Tissue Culture Plantlets Production Model Project Report. Demand for Tissue Cultured Plantlets- A boon for Science & Tech.

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1 Tissue Culture Plantlets Production Model Project Report Introduction Demand for Tissue Cultured Plantlets- A boon for Science & Tech. Entrepreneurs The demand for tissue culture plantlets of commercial plants both in agriculture and horticulture as well as in social forestry is growing day by day, because, the traditional method of propagation is very cumbersome and do not yield much. The emerging scenario on the growing use of tissue culture plantlets predicts that each state in our country should,at least, have ten tissue culture laboratory. It may be pointed out that tissue culture laboratory can also be used to produce biofertilisers like rhizobium, azotobacter azospirillum, phosphate solubilising bacteria culture as well as mushroom spawn culture. The approximate cost of such unit would be RS.3.0 lakh to RS.5.0 lakh for minimum economic out put. The important equipments that are necessary for setting up a tissue culture biotechnology laboratory for commercial purpose are (1) Autoclave, (2) BOD incubator, (3) Laminar Airflow Cabinet, (4) Refrigerator, (5) Air-conditioner and (6) Dehumidifier and all these are indigenously manufactured in India. Tissue Culture Technique Though the description of tissue culture technique is out of the scope of the present article, still an outline of the procedure is given below for entrepreneurs. A healthy and disease.free plant of any commercial variety having a excellent track record of performance is selected for the purpose. Suitable explants like buds, storage tissues, stem section or germinated seedlings are trimmed and then sterilized in detergent solution followed by washing in distilled water. After washing, the explants are placed on suitable culture medium of either a semisolid or liquid form under a sterile laminar airflow cabinet. The single cell could proliferate and divide to form calluses (unorganized tissue masses) which when are supplied with appropriate nutrients and hormonal conditions could then regenerate new plants. After these plantlets are brought out of test tubes are subjected to acclimatize with the soil, natural nutrients and environment. This process is called as hardening. After hardening, the plantlets will be ready for plantation. Important R/D Laboratories working on Plant Tissue Culture SI. No laboratories Plant Species 1. Regional Plant Resource Centre Nayapalli, Bhubaneswar 2. Indo-American Hybrid Seeds Bangalore Banana, Rose, Chrysanthemum Banana, Rose, Orchids, Chrysanthemums, spices. 3. BARC, Bombay Sandal 4. Central Plantation Crop Research' Institute, Coconut Kasargod, Kerala 5. Dept. of Biotechnology, Delhi University. Albizzia lebbeck, Acacia nilotica, Leucaena, leucocephalae, Bamboo 6. Haryana Agril. University Date palm 7. Hindusthan Lever Lab Coconut 8. Indian Inst. of Science, Bangalore Sandal 9. National Bureau of Plant Genetic Resources, Medicinal plants New Delhi 10. National Chemical Laboratory, Pune Bamboo, Teak, Eucalyptus

