You are on page 1of 48

A PROJECT REPORT ON

“Tissue Culture as Best means of Horticulture”

Submitted in partial fulfillment for the award of the degree


Masters In Entrepreneurship And Family
Business
OF
PANJAB UNIVERSITY,Chandigarh

ACADEMIC YEAR
2010 – 2011

Submitted By:-
Submitted To:-
Jasbir Singh Ruzal Ms.Mani
Parti
Roll No- 754
Asstt.Professor
MEFB II Semester Department of Commerce
and Management
GGDSD College GGDSD
College ,
CHD CHD.
..

ACKNOWLEDGEMENT

This project report could not have been prepared, if not for
the help and encouragement from various people. Hence, for
the same reason I would like to thank my guide
Ms. MANI PARTI. It was for her support that I got proper
guidelines for preparing this project. There are many more
people I would like to thank; they are the peoples living in
our society, they helped me with the questions asked and
the answers given by them were up to the mark, they were
confident with their answers.
JASBIR SINGH

DECLARATION

I hereby declare that the project work entitled “Tissue-Culture as Best Means Of
Horticulture” submitted to the PANJAB UNIVERSITY, CHANDIGARH, is a record
of an original work done by me under the guidance of Ms.MANI PARTI and this
project work is submitted in the partial fulfillment of the requirements for the award of
the degree of Masters In Entrepreneurship And Family Business. The results
embodied in this thesis have not been submitted to any other University or Institute for
the award of any degree or diploma.
GGDSD College SIGNATURE
(Chandigarh) (JASBIR SINGH)

CONTENTS
Introduction

Plant Tissue Culture

Methods and Techniques

Major advantages of Tissue-Culture

Commercial prospect

Objective of Tissue-Culture Project

Requirements of Tissue-Culture Projects

Location

Unit Size.

Estimated Cost

Projected Benefit.

Market Development
Terms of Financial Assistance

Repayment

SUMMARY
BIBLIOGRAPHY

INTRODUCTION

Horticulture is the industry and science of plant cultivation including the process
of preparing soil for the planting of seeds, tubers, or cuttings.Horticulturists work
and conduct research in the disciplines of plant propagation and cultivation, crop
production, plant breeding and genetic engineering, plant biochemistry, and plant
physiology. The work basically involves fruits, berries, nuts, vegetables, flowers,
trees, shrubs, and turf. Horticulturists work to improve crop yield, quality,
nutritional value, and resistance to insects, diseases, and environmental stresses.
Horticulture usually refers to gardening on a smaller scale, while agriculture
refers to the large-scale cultivation of crops.The word is composite, from two
words, horti, meaning grass, originating in the Greek χορτον, meaning the same
(grass) and the word "culture".

Horticulturists can work in industry, government or educational institutions or


private collections. They can be cropping systems engineers, wholesale or retail
business managers, propagators and tissue culture specialists (fruits, vegetables,
ornamentals, and turf), crop inspectors, crop production advisers, extension
specialists, plant breeders, research scientists, and of course, teachers.

Disciplines which complement horticulture include biology, botany, entomology,


chemistry, mathematics, genetics, physiology, statistics, computer science, and
communications, garden design, planting design. Plant science and horticulture
courses include: plant materials, plant propagation, tissue culture, crop
production, post-harvest handling, plant breeding, pollination management, crop
nutrition, entomology, plant pathology, economics, and business. Some careers in
horticultural science require a masters (MS) or doctoral (PhD) degree.

Horticulture is practiced in many gardens, "plant growth centres" and nurseries.


Activities in nurseries range from preparing seeds and cuttings to growing fully
mature plants. These are often sold or transferred to ornamental gardens or market
gardens.

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).

MEANS OF HORTICULTURE

Horticulture involves Eight areas of study, which can be grouped into two broad
sections - ornamentals and edibles:

Arboriculture is the study of, and the selection, planting, care, and removal of,
individual trees, shrubs, vines, and other perennial woody plants.

