AN OVERVIEW OF BIOTECH PARK

The State of Uttar Pradesh, a vital hub of scientific activities endowed with rich

biological resources and biodiversity has the unique distinction of having a number of

research institutions, which have expertise and capabilities in biotechnology. In view of

this, Lucknow was declared as the Biotechnology City of India during the 89th session of

the Indian Science Congress on January 3, 2002. These institutions are providing a

great impetus and support in the development of Biotech Park.

The Biotech Park has been set up on 8 acres of land provided by the Department of

Science and Technology, Government of Uttar Pradesh. The thrust areas identified for

the initial phase of the Park are.

Health Care

Agriculture

Environment

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FACILITIES AT BIOTECH PARK:

1. BIO-BUSINESS BLOCK: The bio-business block houses the following facilities:

Business Support Facility

Bioinformatics Centre

Conference Hall

Cafeteria

Business Support Facility: It has been set up to promote business related

activities and to promote companies to work in collaboration with Biotech Park.

Bioinformatics Centre: It has been set up to establish a close network between

various facilities within the Biotech Park and to provide latest information to

industries regarding technological advancements.

Conference Hall: It has been set up to conduct seminars, workshops and lectures

in order to enlighten and motivate the trainees to come up with new ideas and work

towards them.

Cafeteria: A cafeteria is situated at the hub of the block to provide refreshments and

lunch to trainees.

2. SOLVENT EXTRACTION BLOCK: The extraction unit comprises of solid-liquid

solvent extraction system with solvent recovery system for extraction of

Photochemical / Lead molecules from high value medicinal plants and multipurpose

reaction cum hydrolysis and solvent recovery unit along with chromatography. The

extraction unit has the following infrastructural facilities:

Oil fired steam boiler, evaporation capacity: 500 kg/hr and 150 psi steam pressure

for basic heating requirements to main extraction unit.

Hot air tray dryer, High capacity hammer mill and vacuum oven for drying,

grinding/pulverizing of raw materials/finished herbs / medicinal plants.

Refrigerated brine chilling circulation unit for carrying out reactions and chilling of

condenser water.

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Solid liquid extraction unit consisting of two drug holders with agitators of 125 - 150

kg/batch capacity depending on plant bulk density with efficient solvent recovery

system from extract and spent marc.

Silica gel chromatography column for chromatographic separation over silica gel

with solvent recycling system, for enriching and isolation of the photochemical in

high purity. Stainless steel reaction vessel with agitator and solvent reflux / recovery

system for production of semi synthetic drug molecules and for chemical

transformations of the photochemical.

Heavy-duty spray dryer for drying of herbal / aqueous extracts, capacity 10 – 20

kg/hr for making powder of herbal extracts.

Falling film evaporator for concentrating herbal / aqueous extracts for separation of

the extracts from the plant biomass.

Notch type vacuum filtration unit for filtration of aqueous solvent extracts for finer

filtration of the extracts for removal of particulate impurities.

Liquid - liquid counter current extraction equipment for the purification of crude

extracts by liquid partitioning.

Minor equipments like vacuum pumps, metering pumps, weighing machine,

weighing balance, trolleys etc. for auxiliary infrastructure, process and material

handling etc.

Capacity: 250 kg biomass/batch

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3. BIO-FERTILIZER, TISSUE CULTURE AND CENTRAL SUPPORT BLOCK:

Bio fertilizer Unit: This unit has facilities for perfecting the technology and

production of bacterial fertilizers and pesticides, phosphate solubilising bacteria

(PSB), Azotobacter (with a capacity of up to 240 tonnes / annum) Trichoderma (with

a capacity of up to 500 tonnes / annum). The trials runs have been completed in

March 2007 and batch production has started.

Tissue Culture Unit: Tissue culture facility at Biotech Park, Lucknow is spread over

2000 sq. ft. Area that possess the capacity to raise and multiply banana, potato,

Jatropha seedlings. The culture media used in the unit is completely biodegradable

and non-toxic.