2 Biotechnology of Tissue Culture Tissue culture technology is based on the theory of totipotency i.e. the ability of a single cell to develop into whole organism. The major components of the technology include choice of explant (excised part of plant), growing of explant on a defined medium in glass vessel (in vitro), elimination and or prevention of diseases, providing appropriate cultural environment and transfer of plantlets from glass vessel to natural environment (hardening). All these constitute protocol for tissue culture. It varies from species to species and variety to variety within the same species. However, it can be standardized through trial and error and ultimately it should be repeatable and reliable. Steps of Tissue Culture The stages involved in propagation by tissue culture are divided into five. A general account of these stages is outlined below. 1. Choice of Explants - Explants could be shoot tips (meristem), nodal buds, sections from internodes, leaves, roots, centre of bulb, corms or rhizomes, or other organs. The choice depends on the species to be multiplied and the method of shoot multiplication to be followed. Actively growing (shoot tips), juvenile (seedlings) or rejuvenated (Suckers) tissue are preferred. 2. The commercial tissue culture labs commonly use tips of apical or lateral shoots, which contain meristem which are made up of cells dividing actively in an organized manner. They are about 0. 1 mm in. diameter and mm, in length. However, explants should be chosen from typical, healthy, disease free, well tested mother plants cultivated under conditions which reduce contamination and promote growth of tissues to be cultured. If necessary explants may be subjected to virus testing and elimination. The selection of mother plants is very important for commercial success of tissue propagation. The quantity of explant required for propagation by tissue culture is very small. For example, 2 mm thick petiole section from African violet (a flowering herb) could yield 20,000 plantlets per petiole (basal portion of leaf). Foreign/local collaborators with established business may agree to supply explant free of cost. 3. Establishment of Aseptic or Sterile Culture Medium- Excised part of plant is surfaced sterilized and transferred to sterile nutrient medium contained in glass vessel. On an average, about 50 cc. of nutrient medium may be added per glass vessel. The cultures are maintained in growth rooms. If there is no infection and tissues isolated from mother plants survive in the artificial environment, initiation of new growth will take place after a week or so. Thus, germ free culture is established. 4. Production of Shoot Propagules-Once growth is initiated by induction meristematic center, buds develop into shoots by multiplication of cell. There are three types of multiplication systems for production of shoots. 5. Multiplication by Auxiliary Shoots- In this case shoots are produced from excised shoots tips or nodes. Hormones (cytokinins) are used to induce multiple branching. This is the most common method followed in commercial units. However, the rate of multiplication is low. Still is preferred, because auxiliary shoots are likely to be genetically stable and the chances of production of types unlike mothers are less.

3 6. Multiplication by Adventitious Shoots - Explants such as sections of leaves, internodes or roots can produce directly adventitious shoots or other organs. This system has higher multiplication rate, but lesser genetic stability than auxiliary system. 7. Multiplication by somatic Embryos ( Embryoids )- Embryos are usually formed by the union of male and female reproductive cells (zygotic embryo) which ultimately can develop into a,young plant. Embryo-like structures can also be produced from somatic cells. Somatic embryos are independent bipolar structures and are not attached to the tissues of origin. They also can develop to form young plants like zygotic embryos. Somatic embryos may be produced directly from explants such as sections of leaves, internodes or roots on solid culture medium. The formation of young-plants mentioned above, or formation of somatic embryos, mentioned in the preceding paragraph, directly on excised plant parts occurs only in certain species. 8. The most common form of regeneration of plants occurs indirectly from callus. Callus is a mass of undifferentiated dividing cells often formed in tissues cultured in vitro. Callus may give rise either to adventitious shoots, which develop into plantlets, or somatic embryos, which develop into seedlings. Callus is formed even naturally in response to a wound. 9. The formation of callus can be induced by selecting proper tissues and culture medium. This system has the highest multiplication rate and produce complete tiny plants. One gram of explants can produce one lakh somatic embryos., Dormancy can be induced in them or they can be transformed into synthetic seeds. However, callus is genetically unstable or plants spring from it may be unlike mother plants. Such plants re known as off-types. They occur more frequently in callus culture and adventitous shoots culture as compared to axillary shoot culture. Off-types are not desirable in commercial propagation. Regeneration of shoots or intact plants by any one of the multiplication systems described above is influenced by many factors, such as composition of medium (specially concentration of growth regulators), type of tissue, genotype, ploidy level, etc. Normally multiplication cycle Le. the period from incubation of plant parts on medium to formation of shoots varies from 3 to 6 weeks. However, the process is recycled many times by sub-culturing in order to obtain required multiplication rates. After completion of a cycle, shoots are cut separately and transferred to fresh medium. Cutting is done manually by using dissecting tools in laminar flow cabinets, where the air is clean to prevent any contamination. Once the shoots are placed on fresh medium, they are transferred back to the growth rooms. Thus, it may be possible to multiply the shoots 3 to 10 times per cycle of 3 to 6 weeks duration. 10. Preparation of Micro-cutting for Establishment of the Natural Environment- Young axillary or adventitious shoots are finally separated from clusters (micro cuttings) for initiation and development of roots. After separation, they are transferred individually to a medium containing rooting hormone (auxin) and continued to be maintained in the growth rooms until the roots are formed. It may also be possible to transfer the micro cuttings directly to soil or compost in humid green house for root formation. Somatic embryos may directly develop into seedlings. 11. Establishment of Natural Environment - The most critical stage of the propagation by tissue culture is the establishment of the plantlets into the soil. The steps involved are as under:

