

Present Scenario:-
Plant tissue culture has been promoted by Government of India since last 2 decades to enhance the production Land availability of disease free, true to type, quality planting material.

According to the BCIL, (Delhi) report, at the end of 1996 about 76 commercial micro propagation units have registered for the production with Government of India of which 30 are functional within the Country. Total 40 million plants annually are produced against the installed capacity of 110 million plants.

The current progress of the industry is encouraging but the expected rapid growth has not taken place. The primary reason for such a slow growth is because of low awareness level amongst the people about application of this technology. Although within the country the research groups have put lots of effort in standardizing protocols for several plant species, the benefits have not been sufficiently demonstrated to the farmers at the field level Therefore, the technology is not so popular at the grass root level.

At the national level, Department of Bio-technology has promoted the activity with the help of which two pilot plan were set up of the capacity or 1 million plants per annum of economically important plant species. Out of these two units one is Ioc8ted ~t National Chemical Laboratory, Pune, Maharashtra. Extensive efforts have undergone on developing the experts for setting up of the laboratory and developing cost effective indigenous technologies with felid evaluation. This unit is now upgraded to Micro propagation Technology Park to serve the entrepreneurs/industries institutes/universities so king for mass scale production of plants.

Recently national workshop on commercialization of plant tissue culture. Role of Micro propagation Technology park" was convened by DBT, New Delhi on June 141999 with the major objective of creating an awareness and present the concept of Micro propagation Technology Parks.

The potential for the domestic market is enormous and by conservative estimates it is around RS. 150/- cores with the annual growth rate of 10%. In order to fulfill the demand of domestic market even at the meager lever of 10 to 15% efforts needs to concern rate not only to increase the units at private sector but also the back up at National and slate level from the government is necessary.

Currently, the focus of the companies is mainly in the floriculture sector however, micro propagation in banana arid sugarcane is also gaining popularity.

Recently Bhabha Atomic Research Centre, Mumbai have also done initial work on micro propagation Cardamom Mulberry and Sandalwood.

Currently, micro propagation technology is available on the shelf for the crops which can be utilized for field test demonstration and farming. In Maharashtra a research on Biotechnology is under taken in all four agriculture universities. In addition Maharashtra State Seeds Corporation has also established tissue culture laboratory for commercial production of tissue culture saplings of banana and some ornamental plants. The information regarding present production capacity, utilization and cost per plants of tissue culture within the state is given in Appendix-II

There are 17 active public and private sector dealing with production of tissue cultured planets producing Banana, Sugarcane arid Ornamentals & Orchids etc. incidentally, Maharashtra is emerging these crops for shifting their cultivation to such type There are 6 major units in the State producing plantlet, 7 units producing 15.60 lakh plant around 31.30 lakh plants of ornamental crops particularly gerbera carnation and orchids. The units in the State are producing 91.85 lakh tissue Cultured plants every year. This clearly shows that state has already taken lead in this area recognizing a great scope for this industry in future.

Maharashtra is a major banana growing state in the country. Maharashtra tops the list with its higher productivity with area coverage of about 60000 hectares. In order to increase productivity of banana, it is highly essential to adopt new production technology to ensure better quality production. One of the eat that is being posed in recent past is that Sigatoka (Cercospara muse) which would demand replacement of this infected planting material Tissue Culture could be one alternative for faster replacement of planting material. Experimentation has shown that there is a significant increase in the yield in banana and yield parameter litter average number of hands fingers per bunch, length and girth or fingers due to which there is around 30% increase in the yield of banana. Sugarcane also yields 10 -15 0/) increase in productivity due to tissue culture plantlets. Our State occupies 5.2 lakh hectors area under sugarcane with an average productivity or 85.5 MT/ha. Quality planting ma that in sugarcane is also concern a matter of in sugarcane cultivation. Once sugarcane was considered relatively less affected by pests and diseases, however during past few years, a few of rumor pests earlier nave now play a major role Threatening its productivity in future.

There are 13 major pests reported to damage sugarcane crops heavily. A sugar industry in Maharashtra plays a vital role in the rural economy of the State and has successfully boring out economical and social reforms in rural Maharashtra. State produces more than .33O/~ sugar produced in the country. There has been, however, declining trend in the yield for the past few years, major reason being lack of good quality planting material. Using newly developed cost effective protocol of sugarcane through micro propagation large number of sugarcane plantlets are being produced in Maharashtra by various public and private sector units.
One of the major issues apart from planting material has been that of low emergence of eye bud, thereby reducing plant population, and ultimately effecting production of mislabel cane. Tissue Culture technique for production of good quality planting material ensures an adequate and disease free plant to get enhanced yields.

