Plant Propagation By Tissue Culture – Consultancy Project Profile

1. Introduction
Plant tissue culture or micropropagation technology has made invaluable contribution to agriculture by enabling the production of disease free, quality planting material of commercial plants and fruit trees, throughout the year.  It is a technique for in-vitro growth of plantlets from any part of the plant in a suitable nutrient medium under controlled aseptic conditions.

Commercial tissue culture industry is in existence globally since the last 25 years. However, in India, plant tissue culture industry started about 15 years back and is revolutionizing the commercial agriculture sector by enabling mass propagation of quality planting material.

The success of agriculture development hinges on selection of desired types of plants and their multiplication. Traditionally, agriculture crops are multiplied by means of seeds (sexual propagation) or organs other than seeds (asexual or vegetative propagation). These organs are usually stems, roots or modified underground structures. 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 was found commercially successful in the case of orchids. Since then, it has become indispensable in propagation of many valuable crops like banana, sugarcane, papaya etc. Micropropagated plants have been well accepted by farmers all over the country because of its uniform productivity, free from disease, vigorous growth and high yield. Higher yield is contributed by increase in the yield per plant as well as larger number of plants, which can be cultivated per unit area.

 

2.Major advantages of Tissue-Culture

The main advantage of tissue culture technology lies in the production of high quality and uniform planting material that can be multiplied on a year-round basis under disease-free conditions anywhere irrespective of the season and weather. However, the technology is capital, labour and energy intensive. Although, labour is cheap in many developing countries, the resources of trained personnel and equipment are often not readily available. In addition, energy, particularly electricity, and clean water are costly. The energy requirements for tissue culture technology depend on day temperature, day-length and relative humidity, and they have to be controlled during the process of propagation. Individual plant species also differ in their growth requirements.

The commercial advantages of tissue culture technology over its conventional counterpart are summarized below:

  • Tissue culture could be a useful way for circumventing or eliminating disease, which can accrue in stock plants.
  • Tissue Culture 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. 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.
  • Enables storage and maintenance of stock plants/germplasm.

3.Commercial prospects

In India there are approximately fifty established commercial tissue culture units. Their production capacity ranges between 1 million to 5 million and above plants per annum with an aggregate production capacity of 200 million plantlets per year. Most of these tissue culture units are located in Maharashtra, Andhra Pradesh, Karnataka, Tamil Nadu and Kerala. These companies have been concentrating on exploiting local as well as the international markets.

Propagation by tissue culture offers good commercial prospect in ornamental plants, vegetables and also fruit plants, where value of the products is high.
In recent years, the demand for TCPs of elite plant varieties has grown tremendously in domestic market. Still the demand is greater for horticultural and forest species. The Working Group on Horticulture and Plantation Crops for the Eleventh Five Year Plan has projected the total requirement of planting material of fruits, coconut, cashew, black pepper, spices, arecanut etc. as 2000 million by the year 2012. 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. The technologies, which have been perfected for large-scale propagation, are as follows:

Fruits Banana, grapes, cashew, pineapple, strawberry, sapota, watermelon, apple and citrus.
Cash crops Sugarcane, potato and tapioca.
Spices Turmeric, ginger, vanilla, large cardamom, small cardamom, vanilla and clove.
Medicinal plants Aloe vera, geranium, stevia, patchouli, rosemary, gloriosa, tulsi
Ornamentals Gerbera, carnation, anthurium, lily, syngonium, cymbidium, limonium, dracena, philodendron, rose-miniature, caladium, gentiana and cactus.
Trees Teak, white teak, bamboo, eucalyptus, populus, pine and red sanders.
4. Tissue culture technology
Tissue culture technology is based on the theory of totipotency i.e. the ability of a single cell to develop into whole plant. 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.

4.1 Stages of Tissue Culture

Propagation by tissue culture is divided into five stages. A general account of these stages is outlined below.

4.1.1 Choice of explant Explants could be shoot tips (meristem), nodal buds, sections from internodes, leaves, roots, centres of bulbs, 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 tissues (suckers) are preferred.

The commercial tissue culture laboratories commonly use tips of apical or lateral shoots, which contain meristems. Meristems are made up of actively dividing cells in an organized manner. They are about 0.1 mm in diameter and 0.25 – 0.30 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 culture propagation.

4.1.2. Establishment of Germfree (aseptic/sterile) culture
Excised part of plant is surface sterilized and transferred to sterile nutrient medium contained in glass vessel. On an average, about 25 cc nutrient media may be added per glass vessel. The cultures are maintained in growth rooms. If there is no infection and tissue isolated from mother plants survive in the artifical environment, initiation of new growth will take place after a week or so. Thus, germ-free culture is established.