Advantages of propagation by tissue culture:

The elimination of diseases and the production of disease free plantlets.

The rapid production of large numbers of genetically identical plantlets.

Introduction of new varieties and or genotypes.

Preservation of germplasm.

Production of haploid plants which can be used for plant breeding.

Production of plantlets from species in which plant development from seed is

difficult.

Central Support Unit: It was primarily established to provide support to various

other facilities and for large scale production. It is composed of following units:

Distillation unit: The distillation unit has been set up for obtaining essential oil from

aromatic plants such as Menthaarvensis, Menthapiperata, Menthacardiaca,

lemongrass, Palmarosa, Citronella, Basil, Vetiver, and Geranium etc. The recovery

of essential oil from different aromatic plants has been found to be relatively high as

compared to conventional distillation unit used by the farmers at their site. About

1000 kg fresh herbs per batch can be distilled in the unit.

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Vermicomposting unit: This is the fastest and effective way of recycling of organic

waste with the help of earthworms for the production of useful compost. To utilize

the large quantity of agro-waste generated from distillation and solvent extraction

units the vermicomposting unit has been set up at the Park. The unit will serve as

demonstration-cum-training facility for the farmers.

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ABSTRACT

The term plant tissue culture is generally used for aseptic culture of plant tissues,

organs and their components under defined chemical and physical conditions in-vitro.

The basic concept of plant body can be dissected into smaller plants termed as explants

and any explants can be developed into a whole plant. It is a central innovative area of

plant science including agriculture and plant biotechnology. This technique is effective

because almost all the plant cells are totipotent. Each cell possesses the genetic

information and cellular machinery necessary to generate the whole organism. This

technique is used to produce a higher number of plants that is genetically similar to the

parent plant as well as to another.

During the course of training, culturing and sub culturing of different plants like

sugarcane, bamboo and banana was performed. Essential techniques of plant tissue

culture which are sterilization, inoculation and hardening were practised. A number of

plants were grown using MS medium by single explants of the above samples. A variety

of plants were produced using different concentration of hormones.

Keywords: Tissue culture, Explants, Inoculation, Incubation, Sub culturing, hardening.

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INTRODUCTION

Plant tissue involves the use of small pieces of plant tissue (explants) which are

cultured in a nutrient medium under sterile conditions. Using the appropriate growing

conditions for each explants type, plants can be induced to rapidly produce new shoots.

The plantlets can also be divided, usually at the shoot stage, to produce a large number

of new plantlets.

Applications:

Clonal propagation using meristem and shoot culture to produce large number of

identical individuals.

Screening programmes of cells, rather than plants for advantageous characters.

Large scale growth of plant cells in liquid culture has a source of secondary

products.

Production of dihaploid plants from haploid culture to achieve a homozygous

lines more rapidly in breeding programmes.

As a tissue for transformation, followed by either short term testing of genetic

construct or regeneration of transgenic plants.

Removal of viruses by propagation from meristematic tissues.

Sugarcane belongs to the grass family (Poaceae), an economically important seed plant

family that includes maize, wheat, rice, and sorghum and many forage crops. The main

product of sugarcane is sucrose, which accumulates in the stalk internodes. Sucrose,

extracted and purified in specialized mill factories, is used as raw material in human

food industries or is fermented to produce ethanol. Ethanol is produced on a large scale

by the Brazilian sugarcane industry.

Bamboo is one of the fastest-growing plants on Earth, with reported growth rates of

250 cm (98 in) in 24 hours. However, the growth rate is dependent on local soil and

climatic conditions, as well as species, and a more typical growth rate for many

commonly cultivated bamboos in temperate climates is in the range of 3–10 centimetres

(1.2–3.9 in) per day during the growing period. Primarily growing in regions of warmer

climates during the late Cretaceous period, vast fields existed in what is now Asia.

