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Acknowledgement .

I express my heartfelt gratitude to Mr.Sanjay chanday, President SBL Institute of Plant

Biotechnology Biotech park Lucknow, for giving me this wonderful opportunity to work in

his esteemed laboratory.

It is my great pleasure to express my heartfelt gratitude to Dr. Puneet Srivastava, Project

Coordinator, SBL Institute of Plant Biotechnology,Lucknow, for her noble guidance,

thoughtful inspiration, persistent encouragement, constitutive criticism, valuable suggestions

and guidance. He has taught me to think, stay focused and persevere. I will always be grateful

to him for all he has taught to me. It has been a great pleasure working with such a learned

and skilled guide. Words fail me to express my sincere gratitude to him.

I express my gratefulness to Mr.B.K singh and Mr.Anuj Srivastava Dr for all the knowledge

they have given me during my work and for their co-operation and devotion rendered

throughout my training

My thanks to all the members of SBL Institute of Plant Biotechnology for the kind

cooperation extended to me during my project work.

Also, I tend to gratify all my colleagues of this programme for being cooperative and helpful

in the laboratory at SBL.

I also express my sincere thanks to the Head of Department,Biotechnology,Dr.Rajesh tiwari

and all faculties of biotechnology department for their support and encouragement.

Last but not the least I thank my Parents for their everlasting support and continuous

encouragement always.

RATI GUPTA

PLACE- lucknow

DATE- 20-07-10

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DECLARATION

I, RATI GUPTA hereby declare that the project report entitled “In Vitro Studies and Micropropagation of Musa grandnain” is prepared by me under the guidance of Dr. Puneet Srivastava at SBL Institute Of Plant Biotechnology,BiotechPark,Lucknow.

DATE:20-07-10 ratiguptaPLACE-LUCKNOW Signature of Candidate

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CONTENTS

PAGE NO.

1. ABOUT SBL 4

2. LIST OF ABBREVIATION 5

3. INTRODUCTION

INTRODUCTION TO BANANA 6

OBJECTIVE OF MICROPROPAGATION 8

MICROPROPAGATION 11

4. MATERIALS AND METHODS

STERLIZATION TECHNIQUES 15

PREPARATION OF STOCK PLANTS 24

CONTAMINANTS 26

CULTURE MEDIA

STAGES OF INVITRO MICROPROPAGATION OF Musa grandnain 39

FIGURES SHOWING ALL STAGES 48

5. OBSERVATION AND RESULT 59

6. CONCLUSION 62

7. REFERENCES 63

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ABOUT SBL

Sheel Bio Tech ltd is an ISO 9001-2000 Bio-technology, Floriculture, Horticulture and Agro based company founded in 1994 with its office at New Delhi having regional offices all over India. The world class Tissue Culture Laboratory has been established with technical know-how from reputed Netherlands Company M/s Cultiss Holland B.V. is located near International Airport, New Delhi. The company is managed by experts and professionals with outstanding knowledge in the Floriculture, Horticulture, Agriculture, Biotechnology and Green Houses.

Tissue Culture Division – Currentely running two Laboratory at Gurgaon & Lucknow. The lab is biggest in private sector in Northern, Eastern and North East India with a capacity to produce 6 million plants annually and further expanding.Young Plant Nursery Division – at             Gurgoan - Haryana,               Agartalla - Tripura             Lucknow & Bahraich – U.P   Dimapur - Nagaland            Guwahati - Assam,                 Aizwal - Mizoram

Green House Division - Sheel Bio-Tech is a leading company in the design, engineering and production of greenhouses for all crop requirements and climatic conditions with satisfied customers across length and breadth of the country.

Import Division – Importing bulbs of Tulips, Gladilous, Lilium, other flower bulbs and plants of Alstroemeria, Dendrobium, Cymbidium, Anthurium & Strawberry from best breeding companies across the globe.Representing exclusively in India various Holland & New Zealand Breeders.

Project Division – Establishment of turn key projects i.e. Plant Health Clinic, Leaf Analysis Lab, Tissue Culture lab & Model Floriculture Centre.

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LIST OF ABBREVIATION

SL.NO ABBREVIATION CHEMICAL NAME

1 MS Murashige and Skoog,s medium

2 IAA Indole-3-acetic acid

3 IBA Indole-3-butyric acid

4 NAA 1-naphthylacetic acid

5 2,4-D

2,4-dichlorophenoxyacetic acid

6 2,4,5-T 2,4,5-trichlorophenoxyacetic acid

7 BAP 6-benzylaminopurines

8 Kn Kinetin

9 GA3 Gibberellic acid

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INIBAP International Network for the Improvement of Banana and plantain

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INTRODUCTION

Banana is one of the most important major fruit crops grown in India. In respect of area and production, it ranks second only to mango in this country. The banana culture in India is as old as Indian civilization.

It seems that it is one of the earliest fruit crops grown by mankind at the dawn of civilization. In India, bananas are so predominant and popular among people that poor and rich alike like the fruit.

Considering the nutritive value and fruit value of bananas, it is the cheapest among all other fruits in the country. Considering the year round availability of fruits, unlike the seasonal availability of other tree fruits, it has become an inevitable necessity in any household in India, for all functions.

The bananas were grown in Southern Asia even before the prehistoric periods and the world's largest diversity in banana population is found in this area. Hence, it is generally agreed that all the edible bananas and plantains are indigenous to the warm, moist regions of tropical Asia comprising the regions of India, Burma, Thailand and Indo China.

ORIGIN AND DISTRIBUTION

The edible banana is believed to have originated in the hot, tropical regions of South-East Asia. India is believed to be one of the centres of origin of banana. Its cultivation is distributed throughout the warmer countries and is confined to regions between 300N and 300S of the equator.

Banana is also grown, in many other countries of the world namely Bangladesh, the Carribbean Islands, the Canary Islands, Florida, Egypt, Israel, Ghana, Congo, South Africa, Fiji, Hawaii, Taiwan, Indonesia, the Philippines, South China, Queensland and Sri Lanka.

The highest acreage of bananas is in Africa, where bananas reach their maximum importance as starchy food. They are the staple food of the Buganda in Uganda, the Wahaya in Bukoba and the Wachagga in Tanzania.

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IMPORTANCE OF BANANA

Bananas are put into varied uses in India, especially in South India. Almost every part of the plant is used someway or other.

In South India, though hundreds of banana varieties are existing, only eight to ten have attained the status of commercial importance.

The fruits are used for desert purposes as well as culinary purposes. The varieties like Poovan, Rasthali, Robustra, Dwarf Cavendish are grown for table purpose.

The plantains and Monthan group yield fruits which are high in starch content and hence they are used as staple food in some of the African countries.

In India, Nendran is grown mainly for table purposes and for making 'Chips'. The Monthan fruits, both immature and mature ones, are used for culinary purposes as it mixes well with other vegetables in delicious 'curry' preparations of South Indian house holds.

In South India, no festive decoration are said to be complete unless the entrance gates are decorated with full grown banana plants with attractive bunches.

In all auspicious occasions in the life of a South Indian, banana has got important place either as a table fruit or as an offering to God in temples.

The banana male buds are harvested soon after the female phase is over and are sold as a vegetable in the markets.

Similarly, the tender stem, which bears the peduncle, is extracted by removing the leaf sheaths of harvested pseudostem is sold as vegetable.

The corm of developing suckers, rich in carbohydrates, is also cooked and eaten in the form of different curry preparations mixed with pulses by the poor class people.

The lamina of the banana leaves are still widely used as a substitute for dinner plates and is a regular income for the growth.

The dried leaves are used for cup and hat makings by small scale industries. The pseudostem is used for paper manufacture. The dried leaf sheaths give an excellent fibre and are used in garland making as well

as for extracting fibre for cloth making. The banana pseudostem ash is said to be a sure remedy for snake poison as tribals

practice it.

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OBJECTIVE

The objective of micropropagation of banana(musa grandnain) was to obtain disease free elite variety fruit bunching in banana. These plants showed rapid multiplication and easy harvesting. The planting material is of high quality imported from israil.Micropropagation enables the supply of large quantities of plant at a specified planned date.With tissue culture propagation it is possible to propagate and supply plantings of rare and highly wanted plants.The tissue culture plantlets reproduced rapidly and gave fruit in as one year in case of banana.The field establishment is close to 90%.