Floriculture includes the production and marketing of floral crops.


Landscape horticulture includes the production, marketing and maintenance of
landscape plants.

Olericulture includes the production and marketing of vegetables.

Pomology includes the production and marketing of fruits.

Viticulture includes the production and marketing of grapes.

Oenology includes all aspects of wine and winemaking.

Postharvest physiology involves maintaining the quality of and preventing the


spoilage of horticultural crops.
Plant Tissue Culture

Plant tissue culture encompasses culturing of plant parts on an artificial medium.


The plant parts can be a single cell, tissue or an organ. It is also referred to as
micropropagation. Plant tissue culture was practically implemented for the first
time by Haberlandt, a German scientist, in 1902. Later in 1934, Gautheret found
successful results on in-vitro culture of plants. The basic key used in plant tissue
culture is the totipotency of plant cells, meaning that each plant cell has the
potential to regenerate into a complete plant. With this characteristic, plant tissue
culture is used to produce genetically identical plants (clones) in the absence o
fertilization, pollination or seeds.

Methods and Techniques of Plant Tissue Culture

In plant tissue culture, plants or explants such as pieces of leave, stem or root is
cultured in a specific plant medium, which contains essential plant nutrients and
hormones. Other plant growth factors like light and temperature are maintained
and regulated by using artificial conditions. All the procedures of plant tissue
culture are conducted under sterile (aseptic) conditions. The explants then develop
stem, roots and leaves. The generated plantlets are hardened before planting in
outdoor conditions.

To start with plant tissue culture, one should wipe his/her hands, forceps and other
equipments with alcohol or other sterilizer to prevent microbial contamination.
Following this, the explants are surface sterilized by using chemical solutions
such as bleach or alcohol. Though mercury chloride is an effective sterilizer, it is
rarely used due to its potential toxicity. After sterilization, the explants are
introduced into a plant medium, which can be either solid or liquid. The cells
divide and differentiate into plant parts, thus giving rise to a complete plant.

Speaking about the plant medium, liquid type is prepared by mixing inorganic
salts, organic nutrients (sucrose), vitamins, minerals and plant growth regulators
(auxin and cytokinin). In addition to these ingredients, solid medium contains
gelling agent (agar). Nutrient and plant hormones amount vary, depending on the
objective of plant tissue culture. For example, in order to induce more roots, auxin
amount should be high. Plant tissue culture techniques include the culture of
protoplast (a cell without cell wall), meristem, node, anther, ovule, embryo and
seed..

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.

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.

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 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 standardised through trial and error and
ultimately it should be repeatable and reliable (Annexure C).

Stages of Tissue Culture

The stages involved in propagation by tissue culture are dividend into five. A
general account of these stages is outlined below.

a 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. Activity growing (shoot tips), juvenile (seedlings) or rejuvenated
(suckers) tissues are preferred.

The commercial tissue culture labs commonly use tips of apical or lateral shoots,
which contain meristems. Meristems are made up of cells dividing actively in an
organised 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.

The quantity of explant required for propagation by tissue culture is very small.
For example, 2 mm. thick petiole sections 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.

b. Establishment of Germfree (aseptic/sterile) culture

Excised part of plant is surfaced sterilised and transferred to sterile nutrient


medium contained in glass vessel. On an average, about 50 cc. nutrient medium
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.

c. 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 shoots

In this case shoots are produced from excised shoot 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 it is preferred, because axillary shoots are likely to be gentically stable and
the chances of production of types unlike mothers are less.

ii) 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 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
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 under (a) and (b) 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.

The formation of callus can be induced by selecting proper tissue 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 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 o tissue, genotpye, 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.

d. Preparation of micro-cuttings for establishment in the natural


environment.