Some of the largest timber bamboo can grow over 30 m (98 ft) tall, and be as large as

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15–20 cm (5.9–7.9 in) in diameter. Bamboo is one of the fastest-growing plants on

Earth, with reported growth rates of 250 cm (98 in) in 24 hours. However, the growth

rate is dependent on local soil and climatic conditions, as well as species, and a more

typical growth rate for many commonly cultivated bamboos in temperate climates is in

the range of 3–10 centimetres (1.2–3.9 in) per day during the growing period. Primarily

growing in regions of warmer climates during the late Cretaceous period, vast fields

existed in what is now Asia. Some of the largest timber bamboo can grow over 30 m

(98 ft) tall, and be as large as 15–20 cm (5.9–7.9 in) in diameter. However, the size

range for mature bamboo is species dependent, with the smallest bamboos reaching

only several inches high at maturity.

Banana is basically a tropical crop, grows well in temperature range of 13ºC-38ºC. In

India this crop is being cultivated in climate ranging from humid tropical to dry mild

subtropics through selection of appropriate varieties. Soil for banana should have good

drainage, adequate fertility and moisture. Deep, rich loamy soil with pH between 6-7.5

are most preferred for banana cultivation. A soil that is not too acidic and not to alkaline,

rich in organic material with high nitrogen content, adequate phosphorous level and

plenty of potash is good for banana.

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AIMS & OBJECTIVE

Collection of explants.

Preparation of media and study of growth regulators.

Establishment and initiation of culture.

Optimization of media.

Green house technology.

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MATERIALS & METHODS

Instruments Used:

1. HOT AIR OVEN:

Make & Model: JSGW

Hot air ovens are also drying equipment used to

sterilize as well as dry various laboratory

apparatuses. Contrary to an autoclave, it works

on the principle of dry heat sterilization where the

article to be sterilized or dried is subjected to

temperatures ranging from 50-300 °C. Since it

can operate at such high temperatures, it is

primarily used for glass & metal articles. A

thermostat keeps temperatures within set limits

while a fan fitted inside the oven ensures uniform

distribution of heat within.

2. VORTEX MIXER:

Make & Model: Remi CM 101

It is a relatively simpler device which is used to

decrease the time required to dissolve a

substance in a liquid by creating a small vortex

within the container of the liquid. The device

consists of a rubber end which moves in a

circular motion at very high speeds. When the

container of a liquid comes in contact with it,

the motion is transferred to it, creating a vortex

within, thus facilitating rapid mixing

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Figure 1

3. AUTOCLAVE

Autoclaves are widely used in microbiology, medicine, podiatry, tattooing, body piercing,

veterinary science, mycology, dentistry, and prosthetics fabrication. They vary in size

and function depending on the media to be sterilized.

Typical loads include laboratory glassware, other equipment and waste, surgical

instruments and medical waste.

A notable growing application of autoclaves

is the pre-disposal treatment and

sterilization of waste material, such as

pathogenic hospital waste. Machines in this

category largely operate under the same

principles as conventional autoclaves in that

they are able to neutralize potentially

infectious agents by utilizing pressurized

steam and superheated water. A new

generation of waste converters is capable of

achieving the same effect without a

pressure vessel to sterilize culture media,

rubber material, gowns, dressing, gloves,

etc. It is particularly useful for materials which cannot withstand the higher temperature

of a hot air oven.

Autoclaves are also widely used to cure composites and in the vulcanization of rubber.

The high heat and pressure that autoclaves allow help to ensure that the best possible

physical properties are repeatedly attainable.

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4. MILIPORE WATER PURIFICATION SYSTEM:

Make & Model: Milli-Q

It is an ultra purification water system which provides the purest form of water for

laboratory use. The device deploys a series of filtration & de-ionization processes to

remove impurities and unwanted ions from water. It uses minute filters to remove

impurities & ion-exchange resins to exchange unwanted ions with H+ & OH- ions. Milli-Q

systems are used to produce water pure enough to get reproducibility and accuracies

within parts per million.