TISSUE CULTURE IN BANANA INDUSTRY

Tissue culture is one biotechnology that has been extensively and productively used in the banana industry. It has revolutionized the export banana industry and has proven to be a major component in rehabilitating the banana industry of some countries in Asia.

Importance of Tissue Culture in Bananas

Rapid mass production of planting materials

Traditionally, bananas are propagated vegetatively using suckers or corms. Unlike seeded commercial cultivars of other crop species, production of planting materials through suckers take a long time and very few quantity of planting materials are produced per mother plant. Realizing the potential of in-vitro propagation, shoot-tip cultures,Meristem culture have gained popularity the world over for rapid mass multiplication of planting materials. Since the first report of banana in-vitro clonal propagation in 1960s, the tissue culture technology in banana has undergone significant improvement and now used widely in banana production worldwide.

The ability to produce big number of planting materials in relatively short period of time had allowed growing bananas in a bigger parcel of land in a given timeframe, which otherwise be limited by limited availability of suckers. With this technology, one can plan to plant the desired number of plants in a desired area of land.

Efficient distribution and transport of planting materials One distinct advantage of TC over suckers is the ease of transport of large quantity of planting materials with distance. Thousands and even millions of rooted tissue culture plant (meriplants) can be transported in long distances. For instance, in the early 1990s, millions of rooted tissue culture plantlets were sourced fromIsrael by multinational companies to plant vast areas of land in Central America. Several thousands of hectares of banana plantation in southern Philippines also used tissue culture materials from Israel in the late 1990.

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Production of disease-free planting materials

Several major diseases of bananas are known to be transmitted through vegetative planting materials, like suckers and corms. In addition, some pathogens like nematodes and those causing fusarium wilt and moko are also transmitted through the soil and root tissues. Nematodes, fungal and bacterial pathogens are not transmitted through tissue culture. While virus diseases such as BBTV, BBrMV, BSV, and CMV may be transmitted through tissue culture, a procedure maybe used to exclude infected mother plants. Antibodies of these viruses have been developed to detect their presence in a mother plant to be used in TC mass production. This process of virus indexing ensures that the TC materials are free from the pathogens.

Safe exchange of germplasm.

TC technology coupled with virus indexing allows a safe movement and exchange of germplasm. The INIBAP Transit Centre ensures that germplasms requested by various national partners are virus indexed so that germplasm exchanged are free from virus pathogens. TC is the most common material used for germplasm exchange.

Germplasm conservation

Because of seedlessness of many Musa accessions, TC is commonly used in in-vitro conservation. The INIBAP-ITC maintains more than a thousand Musa germplasms in TC under low temperature. While TC has a drawback in germplasm conservation due to its propensity to somaclonal variation as a result of in-vitro culture and repeated sub-culturing, it is still the most common method of in-vitro germplasm conservation, pending the full implementation of cryopreservation in Musa genebanking.

Tissue Culture in Crop Management

Pests and disease management

The ability of producing TC planting materials free from vegetatively transmitted pathogens such as viruses provides a disease management tactic of reducing and even eliminating the initial inocula thereby reducing level of disease incidence and severity.There are several success stories to mention about the value of disease-free TC planting materials in rehabilitating the banana industry that is ravaged by a disease. In India and China, the use of tissue culture planting materials is a very effective control tactic in reducing the incidence of banana bunchy top virus (BBTV) epidemic.

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Tissue culture in cropping systems

Annual cropping/Crop timing

Due to diseases, environmental and market considerations, annual cropping or crop timing were adopted in several countries. Traditionally, bananas are grown as perennial crop where banana farms are perpetuated from year to year through taking care of daughter plants (suckers) after harvesting the mother plant. This type of banana culture, while simple, is vulnerable to build up and subsequent damage by pests and diseases.

Moreover, in countries where the climate is peculiarly adverse at certain season, crop timing maybe resorted. Such is the case of Taiwan and the Philippines where seasonal incidence of typhoon occurs. In China on the other hand, seasonal effect of low temperature due to severe winter can also cause severe crop damage. To alleviate these problems, bananas are grown in such a time that the adverse effects of these climatic factors are minimized or avoided. Hence, annual cropping is adopted.

With this seasonal planting, or annual cropping, TC technology provides large quantities of planting materials in such a narrow window of planting time of large areas of farms.

Crop timing is also used to time the volume of production to coincide with market demands. Market demands and prices follow a certain seasonal trend. Manipulating volume of fruit harvests for this seasonal demand fluctuation require good scheduling of planting of appropriate hectarage. The use of TC planting materials in such a cropping system is essential

TC in Musa Improvement

One of the drawbacks of TC especially during the early phase of its commercial introduction in the 80s is the occurrence of soma-clonal variants. These ‘offtypes’ were mostly morphological in nature, such as shorter plants, narrow leaves, leaf deformation, etc. Refinements of TC production methods have reduced these undesirable soma-clonal variations.

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MICROPROPAGATION

Tissue culture is a term used for the growth of plants or more commonly plant parts in sterile culture. Micropropagation is a method of propagating plants using very small parts of plants that are grown in sterile culture. Micropropagation is not most likely the major use of tissue culture for molecular biologists or plant breeders. However, it is important commercially, and can be used to introduce several concepts that apply to all of tissue culture.

Plant tissue culture techniques have also helped in large- scale production of plants through micropropagation or clonal propagation of plant species. Small amounts of tissue can be used to raise hundreds or thousands of plants in a continuous process. This is being utilized by industries in India for commercial production of mainly ornamental plants like orchids and fruit trees, e.g., banana.

Using this method, millions of genetically identical plants can be obtained from a single bud. This method has, therefore, become an alternative to vegetative propagation. Shoot tip propagation is exploited intensively in horticulture and the nurseries for rapid clonal propagation of many dicots, monocots and gymnosperms.

HOW MICROPROPAGATION IS ACHIEVED

Micropropagation is usually achieved by the release and growth of pre-existing (axillary or lateral) meristems in the initial culture; this is often referred to as shoot culture. A formal definition is clonal in vitro propagation by repeated enhanced formation of axillary shoots from shoot tips or lateral meristems following culture on a medium supplemented with plant growth regulators, in particular cytokinins. The shoots produced are rooted either in vitro or out of culture (ex vitro).

STAGES OF MICROPROPAGATION

Micropropagation is now typically divided into 5 stages. Stages 1-4 were originally proposed by Murashige; Debergh and Maene added Stage 0.

1. Preparative Stage: Donor Plant Selection and Preparation2. Establishment of Explant in Culture3. Multiplication:proliferation of axillary shoots4. Pretransplant (Rooting)5. Transfer to Natural Environment

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Stage 0: Preparative Stage: Donor Plant Selection and Preparation

Explant quality and responsiveness is influenced the physiological phytosanitary condition of the donor plants.

Donor (stock) plants indexed for pathogens. Pathogen-free stock plants maintained in clean conditions (low humidity, drip

irrigation). Vigorous growth is encouraged, but not over-fertilization. Donor plants may be pretreated in certain ways.

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Stage 1: Establishment of Explant in Culture

Surface-sterilization – disinfestation: Must free explant tissues of all contaminating microorganisms, but not cause phytotoxity

Isolation of shoot tip under sterile conditions. Medium - Must contain all components necessary to nourish explant (medium

composition) and to make the explant perform as desired (PGRs). Browning of the medium: Results from oxidation of phenolics leached

out from cut surfaces of explants; often seen with adult woody species. Handled with anti-oxidants, frequent transfer.

Medium formulation is often standard, e.g. M&S, but more complex media may be necessary for smaller explants.

Medium may be semi-solid or liquid; there are advantages and disadvantages of each.

Environmental conditionso Light

o Temperature

o Relative humidity

Culture stabilization

.

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Stage2: Multiplication:proliferation of axillary shoots

Repeated enhanced axillary shoot production. Encouraged by cytokinin in the medium, alone or with a smaller amount of auxin.

Amount of cytokinin and presence and amount of auxin must be determined empirically.

Shoots harvested and shoot clusters transferred to fresh medium at frequent, regular intervals.

Number of subcultures possible from the original culture varies with species/cultivar: reduction of growth, increase in mutations.

Stage3:Pretransplant and rooting

Adventitious rooting of shoots or shoot clusters in vitro. Harvested shoots may be pretreated before rooting: prehardening, elongation,

fulfilling dormancy requirements. For root initiation in vitro, auxins are important. More dilute medium, activated charcoal may be added. Advantages of rooting after removal from culture

o Reduced cost

o Structurally and physiologically better

Stage 4: Transfer to Natural Environment

Acclimatization: Process by which physiologically and anatomically adjust from in vitro to ex vitro conditions.