4 1. Washing of media from plantlets, 2. Transfer of plantlets to compost/soil in high humid green house. 3. Gradual decrease in humidity from 100% to normal over 3-4 weeks, gradual increase in light intensity 12. Plantlets during their growth in laboratory do not photosynthesize and their control of water balance is very weak. They use sugar contained in medium as source of energy. They exist like bacteria (Heterotrophy). They need to be converted to be converted to more plant like existence (autotrophy) i.e. they should be in a position to utilize to carbon-dioxide from the air and solar energy for their food requirement. This acclimatization on the harsh real environment, outside artificial laboratory milieu takes place gradually. 13. Culture Environment - Environment conditions in the growth room which influence cell multiplication are light, day length and temperature. In tissue culture, light is required for synthesis of green pigment (chlorophyll) anddevelopment of organs. The range of light intensities appropriate for culture room varies from 1000 to 5000 lux. Requirement of day length would be in the range of hours. Temperature requirement varies from 20 degree - 30 degree Celsius depending on species of plants. Tropical plants may require higher temperature than temperate plants. 14. Prevention of Contamination- Prevention of contamination in tissue culture is extremely important for commercial success of the unit. The entire production can go waste if the culture is contaminated. Sugar rich culture medium, excised plant tissue and culture environment are all conducive to the growth of pathogens. Therefore, it is essential that all operations are conducted in sterile or aseptic conditions. Various stages involved in prevention of contamination are outlined below: 15. Mother plants should be grown under conditions which do not promote diseases. 16. Explants should be free of disease. Meristem is usually free from disease. Sterilization of explants in solutions of sodium or calcium hypochlorite is necessary. Heat or treatment with certain chemicals may eradicate latent viruses. 17. All equipments and culture media are sterilized by autoclaving at 15 Ib/sq inch pressure at 120 degree Celsius for 15 minutes. 18. The laboratory should be cleaned with disinfectants. Workers should wash their hands and feet with disinfectants before entering the laboratory. They should put on sterilized clothes. 19. Double distilled water should be used for washing explant and preparation of culture medium. 20. UV lamps assist in sterilization of laminar flow cabinets, hatches and instruments. 21. Air handling units are employed for growth rooms, and culture transfer rooms in order to avoid cross contamination between different areas of operation inside the clean area.

5 22. The sterile condition is obtained in laminar air flow cabinets as they are provided with special type of international standard HEPA filters. These filters remove all the dust particles of above 0.3 micro present in the air. Project Report - Tissue Culture Requirement of tissue Culture Laboratory A. Civil Structure ( presumed to be own pre-existing building or else approximately INR /- may be added to the fixed cost ) A. Clean Area Media Store and Production Control Post Autoclave Area Culture Transfer Room Growth Rooms Change Area B. Semi-Clean Area Leg-wash Laboratory/Media preparation/ Autoclave Wash Area Bottle Plant C. Service Area Office Lobby, corridor Scientist Room Computer Room Genset Room Canteen Toilet Other Requirements Auxillary Structures Guard Room, Garage Polyhouse a. Mother Plant area b. Hardening area for Plantlets c. Thermometer d. Hygrometer e. Air cooler Assuming gross area of 140 ft. X 100 ft. i.e.14,000 sq.ft and Includes cost of electrical wiring, plumbing, architects fees, fees to statutory authorities, Electricity Distribution Company etc. N.B : Details of each & every item must be worked out meticulously. Estimated costs are to be supported by designs, rate analysis and quotations wherever necessary.