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5. ANALYTICAL BALANCE:

Make & Model: Saritorus

An analytical balance is a weighing device used

to measure and weigh even minute quantities

with utmost precision (upto 5 decimal points). It

can weigh in grams as well as milligrams. The

device has slits on either sides of the weighing

platform to prevent entry of dust or unwanted

particles in the weighing area.

6. HOT PLATE WITH MAGNETIC STIRRER:

Make & Model: Remi 5MLH PLUS

A hot plate with magnetic stirrer is a laboratory

device that can heat a liquid at constant

temperatures as well as stir it by causing a bar

magnet to rotate within the liquid in presence of a

rotating magnetic field. It is preferred over

mechanical stirrers since it is not prone to wear &

tear and does not interfere with the flow of liquid.

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7. LAMINAR AIR FLOW: Thermo KS-9

A Laminar flow cabinet is a carefully enclosed bench designed to prevent

contamination of semiconductor wafers, biological samples or any particle sensitive

device. Air is drawn through HEPA filter and blown in a very smooth laminar flow

towards the user. The cabinet is usually made of stainless steel with no gaps or

joints where spores might collect. Laminar flow cabinets may use UV-C germicidal

lamp to sterilize the shell and contents when not in use.

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COLLECTION OF EXPLANTS:

We have collected our explants of sugarcane, banana and bamboo from the field of the

Horticulture Department of Biotech Park, Lucknow under the supervision of Mr. R.P.

Sharma. The tissue taken from a plant or seed is transferred to a culture medium to

establish a tissue culture system.

The choice of explants depends upon the methods of shoot multiplication to be

followed. All the plant organs which are nodal segments, inter nodal segments, shoot

tips, root, cotyledons, epicotyls, hypocotyls, leaf, petiole, anthers and ovule etc. are

known to give rise to complete plants.

MEDIA PREPARATION

For achieving success of culture, appropriate composition of media should be prepared.

Different plants vary in their nutritional requirements. Hence each tissue requires

different medium to grow. The basal medium required for the culture of sugarcane is MS

medium. (Musarshige and Skoog 1962).

A number of growth regulators such as alpha-naphthalene acetic acid (NAA), (IAA) and

(BAP) were added at various concentrations to the medium. The concentrated stock

solution of major salts, minor salts and vitamins are prepared to be used in the

preparation of media and stored in refrigerators.

Appropriate amount of stock solutions were added to prepare a culture media. The pH

was maintained to 5.8. The media was poured into washed bottles and then autoclaved

at 121ºC for 15 minutes at 15-lbs psi pressure and transferred into the inoculation room

in aseptic conditions.

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Growth Regulators

Auxins

Auxin regulate the amount, type, direction of plant growth. It was first revealed by a

Dutch scientist Frits Went. They are mostly produced in growth areas such as root

and shoot tips and thus play a major role in cell division and differentiation, fruit

development and formation of roots. The most commonly produced auxins in plant is

indole-3-acetic acid (IAA). 1-Napthelene acetic acid (NAA) is also used as synthetic

auxin.

Cytokinins

Cytokinins are compounds with a structure resembling adenine which promote cell

division. It was so named because of its ability to promote cytokinesis (cell division).

Though it is a natural compound, it is not made in plants (meaning that the hormone

is synthesized somewhere other than in a plant). Kinetin was the first cytokinin

discovered. The most common form of naturally occurring cytokinin in plants today

is called zeatin which was isolated from corn (Zea mays). 6-Benzylaminopurine

(BAP) is the synthetic cytokinin.

Abscisic acid

Abscisic acid (ABA) is one of the most important plant growth regulator. It is used in

plant tissue culture to promote distinct development pathways such as somatic

embryogenesis.

Ethylene

Ethylene is a gaseous phytoharmone that regulates many aspects of plant

morphogenesis. Growth and development of cell in vitro are largely depend on it.