Young axillary or adventitious shoots are finally separated form 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 embryo's may directly develop into
seedlings.
e. Establishment in the 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

- washing of media from plantlets,

- transfer of plantlets to compost/soil in high humid green house,

- gradual decrease in humidity from 100% to normal over 3-4 weeks,

- and gradual increase in light intensity.

Plantlets during their growth in laboratory do not photo synthesise 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 more plant like existence (autotrophy) i.e., they should be in a
position to utilise 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.

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 200 - 300 C depending on species of plants. Tropical
plants may require higher temperature than temperature 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 are 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 of disease. Meristem is usually free from disease. Surface
sterilisation 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 1200 C for 15 minutes. The laboratory should be cleaned with
disinfectants. Workers should wash their hands and feet with disinfectants before
entering the laboratory. They should put on sterilised clothes. 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 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 micron present in the air..

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.

Demonstration of production and performance of elite forest tree species through


tissue culture.

Development and refinement of tissue culture protocols of horticultural and other


economically important species that are either difficult to propagate by
conventional methods or show a marked variability.

To serve as a platform for effective transfer of proven technologies to


entrepreneurs. This included providing the required infrastructure, know-how,
training etc. needed for large-scale production.

To serve as a technology resource centre for upcoming/established tissue culture


units.

To serve as a training centre for large-scale production of plant species.

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 / dug well and parking of vehicles.
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.

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.

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).

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).

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.

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.

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.

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 4, 6.3 and 6.5.

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.

All the requirements of tissue-culture projects, mentioned above could be made


available from local resources.

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 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.

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 19.05
house and green house

equipments (Annexure E)
ii Laboratory Equipments (Annexure F) 27.29
iii Furniture, Fixtures and Office Equipments 6.26
(Annexure H)
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.

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 Sale (No.) Gross Income


(No.) (Rs. lakhs)
1 10,000 NIL NIL
2 1,000,000 750,000 37.50
3 1,250,000 1,000,000 50.00

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.

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.

Terms of Financial Assistance

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.

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.

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.

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.

Security

Banks may obtain such security as per RBI norms.

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).

SUMMARY

Plant tissue culture refers to growing and multiplication of cells, tissues and
organs of plants on defined solid or liquid media under aseptic and controlled
environment. Micropropagation allows rapid production of high quality, disease-
free and uniform planting material. The micropropagation of high quality planting
material of ornamentals, and forest and fruit trees has created new opportunities in
global trading for producers, farmers, and nursery owners, and for rural
employment. The plants can be multiplied under a controlled environment
anywhere irrespective of the season and weather on a year-round basis. However,
micropropagation technology is more expensive than the conventional method of
plant propagation. It is a capital-intensive industry, and in some cases the unit cost
per plant becomes unaffordable. Hence, it is necessary to adopt strategies to
reduce production cost and lower the cost per plant. Plant micropropagation is
primarily based on rapid proliferation of tiny stem cuttings, axillary buds, and to a
limited extent of somatic embryos, cell clumps in suspension cultures and
bioreactors. The cultured cells and tissue can take several pathways. The
pathways that lead to the production of true-to-type plants in large numbers are
the preferred ones for commercial multiplication. The process of
micropropagation is usually divided into several stages i.e., pre-propagation,
initiation of explants, subculture of explants for proliferation, shooting and
rooting, and hardening. These stages are universally applicable in large-scale
multiplication of plants. The delivery of hardened small micropropagated plants to
growers and market also requires extra care.
Low-cost tissue culture technology is the adoption of practices and use of
equipment to reduce the unit cost of micropropagule and plant production. Low
cost options should lower the cost of production without compromising the
quality of the micropropagules and plants. In low cost technology cost reduction
is achieved by improving process efficiency and better utilization of resources.
Low-cost tissue-culture technology is high priority in agriculture, horticulture,
forestry, and floriculture of many developing countries for the production of
suitably priced high quality planting material.
A number of low-cost alternatives can be used to simplify various operations and
reduce the costs in a tissue culture facility. The physical components of a typical
plant tissue culture facility include equipment and buildings with preparation
room, transfer room, culture or growth room, hardening and weaning area, soil-
growing area (greenhouses, plastic tunnels), packaging and shipping area, and
related facilities such as an office, and a store for chemicals, containers and
supplies. The size of the physical components of a tissue culture facility will
vary according to its functional needs, i.e. the volume of production. Careful
planning of a facility can make large savings both in the construction costs and
day-to day operations in the facility. It is recommended that an existing facility
should be visited to view the layout and operational needs before starting a new
facility. Proper choice of media and containers can reduce the cost of
micropropagation. The composition of culture media used for proliferation has a
tremendous influence on production costs. The type of culture vessel influences
the efficiency of transfer during subculture andproduction of propagules per unit
area. The replacement of expensive imported vessels with reusable glass jars and
lids, alternatives to gelling agents, use of household sucrose, and some
components can reduce costs of production. Bulk making of media and storage as
deep frozen stocks also reduces labour costs.

