CHAPTER IV RESULTS AND DISCUSSION

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1 CHAPTER IV RESULTS AND DISCUSSION In this chapter the data collected from the sample population were critically analyzed using different analytical tools and the observations were presented under the following heads: 4.1 To estimate the cost of establishment of tissue culture Lab. 4.2 To workout the economic plants of tissue culture banana plant production. 4.3 To examine survival rate of TCB plant during different stage of production. 4.4 To study the farmer s perception about TCB plants. 4.5 To develop bankable project of planting material for production TCB. 4.1 ESTABLISHMENT COST OF TISSUE CULTURE LAB The establishment cost of tissue culture laboratory of Department of Breeding and Genetics in Junagadh Agricultural University. Commercial plant tissue culture unit consists of the following components: Storage Room for Chemicals It is advisable to have a separate area for storage of chemicals, apparatus and equipments. Chemicals required in small amounts should not be purchased in large quantities as they may lose their activity, pick up moisture or get contaminated. Such problems can be overcome by purchasing small lots on a regular basis Washing and Media Preparation Room The glassware washing area should be located near the sterilization room. This area should have at least one large sink but two sinks are preferable with running tap water. Adequate workspace is required on each sides of the sink; this space is used for glassware soaking and drainage. Plastic netting can be placed on surfaces near the sink to reduce glassware breakage and enhance water drainage. The outlet pipe from the sink should be of PVC to resist damage from acids and alkalis. Both hot and cold water should be available and the water still and de-ionisation unit should be located nearby. The washing room should be swapped periodically. Mobile drying racks can be used and lined with cheesecloth to prevent water dripping and loss of small objects. Ovens or hot air-cabinets should be located close to the glassware washing and storage area. Dust-proof cabinets and storage containers should be installed to allow for easy access to glassware. When culture vessels are removed from the 26

2 growth area, they are often autoclaved to kill contaminants and to soften semi-solid media. It should be possible to move the vessels easily to the washing area. The glassware storage area should be close to the wash area to expedite storage and access for media preparation. The media preparation room should have smooth walls and floors, which enable easy cleaning to maintain a high degree of cleanliness. Minimum number of doors and windows should be provided in this room but within the local fire safety regulations. Media preparation area should be equipped with both tap and purified water. An appropriate system for water purification must be selected and fitted after careful consideration of the cost and quality. A number of electrical appliances are required for media preparation; hence, it is essential to have safety devices like fire extinguisher, fire blanket and a first aid kit in the media preparation room. A variety of glassware, plastic ware and stainless steel apparatus is required for measuring, mixing, and media storage. These should be stored in the cabinets built under the worktables and taken out for use as and when required. The water source and glassware storage area should be in or near the media preparation area. The workbench tops should be made with plastic laminate surfaces that can tolerate frequent cleaning. Media storage room should have capacity to storage the media for at least 7 days. Sterility Class 1,00,000 is desirable for media storage room Inoculation Room The most important work area is the Inoculation room where the core activity takes place. The transfer area needs to be as clean as possible with minimal air disturbance. Walls and floors of the inoculation room must be smooth to ensure frequent cleaning. The doors and windows shoube minimal to prevent contamination, but within local safety code. There is no special lighting requirement in the transfer room. The illumination of the laminar airflow chamber is sufficient for work. Sterilization of the instruments can be done with glass-bead sterilizers or flaming after dipping in alcohol, usually ethanol. The culture containers should be stacked on mobile carts (trolleys) to facilitate easy movement from the medium storage room to the transfer room, and finally to the culture room. Fire extinguishers and first aid kits should be provided in the transfer room as a safety measure. Special laboratory shoes and coats should be worn in this area. Ultraviolet (UV) lights are sometimes installed in transfer areas to disinfect the room; these lights should be used only when people and plant material are not in the room. Sterility Class 1,00,000 is desirable for 27