Relatively slow process, may take weeks, starch reserves important. Must adjust from high to lower relative humidity (e.g. from 98-99% to 20 - 60%):

development of sufficient defenses to control water loss. Poor cutilcle development: epicuticular wax needs to be formed. Abnormal stomatal development and function.. From clean vs. presence of pathogens. Must adjust from low light to high light: from low photosynthetic competence

(heterotrophic nutrition) to photosynthetic competence.o Poorly differentiated leaf structure.

o Poorly developed chloroplasts.

o Supplied carbohydrate source to independent carbon fixation.

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STERLIZATION(ASEPTIC) TECHNIQUES

OBJECTIVE The most important and rather difficult aspect of the in vitro techniques is the requirement to carry out various operations under aseptic conditions. Bacteria and fungi are the most common contaminants observed in cultures. It is found that contaminants grow much faster than the cultured tissue and may also give out metabolic wastes which are toxic to plant tissues. Therefore, sterilization is essential.

Sterilization of Culture Vessels and Instruments

1. Glasswares, metal instruments and aluminium foil can be sterilized by exposure to hot dry air (160oC – 180oC) for 2-4 hr. in a hot- air oven. All items should be properly sealed before sterilizing.

2. Autoclaving is a method of sterilizing with water vapour under high pressure. Nearly all microbes are killed on exposure to the superheated steam of an autoclave. Normally, glasswares, cotton plugs, gauze, plastic caps, filters or pipettes are autoclaved at 121oC and 15 psi for 15-20 min. Some types of plastic labware can also be repeatedly autoclaved.

3. Flame Sterilization: Metallic instruments like forceps, scalpels, needles and spatulas are sterilized by dipping in 95% ethanol, followed by flaming and cooling.This technique is called flame sterilization. Autoclaving of metallic instruments is generally avoided as they may rust and become blunt.

4. Dry Sterilization: Now a days, in place of flame sterilization, dry sterilization of instruments using ‘steri- pots’ is practised in order to avoid instant fires caused by alcohol.

STERIIZATION OF NUTRIENT MEDIA:

4.Autoclaving: Culture media in glass containers is sealed with cotton plugs, aluminium foils or plastic closures and autoclaved at 15 psi and 121oC for 15-40 min. from the time the medium reaches the required temperature. Exposure time depends on the volume of the liquid to be sterilized. The pressure should not exceed 20 psi,a higher pressure may lead to decomposition of carbohydrates and other components of a medium.

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5.Filter Sterilization: Vitamins, amino acids, plant extracts, hormones and carbohydrates are ‘thermolabile’ and may decompose during autoclaving. So, they are filter sterilized where the solutions are passed through a bacteria proof membrane- filter under positive pressure. A ‘Millipore’ or ‘Seitz filter’ with a pore size of not more than 0.2 um is generally used in filter sterilization.

Plant tissue culture media are generally sterilized by autoclaving at 121 °C and 1.05 kg/cm2 (15-20 psi).

The time required for sterilization depends upon the volume of medium in the vessel. The minimum times required for sterilization of different volumes of medium are

listed below.

Minimum Autoclaving Time Forplant Tissue Culture Medium

*Minimum autoclaving time includes the time required for the liquid volume to reach the sterilizing temperature(121 °C) and 15 min. at 121 °C (Burger, 1988)

It is advisable to dispense medium in small aliquots whenever possible as many media components are broken down on prolonged exposure to heat. There is evidence that medium exposed to temperatures in excess of 121 °C may not properly gel or may result in poor cell growth.

Several medium components are considered thermolabile and should not be autoclaved. Stock solutions of the heat labile components are prepared and filter sterilized through a 0.22 μm filter into a sterile container.

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Sterilization of Culture Rooms and Transfer Area:

Initially, the culture rooms are cleaned by gently washing all floors and walls with a detergent soap. This is followed by carefully wiping then with 2% sodium hypochlorite solution or70% ethyl alcohol.

The transfer area is also sterilized once or twice a month by washing with a commercial brand of antifungal spirocyte. Larger transfer rooms are best sterilized by exposure to UV light, when there are no experiments in progress (since UV is harmful to eyes). HEPA filter ventilation unit can also be installed in the room.

Laminar air-flow hoods are usually sterilized by switching on the hood and wiping the working surface with 70%ethyl alcohol, 15 min. before starting any operation under the hood.

AUTOCLAVE

APPLICATION OF MOIST HEAT

It is designed to use steam under regulated pressure.It is essentially a double jacketed steam chamber equipped with devices which permit the chamber to be filled with saturated steam and maintained at a designated temperature and pressure for any period of time.

PRINCIPLE

Heat in the form of saturated steam under pressure is used.steam under pressure

provides temperatures above those obtainable by boiling. Steam is both dry and

saturated.Dry in the sense that it does not contain water droplet and saturated as it is at

a phase where it is holding all the water it can in form of vapour. Three stages of

autoclave processing are:-

i. Heating :- Time in which the autoclave reaches the desired temperature

ii. Holding :- Time duration for which autoclave maintains the

temperature for killing of microbes.

iii. Cooling :- Time which an autoclave takes to come back to normal temperature

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512 × 768 - Vertical Commercial autoclave

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The efficiency of autoclave can be checked in several ways:

Autoclave Tape

The most efficient way is to use an autoclave tape. When the autoclave tape is autoclaved, a reaction causes dark diagonal strips to appear on the tape indicating that it is autoclaved.

Biological Indicators

Biological indicators are recommended as an adjunct to the daily monitoring of autoclave cycle parameters.

Biological indicators can be used to confirm thermocouple data when checking heating profiles and validating autoclave operational parameters. They should not be used in isolation as a measure of sterilisation efficacy.

Manufacturers of biological indicators using Bacillus stearothermophilus claim that spores will survive 5 minutes at 121⁰C but not 15 minutes at 121⁰C.

Check each lot by confirming survival for 5 minutes at 121⁰C.

Biological indicators have several limitations:

• Results are retrospective (several days). Autoclaved materials may be required earlier.

• They cannot be used for checking the centre of large liquid loads. • They are less accurate in determining holding times than temperature measurement. • They cannot be used for sterilisation cycles below 121⁰C.

Chemical Indicators

There are many types of chemical indicators in use and one must check the performance of a particular type and use it in conjunction with temperature and time measurements only.

Air Removal

For efficient sterilisation exclusion of air is a primary goal. If air is present steam may become superheated with a relative humidity less that 100% and sterilisation efficiency is decreased.

Air removal is achieved by:

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1. Downward displacement of steam, or2. Evacuation by pump prior to sterilisation cycle

Downward Displacement Steriliser

As steam enters the chamber, it fills the upper areas as it is less dense than air. This compresses the air at the bottom, forcing it out through the strainer and drain pipe past the temperature sensing device to waste. Only when air evacuation is complete should discharge stop. (This can be done manually or automatically).

High Vacuum Autoclaves

High Vacuum Autoclaves are not suitable for the sterilisation of liquids and are primarily used for non aqueous materials or porous loads where air is likely to be trapped in cavities or gaps. The vacuum line in pre-vacuum sterilisers should be fitted with appropriate air filters to prevent the release of infectious aerosols into surrounding areas.

Precautions taken in operating autoclave before and after process

Excessive autoclaving should be avoided as it will degrade some medium components, particularly sucrose and agar breakdown under prolonged heating. Especially when under pressure and in an acidic environment. A few extremely thermoduraic microorganisms exist that can survive elevated temperature for sometime. But 15-30 minutes kill even those.

At the bottom of the autoclave the level of water should be verified.

Not to accelerate the reduction of pressure after the required time of autoclaving. If

the temperature is not reduced slowly, the media begin to boil again. Also the medium in the containers might burst out from their closures because of the fast and forced release of pressure.

Bottles, when being autoclaved, should not be tightly screwed and their tops should be loose. After autoclaving these bottles are kept in the laminar air-flow and the tops of these bottles are tightened on cooling.