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

Calathea, Hyacinth, Heliconia, Anemone, Syngonium, Begonia, Dracaena,


Eucharis, Peperomia, Caladium, Euphorbia, Carnation, Pelargonium,
Chrysanthemum, Platycerium, Gerbera, Pteris, Saintpaulia, Davallia,
Streptocarpus, Osmunda, Anthurium, Mammillaria, Aechmea, Christmas Cacti,
Cyclamen, Easter Cacti, Kalanchoe, Agapanthus, Episcia, Asparagus,
Spathiphyllum, Gloxinia, Alstroemeria, Guzmania, Hamamelis, Cordyline,
Hemerocallis, Schefflera, Dendrobium, Liriope, Cymbidium, Strelitzia, Cattleya,
Nandina, Odontoglossum, Rhododendron, Phalaenopsis, Bougainvillea, Vanda,
Buddelia, Weigela, Magnolia, Ribes, Deutzia, Epidendrum, Crocsmia, Forsythia

B. Vegetables

Onion, Asparagus, Brassica, Tomato, Chicory, Capsicum etc.

C. Fruit plants

Banana, Strawberry, Apple, Pear, Cherry, Citrus, Rubus, Gooseberry, Grapes,


Papaya, Pineapple, Ananas etc.

D. Forest species

Poplar, Eucalyptus, Bamboo, Pinus, Cupressus, Thuja, Sequoia, Ulmus, Spiraea

Betula, Salix, Ilex, Fagus, Picea etc.

E. Others

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 400

Preparation/Auto Clave
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. Particulars Area Rate Amount


(Rs.)
No (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) 3000 sq.ft. 14/sq.ft 42000

for Plantlets
(iii) Polytunnels 25 no.s each of 40 5/sq.ft 5,000.00
sq.ft.
(iv) Wirenet for polyhouse 3600 sq.ft. 36,000.00
walls
(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 1000.00

Hygrometer etc
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.lakhs)


Rs./pc (4) (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) 8 30,000 3.90

(For about 1500 sq.ft. area, with two


standby)
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 25,000 0.25
instruments
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 1 144,000

(Scientist-in-charge)
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.

Estimated Cost = Rs. 4.50 lakhs.