Hence, modification in composition and its interaction with the nutrient medium has

been used as the primary strategy to manipulate plant morphogenesis.

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Gibberellins

Gibberellins (GAs) are plant hormones that regulate growth and influence various

developmental processes, including stem elongation, germination, dormancy,

flowering, sex expression, enzyme induction, and leaf and fruit senescence. It was

first recognized in 1926 by a Japanese scientist, Eiichi Kurosawa and was first

isolated in 1935 by Teijiro Yabuta and Sumuki.

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Media preparation

The appropriate composition of the medium largely determines the success of cultures.

Plant material does vary in their nutritional requirements and therefore it is often

necessary to modify the medium to suit a particular tissue. The basal medium employed

for the culture is MS medium (Murashige and Skoog, 1962). A variety of growth

regulators such as Kinetins, 1-Napthelene acetic acid (NAA) and 2,4-dichlorophenoxy

acetic acid (2,4-D) were added to the medium in combination at various combinations

and were used for initiating different experiments. The concentrated stock solutions of

the major salts, minor salts and vitamins are prepared to be used in preparation of the

media which can be stored by refrigeration.

The medium was prepared by adding appropriate quantities of the stock solutions and

correct volume was made up by using distilled water. The pH was adjusted in all cases

to 5.8 using 1N NaOH and 1N HCl. Agar was also used for semi solid medium for

culture initiative/establishment. Before autoclaving media was poured into washed and

dried glass bottles which were capped and labelled properly. These were then

autoclaved at 121°C for 15 minutes at 15- lbs psi pressure and transferred to the

inoculation room where they were stored under aseptic conditions.

Composition of MS media.

Macronutrients and Micronutrients.

Ammonium nitrate (NH4NO3) 1,650mg/l

Boric acid (H3BO3) 6.2mg/l

Calcium chloride (CaCl2.2H2O) 440mg/l

Cobalt chloride (CoCl2.6H2O) 0.025mg/l

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Magnesium sulphate (MgSO4.7H2O) 370mg/l

Cupric sulphate (CuSO4.5H2O) 0.025mg/l

Potassium phosphate (KH2PO4) 170mg/l

Ferrous sulphate (FeSO4.7H2O) 27.8mg/l

Potassium nitrate (KNO3) 1,900mg/l

Sodium molybdate (Na2MoO4.2H2O) 0.25mg/l

Zinc sulphate (ZnSO4.7H2O) 8.6mg/l

Na2EDTA.2H2O 37.2mg/l

Vitamins and Supplements

Inositol 100mg/l

Thymine.HCl 0.1mg/l

Pyridoxine.HCl 0.5mg/l

Nicotinic acid 0.5mg/l

Sucrose 30g/l

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Stock solution

Contents added. Concentration of stock solution.

BAP 5mg/l

IAA 0.25mg/l

IBA 0.25mg/l

Calcium chloride 2.2gm/50ml

Media for different plants:

Sugarcane

For sugarcane 100ml of MS media was prepared. Synthetic hormones are now added

with the concentration of 10µl of NAA, 400µl of BAP, 1ml of calcium chloride from the

stock solution and 10mg ascorbic acid was also added.

Bamboo

100ml of MS media was prepared in 3 different concentrations of BAP (30µl, 50µl and

80µl) in 3 conical flasks. 5µl of IBA, 1ml of calcium chloride was added from stock

solution and 10mg of ascorbic acid was also added in all the 3 flasks.

Banana

For banana plant tissue culture, 100ml MS media was prepared to which 500µl of BAP,

25µl of IAA, 25µl of IBA from the stock solution was added. 10 mg of ascorbic acid and

1ml of calcium chloride was further added to the media.

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STERILIZATION

Explants sterilization

Select the disease free and young and healthy explants as their cells will be

more likely to have retained their totipotency.

The collected explants are partially trimmed off and then washed thoroughly

under running tap water for 3-4 minutes to wash off external contamination.