To ensure that the air- exhaust is functioning normally. To ensure that lid of the autoclave is properly closed

LAMINAR AIR FLOW

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Laminar air hoods are used in commercial and research tissue culture labs. A horizontal laminar flow unit is designed to remove the particles from the air. Room air is pulled into the top of the unit and pushed through a HEPA (High Energy Particle Air) filter with a uniform velocity of 90 ft/min across the work surface. The air is filtered by the HEPA filter so nothing larger than 0.2 um (micrometer) can pass through. This renders the air sterile. The flow of air from the unit discourages any fungal spores of bacteria from entering. All items going inside the unit should be sterile or sprayed with ethanol or isopropyl alcohol. They will remain sterile unless you contaminate them.

288 × 267 - Lunaire Model HLF3072BT Horizontal laminar flow with steri-pots

Working in the Transfer Hood

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The hood should remain on continuously. If for some reason it has been turned off, turn it on and let it run for at least 15 minutes before using.

Make sure that everything needed for the work is in the hood and all unnecessary things are removed. As few things as possible should be stored in the hood.

Check the bottom of the hood to make sure there is no paper or other debris blocking air intake.

Remove watches, etc., roll up long sleeves, and wash hands thoroughly with soap (preferably bactericidal) and water.

Spray or wipe the inside of the transfer hood (bottom and sides, not directly on the filters)

with 70% EtOH. Others use disinfectants such as Lysol®. Wipe the work area and let the spray dry.

Wipe hands and lower arms with 70% EtOH.

Spray everything going into the sterile area with 70% ethanol. For example, spray bags of petri dishes with 70 % alcohol before you open them and place the desired number of unopened dishes in the sterile area.

Work well back in the transfer hood (behind the line). Especially keep all flasks as far back to the back of the hood as possible. Movements in the hood should be contained to small areas. A line drawn across the distance behind which one should work is a useful reminder.

Make sure that materials in use are to the side of your work area, so that airflow from the hood is not blocked.

Don’t touch any surface that is supposed to remain sterile with your hands. Use forceps, etc.

Instruments (scalpels, forceps) can be sterilized by flaming - dipping them in 95% EtOH and then immediately placing them in the flame of an alcohol lamp or gas burner. This can be dangerous if the vessel holding the alcohol tips over and an alcohol fire results.

Arrange tools and other items in the hood so that your hands do not have to cross over each other while working. For a right-handed person, it is best that the flame, alcohol for flaming, and tools be placed on the right. The plant material should be placed to the left. All other items in the hood should be arranged so that your work area is directly in front

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of you, and between 8 and 10 inches in from the front edge. No materials should be placed between the actual work area and the filter. Keep as little in the hood as possible.

Plant material should be placed on a sterile surface when manipulating it in the hood. Sterile petri dishes (expensive), sterile paper towels, or sterile paper plates work fine. Pre-sterilized plastic dishes have two sterile surfaces-the inside top and inside bottom.

Sterilize your instruments often, especially in between individual petri plates, flasks, etc.

The tools should be placed on a holder in the hood to cool or should be cooled by dipping in sterile water or medium before handling plant tissues.

Wipe up any spills quickly; use 70% EtOH for cleaning. Clean hood surface periodically while working.

Use of glass or plastic pipettes: Glass pipettes are put into containers or wrapped and then autoclaved. Plastic pipettes are purchased presterilized in individual wrappers. To use a pipette, remove it from its wrapper or container by the end opposite the tip. Do not touch the lower two-thirds of the pipette. Do not allow the pipette to touch any laboratory surface. Insert only the untouched lower portion of the pipette into a sterile container.

Remove items from the hood as soon as they are no longer needed. All cultures must be sealed before leaving the hood.

When transferring plant cultures, do contaminated cultures last. Situate the cultures so that the contaminated part is closest to the front of the hood. When finished in the hood, clean up after yourself. Remove all unnecessary materials and wipe the hood down with 70% EtOH.

Be sure when you are finished that you turn off the gas to the burner!

PREPARATION OF STOCK PLANTS

Prior good care of stock plants may lessen the amount of contamination that is present on explants. Plants grown in the field are typically more “dirty” than those grown in a greenhouse or growth chamber, particularly in humid areas like Florida.

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Overhead watering increases contamination of initial explants. Likewise, splashing soil on the plant during watering will increase initial contamination. Treatment of stock plants with fungicides and/or bacteriocides is sometimes helpful.

It is sometimes possible to harvest shoots and force buds from them in clean conditions. The forced shoots may then be free of contaminants when surface-sterilized in a normal manner.

Seeds may be sterilized and germinated in vitro to provide clean material. Covering growing shoots for several days or weeks prior to harvesting tissue for culture may supply cleaner material.

Explants or material from which material will be cut can be washed in soapy water and then placed under running water for 1 to 2 hours.

Various chemicals used for surface sterlisation

1.Sodium Hypochlorite

Sodium hypochlorite, usually purchased as laundry bleach, is the most frequent choice for surface sterilization. It is readily available and can be diluted to proper concentrations. Commercial laundry bleach is 5.25% sodium hypochlorite. It is usually diluted to 10% - 20% of the original concentration, resulting in a final concentration of 0.5 - 1.0% sodium hypchlorite. Plant material is usually immersed in this solution for 10 - 20 minutes. A balance between concentration and time must be determined empirically for each type of explant, because of phytotoxicity.

2. Ethanol (or Isopropyl Alcohol)

Ethanol is a powerful sterilizing agent but also extremely phytotoxic. Therefore, plant material is typically exposed to it for only seconds or minutes. The more tender the tissue, the more it will be damaged by alcohol. Tissues such as dormant buds, seeds, or unopened flower buds can be treated for longer periods of time since the tissue that will be explanted or that will develop is actually within the structure that is being surface-sterilized. Generally 70% ethanol is used prior to treatment with other compounds.

3. Calcium Hypochlorite

Calcium hypochlorite is used more in Europe than in the U.S. It is obtained as a powder and must be dissolved in water. The concentration that is generally used is 3.25 %. The solution must be filtered prior to use since not all of the compound goes into solution. Calcium hypochlorite may be less injurious to plant tissues than sodium hypochlorite.

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4. Mercuric Chloride

Mercuric chloride is used only as a last resort in the U.S. It is extremely toxic to both plants and humans and must be disposed of with care. Since mercury is so phytotoxic, it is critical that many rinses be used to remove all traces of the mineral from the plant material.

5. Hydrogen Peroxide

The concentration of hydrogen peroxide used for surface sterilization of plant material is 30%, ten times stronger than that obtained in a pharmacy. Some researchers have found that hydrogen peroxide is useful for surface-sterilizing material while in the field.

Enhancing Effectiveness of Sterilization Procedure

• Surfactant (e.g.Tween 20) is frequently added to the sodium hypochlorite. • A mild vacuum may be used during the procedure. • The solutions that the explants are in are often shaken or continuously stirred.

Rinsing

After plant material is sterilized with one of the above compounds, it must be rinsed thoroughly with sterile water. Typically three to four separate rinses are done.

Use of Antibiotics and Fungicides in Vitro

It is found that the use of antibiotics and fungicides in vitro is not very effective in eliminating microorganisms and these compounds are often quite phytotoxic.

CONTAMINANTS

The 3 main types of microbial contaminants in tissue culture are: Bacteria and Fungi Mycoplasma Viruses

Bacterial and Fungal Contamination

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Bacterial contamination is generally visible to the naked eye and detected by a sudden increase in turbidity and color change of the culture medium as the result of a change in pH. The cell culture may survive for a short time but the cells will eventually die.

Mycoplasma Contamination

Mycoplasmas are the smallest free-living self-replicating prokaryotes. They lack a cell wall and lack the ability to synthesize one. They are 0.35m in diameter and can be observed as filamentous or coccal forms. There are 5 major species that are tissue culture contaminants, namely M. hyorhinis, M. arginini, M. orale, M. fermentans and Acholeplasma laidlawii.The effects of mycoplasma infection are more insidious than those of bacteria and fungi inducing several long term effects. These include:

Reduced growth rate Morphological changes Chromosome aberrations Alterations in amino acid and nucleic acid metabolism

Viruses

Viral contaminators are virtually unseen and undetectable and difficult to remove from a culture, because they are so small. They require the machinery of a host cell to replicate and may or may not cause harm to the host cell culture they are invading. If not, they will go unnoticed, but if they are cytopathic (toxic), the cell culture will be destroyed, seemingly for no reason.

Chemical ContaminationChemical contamination is the presence of any non-living substance in the cell culture that causes adverse effects to the cells. This may include impure media or even water. Chemical contaminants could also come from unclean storage vessels.

CONTAMINATION CONTROL AND CURATING CULTURES

Many of the problems can be resolved by better and careful handling of cultures, materials and equipment , and the general aseptic condition of the laboratory.

