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 PartiRoll 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

(Annexure H)6.26

iv Water supply system (Annexure G)0.63

v Training1.00

vi Consultancy / Know-how fees2.00

   56.23

B) Contingency3.77

   60.00

C) Recurring Cost (Annexure K)  

  Year 113.35

  Year 225.55

  Year 327.08

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

(No.)

Sale (No.) Gross Income

(Rs. lakhs)

1 10,000NIL 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

NH4NO31650

KNO31900

CaCL22H2O440

MgSo47H2O370

KH2PO4170

FeNaEDTA36.7

Minor Elements  

H3BO36.2

MnSO44H2O22.3

ZnSO47H2O8.6

KI0.83

Na2MoO42H2O0.25

CuSO45H2O0.025

CoCI26H2O0.025

Organic Compounds  

Thiamine HCI0.1

Inositol100.0

Nicontinic Acid0.5

Pyridoxine HCI0.5

Glycine2.0

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 Control200

 

  2. Post Autoclave Area150

 

  3. Culture Transfer Room300

 

  4. Growth Rooms    

  (i)250

 

  (ii)250

 

  (iii)250

 

  5. Change Area100 1500

       

B. Semi-Clean Area    

  6. Legwash100

 

 7. Laboratory / Media

Preparation/Auto Clave

400 

  8. Wash Area    

  (i) Bottle200

 

  (ii) Plant200

 

  9. Store (consumables)250 1150

       

C. Service Area    

  10. Office Lobby, corridor550

 

  11. Scientist Room200

 

  12. Computer Room100

 

  13. Genset Room150

 

  14. Canteen200

 

  15. Toilet150 1350

  Total   4000 Sq.ft.

  Covered Area (approx.)   5000 Sq.ft.

 

Annexure E

CIVIL STRUCTURES  

Sl.

No

Particulars Area Rate

(Rs.)

Amount

(Rs.)

1Boundary Wall (a) 480 sq.ft. 270/sq.ft. 129,600.00

2Laboratory (b) 5000 sq.ft. 300/sq.ft. 1,500,000.00

3Auxilary Structure 500 sq.ft. 200/sq.ft 100,000.00

4Polyhouse      

  (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) Trays400

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)

1Autoclave 2 184,000 3.68

2Balances 2 45,000 0.90

3pH meter 2 7,000 0.14

4Laminar airflow 2 138,700 2.77

5Distillation set 2 36,650 0.73

6Computer System   200,000 2.00

7Air-conditioners :      

  a) 1.0 tonnes 6 25,000 

  b) 1.5 tonnes (2 standby)

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

8 30,000 3.90

standby)

8Microscopes   20,000 0.20

9Bottle washing unit 1 300,000 3.00

10Media cooking unit 1 100,000 1.00

11Growth room racks (6 racks/room) 18 6,000 1.08

12Trays (4 trays/shelve) 600 80 0.48

13Trolleys 8 1,500 0.12

14Diesel Genset (62.5 KVA) 1 265,000 2.65

15Dissecting Kits and Inoculation

instruments

  25,000 0.25

16Refrigerator 1 20,000 0.20

17Air filters 2 18,000 0.36

18Oven 1 15,000 0.15

19Rotary Shaker 2 25,000 0.50

20Bottles 30,000 3 0.90

21Lab clothes - 25,000 0.25

22Washing machine 1 15,000 0.15

23Incinerator 1 25,000 0.25

24Fire fighting equipment - 20,000 0.20

25Stabilizers (10) - 28,000 0.28

26Miscellaneous Glassware - 25,000 0.25

27Tubelights for growth rooms 600 150 0.90

        27.29

 

Annexure G

Water Supply System

(Rupees)

 

1STW - 2" 9,000

2Overhead Tank (1000 litres) 6,000

3Pumpset (3 HP) 5,000

4Pump House 5,000

5Mist system for Green house 38,000

    63,000

 

Annexure H

Furniture, Fixtures and Office Equipments

Furniture and Fixtures (Rupees)  