Tissue Culture in ornamental Plants constitute a major part of the total lancing material produced in state out of 31.25 lakh ornamental and other plantlets, gerbera a alone is a major flower crops produced in the State.
Despite increasing production of Tissue Cultured plants in the State, the total plants produced by various units are only 73% of the total capacity created for production. Considering huge requirement of tile State, it is quite essential to augment these industries by strengthening existing units and also creating new infrastructure for further production of Tissue Culture of various high value crops.

Although, this technology has been adopted by the farmers, considering area covered, adoption is at a very low level. Banana growers used this technology on about 800 hectors, whereas spread of sugarcane tissue cultured plantlets is only on 1 W hectare. One of the constraints for adoption of this technology has Ken an initial high cost of tissue cultured plants. The initial cost riot bei~4g affordable to farmers compared to the cost of traditional planting material, there is slow increase in the demand. The Tissue Culture Units also fail in reducing production cost of planting material mainly du2 to heavy initial capital investment. The private sector has good tissue culture plants, but they are not ready with relevant standardized package of practices to meet field requirement raising field crop with tissue cultured plants. Lack of an adequate R&D infrastructure at unit level makes such type of recommendation impossible to private units arm an turn, grower has accepted to use tissue cultured plaids with traditional package of practices. This situation not only leads to wastage market purchased inputs, but also reduces per rupee net return.

Potential:-
Horticulture is an emerging sector of economic importance in Maharashtra. Similarly sugarcane is also an important industrial crop of the state. Floriculture has already established i~ place in economic development of farmers of Maharashtra. Some awareness has been created in use of micro propagation plants in the cultivation of banana, and sugarcane. However, coverage under micro propagation plants is hardly to the extent of 800 hectors in case of banana and very little in case of sugarcane. Considering the area cove age of banana and inherent potential of technology, there is tremendous scope to use micro propagated plants in banana. Similarly sugarcane also offers scope for use of micro propagated plantlets on a large scale for enhancement of productivity and better sugar recovery.

(1)           Banana:-
Banana covers 60000 hector area present production of micro propagated seedlings is 36.15 lakhs in banana covering approximately 800 hectors It is proposed to cover 1000 hectors area the first year and reach the level of 5000 hector in the 5th year with an addition of 1000 ha area per annum. This would require 45 lakhs tissue cultures seedlings in the first year and the state will have to reach a production level of 225 lakhs pantalets in the 5th year. In totality this will and to a spread of tissue culture plants on an area of 15000 hectors considering the use of daughter suckers in the ration of 1:3. Thus 25 percent of banana in the state can be covered under this technology which All help to enhance the productivity of banana in Maharashtra. Even though it is possible to have larger coverage of area, high cost of planting material becomes prohibitive for average farmer. It is thus necessary therefore, to reduce the production cost so that the average farmer can have a access to this technology. One of [lie reasons for high production cost is the heavy capital investment, for which the unit holder has to, pay heavy interest. This interest can be subsidized and converted ii. to soft loan at the interest rate of.10%. This difference in the rate of interest may be borne by the state government. For strengthening the existing units a grant towards interest subsidy may be given to the units to produce additional 25 percent plants over the existing production capacity if they agree to reduce the rate by Rs.2/- per plant.

(2)           Sugarcane:-
In sugarcane the strategy for increasing are under tissue culture plants would be three fold. Initially the nurseries of breeder seed would be prepared by the factories using plants produced by using meristems from the mother source garden on 100 hector. which will require 15 lakhs plantlets © 15000 plants/hector for planting the area. The multiplication ratio being approximately 1:20, the production of sets would be around 300 lakhs sets sufficient to over an area of about 1200 hectors, under foundation stage seed production. Further the sets for this foundation stage plants would be used to produce certified stage seed sets sufficient to cover the area of 12000 hector at an expected multiplication rate of 1:10. Further the sets produced from such certified area will be distributed to farmers for growing commercial crop of sugarcane on an area of about 120000 hectors. This program would be maintained by VSI through the selected sugar factories.
Instead of producing new plants, the existing tissue culture units will have to be brought under proposed quality control mechanism to ensure production of good quality and true to type plantlets and subsequent enhanced production. However, the production capacity upgradation may be thought of after the midterm review of the program during third year.