However, occasionally contamination will be found in a valuable culture that cannot be replaced and attempts have to be made to save it.

Endophytic bacterial contamination is usually managed by antibiotic treatments.

An ideal antibiotic should be soluble, stable, unaffected by pH and the media, without side effects, broadly active , bactericidal , non-resistance inducing , inexpensive , active in combination with other antibiotics , and non-toxic to human health.

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Carbenicillin,cephalosporin,gentamicin,polymixin,rifampicin,streptomycin,andtimentin are commonly used.

As an alternative for sterilization by autoclaving, disinfection protocols for vessels and media using sodium hypochlorite (NaOCl) were developed for banana (Musa spp.) shoot

The NaOCl concentration of 0.002% was sufficient to prevent microbial growth when the disinfested vessels were incubated with a modified MS medium for banana shoot cultures. Culture media disinfected by NaOCl were successfully achieved for banana.

For plant tissue cultures, non-autoclavable vessels are suggested to be disinfected by the solution of 0.01% NaOCl at pH 5.4, and culture media by supplementing with 0.002% NaOCl. Beforesubculturing or multiplication the explants are diped in NaOCl solution for saving the cultures

PLANT PRESERVATIVE MIXTURE: PPM is a proprietary broad spectrum biocide,which can be used to control contamination in plant cell cultures,either during the sterilization procedure or as medium component.

• PPM comes in an acidic liquid solution (pH 3.8).• The recommended dose is 0.5-2.0 ml of PPM per litre of media. • It is effective against fungi as well as bacteria,thus it can be substituted for a cocktail

of antibiotics.• PPM is less expensive than antibiotics,which makes it affordable for wide and

routine use• Seeds and explants with endogenous contamination can be sterilized at doses of 5-

20ml/L of PPM .

CULTURE MEDIA

When cultured in vitro, all the needs, both chemical and physical, of the plant cells have to be met by the culture vessel,the growth medium and the external environment (light,temperature, etc.). The growth medium has to supply all theessential mineral ions required for growth and development.

In many cases (as the biosynthetic capability of cells cultured invitro may not replicate that of the parent plant), it must also supply additional organic supplements such as amino acids and vitamins.

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Many plant cell cultures, as they are not photosynthetic,also require the addition of a fixed carbon source in theform of a sugar (most often sucrose).

One other vital component that must also be supplied is water, the principal biological solvent. Physical factors, such as temperature, pH, the gaseous environment, light (quality and duration) and osmotic pressure, also have to be maintained within acceptable limits.So the growth and morphogenesis of plant tissue in vitro largely depend on the composition of the culture media. No single medium can be suggested for all types of plants and organs. Thats why the details of culture medium have to be worked out for each plant material separately.

Commonly used media are

i. Murashige & Skoog’s medium (MS medium)ii. Gamborg et al. (B5 medium).iii. White’s medium.

The most important of these media is MS medium.

Media Composition

The major constituents of most plant tissue culture media are:i. Inorganic nutrients (macro - and micro-nutrients)ii. Carbon source (sugar)iii. Organic supplementsiv. Growth regulatorsv. A gelling / solidifying agent (agar).

Inorganic nutrients

A variety of mineral elements (salts) supply the needed macro and micronutrients required in the life of a plant. Elements required in concentrations greater than 0.5 m mol L -1 are referred to as Macronutrients and those less than 0.05 mM/L as Micronutrients.

Macronutrients

They include six major elements:Nitrogen(N), Phosphorus(P), Potassium(K), Calcium (Ca),Magnesium (Mg) and Sulphur (S), present as salts in the media.All are essential for plant cell & tissue growth. Culture media should contain at least 25 mmolL-1 nitrate and potass

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Micronutrients:

These are Iron (Fe), Manganese (Mn), Zinc (Zn), Boron(B),Copper (Cu) and Molybdenum (Mo). Chelated forms of Fe and Zn are commonly used in preparing culture media eg. For embryo induction, EDTA-iron chelate is used in place of iron citrate.

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TABLE -Some of the elements important for plant nutrition and their physiological

function.These elements have to supplied by the culture medium in order to support the growth of culture.

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Carbon and Energy Source

The most preferred carbon source in plant tissue culture is ‘Sucrose’. Glucose supports equally good growth, while fructose is less efficient. Sucrose, while autoclaving the medium is converted to glucose and fructose. In the process, first glucose is used and then, fructose. Plant cells and tissues in the culture medium lack autotrophic ability and therefore, need external carbon for energy. The addition of an external carbon source to the medium enhances proliferation of cells and regeneration of green shoots.

Organic Supplements

1.Vitamins:

Plants synthesise vitamins endogenously and these are used as catalysts in various metabolic processes. When plant cells and tissues are grown in vitro, some essential vitamins are synthesized but only in sub- optimal quantities. Hence, it is necessary to supplement the medium with required vitamins and amino acids to achieve the best growth of the tissue. Thiamine (B1), nicotinic acid (B3),pyridoxine (B6), calcium pantothenate (B5) and myo inositol are used more often, in the range of 0.1- 10.0 mg/l.

2.Amino Acids:

Cultured tissues are normally capable of synthesizing the amino acids necessary for various metabolic processes. In spite of this, the addition of amino acids to the media is important for stimulating cell growth in cultures and for establishing cell lines. Casein hydrolysate (0.05- 0.1%), L- glutamine (8 mmol/l), Lasparagines(100 mmol/l), L- glycine (2mmolL-1), L-arginine and L-cysteine (10mmolL-1) are common sources of organic nitrogen used in culture media.

.3.Other Organic Supplements:

These include organic extracts eg. protein (Casein) hydrolysates, coconut milk, yeast and malt extracts, ground banana, orange juice and tomato juice.They are of undefined nature. In tissue culture, success achieved with coconut milk (5 to 20%) and protein (casein) hydrolysate (0.05 to 1.0%) has been significant. Potato extract has been found a suitable medium for anther culture.

4.Activated Charcoal:

The addition of activated charcoal (AC) to culture media stimulates growth and differentiation (rooting). AC adsorbs inhibitory compounds and darkening of the medium occurs. AC is generally acid-washed and neutralised before its addition at concentrations of

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0.5- 3% to the culture medium. It also helps to reduce toxicity by removing toxic compounds (eg.Phenols) produced during the culture and permits unhindered cell growth.

5. Antibiotics:

Some plant calls have a systemic infection of microorganisms. To prevent the growth of these microbes,it is essential to enrich the media with antibiotics, eg. Streptomycin or kanamycin at low concentration effectively controls systemic infection and do not inhibit the growth of cell cultures.

Growth Regulators

These include:i)Auxins ii) cytokinins, iii) Gibberellins, iv) Abscisic acid and, (v) Ethylene. The growth, differentiation and organogenesis of tissues occurs only on the addition of one or more of these hormones to the medium.

Auxins

Commonly used auxins are : i. Indole-3 - acetic acid (IAA).ii. Indole –3- butyric acid (IBA)iii. 2,4-dichlorophenoxyacetic acid (2,4-D)iv. Naphthoxyacetic acid (NOA)IAA occurs naturally in the plant tissues. Auxins have the property of inducing cell division. In nature, hormones of this group are involved in elongation of stem, internodes, tropism, apical dominance, abscission and rooting. 2, 4-D is mostly employed to induce callus production. Auxins are generally dissolved either in ethanol or dilute NaOH.

Cytokinins

These are adenine derivatives which are mainly concerned with cell division, modification of apical dominance and shoot differentiation, in the tissue culture. Commonly used cytokinins include: i. 6-Benzylaminopurine (BAP)ii. Isopentyl-iii. Furfurylamino purine (Kinetin).iv. Zeatin.v. 6- Benzyladenine (BA).

The ratio of auxins and cytokinins is important with respect to morphogenesis in the culture system. For embryogenesis, callus initiation and root initiation, the ratio of auxins to

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cytokinin is high, while the reverse leads to axillary and shoot proliferation. Equally important is concentration of the two hormones.

Eg.2,4-D and BA at a concentration of 5.0 mgL-1 promote callus formation, but if used at 0.1 mg L-1 concentration, they promote shoot formation. Cytokinins have been shown to activate RNA synthesis and to stimulate protein and enzyme activity in certain tissues.