The collected explants were properly wiped with 70% ethanol.

Remove the young leaves from the top portion of plant and excise the spindle

from the top.

The explants were taken inside the laminar hood for sterilisation. Inside the

laminar, sterilisation with 0.1% mercuric chloride is done for approximately 8-10

minutes. After that, it is removed from mercuric chloride soln. and rinsed three

times in sterile, autoclaved distilled water to completely remove any remaining

mercuric chloride.

The last step is performed inside a laminar flow hood to maintain the sterilized

condition of explants and to prevent the re-introduction of contaminating

microbes. Finally all extra water is removed from the explants and the explants

are ready to be trimmed.

Media Sterilization

To sterilize the prepared MS media for plant tissue culture

The media was poured in different glass bottles.

The caps of the bottles should be closed tightly so that no contamination may

occur.

The bottles were then kept in the autoclave at 121ºC for 15-20 minutes at 15 psi.

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INOCULATION

INITIATION AND CULTURING OF THE EXPLANT FOR SHOOTING

Before starting the inoculation the laminar airflow chamber should be U.V. sterilized for

20 minutes.

The sterilised plants were transferred aseptically from the conical flasks to sterile

glass plates under laminar hood in inoculation chamber for making them in sizes

of 4cm. The outer part of the explants should be removed with sharp blade with

appropriate precautions.

Rinse the forceps in 70% ethanol and flame the forceps. Sterilise the Petri plates

so that the inoculating explants can be kept on it timely.

Remove the lid from one bottle with the long forceps without touching the rim of

the bottle and place the explants into the bottles.

Return the forceps to the ethanol and mark the variety of explants (whether

banana, sugarcane or bamboo) on the bottles along with the date.

Repeat the same procedure for all explants available and put the bottles in the

incubator with temperature maintained at 22ºC.

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MULTIPLICATION OF SHOOTS

Concept of multiplication:

The multiplication of explants is very crucial stage in the propogation of any species for

commercial purposes. BAP and NAA is important for the multiplication process.

BAP: 6-Benzylaminopurine

NAA: Indole acetic acid

Protocol for multiplication:

Transfer the shoots from the culture bottle to a sterile glass plate using flamed

forceps. Remove the brown coloured leaves from the clump and subdivide the

clump into smaller clumps.

Transfer the shoot into a new medium flask containing multiplication medium.

Incubate the flask in the incubator under light and repeat the step 2 to 4 times

every 2 to 3 weeks so that the shoots are formed.

If a sterile environment was not maintained,contamination will be obvious within 3

to 4 days.

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ROOTING OF PLANTLETS

Concept of Rooting

Portion of explants that contains one or more than one shoots could be transferred

to a medium that contains higher concentration of auxins, resulting in root formation,

once the sufficient number of shoots have been generated.

The production of roots is easily achieved in some species by reducing the

cytokinins level or in MS basal medium with or without the addition of extra root

promoting auxins, especially IAA and IBA.

IAA: Indole acetic acid

IBA: Indole butyric acid

Protocol for Rooting:

In the laminar flow, under sterile conditions, remove the para-film and cap

from the culture bottle and use the forceps to carefully remove the

explant from the multiplication medium.

Place the multiplied shoot mass on a sterile petri dish or on the sterile

glass plate. Using a sterile scalpel carefully remove or cut plantlets away

from the mass of shoots/clumps. Remove the undesirable portion of the

explant and using sterile forceps rinsed in 70% ethanol and flamed,

carefully place plantlets into the rooting medium.

Note that lower side of the shoot remains in contact with the media and

shoot remains straight. Carefully cap the culture bottle, label them properly

and return them to the rack for 1-2 weeks under the same condition.

During this time, the shoots will continue to grow, however, most of the

plant’s energy will be focused into producing roots.

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RESULT AND DISCUSSION

INCUBATION

FIRST WEEK:

After the incubation of 1 week, explants in the controlled temperature and humidity, it

was observed that there was no growth of explants.