Annexure K

RECURRING EXPENDITURES

(Rs. lakhs)
S.No. Item YEARS
1 2 3 onwards
i Salaries and wages 5.67 11.34 11.34
ii Laboratory consumables 0.25 0.50 0.50
iii Greenhouse rooting media 0.15 0.30 0.30
iv Mother plant 0.10 0.10 0.10
v Power 1.25 3.50 4.50
vi Fuel 0.25 0.40 0.50
vii Packaging - 0.75 1.00
viii Air freight - 0.52 1.05
ix Administrative
a. Printing & Stationery 0.07 0.15 0.15
b. Postal, Telephone Telex 0.25 0.50 0.50
c. Travels 0.50 0.50 0.50
d. Books and periodicals 0.25 0.25 0.25
x Market Development
a. Foreign visits for marketing contract 1.50 1.50 -
b. Publicity abroad 0.50 0.50 0.50
c. Distribution of free samples 0.50 0.50 -
xi Repairs and Maintenance(including - 1.00 2.65
replacement of polythylene)
xii Insurance 1.06 2.12 2.12
xiii Breakage of Glasswares and bottles(10% p.a.) 0.05 0.12 0.12
xiv Contingency (includes replacement of roofing 1.00 1.00 1.00
material, LDPE for greenhouse)
13.35 25.55 27.08

Annexure L

estimated production

Sl.No. Item Details


1 No of work stations in Laminar Flow cabinets (2) 16 (in two shifts)
2 Total Floor spacein Growth Rooms 750 sq.ft.
3 Total space in 18 Racks each with 8 shelves 8x18x18
(each shelve 18 sq.ft.) = 2592 sq.ft.
4 Total No. of culture bottles (About 200 bottles/shelve) 200x8x18
= 28,800
5 No. of bottles to be used for shoot multiplication cycle 25,000
(Assumption 86% capacity utilisation)
6 No. of shoots to be produced/multiplication 125,000
cycle(Assuming multiplication ratio as 5)
7 Total production of shoots per year assuming 10 12,50,000
multiplication cycles
8 Total exportable production (Assuming 80% of Total) 1,000,000

Annexure M

financial analysis

(Rs. lakhs)

S.No Item YEARS


1 2 3-15
1 Fixed Cost 60.00 - -
2 Recurring Cost 13.35 25.55 27.08
3 Total Cost 73.35 25.55 27.08
4 Benefit - 37.50 50.00
5 PW of Cost at 15% DF 63.81 19.31 114.3

=197.42
6 PW of Benefit at 15% DF - 28.35 211.05
BCR = 1.21 239.4
7 Incremental Benefit -73.35 11.95 22.92
8 PW of Incremental -58.68 7.65 55.44

Benefit at 25% DF =4.41


9 PW of Incremental -55.41 7.07 43.71

Benefit at 30% DF = -5.63

IRR = 25+5(4.41 )

(4.41+5.63)

= 27%

Annexure N

Repayment Schedule- Plant Tissue Culture

(Amount in Rs.Lakhs)

Year Incremental Bank Loan Repayment Outstanding Surplus


benefit Outstanding at the end of
the year
Interest Principal Total
at 12% outgo
1 -73.35 55.00 - - 0.00 55.00 -79.95
2 11.95 55.00 6.60 - 6.60 55.00 5.35
3 22.95 55.00 6.60 - 13.20 55.00 9.72
4 22.95 55.00 6.60 9.00 15.60 46.00 7.32
5 22.95 46.00 5.52 10.00 15.52 36.00 7.40
6 22.95 36.00 4.32 11.00 15.32 25.00 7.60
7 22.95 25.00 3.00 13.00 16.00 12.00 6.92
8 22.95 12.00 1.44 12.00 13.44 0.00 9.48

Note: 1) Interest in the I year is paid on the III year since one year grace period
for payment of interest

2) Grace period for repayment of principal is 3 years

DISCLAIMER

"The techno-economic parameters including cost, economics, repayment schedule


and other terms & conditions are only indicative. While formulating or submitting
the projects to NABARD, the entrepreneurs/banks should revise their projects
according to the specific situation obtaining in project areas. The rates of interest
charged to ultimate borrowers, rate of interest on refinance charged to
participating banks by NABARD, quantum of refinance, etc. wherever mentioned,
are as applicable at the time of preparation of the model schemes and as such, the
latest position in this regard can be ascertained from the circulars/guidelines
issued by NABARD from time to time. Further the financial viability and
bankability of the scheme may have to be reworked taking in to account the
present tax structures and other levies wherever applicable".

