The success of Horticulture development hinges on selection of desired types of plants and their multiplications. Selection of desired types is based on evaluation of the quantitative and qualitative performance of plants and also in some cases their aesthetic appeal. Over the years, the horticulturists have developed various techniques for selection of desired types of plants and their multiplication. Recently interesting developments have taken place in the field of plant multiplication which involves culture of cells or tissues in laboratory.
Traditionally, horticultural plants are multiplied by means of seeds (sexual propagation) or organs other than seeds (asexual or vegetative propagation). These organs are usually stems, leaves or roots. Though multiplication by seeds is the cheapest method, it suffers form certain disadvantages. Plants raised from seeds may not repeat good performance of mother plants. Many horticultural plants take a long time to produce seeds/fruits and many of them do not produce viable seeds or desired quality of seeds. Plants propagated vegetatively do not suffer from these disadvantages. However, vegetative propagation is rather a slow, time and space consuming process. Besides, it is usually infected with latent diseases. Some plants are also not amenable to vegetative method of propagation, for example, coconut, papaya, oil palm, clove etc.
Therefore, scientists started a quest for an alternative method of plant propagation which could overcome the disadvantages of both the methods described above. After many trials and errors in the sixties, plant propagation by tissue culture method, which could overcome disadvantages of propagation by seeds or vegetative organs, was found commercially successful in the case of orchids. Subsequently, the method has been perfected for many other plants (Annexure A). The method (also known as micro-propagation) involves the culture of whole organism from cells or tissues or plant parts in glass (in vitro) on a defined medium under germ free conditions (sterile or aseptic), whereas conventional method of vegetative propagation (macro-propagation) involves culture of parts into whole organisms in natural conditions (in vitro).
2. Major advantages of Tissue-Culture :
A large number of true to the type plants could be propagated within a short time and space and that too throughout the year. For example, it may be possible to propagate 2-4 lakhs of Tissue Cultured Plants (TCP) from a single bush of rose against 10 to 15 plants by vegetative means. Also, it may take about 2-4 months to produce healthy planting materials by tissue culture means, whereas a minimum of 6-8 months is required for most species by the latest method of vegetative propagation.
Tissue-culture could be a useful way of circumventing or eliminating disease which can accrue in stock plants.
TCPs may have increased branching and flowering, greater vigour and higher yield, mainly due to the possibility of elimination of diseases.
The method may succeed to propagate plants where seeds or vegetative propagation is not possible or difficult or undesirable.
The method saves space and energy. For example, 2500m2 of heated green house can be replaced by a climatised room of 10m2
The flexibility of nurseries can be improved. As the capital investment on mother plants is reduced to almost zero, it may be easier to adapt to changing conditions. Additionally, a better programming of the production is possible, because of the greater plant uniformity and the availability in the mass at any time.
Tissue culture can be utilised for breeding new varieties, preservation of germplasm and in vitro synthesis of metabolites.
3. Commercial prospect
Propagation by tissue-culture offers good commercial prospect in ornamental plants, vegetables and also fruit plants, where value of the products is high. The technique has reportedly been successful in more than 100 species of plants. It has been estimated that more than 350 million TCPs are being produced annually through tissue culture.
In India there are at least ten commercial organisations, which have developed technical competence for tissue culture with or without foreign tie-ups. The present installed capacity is about 50 million TCPs and the export is of the order of Rs. 5 crores. The working group appointed by the Ministry of Commerce has proposed an export target of Rs. 30 crores i.e. about 60 million TCPs over a five year period as against the present production of 8-10 million TCPs. Tissue culture method of propagation is highly labour intensive, 55-60% of the cost is on account of labour. India's potential for export of tissue culture plants is rated very high because of abundant and cheap labour.
The domestic market for TCPs, though nascent at present, is likely to develop, since tissue culture method of propagation can multiply an elite plant very rapidly. It is well known that one of the major constraints of horticultural development in our country is inadequate availability of quality planting materials. The demand of planting materials for major fruit crops has been estimated to be of the order of Rs. 175.4 crores during the Eighth Plan period.
It may not be possible to meet this requirement by conventional nurseries. It would, therefore, be desirable to encourage commercial tissue culture labs to supplement the production of planting material by conventional means.
4. Tissue-Culture Technology
The details of technology are given in the Annexure O.
5. Objective of Tissue-Culture Project
The primary objective 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.
6. 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:
6.1 Land:
It is required to set up laboratory, mother plant unit, green house and office. Space may also be required for installing tube well / dug well and parking of vehicles.