SECOND WEEK:

If was found that few samples of explants have showed contamination whereas some

have shown some sprouting. It was found that there was an increase in the size of the

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explants and whole of the explants had turned green. The medium at the base of

explants turned brown which is due to the release of phenolics in the medium.

Table 1: Observation of sugarcane explants establishment.

TIME DURATION OBSERVATION

BOTTLE 1 BOTTLE 2

1st WEEK no sprouting no sprouting

2nd WEEK no sprouting tiny sprouts

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Table 2: observation of bamboo explants establishment.

TIME DURATION OBSERVATION

0.3mg/l 0.5mg/l 0.8mg/l

1st WEEK no sprouting no sprouting no sprouting

2ndWEEK no sprouting contamination Sprouting

Table 3: observation of banana explants establishment.

TIME DURATION

OBSERVATION

Bottle 1 Bottle 2

1st WEEK no sprouting no sprouting

2nd WEEK callus formation Contamination

SUB CULTURING:

After a period of time it becomes necessary to transfer the tissue cultured shoots into a

fresh medium, due to depletion of essential nutrients and drying out of the gel.

Transfer several shoots from the culture bottles to a sterile glass plate using

flame sterilized forceps. Remove the brownish parts from the clump and sub-

divide the clump into smaller clumps.

Transfer the mass into a new culture flask containing multiplication medium.

Incubate the flask into incubator under light and repeat the steps 2-4 times every

2-3 weeks so that a mass of shoot is formed.

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If sterile environment was not maintained, contamination will be obvious within 3-

4 days.

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HARDENING

The term “hardening” refers to any treatment that results in the firming or hardening of

the plant tissue. Such a treatment reduces the growth rate, thickens the cuticle and

waxy layers, and reduces the percentage of freezable water in the plant. Such plants

often have smaller and darker green leaves than non- harden plants. Hardening results

in an increased layer of carbohydrate in plants permitting a more rapid root

development than occurs in non-harden plants.

Hardening is generally done in 2 steps:

PRIMARY HARDENING.

After 15-20 days of culture in MS media, the rooted plantlets were transplanted

for primary hardening for about 45 days in a polyhouse which is covered with a

agronet. The temperature of the agronet is maintained at 28ºC.

The plantlets were first washed with distilled water and fungicide. The fungicide

may be 5% tricoderma, 5% M45 or 2% bavistine.

The plantlets are then fixed into a protray containing cocopeat.

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SECONDARY HARDENING.

The small plants are then transfered to a nethouse where secondary hardening is

performed.

The plants are fixed into polybags containing 75% soil, 25% vermicompost and a

less percentage of fungicide.

Sunlight is an essential factor in secondary hardening as the temperature needed

for the growth of plant should be maintained at 30-35ºC.

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TRANSPLANTATION TO THE MAIN FIELD.

Only after eight to twelve of hardening the plants were ready to withstand the natural

atmospheric conditions and high temperature. Thus, they were transplanted into the

main field for commercial production.

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CONCLUSION

In this present study, we have attempted to work on Tissue culture protocol for

Sugarcane, Bamboo and banana. Though there are several problems associated with

these crops in relation with cultivation, identification of suitable variety in different agro

geographical regions.

Sugarcane, which is an important crop for Punjab farmers. But due to difficulty in

cultivation of sugarcane because of variation in temperature and other atmospheric

conditions, there is a decline trend of sugarcane cultivation in Punjab. But with a

renewed approach in combination of better package of services, better planting material

and combination of Plant Biotechnological approaches for mass propagation will

certainly bring a change in the whole scenario. Mass propagation of Sugarcane through

tissue culture approach will definitely result in availability of disease free germplasm,

dissemination of newer germplasm with improved agronomic characters for the ultimate

benefit of the farmers.