3 inoculation room which can be achieved through installation of pressurized air module or air handling unit Growth Room Culture room is an equally important area where plant cultures are maintained under controlled environmental conditions to achieve optimal growth. It is advisable to have more than one growth room to provide varied culture conditions since different plant species may have different requirements of light and temperature during in vitro culture. Also, in the event of the failure of cooling or lighting in one room, the plant cultures can be moved to another room to prevent loss of cultures. In the growth room, the number of doors should be minimal to prevent contamination. The culture containers can be placed on either fixed or mobile shelves. Mobile shelves have the advantage of providing access to cultures from both sides of the shelves. The height of the shelves should not exceed 2m Cost Details Profile of a self contained unit The project profile of a micro-propagation unit with an annual production capacity of 1 million plantlets is discussed below. Location The tissue culture laboratory should be preferably located in a moderated climate condition having uninterrupted supply of water and power. The tissue culture operations have to be carried out under controlled conditions of temperature. Extreme climatic condition adds to the cost of maintenance. Project cost Table 4.1: Fixed cost of tissue culture lab Sr. No. Head Cost ( Rs. In lakhs) 1 Land Building Utilities Equipment Green house and shade house 6 Miscellaneous fixed asset 0.92 Total

4 Land Approximate 4 vigha land should be adequate for setting up a TC unit with the above capacity. Cost of land is assumed at Rs Lakhs Building and civil works The building of about 2900 sq.ft includes class 35 clean rooms and areas with comfort AC for laboratory, growth rooms and office space. The following facilities would be required in the building. a) Storage room for chemicals b) Washing and Media preparation room c) Sterilization room d) Inoculation room e) Culture room The total cost is estimated at Rs Rs. 400/sft. Green house A green house of 2500 sq.ft. and a shade house of 27,000 sq.ft. have been assumed at a cost of Rs lakhs and 2.67 lakhs (total Rs. 10 lakhs) respectively. The greenhouse should be provided with heating equipment, fans and cooling systems. Equipment IV. Major equipment and instruments required for the plant are given in annexure Variable cost requirement (I) Raw material The basic inputs for the production of micro-propagated plantlets include meristems of elite and disease free plants, ready to use culture medium, sucrose and agar. (II) Manpower The unit with the proposed capacity may need people at various positions including managerial, supervisory, skilled and unskilled. 29

5 Table 4.2: Variable cost in tissue culture lab Head (Rs. lakhs) Raw Material 0.83 Manpower 0.80 Utilities (power, water) 0.15 Contingencies (marketing, office expense, repair etc) 0.14 Total 1.92 Recurring expenses (per annum) Rs lakhs Rs lakhs Capital investment for establishment of tissue culture laboratory is presented in table 4.3. establishment of TC lab is Rs lakhs which included the fixed cost, and variable cost. Table 4.3: Establishment cost of tissue culture lab Head (Rs. lakhs) Fixed cost Variable cost (3 months) 5.76 Total ECONOMICS OF TCB PLANTS The following table 4.4 provides an overview of economic of tissue culture banana plant production, in which included fixed cost of land, machinery, greenhouse, net house and office equipment as well as variable cost of raw materials, utilities (water, power), salaries, miscellaneous expenses and Interest of working capital. The analyses of economics cost of TCB is calculated for 10,000 plants. Finally, conclude the cost of TCB plant is Rs 35.57/plant. Fixed cost of tissue culture laboratory cost was described in Annexure III. Table 4.4: Economics of tissue culture banana plant production Cost Component Unit Cost (Rs.) A. Fixed Cost 1. Land (rental value) Square feet Machinery and Equipment

6 3. Greenhouse & Net House Office equipment and furniture Total fixed Cost (A) B. Variable Cost 1. Raw material Utilities Salaries Mandays Miscellaneous Expenses Interest of working capital 10% Total variable cost (B) Total Cost (A+B) Price of TCB plant (Total cost/ No. of plants) SURVIVAL RATE Survival rate of tissue culture banana in different stages like laboratory, greenhouse, net house and finally planting in the farm was presented in table The highest per cent of TCB plants survived per cent in year of 2016 and lowest per cent of TCB plants survived per cent in year of Overall observation it can be conclude that the highest loss in incurred in green house and lowest loss in farm. 4.4 FARMER S PERCEPTION For the study of farmers perception about TCB plants consider following parameter. 1. Pest tolerance 2. Disease tolerance 3. Yield potential 4. Sweetness 31