Gibberellins and Abscisic acid: GA3 is the most common gibberellin used. It promotes the growth of cell cultures at low density, enhances callus growth and induces dwarf or stunted plantlets to elongate. GA3 is used to induce plantlets formation from adventive embryos formed in culture.

Abscisic acid (ABA) in the culture medium either stimulates or inhibits the callus growth depending on the species.

Ethylene

Ethylene is a gaseous, naturally occurring, plant growth regulator most commonly associated with controlling fruit ripening in climacteric fruits, and its use in plant tissue culture is not widespread. It does, though, present a particular problem for plant tissue culture. Some plant cell cultures produce ethylene, which, if it builds up sufficiently, can inhibit the growth and development of the culture. The type of culture vessel used and its means of closure affect the gaseous exchange between the culture vessel and the outside atmosphere and thus the levels of ethylene present in the culture.

Solidifying Agents

Gelling or solidifying agents are commonly used for preparing semi-solid or solid tissue culture media. Agar (a polysaccharide obtained from seaweeds) is used to provide solid surface for growth because in the liquid medium, the tissue will be submerged and die due to lack of available oxygen.

pH:

Plant cells and tissues require optimum pH for growth and development in cultures. The pH affects uptake of ions and for most of the culture media pH 5.0 to 6.0 before sterilization is considered optimal. Higher pH is likely to give a hard medium while a low pH results in unsatisfactory solidification of agar. While preparing a medium, the pH can be adjusted to the requirement of an experiment.

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PROCEDURE OF MEDIA PREPARATION

1. Measure approximately 700 ml of tissue culture grade water .2. While stirring the water, add Macronutrient Solution.3. Continue stirring the mixture while adding Micronutrient Solution.4. Add desired heat stable supplements (e.g. sucrose, gelling agent, vitamins, auxins, cytokinins, etc.).5. Add additional tissue culture grade water to bring the medium to the final volume.6. While stirring, adjust medium to desired pH using NaOH, HCl or KOH.7. If a gelling agent is used, heat until the solution is clear.8. Dispense the medium into the culture vessels before (or after) autoclaving according to your application. Add heat labile constituents after autoclaving.9. Sterilize the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121 °C, for the time period described under Sterilization protocol.

MURASHIGE AND SKOOG’S (MS)MEDIUM COMPOSITION

MS MEDIA COMPOSITION FOR INITIATION OF Musa grandnain

S.NO. COMPOUND AMOUNT IN gm OR ml PER litre

1. NH4NO3 1.65 gm2. KNO3 1.90 gm3. MgSO4 0.37 gm4. CaCl2 0.44 gm5. KH2PO4 0.17 gm6. NaH2PO4 0.072 gm7. Meso Inositol 0.100 gm

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8. MN 5 ml9. MR 1 ml10. Iron 5 ml11. Glycine 1 ml12. Pyridoxine 0.5 ml13. Nicotinic acid 05 ml14. Thiamine 0.1 ml15. BAP 2 ml16. Adenine Sulfate 0.048 ml17. L-Tyrosine 0.072 ml18. IBA 0.75 ml19. Sucrose 30 gm20. Agar 9.0 gm21. Charcoal 0.6 gm22. Water pH 5.023. pH of media 5.8

MS MEDIA COMPOSITION FOR MULTIPLICATION OF Musa grandnain

S.NO. COMPOUND AMOUNT IN gm OR ml PER liter

1. NH4NO3 1.65gm2. KNO3 1.90gm3. MgSO4 0.37gm4. CaCl2 0.44gm5. KH2PO4 0.167gm6. NaH2PO4 0.072gm7. Meso Inositol 0.100gm8. MN 5 ml9. MR 1 ml10. Iron 5 ml11. Glycine 1 ml12. Pyridoxine 0.5 ml13. Nicotinic acid 0.5 ml14. Thiamine 0.1 ml15. BAP 5 ml16. Adenine Sulfate 0.048 ml17. L-Tyrosine 0.072 ml18. IBA 0.75 ml19. Sucrose 30gm20. Agar 9.0gm21. Charcoal 0.6gm22. Water pH 5.023. pH of media 5.7

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MS MEDIA COMPOSITION FOR SHOOTING AND ROOTING OF Musa grandnain

S.NO. COMPOUND AMOUNT IN gm OR ml PER liter

1. NH4NO3 1.65 gm2. KNO3 1.90 gm3. MgSO4 0.37 gm4. CaCl2 0.44 gm5. KH2PO4 0.17gm6. NaH2PO4 0.096gm7. Meso Inositol 0.100 gm8. MN 10 ml9. MR 1 ml10. Iron 5 ml11. Glycine 1 ml12. Pyridoxine 0.5 ml13. Nicotinic acid 0.5 ml14. Thiamine 0.1 ml15. BAP 3 ml16. Adenine Sulfate 0.048 ml17. L-Tyrosine 0.072 ml18. IBA 0.75 ml19. Sucrose 30 gm20. Agar 8.9gm21. Charcoal 0.6 gm22. Water Ph 5.023. pH of media 5.7

MODIFIED MS MEDIA FOR SHOOTING

MEDIA NO. MEDIA CONC.(mg/lit)M1 MS+BAP(0.5 mg/l)M2 MS+BAP(1 mg/l)M3 MS+BAP(1.5 mg/l)M4 MS+BAP(2.5mg/l)M5 MS+BAP(1.0mg/l)+IAA(0.5

mg/l)M6 MS+BAP(1.5mg/l)+IAA(0.5mg/l)

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MODIFIED MS MEDIA FOR ROOTING

SL.NO. MEDIUM CONC.

(mg/l)R1 ½ MSR2 ½ MS + IAA

(0.5mg/l)R3 ½ MS + IAA

(1.0mg/l)R4 ½ MS + NAA

(0.5mg/l)R5 ½ MS + NAA (1mg/l)

INVITRO CULTURE OF MERISMATIC TISSUE AND MICROPROPAGATION OF Musa grandnain

The process of invitro culture and micropropagation of Musa grandnain is carried out in following stages:

STAGE 1-INITIATION

SOP FOR INITIATION

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Selection of explant:Generally apical meristem part of the plant is taken as explant.Preparation of explant:The side shoots and the leaves and hairs of the explant are removed by scraping with the blade.Treatment of explant:Wash the explant with water.Wash the explant with surfactant and fungicide for about 1 hours.Treatment inside the laminar flow cabinet:Treat the explants with the chlorine soln 3% for about 3-10 minThen treat with 70% alcohol for about 1 minRinse thrice with autoclaved waterGive fresh cut to treated explantAt last incubate the explants in the desired media.

SOP FOR INITIATION INSIDE LAMINAR FLOW

Wear lab coat, mask and cap before opening the laminar flow Switch on the laminar flow. On the UV light 1 hour before working. Wipe hands and laminar surfaces properly with 70% alcohol. Take forceps, scalpels(blades inserted) inside in bead sterilizer (with a

temperature of 280ºC) On the laminar flow ,take alcohol and then fire the match stick on floor(for

sterilization) Then wipe the culture bottles and media bottles with alcohol before allowing

them in the laminar hood. While working (dissection) forceps are to be used for handling instead of hands. There should not be any cross movement across the cultures in the laminar. The mouth of culture bottles should be kept towards the laminar air. Then after working put the tape properly without any air gap and then clean the floor.

COLLECTION OF EXPLANTS

Shoot cultures of banana start conventionally from any plant part that contains a shoot meristem, i.e. the parental pseudostem, small suckers, peepers and lateral buds . The apex of the inflorescence and axillary flower buds are also suitable explants for tissue culture initiation. Overall, it is important to select explant material from preferably mature individuals whose response to environmental factors is known, and whose quality traits governed by genotypic and environmental effects have been identified.

Most commonly used explants -For rapid in vitro multiplication of banana, shoot tips from young suckers of 40-100 cm height are most commonly used as explants.

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Treatment of explants

The top portion is discarded and bottom portion i.e. bud is taken of approximately 10 cm.Wash the bud with tap water to remove all the dirt and dust.Then cut the outside layers of the bud keeping the middle portion intact with three to four layers around.

Dip the buds in DM water containing bavistin (3gm /l) and streptocyclin (antibacterial in action) (0.1gm/l).

Shake continuously for about an hour.

Wash with DM water twice.

Treat with DM water containing ascorbic acid (0.04gm/l) and few drops of Tween 20 and shake well for 10-20 minutes.

Take them into the laminar.

Wash with sterile water twice.

Dip in sodium hypochlorite solution (30% for smaller buds or 35% for larger ones) and shake well for about ½ an hour.