6 B: Laboratory Equipment Sl. No Items Requirement Approx. cost in INR Autoclave Electronic Balances PH meter Laminar airflow Distillation set Computer System Airconditioners: tones tones - 2 Microscopes Bottle washing unit Media cooking unit Growth room racks (6 racks/room) Trays (4 trays/shelve) Trolleys Diesel Genset (62.5 KV A) Dissecting Kits and Inoculation instruments Refrigerator Air filters Oven Rotary Shaker 20. Bottle Washing machine Incinerator Fire fighting Stabilizers Miscellaneous Glasswares Fluorescent Lights for Growth rooms Total C: FURNITURE & FIXTURE / OFFICE EQUIPMENTS Tables for GM and Assistant Managers Clerk tables Lab Tables Chairs and Sofa set (visitors 1Sofa +10Chairs area) Cupboard Lab. Racks Fluorescent light for offices, lobby etc. Fans (6) Fax Machine Telephone Office PC with printer, Scanner Pick up Van Total

7 D: Water Supply System 1. Shallow Tude Well -4" dia 2. Overhead Tank (1000 litres) 3. Pumpset (3HP) 4. Pump House 5. Mist System for Green house E Overheads / Recurring Expenditure Salary General Manager (Scientist-in-charge) Asst. Managers Laboratory Asst. Manager Greenhouse Asst. Manager Marketing Operators Helpers Clerk-cum- Typist Guards Driver Mechanic Contingencies: Per Month Electric Bill, Telephone, POL, Hospitality for Customers, Misc. contingencies, Advertisement cost TOTAL per month Total Project Cost: Fixed Cost : A B C D = Recurring Cost: E X three months= X 3 = POWER REQUIREMENT (1) Fluorescent lights for growth Room Assuming 32 fluorescent lights/rack (4 in each shelve, total number of fluorescent lights for 18 racks in 3 rooms is 576, say 600. b. Therefore 600 X 40 watt X 18hr. X 365 days= 1,57,680 k Watt hr say 1,58,000 units. 2. Airconditioners A.C. 1 tone, 6 x 1.5 K.W. = 9KW A.C. 1.5 tone 6 x 2.5 KW = 15 KW = 24KW Therefore 24 KW x 24 hrs x 365 days =2,10,240 KW hr. 2,10,000 units. 3. Exhaust Fan for Green house 4 no. of fans (24" ) x 0.5 KW x 12 hr. x 365 days= 8760 units, say 9000 units 4 General Lighting 25 (No. of tubelights x 40 x 12 hr x 365 days = 3600 units

8 Fans 6 x 60 x 12 hrs x 300 = 1300 units Total 4900 units, say 5000 units Total = lakh units =3.82 lakh units Cost may be calculated on the basis of prevailing electricity tariff for industries. Estimated Production 1. No. of work station with Laminar Airflow 16 Nos. ( for 2 shifts) No. of Flow cabinet 2 2. Total Floor space in Growth Rooms 750 sq.ft. 3. Total space in 18 Racks each with 8 shelves 8x18x18 = 2592 ft 3 each shelve 18 ft 3 4. Total No. of culture bottles - About X8X18 = 28,000 bottles per shelve. 5 No of bottles to be used for shoot 25,000 multiplication cycle - Assumption: 86% capacity utilization 6. No. of shoots to be produced/ multiplication 1,25,000 cycle - Assuming multiplication ratio as Total production of shoots per year assuming 10 multiplication cycles Total exportable production -Assuming 80 % of Total. 12,50,000 10,00,000 Financial analysis including Break Even Analysis may be done as per the present prices and tariff.