(3)           Flowers:-
Existing laboratories are also producing flowering species. Since number of farmers for ploy house horticulture is increasing annually there is a scope for various use of tissue culture planting material of flowering plants and vegetable crops. To augment the growing requirement or planting material, production capacity of existing tissue culture units involved in production of tissue culture seedlings of flowering species like Gerbera, Carnation, Lilly, arcadias etc. will have to be increased. With the existing production of (31 30 laths flowering plants, hardly an area of 50 hect is covered V the green house. Considering the increasing number or green houses in the state there is a scope of expansion of unites for production of floriculture plants. It is proposed to reach a level of 100 hectors iii the next 5 years for which production of 65 lakhs plants is estimated. There are many small scale floriculture units already established in Maharashtra. Most of the units are importing planting material horn abroad which consumes very heavy foreign exchequer. Indigenous production of such Plants would help reducing the burden on foreign exchequer and also help to reduce capital investment on large scale iii floriculture units.

(4)           Other Potential Crops:-
There are some species of economic importance grown in the state, turmeric, ginger are the crops where produce has already been developed in the state. Micro propagation of elite varieties of these crops can be taken up in the tissue culture laboratories to benefit the farmers. Propagation of medicinal plants like Saved Musali Asparagus through tissue culture is also possible. To enhance the utility of infrastructure established for tissue culture laboratories can be diversified to range of crops so that units also run in profit.

Strategy for establishment of Tissue Culture

Laboratories
In order to meet the projected requirements. the state government will have to
(a)             Strengthen the existing units arid
(b)             Encourage & establishment of new tissue culture units.

(a)           Strengthen the existing units
On an average the existing units are running with 75 percent capacity. The production cost of tissue culture plants is very high and ranges from Ps. 5.5 to 16 depending upon the crop species. Obviously such a high cost becomes prohibitive for an average farmer. It is thus necessary therefore to reduce the production cost so that the average farmer can have a free access to this technology. One of the reasons for high production cost is heavy capital investment for which the unit holder has to pay heavy interest. This interest cap be subsidized and converted in to soft loan at the interest rate of 10.This difference in the rate of interest may be borne by the state government for a period of next three to five years.

For strengthening the existing units a grant ray be given to the units so as to produce additional 25 percent plants over the existing production capacity at a rate lower by at least 2/- per plant. This assistance could be in the form of interest subsidy on the present interest burden.

(b)           Encourage & Establishment of new tissue culture units
Establishment of a new tissue culture units will be treated at par with other industrial units so that inherent benefit of the industry will automatically flow to such units also. The zoning declared for the general industries should also be made applicable to tissue culture units with little modification treating, all zones as D± Zones. Since the pressure on industries department is very heavy for giving incentives to different small scale industries, 100% requirement of incentive amount should be made available for tissue culture units and should be placed at the disposal of agriculture department for next five years. In addition to this, there should be space reservation in the industrial area for tissue culture laboratories for at least a period of five year.

In order to make these units viable certain policy decision need to be taken up by the State Government-
i)       Tissue culture plants are charged Sales Tax. Although the producer has to pay the tax, the ultimate burden o~ this tax is shifted on the farmer. it is proposed that the sales tax on tissue culture plantlets should be waved at least for initial 10 years.
ii)      The electricity tar ii r needs to be brought at par with tariff for agriculture purpose. For this specific order from government needs to be issued with an express mention that tissue culture is an agricultural activity.
iii)    The tissue culture units extending hardening facilities in captive areas, should be given an incentive for polenta paining. Sheds at the rate or 50 percent of erection cost.
iv)   For establishment of new unit in private sector, grant of Rs. 10 lakhs may be given for the production capacity o~ minimum 5 lakhs plant per annum.
v)    The job it venture may be encouraged with the help of agriculture universities, on the governments lands like TSF,TCDs, and university farms
vi)   100 percent assistance may be given to the unit holder for preparation of a project report production capacity.
vii) Wide publicity for such incentives from government may be given in the state so that more member of entrepreneurs can avail benefit of this opportunity.

Extension Plan
The extension system today is partially successful in creating general awareness among farmers about benefits of tissue culture However, farmers are not clear about me entire package of tissue culture technology. The presumption that tissue cultured seedlings itself will increase the yield needs to be addressed in effective manner. Entire package of practices required for getting optimum yield needs to be demonstrated effectively on the farmers fields In order to spread the proper message of benefits of micro propagation, extension machinery itself needs to be equipped and trained in this area. The entire extension program for bio technology has to be organized in such a manner that extension workers and the farmers get simultaneous training and could upgrade their skills for quality production from tissue culture plantlets. Agronomic practices for tissue culture plantlets have to be finalized through research and then have to tb propagated through extension machinery of the state.

Training
Intensive training program for extension staff may be organized first to train the master trainers and generate in louse resource persons.