Wash with sterile water thrice.

Note-Variants of this decontamination protocol exist. They differ in explant type and size, disinfection procedure (single or double sterilisation) , type of disinfectant (calcium hypochlorite instead of sodium hypochlorite) and its concentration and treatment duration.

INITIATION INSIDE THE LAMINAR HOOD

Wear lab coat, mask and cap before opening the laminar flow

Switch on the laminar flow.

On the UV light 1 hour before working.

Wipe hands and laminar surfaces properly with 70% alcohol.

Take forceps, scalpels(blades inserted) inside in bead sterilizer (with a temperature of 280ºC)

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On the laminar flow ,take alcohol and then fire the match stick on floor(for sterilization)

Then wipe the culture bottles and media bottles with alcohol before allowing them in the laminar hood.

While working (dissection) forceps are to be used for handling instead of hands.

There should not be any cross movement across the cultures in the laminar.

The mouth of culture bottles should be kept towards the laminar air.

The layers of the explant have to be slowly and carefully removed so that we are left with the meristem inside.

Then it is to be inoculated in the banana initiation media in jam jars (prepared, poured and autoclaved earlier).

Finally the jam jars are sealed tightly with parafilm around the neck with no air gap.

The explants (designated as S0) was incubated at 26ºC to 28ºC with 14 hours light/dark cycle (daylight fluorescent tubes) (photosynthetic flux PPF of about 60µE/m2s).

Usually two types of growth regulators, a cytokinin and an auxin, are added to the banana growth medium. Their concentration and ratio determines the growth and morphogenesis of the banana tissue.

We routinely add 2.25 mg/l 6-benzyladenine (BA) and 0.175 mg/l indole-3-acetic acid (IAA) to the initiation medium.

In most banana micropropagation systems, semi-solid media are used. As a gelling agent agar (5-8 g/l) is frequently added to the culture medium but our preference is for Gelrite (2-4 g/l) because of its higher transparency, allowing much earlier detection of microbial contamination.. 

STAGE 2-SUBCULTURING AND MULTIPLICATION

After a period of time, it becomes necessary, chiefly due to nutrient depletion and medium drying, to transfer tissues to fresh media. This is particularly true of tissue where a portion of tissue is used to inoculate new culture tubes or flasks; this is known as subculturing.

Contamination free explants are further cultured on multiplication media supplemented with plant growth hormones(cytokinins) which help in proliferation of axillary buds into multiple shoots. These shoots are divided and multiplied to bulk up

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the multi culture stock. The multiplication cycles are restricted to 8 because Banana is genetically highly unstable.

Multiplication rate of bud depend upon the cultivar,concentration of cytokinin and number of subsubcultures.Number of subcultures should be limited to 7-8 in order to reduce the incidence of arising somaclonal variations.

Initial subculturing was done when the explant produced some shoots. For sub-culturing, the entire samples of in vitro shoot were cut into small pieces so that each piece would contain about one shoot Each piece was inoculated into a similar fresh multiplication media.There were six different combination of media(M1,M2,M3,M4,M5,M6).Approx 12 cultures are subcultured in each media.

Leaf and blackish or brownish basal tissues were removed to expose the meristems.

Alternatively, freshly initiated cultures can be kept in complete darkness for one week. Antioxidants, such as ascorbic acid or citric acid in concentrations ranging from 10–150 mg/l, are added to the growth medium to reduce blackening, or the explants are dipped in antioxidant solution (cysteine 50 mg/l) prior to their transfer to culture medium

Plant cell and tissue cultures may be maintained indefini1iely by serial subculturing.

The multiplication rate is referred to as the increase in number of propagule(each consists of 2 – 3 shoots/bud)obtained from one piece of shoot/bud cluster from the previous subculture.

The percentage of elongated shoots referred to those shoots above 1 cm tall with small leaves , globular buds are short and round with scalar like leaves.

Multiplication medium consisted of MS macro and micro salts supplemented with thiamine HCl (0.4 mg/ml),myo-inositol and L-Tyrosine (both at 100mg/l),sugar (30g/l),agar(5.5 gm/l) and adenine sulfate (80mg/l).pH was adjusted to 5.8 before autoclaving.

.

Each glass vessel contains one piece of explant (sucker) and two to three pieces of propagule in later cycles of subculture.

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Cultures were incubated at 25+2 C with 8 hours photoperiod.cultures were transferred to fresh media after 15 to 20 days.After optimum growth they are transferred to another media for rooting and shooting.

CULTURE ROOM

The growth and development of tissues cultured plants in vitro is generally monitored by observing cultures at regular intervals, in the culture room. Plants regenerated from in vitro tissue cultures are transplanted to soil in pots. The potted plants are ultimately transferred to green houses or growth cabinets and maintained for further observations under controlled conditions of light, temperature and humidity.

All types of plant tissues are incubated under conditions of well-controlled temperature, humidity, illumination and air circulation. These enviormental factors may influence the growth and differentiation process directly during culture and indirectly by affecting their response in subsequent generation.

Typically culture room has light and temperature control for 24 hours.

Usually, air-conditioners and heaters are used to maintain the temperature around 25± 2o C.

Cultures are generally grown in diffused light. Other requirements are a humidity range of 20-98%, controllable to ±3% and uniform forced–air ventilation.

Culture room should have enough shelves illuminated by a set of fluorescent tubes for storing cultures.

All the cultures should be labelled, giving details of the experiment (name of the plant, explant, medium, date of culture and other information) to ensure identity and for recording monitored results.

Shakers with controlled temperature and light are also installed in a culture room.

Characterstics things required in culture room

Temperature control (17-27°C). Electricity supply essential for lighting, cooling and heating.

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Shelves for culture racks. Fluorescent tubes for lighting. Timer for regulating day-length. Racks for culture vials. Rotary shaker. Observations table.

SOP FOR CHECKING OF CULTURES

Only clean cultures should be sent to the inoculation room. All the contaminated cultures should be discarded. The cultures should be thouroughly checked in the checking table.

Quality parameters for dispatching of cultures to the green house

Only good grown cultures should be sent to the green house. Cultures should have proportionate root and shoot. Abnormal cultures(i.e.)

Rootless culturesBushy growth culturesNon vigourous shootsAlbino types

Grading of shootsA grade:7-9 cm ht. 4-5 leavesB grade 6-8 cm ht 3-4 leaves

STAGE3-SHOOTING AND ROOTING

SHOOTING

Auxins and cytokinins are the most widely used plant growth regulators in plant tissue regulators in plant tissue culture and are usually used together,the ratio of the auxin to the cytokinin determining the type of culture established or regenerated.

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Shooting is the third step of invitro culture of Musa grandnain.This process take place in shooting media (M1,M2,M3,M4,M5,M6)the composition of media has described earlier.This process takes place 3-4 weeks.A high cytokinin to auxin ratio favours shoot formation.

The established cultures on shoot induction medium were routinely transferred after every 3 weeks by subdividing the shoots into single shoots.

Before each subculturing,at the micropropagation stage,the number of shoots per explants(determined by counting the number of shoots/explants) and average shoot length were measured.

ROOTING

Rooting is next step after completion of shooting.a high auxin to cytokinin ratio generally favours root formation.

For rooting, half strength MS supplemented with auxin combination was used(described earlier in media used) named R1,R2,R3,R4,R5.

When the shoots grew about 3-5 cm in length with 3-6 well developed leaves, they were rescued aseptically from the culture tubes and were separated from each other and again cultured on freshly prepared medium containing different combinations of hormonal supplements for root induction

Regenerated shoots were taken out carefully from the culture vessels and individual shoots were separated and implanted in the rooting media containing half strength MS with different concentrations (0.2 - 2.0 mg/l) and combinations of auxins (IAA and NAA) .

STAGE4-HARDENING

PRIMARY HARDENING

Green house for primary hardening

They are constructed with polysheet(200um),equipped with water repellent benches,four way misting system,thermal shade net,thermometer,hygrometer and luxmeter to monitor temperature ,humidity and light intensity.