BIBLIOGRAPHY
Ammirato, P.V. 1983. Embryogenesis. In: Handbook of Plant Cell Culture.
Evans, D., Sharp,
W.R., Ammirato, P.V. and Yamada, Y. (Eds.) MacMillan, New York.
Pp.82-123.
Bajaj, Y.P.S. 1995. Somatic Embryogenesis and Synthetic Seed. In:
Biotechnology in
Agriculture and Forestry. Vol. 30. Springer-Verlag, Berlin.
Bhojwani, S.S., and Razdan, M.K. 1983. Plant Tissue Culture: Theory and
Practice. Elsevier
Science Pub. Amsterdam.
Cyr, D.R. 2000. Seed substitutes from the laboratory. In: Seed Technology
and its Biological
Basis. Black, M. and Bewley, J.D. (Eds.). Sheffield Acad. Press, Sheffield.
Pp.326-372.
Cutter, E.G. 1965. Recent experimental studies of the shoot apex and shoot
morphogenesis.
Bot. Rev. 31: 7-113.
Dobrev, P., Motyka, V., Gaudinová, A., Malbeck, J., Trávnílková, A.,
Kamínek, M. and
Valková, R. 2002. Transient accumulation of cis- and trans-zeatin type
cytokinins and
its relation to cytokinin oxidase activity during cell cycle of synchronized
tobacco BY-2
cells. Plant Physiol. Biochem. 40: 333-337.
George, E.F. 1993. Plant Propagation by Tissue Culture. Part 1. The
Technology. Exegetics
Ltd., Edington.
Guri, A.Z. and Patel, K.N. 1998. Compositions and methods to prevent
microbial
contamination of plant tissue culture media. U. S. Patent No. 5,750,402.
Hanning, G.E. and Conger, B.V. 1986. Factors influencing somatic
embryogenesis from
cultured leaf segments of Dactylis glomerata. J. Plant Physiol. 123: 23-29.
Holdgate, D.P and Zandvoort, E.A. 1997. Strategic considerations for the
establishment of
microorganism free tissue cultures for commercial ornamental
micropropagation. In:
Pathogen and Microbial Contamination Management in Micropropagation.
Cassells,
A.C. (Ed.) Kluwer Acad. Pub. Dordrecht. Pp.15-22.
Jayasankar, S., Bondada, B.R., Li, Z. and Gray, D.J. 2002. A unique
morphotype of grapevine
somatic embryos exhibits accelerated germination and early plant
development. Plant
Cell Rep. 20: 907-911.
Kitzinger, E.M., Jansen Van Vuuren, R., Woodward, B., Rong, I.H., Spreth,
M.H.and
Slabbert, M.M. 1997. Elimination of external and internal contaminants in
rhizomes of
Zantedeschia aethiopica with commercial fungicides and antibiotics. In:
Pathogen and
Microbial Contamination Management in Micropropagation. Cassells, A.C.
(Ed.)
Kluwer Acad. Pub. Dordrecht. Pp.61-167.
Lee, E.K, Cho, D.Y. and Soh, W.Y. 2001. Enhanced production and
germination of somatic
embryos by temporary starvation in tissue cultures of Daucus carota. Plant
Cell Rep.
20: 408-415.
Leifert, C. and Woodward, S. 1998. Laboratory contamination management:
the requirement
for microbiological quality assurance. Plant Cell Tiss. Org. Cult. 52: 83-88.
Leifert, C. and Cassells, A.C. 2001. Microbial hazards in plant tissue and
cell cultures. In
vitro cellular and developmental biology. Planta 37: 133-138.
Mamiya, K. and Sakamoto, Y. 2001. A method to produce encapsulatable
units for synthetic
seeds in Asparagus officinalis. Plant Cell Tiss. Org. Cult. 64: 27-32.
www.nabard.com
www.rbi.com