4.1.3. Production of shoots/propagules Once growth is initiated by induction of meristematic centres, buds develop into shoots by multiplication of cells. There are three types of multiplication systems for production of shoots.

i) Multiplication by axillary shootsIn this case shoots are produced from excised shoot tips or nodes. Commonly hormones (cytokinins) are used to induce multiple branching wherein, the rate of multiplication is low. Still it is preferred, because axillary shoots are likely to be genetically stable and the chances of production of types unlike mothers are less.

ii) Multiplication by adventitious shootsExplants 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 axillary system.

iii) 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 can also 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 under (i) and (ii) above, or formation of somatic embryos, mentioned in the preceding para, directly on excised plant parts occurs only in certain species.

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 wound.

Selecting proper tissue and culture medium can induce the formation of callus. 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 arising from it may be unlike mother plants. Such plants are known as off-types. They occur more frequently in callus culture and adventitious shoot culture as compared to axillary shoot culture. Off-types are undesirable 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 i.e., 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.

4.1.4 Preparation of micro-cuttings for establishment in the natural environment.
Young axillary or adventitious shoots are finally separated from clusters (micro cutting) 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.

4.1.5 Establishment in the natural environment
The most critical stage in propagation by tissue culture is the establishment of the plantlets in the soil. The steps involved are as under
– washing of media from plantlets,
– transfer of plantlets to compost/soil in high humid green house,
– gradual decrease in humidity from 100% to ambient levels over 3-4 weeks,
– and gradual increase in light intensity.
Plantlets during their growth in laboratory do not photo synthesize and their control of water balance is very weak. They use sugar contained in medium as source of energy. They exist like bacteria (heterotrophs). They need to be converted to more plant like existence (autotrophs) i.e., they should be in a position to utilize carbon-di-oxide from the air and solar energy for their food requirement. This acclimatization on the harsh real environment, outside artifical laboratory milieu takes place gradually.

4.2. 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) and development 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 16-18 hours. Temperature requirement varies from 20 – 30°C depending on species of plants. Tropical plants may require higher temperature than temperate plants.

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 be conducted in sterile or aseptic conditions. Various stages involved in prevention of contamination are outlined below: – Mother plants should be grown under conditions which do not promote diseases. Explants should be free from disease. Meristem, used as an explant, is usually free from disease. Surface sterilization of explants in solutions of sodium or calcium hypochlorite is necessary. Heat or treatment with certain chemicals may eradicate latent viruses. All equipments and culture media are sterilised by autoclaving at 15-lb/sq. inch pressure at 121°C for 15 – 20 minutes. Double distilled water should be used for washing explant and preparation of culture medium. UV lamps assist in sterilisation of laminar flow cabinets, hatches and instruments. 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. The sterile condition is obtained in laminar airflow cabinets as they are provided with special type of international standard HEPA filters. These filters remove all the dust particles of above 0.3 micron present in the air.

5. Objective of Tissue-Culture Project

The primary objective of tissue culture projects could be propagation of large quantity of good quality planting material from elite mother plants within a short period of time and space.

6. Requirements of Tissue-Culture Project

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, 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.  Correct design of a laboratory will not only reduce contamination, but also achieve high efficiency in work performance. Properly planned and designed laboratories can reduce both the operational and energy costs. A tissue culture laboratory must be designed to accommodate the equipment and its use in the various stages of micropropagation in the most efficient manner.

 

 

Annexure – 1(b)
Civil Structures- Specification of laboratory
  Particulars Floor Area (sq.ft)
A. Clean Area  
 1 Media Store and Production Control 200
 2 Post Autoclave Area 200
 3 Culture Transfer Room 500
 4 Growth Rooms
(i) 750
(ii) 750
(iii) 750
5 Change Area 300 3450
B. Semi-Clean Area
6 Legwash 100
7 Laboratory / Media prepration /Autoclave 500
8 Wash Area
    (i) Bottle 250
    (ii) Plant 250
9 Store (consumables) 300 1400
C. Service Area
10 Office Lobby, corridor 550
11 Scientist Room 400
12 Computer Room 200
13 Genset Room 150
14 Canteen 200
15 Toilet 150 1650
Total 6500
Covered Area (approx.)   6500 Sq.ft.