6.2 Source of technology:
It would be evident from the general outline of the technology, given in the Annexure- O 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 are recruited, it may be possible to set up such units without any tie-up.
6.3 Mother Plants:
Mother Plants would serve as source of tissues (explant). 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 cost. Otherwise, collection, maintenance and testing of superior mother plants would be necessary.
6.4 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 D).
6.5 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 (Annexure B).
6.6 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.
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.
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.
6.7 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% export- oriented projects, it may be possible to export the plantlets directly from the laboratory without subjecting them to hardening.
6.8 Electricity:
As it would be evident form 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.
6.9 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.
6.10 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 4, 6.3 and 6.5.
6.11Skilled 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.
All the requirements of tissue-culture projects, mentioned above could be made available from local resources.
7. 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.
8. 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 million to 20 million TCPs per year. The size envisaged in the present model is 1.25 million TCPs per year. It has been estimated that, to produce 1.25 million TCPs, a laboratory of 5000 sq. ft. would be required. A green house facility of 5000 sq. ft. for maintenance of mother plants and hardening tissue cultured plants would be helpful.
9. Estimated Cost.
The estimated cost for production of 1.25 million plants is indicated below:
No.
Particulars
Cost (Rs. Lakhs)
A)
Fixed Cost
i
Tissue culture laboratory including green house and green house
equipments (Annexure E)
19.05
ii
Laboratory Equipments (Annexure F)
27.29
iii
Furniture, Fixtures and Office Equipments (Annexure H)
6.26
iv
Water supply system (Annexure G)
0.63
v
Training
1.00
vi
Consultancy / Know-how fees
2.00
56.23
B)
Contingency
3.77
60.00
C)
Recurring Cost (Annexure K)
Year 1
13.35
Year 2
25.55
Year 3
27.08
D)
Capital Cost (Rs.60.00 + Rs.13.35 Lakhs)
73.35
The recurring expenditure of only 1st year may be capitalised. Thus, the total cost may amount to Rs. 73.35 lakhs.
The estimated cost does not include cost of land. For 100% export-oriented units, land cost, may be included in the above estimate depending on merit of each case subject to a limit.
However, it may be noted that above estimates are subject to actual drawings and rate analysis by competent architects for all civil structures and quotations from accredited dealers for all equipments, furniture, etc.
10. Projected Benefit.
Of the total production of 12,50,000 TCPs, about 10,00,000 TCPs (80%) may be expected to be of exportable quality.
According to a research report of the University of Georgia, U.S.A., published in 1988, the price of TCPs in U. S. A. varied as under:
Stage III -- US$ 0.16 - 0.30/unit depending on species.
Stage IV -- US$ 0.29 - 0.40 " " "
Stage V -- US$ 0.39 - 0.69 " " "
Therefore, a net average price of Rs. 5.00/unit (net of expenses abroad on transport, commission, etc.) may be considered as reasonable.
The setting up of laboratory and other facilities and standardisation of the tissue-culture protocol may take about one year. However, 20,000 TCPs may be produced in the 1st and 2nd year for distribution as free samples.
Therefore, no income has been projected in the 1st year. Thus the benefit ( Annexure L) is projected as under:
Year
Total Production (No.)
Sale (No.)
Gross Income (Rs. lakhs)
1
10,000
NIL
NIL
2
1,000,000
750,000
37.50
3
1,250,000
1,000,000
50.00
11. Market Development
The commercial prospect of tissue culture has been mentioned under paragraph 3. Presently, export-oriented units in tissue-culture enter into buy back arrangements with foreign collaborators. Under these arrangements high cost equipments are imported and high fees are paid on know-how even though these are locally available. The buy-back is available only for a limited period of 2-3 years.
In the present model it has been assumed that the beneficiaries will develop their overseas markets by visits, publicity, distribution of free samples, etc. Since all materials, equipments and know-how are locally available, it might be possible to produce high quality TCPs at a comparatively low cost.
12. Financial Viability
Financial analysis based on Discounted Cash Flow Technique (Annexure M) indicates that the project is financially viable, as would be evident from the following data:
NPW at 15% DF -- Rs. 41.98 lakhs
BCR " " -- Rs. 1.21 lakhs
IRR " " -- 27%
13. Financial Assistance
The tissue-culture export-oriented projects are eligible for refinance support by NABARD. Banks may provide loan for the activity provided the scheme is technically feasible and financially viable.
14. Terms of Financial Assistance
14.1 Nature of Beneficiaries
The beneficiaries under the project could be qualified professional entrepreneurs/sole proprietary concerns/partnership firms/public and private limited companies/co-operatives.