7 Table 4.5: Survival rate of TCB at the different stages Year No. of plants in lab No. of plants in greenhouse Survival in greenhouse No.of plants in nethouse Survival in nethouse No. of plants in farm Survival in farm

8 5. Cook ability 6. Lodging 7. Suckering ability 8. Finger size 9. Finger length 10. Bunch size 11. Feed potential 12. Drought tolerance 13. Maturity period 14. Ripening quality 15. Storability Table 4.6 shows farmers perception about TCB plants, it says 66 percent farmers were perceived medium and 32 per cent farmers were perceived good quality of TCB plants. Anyone farmers can t says the excellent and very poor quality of TCB plant. Table 4.6: Farmers perception about tissue culture banana Parameter Scale No. of Farmer Percentage Excellent 0 0 Good Medium Poor 1 2 Very poor 0 0 Source: Compiled from field survey 33

9 2. 2% % % Excellent Good Medium Poor Very poor Fig. 1 : Farmers perception about tissue culture banana 4.5 BANKABLE PROJECT OF TCB Aim of Tissue-Culture Project The primary aim of tissue culture projects could be propagation of large quantity of good quality planting materials from elite mother plants within a short period of time and space Requirements of Tissue-Culture Projects In line with the technology and objective of tissue cultural propagation, various facilities may also be required for such projects which are indicated below: Land It is required to set up laboratory, mother plant unit, green house and office. Space may also be required for installing tube well and parking of vehicles Source of technology It would be evident from the general outline of the technology, given in the introduction (1.7.1) that propagation by tissue culture is much more sophisticated than other types of plant propagation; A tie-up with reputed laboratories, Indian or foreign, could be helpful. However, if well qualified and experienced staff is recruited, it may be possible to set up such units without any tie-up Mother Plants Mother Plants would serve as source of tissues (explants). Their performance should be tested before use as source of explant. In case of tie-up with well established laboratories, explants from tested mother plants could be available free of 34

10 cost. Otherwise, collection, maintenance and testing of superior mother plants would be necessary Laboratory: A tissue-culture laboratory generally comprises of media preparation room, media store room, inoculation room, growth room, culture transfer room, sterilization area, washing area, etc. The floor plan should be designed to promote maximum efficiency. The design should facilitate maintenance of optimum temperature, humidity, illumination and ventilation. Inside air of laboratory should be free from dust particles (Annexure IV) Culture Media: The medium in which plant tissue grows is made up of various salts (containing all the major and micro elements essential for growth of plants), vitamins, sugars (usually sucrose) and growth regulators at appropriate concentration. Of the various constituents of the medium, the concentration of growth regulators is critical. The plant growth is virtually controlled by the ratio between two groups of growth regulators. Cytokinin group favours shoot growth, whereas auxin group favours root growth. The ratio varies between species and even between varieties within a specie. Sugar is the source of energy since tissues and shoots, while in the laboratory do not normally photosynthesize. Other nutrients perform their usual structural, functional and catalytic role. The agar is added to solidify the medium. Commercial tissue culture received major boost with the development of an improved medium in early sixties by Murashige and Skoog (Table 1.2) Equipments Propagation by tissue-culture needs a good number of laboratory equipments. The various equipments and their functions are outlined below: i) Autoclave: Sterilisation of all glass apparatus and culture media can be accomplished by means of steam generated in the autoclave. ii) Analytical/Top Pan balances: For accurate measurement of various constituents of culture media, these balances would be required. Top pan balance is used for measuring larger quantities, while analytical balance is used for measuring smaller quantities. 35