 

 

LABORATORY EQUIPMENTS
Sl.No. Particulars No. Rate per unit Total
1 Autoclave 2 475000 950000
2 Balances 4 50000 200000
3 pH meter 5 10000 50000
4 Laminar airflow 10 90000 900000
5 Distillation unit 2 150000 300000
6 Computer System 5 50000 250000
7 A.C. 1.0 tonnes 4 15000 60000
8 Microscopes 4 30000 120000
9 Bottle washing unit 1 200000 200000
10 Media cooking unit 1 200000 200000
11 Growth room racks (20 racks/room) 60 12000 720000
12 Trays (6 trays/shelve) 2880 120 345600
13 Trolleys 15 2000 30000
14 Diesel Genset (62.5 KVA) 1 265000 265000
15 Dissecting Kits and Inoculation instruments 12 15000 180000
16 Refrigerator 2 20000 40000
17 Air handling unit 2 200000 400000
18 Oven 2 25000 50000
19 Rotary Shaker 2 130000 260000
20 Bottles 200,000 5 1000000
21 Lab clothes 30 750 22500
22 Washing machine 1 18000 18000
23 Incinerator 1 25000 25000
24 Fire fighting equipment 5 20000 100000
25 Stabilizers 10 5000 50000
26 Laboratory glass wares 200000
27 Cryopreservation unit 2 250000 500000
28 Tubelights for growth rooms 1920 150 288000

 Total

7964100
Maintenance of plant & Machinery 6741600
 
FURNITURE, FIXURES AND OFFICE
  EQUIPMENTS (in Rupees)
S.No Items
1 Tables, Chairs and other furnitures 150000
2 Electrical fittings 750000
3 Tube light for offices,lobby etc., (25) 4000
4 Computer Systems (2 units) 100000
5 Fans 7500
6 Fax Machine 30000
7 Telephone 10000
8 Intercom 16000
9 Pick up Van 440000
10 Miscellaneous 10000
Total 1517500
WATER SUPPLY SYSTEM
S.No Items (in Rupees)
1 Shallow Tube Well 24000
2 Overhead Tank (1000 litres) 30000
3 Pumpset (3 HP) 30000
4 Pump House 10000

Total

94000

 

Our Happy Clients

 

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1  Mr. Ajit P. Soman Pune Futuretech Foods India Pvt. Ltd.202, Phatak Park Apts., Behind RankaJewellers,Karve Road, Pune – 411 029 Food Processing : Vacuum Fried & Vegetable Chips 15 Product is ready to introduce in market , Ready to eat vacuum fried chip product in market
2  Dr. Sandeep P. Nikam Pune ZeFi Biotech,C-2, 403, Shivaranjan Towers, Someshwarwadi, Off Baner Road, Pune – 411 045. Advanced Biotechnology : Zebrafish Model 7 Automated sample holder system for zebrafish screening Prototype manufactured and trying to resolved the operational issues
3  Mr. ChitrankBapat Pune Fabtech Technologies International Pvt. Ltd.Fablife, 208, 21, Harmony, 759/21, Lane No.4B,D.B.Deodhar Road, Deccan Gymkhana, Pune – 411 004 Production of Tetanus Vaccine using Animal Free base medium 17 Formulation of animal free base medium for vaccine manufacturing Achieved the yield of 60 – 80 LF / mL. Tetanus toxoid produced from meat free media is under animal testing at National Toxicology Centre, Pune.
4  Dr. S. B. Pandit Pune ArushiSpeciality Chemicals Pvt. Ltd. “Shreeprasad”, Bunglow No.4, ShyamsunderSociety,NaviPeth, Pune – 411 030. Amphoteric products for Leather Kelsoft AMP 10 Manufacturing of Amphoteric softener for leather industry Kelsoft AMP 30,Product is fully developed.
5  Mr. Rajendra V. Ladkat, Pune FreezCool,Shop No.1 & 2, Akash Building,396, KasbaPeth, Pune 411 011. Hot & Cold Storage 11.5 Hot and cold storage unit manufacturing technology is under Testing and trail of Developed Hot and Cold unit in summer,winter.
6  Ms. Jyotsna Ravi Gokhale Pune B-4, 305, PS Villa, Pune Solapur Road, Near Manjri Stud Farm, Pune Production of Microbial Pectinase using EichhorniaCrassipes 5 Working on different processes like media standardization, temperature etc to increase yield.
7  Dr. Mahesh Kshirsagar, Shrirampur TrimurtiAyurherbal Pvt. Ltd.Ward no.6 ShivajinagarNewasa road, Shrirampur Pin-413709 Nano-technology in Ayurveda 14 Trimurtiayuherbalpvt ltd have FDA approved Suvarnaprash product in dropper bottle ready for market also got the   FDA approval for churn , tablet , oil, capsule vale sections.
8 ShahsankWagh Pune Keshava ventures, 1002, city crown, sr. no 1/11, CTS 432(part), near Karve statue, Karve Road, Pune- 411038 Ice rink manufacturing with Aluminium Groove base 15 Ice rinks with innovative solution to bring freezing time for ice skating system,Ice rinks are manufactured as per the innovation basis and ready for commercialization.

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