Similarly, short supply of seeds due to number of reasons in case of bamboo has

overcome with tissue culture method to great extent at reasonable cost and good

success rate. Using the method of plant tissue culture large scale propagation of

bamboos is possible at minimal cost and with the lowest labour input. By this method,

one seed can “plant” a whole and perhaps several hectares. If the plant prove to be of

good quality, a hectare of monoclonal superior plants will be available for further

propagation by conventional methods.

Whereas, Banana  being a crop  that  is  conventionally  propagated  by  vegetative 

means, can  easily  be  adopted  for  tissue culturing  in a laboratory  and  the  inherent 

benefits  of  the  system  can  be  successfully  employed  to  fully  exploit  the 

economic  potential  of  source  of   donor  or  mother  plant  material  which  are 

selected  from a areas  that  have  recorded  disease  free  cropping   several  years. 

From  such  source, healthy,  vigorous, high  yielding  donor  plants  of  the  desired 

banana  cultivars  are  identified  in  the  clonal  selection  process.

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We observe and conclude the following in our studies:-

1) In identifying the sterilization protocol for the explants (sugarcane, bamboo and

banana), our experience with the followed protocol is satisfactory.

2) We have also observed that the explants have to be sub cultured for 3-4 times at

a interval of 2-3 weeks.

3) The media combination used for initial establishment phases is MS basal +

Growth Regulators + 440mg/l Calcium chloride + 10mg/l agar.

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GLOSSARY

ABBREVIATION NAME

% Percentage

MS Murashige and Skoog

IBA Indole butyric acid

NAA Naphthalene acetic acid

IAA Indole acetic acid

BAP 6-benzylaminopurine

2,4-D 2,4-dihydroxyphenoxy acetic acid

mg/l Milligram per litre

µl Micro litre

ºC degree Celsius

34

LECTURES AND WORKSHOP ATTENDED

I attended a lecture on “Microbes in Biotechnology” by Dr. Pooja Pal on 15th

May,2014. We got to learn about different micro-organisms and their applications

in biotechnology and various other fields. Recombinant DNA technology is the

most efficient application used in the present scenario. She also gave knowledge

about the process of bioremediation which is very effective technique for cleaning

up the environmental pollutants such as oil, harmful gases and heavy metals.

During the course of training, Biotech park has also give us the opportunity to

attend the workshop which was held on “World Accreditation Day” on 9th

June, 2014. It was a global initiative jointly established by the International

Accreditation Forum (IAF) and International Laboratory Accreditation

Corporation (ILAC) to raise awareness of the importance of accreditation related

activities.

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REFERENCES

Brandes (1985). History and development of sugarcane as a world crop . in The

Sugarcane:1-5.

Cheema, G. S. et al (1995). Current Science,68, 6 : 650-653.

Bhau, Brijmohan Singh (1999). Hairy root culture and secondary

metabolite production. Role of biotechnology in medicinal and aromatic

plants, Vol II, Ukaaz Publications, Hyderabad.

Agnihotri(1998). Evaluation of fungal and bacterial antagonists against

Colletotrichum falcatum, causing red rot of sugarcane in Sugarcane and

its problems. Indian sugar oct.2000 423-425

Ajinder Kaur, Gosal, S. S, Raman Gill, Thind, K. S. (2001). Induction of

plant regeneration and somaclonal variation for some agronomic traits in

sugarcane (Saccharum officinarum L.). Crop Improvement, 28, No.2 : 167-

172, 6 ref.

Barber (1931). History and development of sugarcane as a world crop .

in The Sugarcane:1-5.

E.C. Cocking. Somatic embryogenesis and plant regeneration from rice

protoplasts Plant Genetic Manipulation Group, Embryogenesis in Barny

yard grass, Plant science : 161-163.

Enriquez, G. A., Arencibia, A. Selman-Housein, G. Hernandez, L. Tiel,

K., Fuentes, A. D. (2001). Banana biotechnology and genetic

engineering experience at CIGB: 578-579.

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