14.2 Margin Money
The entrepreneurs should meet 25% of the project cost out of their own resources. However, NABARD could consider selectively, provision of margin money assistance on soft terms as per the guidelines, vide circular No. DPD. 67/92-93 (Ref:No.3708/NFS. 85/92-93) dated 27 February 1993.
14.3 Interest Rate
Interest rate will be as indicated by RBI / NABARD from time to time. The current rate of interest of the ultimate beneficiary is 15% p.a. for the size of loan exceeding Rs. 2 lakhs. However, the repayment programme has been worked out at 17% interest to take care of interest tax and other charges.
14.4 Quantum of Refinance
In view of priority attached to export, NABARD would provide refinance to the extent of 90 per cent of the loan for all 100% EOPs.
14.5 Security
Banks may obtain such security as per RBI norms.
15. Repayment
The interest payment will start from 2nd year and the repayment of principal from the 4th year. The entire loan with interest may be repayable over a period of 8 years. (Annexure N).
Annexure A
Plant Species Reported to respond to propagarion by Tissue Culture
A. Ornamental plants
Philodendron, Gladiolus, Diffenbachia, Lily, Monstera, Kalanchoe, Maranta, Pentunia, Alocasia, Narcissus, Aloe vera, Rose, Fern, Mimosa, Ficus benjamina, Gypsophila, F. elastica, Aster, F. lyrata, Aglaonema, Ficus robusta, Hydrangea, Ficus mini,Amarylis, F. compacta, Nerine, F. foliole,Tulip, Iris, Nephrolepis, Freesia
Coffee, Cardamom, Ginger, Turmeric, Vanilla, Hops, Oil Palm etc.
Murashige and skoog medium
Major Elements
mg/1
NH4NO3
1650
KNO3
1900
CaCL22H2O
440
MgSo47H2O
370
KH2PO4
170
FeNaEDTA
36.7
Minor Elements
H3BO3
6.2
MnSO44H2O
22.3
ZnSO47H2O
8.6
KI
0.83
Na2MoO42H2O
0.25
CuSO45H2O
0.025
CoCI26H2O
0.025
Organic Compounds
Thiamine HCI
0.1
Inositol
100.0
Nicontinic Acid
0.5
Pyridoxine HCI
0.5
Glycine
2.0
Glusose/Sucrose
Agar
Auxins (IBA/NAA)
Cytokinins (BAP/Kinetin)
Annexure D
TISSUE CULTURE LABORATORY
Civil structures
A.
Clean Area
Floor Area
(Sq.ft.)
1. Media Store and Production Control
200
2. Post Autoclave Area
150
3. Culture Transfer Room
300
4. Growth Rooms
(i)
250
(ii)
250
(iii)
250
5. Change Area
100
1500
B.
Semi-Clean Area
6. Legwash
100
7. Laboratory / Media
Preparation/Auto Clave
400
8. Wash Area
(i) Bottle
200
(ii) Plant
200
9. Store (consumables)
250
1150
C.
Service Area
10. Office Lobby, corridor
550
11. Scientist Room
200
12. Computer Room
100
13. Genset Room
150
14. Canteen
200
15. Toilet
150
1350
Total
4000 Sq.ft.
Covered Area (approx.)
5000 Sq.ft.
Annexure E
CIVIL STRUCTURES
Sl.
No
Particulars
Area
Rate (Rs.)
Amount
(Rs.)
1
Boundary Wall (a)
480 sq.ft.
270/sq.ft.
129,600.00
2
Laboratory (b)
5000 sq.ft.
300/sq.ft.
1,500,000.00
3
Auxilary Structure
500 sq.ft.
200/sq.ft
100,000.00
4
Polyhouse
(i) Mother plant area (c)
2000 sq.ft.
14/sq.ft.
28,000.00
(ii) Hardening area (c)
for Plantlets
3000 sq.ft.
14/sq.ft
42000
(iii) Polytunnels
25 no.s each of 40 sq.ft.
5/sq.ft
5,000.00
(iv) Wirenet for polyhouse walls
3600 sq.ft.
36,000.00
(v) Sunshade net
2000 sq.ft.
2/sq.ft.
4,000.00
(vi) Exhaust Fans
4 nos.
5000/pc
20,000.00
(vii) Trays
400
100/pc
40,000.00
(viii) Thermometer
Hygrometer etc
1000.00
1,905,600.00
Say Rs.19.05 lakh
(a) Assuming gross area of 140 ft. x 100 ft. ie. 14,000 sq. ft.
(b) Includes cost of electrical wiring, plumbing, architects fees, fees to Statutory authorities, Electricity Boards etc.