11 iii) ph meter: It is used for measuring and adjusting hydrogen ion concentration of the culture media or solution. Hydrogen ion concentration needs to be maintained accurately for achieving optimum growth of plants. iv) Laminar Air-flow cabinets: In these cabinets shoots developed on explants are separated from clusters and transferred to fresh medium under sterile condition. Inoculation can also be done here. v) Distillation sets: Water to be used for preparation of culture media should be free from all impurities and salts. This can be accomplished by double distillation of water. vi) Computer System: Computerisation of laboratory in the following aspects would be helpful. Production Planning Time scheduling of Sub-culturing. Quality control of plantlets, Growth room status, Material requirement, Market planning. vii) Air Conditioners with Stabilizers: Maintenance of desired temperatures in growth room, inoculation room/culture transfer room would be possible by air conditioning these areas. viii) Microscopes: a) Stereo Microscope: This would enable dissecting out small size meristem from shoot tips by removing the protective covers of leaf primodia. b) Compound microscope: This enables detection of bacteria and fungi in culture and plant tissues. ix) Bottle Washing Unit: Since a large number of bottles or vessels in which plants will be grown are required to be washed repeatedly before use, an automatic bottle washing unit would be helpful. x) Media Cooking Unit: Culture media, which contains all the essential nutrients, sugar and agar, needs to be cooked before use. A media cooking unit for a large scale commercial unit is, therefore, desirable. xi) Growth room racks: These hold the culture bottles in trays. They are mobile over a set of rails in order to maximise utilisation of space. 36

12 xii) Trays: Supporting structure for culture bottles/vessels. xiii) Hatches : Pass through boxes used as gateway between clean area and semi-clean area for exchanging materials. xiv) Tube lights: Fluorescent tube lights are mounted on the bottoms of the shelves so that culture bottles containing explants/growing tissues receive requisite intensity of lights. xv) Dissecting Kits These are necessary for separation of shoots and preparation of micro cuttings. Apart from the above, equipments such as refrigerator, rotary shakers, a stand by Genset, fire extinguisher, oven, air filters and furniture would be necessary. The office should have facilities such as Fax Machines, Telephone, Typewriters etc Green House In tissue-cultural propagation, green house may be required primarily for the undernoted reasons: To raise and maintain mother plants so that growth of organs suitable for tissue culture is maximum particularly in case of ornamentals. To harden the plantlets gradually in natural environment. Green house will enable the control over light intensity and humidity, which is necessary for hardening of plants. However in case of 100 per cent export- oriented projects, it may be possible to export the plantlets directly from the laboratory without subjecting them to hardening Electricity As it would be evident from the preceding paragraphs, no tissue-culture laboratory can operate without electricity. It is required for the following purpose: To illuminate tissues and shoots while they are in laboratory. To operate various equipments and facilities, which include air conditioners Water The various purposes for which water is essential are indicated below: Water for growing mother plants, hardening of plantlets, washing, canteen, toilets, etc. Distilled water for preparation of culture media and reagents Raw materials Raw materials required for tissue-culture project, apart from explant, are various constituents of culture media. These have already been discussed under paragraphs and

13 Skilled Manpower Tissue-culture is a highly skilled operation. It would, therefore, be essential that laboratory and green house workers are well qualified and experienced in the technology. Their training in well established commercial laboratories would be helpful Location Tissue culture project for export may be located near to Airports. The site should be well connected with roads. Assured source of water and supply of electricity to the site are essential Unit Size The size of tissue-culture project could be expressed in terms of the capacity of production of tissue-cultured plants (TCPs). The projects so far set up in our county with assistance from financial institutions have production capacities, which vary from 1 lakhs to 1.5 lakhs TCPs per year. The size envisaged in the present model is 1 lakhs TCPs per year. It has been estimated that, to produce 1 million TCPs, a laboratory of 4 vigha would be required. A green house facility for maintenance of mother plants and hardening tissue cultured plants would be helpful Projected Benefit. Table 4.8 describes the total production, sales and income of TCB plants of estimated 25 years. The production increase till 7 years and remains stable for rest of the years. There was 5 per cent of loss bared every year from total production. Estimated cost for first three years was Rs.15 per plant. Gross income also increases on the basis of production and sales incurred every year. Table 4.7: Production, sales and income of TCB plants Year Total Production (No.) 25,000 35,000 40,000 50,000 55,000 60,000 65,000 75,000 Sales (No.) 23,750 33,250 38,000 47,500 52,250 57,000 61,750 71,250 Gross Income (Rs. lakhs)