This training may be organized for identified persons in the institutes of repute. This training should be a season long training so that the ‘technology per-se and it’s on farm application could be simultaneously observed and studied by the trainee. In second phase this trained manpower or resource persons will train the field functionaries so that the field level workers will also be able to develop technology. During the first year, there has to be a major thrust this training of staff.

The farmers also need training in this aspects of bio technologies.

i)       It is experienced that the farmers who use tissue culture plants is may it be open land cultivation or the green house cultivation, they are not fully aware of the crop agronomy in a changed situation. Their training needs to be concentrated on the production aspects particularly the agronomy and nutritional management. Agriculture univer5ti~s may not be able to train such huge number of farmers. Private se 4cr also needs to be involved in the process of farmers training. Training farmers should be mandatory on the part of entrepreneur for which 10 percent financial assistance on the amount of effective sale may be given as assistance from the Government. This training will also help hi generating demand for tissue culture plants.
ii)      Sugar industry should also be actively involved in tissue culture technology. Sugar factories may be persuaded U establish small tissue culture laboratories in their operational area VSI can take lead and coordinate these activities.
iii)    There needs to be a regular interaction arid continuous dialogue between the develop in en t agencies, extension workers and Regular work shops, seminars, symposium needs to be organized for making farmers aware and also getting feed back from them. electronic media will play a vital role in propagating this technology There is a need to produce video films and CDs detailing the production technology along with success story.
iv)   Technology Demonstrations
Through there is an awareness about use of tissue culture in agriculture there is hardly a demonstration on tissue culture technology exhibiting total package of practices .Extension machinery and the private sector will have to be aggressive in organizing demonstration on the farmers fields .Listing out the technology generated in various crops will be the first stage in this process. Identification of user farmers will be the second stage. There should be a cross section of farming community representing the different tissue culture trails. The demonstration should be minimum 0.2 hector area so that impact of the technology will be visible.
The assistance for organizing demonstrations should be given by the state government and by respective private sector. There should be a fan mer rally on the demonstrations plots to acquaint farmers with the result of this technology. Farmers trips needs to be organized from the areas where technology adoption is low to the areas with high level of technology adoption may it be in the state or out of the stale.

Quality Control Mechanism
Although tissue culture laboratories have established in the state during last decade producing tissue culture o plants for distribution to the farmers, there is no mechanism to checked the quality of planting material produced by them. Since the quantum of tissue culture plant would be increasing at higher rate The quality of plants will have to be ensured for getting desired type of quality planting material. It should be mandatory for all the tissue culture units to register themselves with the competent authority designated by the state 90\ eminent. This will help monitoring the production and also the quality of sources of multiplication. The plants produced through tissue culture technology will also be subjected to the provisions under existing Seed laws, Rules and Orders Present enforcement agency of the state government. would be deployed to undertake regular inspections and checks to ensure supply of quality planting material. The purity standards of a particular clone/variety should be ascertained by raising the mother clones in the genetic garden which will be maintained by the state agriculture universities aid institutes like VSI. There should be a mechanism of regular inspection of laboratories by a team constituted by the Government. This team will fix the cities for the proper working of laboratory, sanitation of [lie equipment, (All production related infrastructure and undertake regular ii inspections of the production units.

One central quality testing labor Atari government in public sector.  Those LII laboratory will avail this facilities by pay as fixed by state government. One laboratory should be established by the to test the samples drawn by info amendment should be made in the exit will established by state its without the quality testing lug app operate service charges central Fever quality testing qovemnierit under public sector icei’iient autlmrity. Appropriate sting seed laws to incorporate provisions for quality control of tissue culture.

Future Research and developed needs
At present the cost of tissue culture planted are quite high due to how multiplication rates in many protocol efforts need to take place in this field for example, banana plants multiplication needs to be taken from 2.5 - 3.0 at last 5.00 Various issues regarding decrease in cost needs to be concentrated for at least next five years. Secondly agronomic practices commensurable with tissue culture technology have not been finalized so far. This need to be taken up by agricultural universities and Vasantdada Sugar institute with strategy to propagate it amongst farmer in nest five year. Thirdly new protocol. need to be firmed up and propagated for these crops which have tremendous potential for Maharashtra, those may include medicinal and aromatic plants , spices, mango and cashew nut for state ’s agro climatic conditions. Fourthly, be spelled out and quality testing facilities standardization of such quality parameters so that provision of seed Act, Rules and Seed Control Order could be effectively enforced is a pre-requisite for quality control measures, Fifth import replacement substitutes need to be developed so that quality outflow of royally could be eliminated in may of the flower varieties. If would also save precious for exchange . Finally extension methodologies and post harvest measures need to be addressed so that tissue culture technology expands and quality product reach up to the consumer end of good economic returns .