(c) For details of the estimates, vide model on carnation project.
N.B.: Estimated costs are to be supported by designs, rate analysis and quotations wherever necessary.
ANNEXURE F
LABORATORY EQUIPMENTS
Sl.No.(1)
Particulars (2)
No. (3)
Rate Rs./pc (4)
(Rs.lakhs) (5)
1
Autoclave
2
184,000
3.68
2
Balances
2
45,000
0.90
3
pH meter
2
7,000
0.14
4
Laminar airflow
2
138,700
2.77
5
Distillation set
2
36,650
0.73
6
Computer System
200,000
2.00
7
Air-conditioners :
a) 1.0 tonnes
6
25,000
b) 1.5 tonnes (2 standby)
(For about 1500 sq.ft. area, with two standby)
8
30,000
3.90
8
Microscopes
20,000
0.20
9
Bottle washing unit
1
300,000
3.00
10
Media cooking unit
1
100,000
1.00
11
Growth room racks (6 racks/room)
18
6,000
1.08
12
Trays (4 trays/shelve)
600
80
0.48
13
Trolleys
8
1,500
0.12
14
Diesel Genset (62.5 KVA)
1
265,000
2.65
15
Dissecting Kits and Inoculation instruments
25,000
0.25
16
Refrigerator
1
20,000
0.20
17
Air filters
2
18,000
0.36
18
Oven
1
15,000
0.15
19
Rotary Shaker
2
25,000
0.50
20
Bottles
30,000
3
0.90
21
Lab clothes
-
25,000
0.25
22
Washing machine
1
15,000
0.15
23
Incinerator
1
25,000
0.25
24
Fire fighting equipment
-
20,000
0.20
25
Stabilizers (10)
-
28,000
0.28
26
Miscellaneous Glassware
-
25,000
0.25
27
Tubelights for growth rooms
600
150
0.90
27.29
Annexure G
Water Supply System
(Rupees)
1
STW - 2"
9,000
2
Overhead Tank (1000 litres)
6,000
3
Pumpset (3 HP)
5,000
4
Pump House
5,000
5
Mist system for Green house
38,000
63,000
Annexure H
Furniture, Fixtures and Office Equipments
Furniture and Fixtures (Rupees)
1
Tables for GM and Assitant Managers
25,000
2
Clerk
2,000
3
Lab. Tables
20,000
4
Chairs and Sofa set (visitors tables)
35,000
5
Cupboard
15,000
6
Lab. racks
5,000
7
Miscellaneous
3,000
105,000
8
Tube light for offices,lobby etc., (25)
4,000
9
Fans (b)
6,000
10
Fax Machine
30,000
11
Telephone
10,000
12
Typewriter
15,000
13
Intercom
16,000
14
Pick up Van
440,000
521,000
Total
626,000
Annexure I
Overheads
A.
Salary
Salary/year (Rs.)
1
General Manager 1
(Scientist-in-charge)
144,000
2
Assistant Managers (Rs.6,000 p.m.)
216,000
a. Laboratory - 1
b. Greenhouse - 1
c. Marketing and office - 1
3
Operators (Rs.3,000 p.m.)
16
576,000
4
Helpers (Rs.1,000 p.m.)
10
120,000
5
Clerk-cum-Typist
1
24,000
6
Guards
3
27,000
7
Driver
1
10,000
8
Mechanic / Overseer
1
12,000
9
Contingencies
5,000
1,134,000
Annexure J
Power requirement
1. Tubelights for growth room
Assuming 32 tubelights / rack (4 in each shelve, total number of tubelights for 18 racks in 3 rooms is 576, say 600.
Therefore 600x40 Watt x 18 hr. 365 days = 1,57,680 k.Watt.hr. Say 1,58,000 units.
2. Air conditioners
A.C. 1 tonne, 6x1.5 KW = 9 KW
A.C. 1.5 tonnes 6x2.5 KW = 15 KW
--------
24 KW
Therefore 24 KW x 24 hrs x 365 days = 2,10,240 KW hr. = 2,10,000 units
3. Exhaust Fans 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 hrs. x 300 days) = 3600 units
Fans 6 x 60 x 12 hrs. x 300 = 1300 units
Total = 4900 units, Say 5000 units
Total 1.58 + 2.10 + 0.09 + 0.05 lakh units = 3.82 lakh units
Consumption for misc. laboratory equipments, 0.68 lakh units (on lumpsum basis). Therefore, total consumption may be about 4.50 lakh units. Assuming electricity tariff of Rs. 1.00/unit for agricultural purposes.