Media ingredients for primary hardening

Cocopeat(N 2.375%,P2O5 0.07%,k2O 1.14%,pH 5.5)

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Perlite and fungicides,bactericides,insecticides etc

In vitro grown plants were delicate because of Poorly developed cuticle Poor stomatal activity Limited mesophyll Plenty of intracellular activities

Hence,they have to undergo several anatomical and physiological changes for their establishment in green house conditions.For this purpose,in this growth phase,they undergo adaptation from heterotrophic to autotrophic condition.This transition is achieved by gradually exposing the plantlets from condition of highest humidity(90%) and diffused light,to decreasing humidity(70%) and increasing light intensity.To achieve this ,these plantlets were maintaind at 70% humidity and 25 c for 40 -45 days,after which they were shifted to shade houses for secondary hardening.

METHOD

In this process the plantlets under aseptic conditions of laboratories have to be shifted to glasshouse.

They are first cleaned with tap water to remove any agar sticking to them,then washed with fungicide(Bavistin) and bactericide (Streptocycline).

The coccopeate and perlite mixed in the ratio of 3:1 and filled into plant tray of 52 cavity or 98 cavity size.

Plantlets are then pressed into this mixture without leaving any air gap.

Conditions were: HUMIDITY: 65 to 80%

TEMPERATURE: 20-29ºC TIME: 40 days

SECONDARY HARDENING

Shade house for secondary hardening

Shade house constructed with 50% light cutout black shade net,equipped withdrip irrigation system,supplying water/fertilizers to every plant with an individual dripper ,fertigation equipment and microsprinklers.

Media ingredients for secondary hardening

River bed soil

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Sand farmyard manureVermicompost

After the primary hardening, the plantlets(not exposed to biofertilizers) were transferred to secondary hardening in polyhouses or greenhouses.

METHOD Here soil , sand, farmyard manure and vermin compost are mixed in the ratio of

1:1:1:1 , then Trichoderma : 0.05 gm, Azotobacter : 0.025 gm, Vereria : 0.025 gm , PCB:0.025 gm are mixed

Above constituents are added per gram of mixture.

These all are mixed and filled in polybags of 4X6 inch and then watered. Carefully the plantation is done.

Shade house with drip irrigation system supplying water/fertilizers to every plant with drippers,fertigation equipment and micro sprinklers was used for secondary hardening.

Here the plantlets are kept for 30-40 days at same temperature and humidity which then become ready to be grown into soil.

STEPS INVOLVED IN INTIATION OF Musa grandnain

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Figure 1A Suker selected for isolation of Meristem

Figure 2 Isolation of merismatic tissue

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Figure 3 Inoculated in MS media

Figure 4 Inoculated tissue after 1 week

MULTIPLICATION STAGE INVOLVED IN Musa grandnain

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Figure 5 Plantlets divided into single parts for multiplication and subculturing

Figure 6 Growth in plants after multiplication

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Figure 7 Cultures after first subculturing

Figure 8 Multiplication stage after 3 weeks

SHOOTING AND ROOTING IN Musa grandnain

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Figure 9 Shoot formation under in vitro conditions

Figure 10 Fully developed shoots in invitro conditions

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Figure 11 Root formation in plants

Figure 12 Well developed roots in plants and plants were ready for primary hardening.

HARDENING OF Musa grandnain PLANTS

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Figure 13 Filling of plant tray with primary hardening mixture

Figure 14 Planting under glass house conditions in plastic tray for primary hardening

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Figure 15 Primary hardened plants transfer to net house

Figure 16Filling of poly bags for Secondary hardening in green house

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Figure 17 Plants under green house conditions in polybags for secondary hardening

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CONTAMINATION IN CULTURE

Figure 18 Bacterial contanination

Figure 19 Fungal contamination

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Figure 20 Mix contamination

Figure 21 Badly contaminated plants(discardable plants)

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OBSERVATION AND RESULTS

. The in vitro culture of Musa grandnain was done in three steps

INITIATION MULTIPLICATION SHOOTING AND ROOTING

observation or results of these steps are listed as below.

INITIATION AND MULTIPLICATION

There were six different media combination in which total number of culture was 12. The conc. of first media MS+BA (0.5 mg/lit),second media MS+BAP (1 mg /lit),third

media MS+BAP(1.5 mg/lit.),fourth media MS+BAP(2.5 mg/lit),fifth media MS + BAP(1 mg/lit) + IAA (0.5 mg/lit) and sixth media MS+BAP (1.5 mg/lit)+ IAA(0.5mg/lit)

We got best percentage of culture response in M5 and M6 media within 5-6 days and that was 89.24% and 91.24% respectively.

TABLE :Effects of different media conc. On the percentage of culture response.

MEDIA NO.

MEDIA CONC.(mg/lit) TOTAL NO. OF CULTURES

% OF CULTURE RESPONSE

DAYS

M1 MS+BAP(0.5 mg/l) 12 59.52% 21M2 MS+BAP(1 mg/l) 12 79.51% 20M3 MS+BAP(1.5 mg/l) 12 87.23% 19M4 MS+BAP(2.5mg/l) 12 71.24% 22M5 MS+BAP(1.0mg/l)+IAA(0.5

mg/l)12 89.24% 21

M6 MS+BAP(1.5mg/l)+IAA(0.5mg/l) 12 91.24% 21

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SHOOTING

From above data there were six different cultures.In serial no.1 and 2 the total no. of cultures are 12 and no.of shoot is 5 and shoot height was 7.75 cm.In serial no. 3 and 4 the total no. of cultures is 12 and no. of shoot is 6 and shoot height was 8 cm.In serial no. 5 and 6 the total no. of cultures was 12 and no. of shoot was 7 and shoot height was 9.25.The best response was shown by serial no. 3The poor response was shown by serial no. 5

TABLE:The number of shoots and height of banana plantlets in shooting media.

SI.NOTOTAL NO.OF

CULTURES

NO.OF SHOOTSSHOOT

HEIGHT(CM) RESPONSE

1. 12 5 7.75 ++++2. 12 5 7.75 ++++

3. 12 6 8.0 +++++

4. 12 6 7.8 +++

5. 12 7 9.00 ++

6. 12 7 9.25 +++

NOTE : +++++ EXCELLENT,++++ VERY GOOD,+++GOOD,+POOR

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ROOTING

The best response was seen by cultured plants in R5 media and rest were satisfactory except R1 media containing only ½ MS.

TABLE: THE EFFECT OF DIFFERENT ROOTING MEDIA IN THE RESPONSE OF ROOTS IN Musa grandnainTISSUE CUTURE PLANTLETS

SL.NO. MEDIUM DAYS RESPONSER1 ½ MS 25 +++R2 ½ MS + IAA

(0.5mg/l)25 ++++

R3 ½ MS + IAA (1.0mg/l)

25 ++++

R4 ½ MS + NAA (0.5mg/l)

25 ++++

R5 ½ MS + NAA (1mg/l)

25 +++++

NOTE:+++++ EXCELLENT ++++ VERY GOOD+++GOOD+POOR

TABLE: CONTAMINATION RECORD

SI.NO DAYS NO.OF SHOOTS

NO.OF CULTURE/BOTTLE

OBSERVATION AND CONTAMINATION

1. 22 8 6 All plants were healthy.

2. 23 9 5 All plants were healthy.

3. 24 11 5 All plants were healthy.

4. 24 15 7 All plants were healthy but leaves were infected.

5. 25 14 8 All plants were healthy

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CONCLUSION

The experimental results indicated that the types of cytokinin and their concentration significantly influenced shoot multiplication and elongation.Moderate conc. Of cytokinin increases the shoot multiplication rate but very high conc. Decreases multiplication especially depressed shoot elongation.

It was observed that all the explants did not behave similar in vitro in terms of multiplication

BAP conc. ranges from 0 .1 mg/l to 1 mg/l did not improve much shoot multiplication or elongation but above 1.5mg/l reduces shoot elongation.

Supplementation of 1.5mg/l BAP with MS medium produced the best multiplication and elongation in all cultures.

The joint effect of BAP and IAA increases shoot elongation better compared to BAP alone.

Rooting can be stimulated when individual shoots are transferred to basal medium alone.However auxins may increases further root intiation.

It was found that NAA was more effective than IAA.

Optimum NAA conc. was found to be 1mg/l.

Our results show that when active charcoal was added to medium it was not necessarly to include IBA and NAA for rooting.

Using active charcoal alone will reduce the cost of producing plantlet for field.

Recommended conc. Of activated charcoal is 0.1 to 0.25%

Overall it is recommende that the multiplication of banana explants on MS medium in combination with 1.5mg/l BAP and .5 mg/l IAA followed by rooting with activated charcoal only.

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SBL INSTITUTE OF PLANT BIOTECHNOLOGY standard protocols and media formulations.

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