1Tables for GM and Assitant Managers 25,000 

2Clerk 2,000 

3Lab. Tables 20,000 

4Chairs and Sofa set (visitors tables) 35,000 

5Cupboard 15,000 

6Lab. racks 5,000 

7Miscellaneous 3,000 105,000

       

8Tube light for offices,lobby etc., (25) 4,000 

9Fans (b) 6,000 

10Fax Machine 30,000 

11Telephone 10,000 

12Typewriter 15,000 

13Intercom 16,000 

14Pick up Van 440,000 521,000

  Total   626,000

 

Annexure I

Overheads  

A. Salary   Salary/year (Rs.)

1General Manager 1

(Scientist-in-charge) 

144,000

2Assistant Managers (Rs.6,000 p.m.)   216,000

  a. Laboratory - 1    

  b. Greenhouse - 1    

  c. Marketing and office - 1    

3Operators (Rs.3,000 p.m.) 16 576,000

4Helpers (Rs.1,000 p.m.) 10 120,000

5Clerk-cum-Typist 1 24,000

6Guards 3 27,000

7Driver 1 10,000

8Mechanic / Overseer 1 12,000

9Contingencies   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 Y E A R S

    1 23

onwards

i Salaries and wages5.67 11.34 11.34

ii Laboratory consumables0.25 0.50 0.50

iii Greenhouse rooting media0.15 0.30 0.30

iv Mother plant0.10 0.10 0.10

v Power1.25 3.50 4.50

vi Fuel0.25 0.40 0.50

vii Packaging -0.75 1.00

viii Air freight -0.52 1.05

ix Administrative      

  a. Printing & Stationery0.07 0.15 0.15

  b. Postal, Telephone Telex0.25 0.50 0.50

  c. Travels0.50 0.50 0.50

  d. Books and periodicals0.25 0.25 0.25

x Market Development      

  a. Foreign visits for marketing contract1.50 1.50

-

  b. Publicity abroad0.50 0.50 0.50

  c. Distribution of free samples0.50 0.50

-

xi Repairs and Maintenance(including

replacement of polythylene)

-1.00 2.65

xii Insurance1.06 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

material, LDPE for greenhouse)1.00 1.00 1.00

   13.35 25.55 27.08

 

Annexure L

estimated production  

Sl.No. Item Details

1No of work stations in Laminar Flow cabinets (2) 16 (in two shifts)

2Total Floor spacein Growth Rooms 750 sq.ft.

3Total space in 18 Racks each with 8 shelves

(each shelve 18 sq.ft.)

8x18x18

= 2592 sq.ft.

4Total No. of culture bottles (About 200 bottles/shelve) 200x8x18

= 28,800

5No. of bottles to be used for shoot multiplication cycle

(Assumption 86% capacity utilisation)25,000

6No. of shoots to be produced/multiplication

cycle(Assuming multiplication ratio as 5)125,000

7Total production of shoots per year assuming 10

multiplication cycles12,50,000

8Total exportable production (Assuming 80% of Total)

1,000,000

 

Annexure M

financial analysis

(Rs. lakhs)

 

S.No Item Y E A R S

    1 23-15

1Fixed Cost

60.00- -

2Recurring Cost

13.35 25.55 27.08

3Total Cost

73.35 25.55 27.08

4Benefit -

37.50 50.00

5PW of Cost at 15% DF

63.81 19.31114.3

=197.42

6PW of Benefit at 15% DF

BCR = 1.21

-28.35

211.05

239.4

7Incremental Benefit -73.35

11.95 22.92

8PW of Incremental

Benefit at 25% DF

-58.687.65

55.44

=4.41

9PW of Incremental

Benefit at 30% DF

IRR = 25+5(4.41 )

(4.41+5.63)

= 27%

-55.417.07

43.71

= -5.63

 

Annexure N

Repayment Schedule- Plant Tissue Culture

(Amount in Rs.Lakhs)  

 

Year Incremental

benefit

Bank Loan

Outstanding

Repayment Outstanding

at the end of

the year

Surplus

      Interest

at 12%

Principal Total

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".

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