14 Financial Viability Financial analysis based on Cash Flow Statement indicates that the project is financially viable, as would be evident from the following tables 4.8 and 4.9. The estimated cost for production of 1 lakh plants per year. The estimated cost does not include cost of land. Recurring cost increases 1 per cent every year based on previous year and production of plants based on availability of raw materials and manpower. NPV is the sum of the present value of all future cash flows. The present value refers to discounted value of cash flows at future dates. A project is considered for investment if its NPV is positive. NPV was calculated for this project is Rs lakhs. Break-even is the point of balance making neither a profit nor a loss. The break-even point is achieved when the generated profits match the total costs accumulated till the date of profit generation. Establishing the break-even point helps businesses in setting plans for the levels of production which it needs to maintain be profitable. In the project break-even point reached at lakh plants of tissue culture banana. Generally speaking, higher the ratio better, here, DSCR was more than 1. DSCR was calculated for first year, it is 6.52 per cent so this project is financial viable. Overall concluded the project is financially viable through NPV, BEP, and DSCR. 39

15 Table 4.8: Investment expenses of tissue culture lab Sr. Details No A Investment Cost 1 Land Building Utilities Machinery and equipment Green house and net house 10 6 Miscellaneous fixed asset 0.92 Total

16 Table 4.9: Cash flow statement of tissue culture banana plants production (Year 1-6) Sr. No. Details Year B Sales Revenue Total Revenue C Cost of Production Raw material purchase Total cost of production D Other Expenses Salary Power charges

17 Maintenances and repair Total cost of other expenses E Depreciation Cost Depreciation on machinery and equipment Depreciation on greenhouse & net House Depreciation on Office equipment and furniture Total cost of depreciation F PBIT (Profit Before Interest and Tax) G Interest (15%) H PAT (Profit after tax) or Cash flow I Tax (() %)

18 J Net Cash flow (Profit) K Cumulative Cash Flow Table 4.10: Cash flow statement of tissue culture banana plants production (Year 7-25) Sr. No. Details Year B Sales Revenue Total of B C Cost of Production Raw material purchase

19 Total cost of production D Other Expenses Maintenances and repair Power charges Salary Total cost of other Expenses E Depreciation Cost Depreciation on machinery and equipment

20 Depreciation on greenhouse & net House Depreciation on Office equipment and furniture Total cost of depreciation F PBIT (Profit Before Interest and Tax) G Interest (15%) H PAT (Profit after tax) or Cash flow I Tax ()%) J Net Cash Flow (Profit) K Cumulative cash flow

21 Net Present Value (NPV) NPV = Cumulative Cash Flow Total Investment = = lakhs Break Even Point (BEP) (BEP) = Fixed Cost Total Sales Variable cost = ( ) = lakh plants Debt Service Coverage Ratio DSCR = PAT + Interest + Non cash expenses Installm en t cost = % = 6.52 % 46

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Selection of mother plant. Preparation of explants. Fresh inoculation. Multiplication. Rooting. Planting out. Primary hardening. Secondary hardening

Selection of mother plant. Preparation of explants. Fresh inoculation. Multiplication. Rooting. Planting out. Primary hardening. Secondary hardening 7.6 Protocol for micropropagation of banana 153 PROTOCOL FOR MICROPROPAGATION OF BANANA Joy P. P., Anjana R. & Prince Jose, Pineapple Research Station (Kerala Agricultural University), Vazhakulam-686670,