Establish Protocols

7/30/2019 Establish Protocols

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TO ESTABL ISH PROTOCOLS FOR

ANDROGENESIS, MICROPROPAGATION

&SOMATIC EMBRYOGENESIS

IN SELECTED PLANTS

Submit ted to: Subm it ted by:

Department of Biotechnology Swati Sharma

GGDSD College B.Sc Biotechnology(Hons.)-

Affliated to Panjab University G.G.D.S.D college

CHANDIGARH CHANDIGARH


2/17

Certificate 1

This is to certify that the project entitled TO ESTABL ISH PROTOCOLS FOR

ANDROGENESIS, MICROPROPAGATION & SOMATIC EMBRYOGENESIS IN

SELECTED PLANTShas been carried out by ______________ under supervision

of ______________________ in the Department of Biotechnology, GGDSD

College, Chandigarh.

Dr. Navneet Batra

Co-ordinator

Dept. of Biotechnology

G.G.D.S.D College, Chandigarh


3/17

Certificate 2

This is to certify that the project entitled TO ESTABL ISH PROTOCOLS FOR

ANDROGENESIS, MICROPROPAGATION & SOMATIC EMBRYOGENESIS IN

SELECTED PLANTShas been carried out by ______________ under supervision

of ______________________ in the Department of Biotechnology, GGDSD

College, Chandigarh.

Mrs Samriti Dhawan

Project supervisor

Lecturer

Dept. of Biotechnology

G.G.D.S.D College, Chandigarh


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ACKNOWLEDGEMENTS

I express my heartfelt gratitude to all the people involved in this project, without whom

completion of this project would not have been possible. Firstly, I submit my sincere

thanks to Department of Biotechnology, New Delhi, for encouraging science students

by pursue research by offering such benovalent opportunities in the form of short term

Summer Training Projects. I also owe my gratitude to our College principal, Dr A.C.

Vaid for initiating efforts, so that such opportunities reach the students.

I thank Dr Navneet Batra, Head of Department and Mrs Samriti Dhawan my project

supervisor, for their able guidance throughout the project. I am grateful to the laboratory

staff for their support, co-operation and patience.

At last however not the least, I thank God, my parents and college teachers with whose

help I was in a position to grab this opportunity and fulfill my dream.


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LIST OF ABBREVIATIONS

vs versus

& and

mg milligrams

l litre

lab laboratory

lb pounds

sq square

0C degree centigrade

temp temperature

NAA -Naphthalene acetic acid

2,4-D 2,4 dichloro acetic acid

Kn Kinetin

BAP Benzyl amino purine

pH hydrogen ion concentration

conc concentration

viz namely

sp species

HEPA high efficiency particulate air

m micrometer

% percentage

psi per square inch


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min minute

hrs hours

ml milli litre

iP iso pentyl adenine

UV ultra violet

EtOH ethyl alcohol

IAA Indle-3-acetic acid

IBA Indole-3-butyric acid

mg milli gram

NaOH sodium hydroxide

HCl hydrochloric acid

gm gram


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CONTENTS

1. Introduction

2. layout of a Plant Tissue Culture Laboratory

3. Sterile techniques

4. Preparation of Stock Solutions

5. Preparation of Media

6. Culture Techniques:

Anther culture (Androgenesis)

Micropropagation

Embryo culture

Somatic Embryogenesis


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INTRODUCTION

Green plants owing to there unique ability to perform photosynthesis, are the source ofall the energy to all. Plants provide food, feed, fiber, fuel pharmaceuticals, timber, and a

variety of other materials. With such divergent uses there is a continuous effort in

improving plant varieties. Advent of Plant tissue culture practices has been a great

breakthrough. Use of old world Conventional breeding methods for improvement are

now of limited use as compared to new world Plant Biotechnology techniques.

CONVENTIONAL BREEDING vsPLANT BREEDING

Modification of crop genotypes to make them more useful to humans constitutes plant

breeding. It involves the following steps:

1. Introduction2. creation of variations3. Selection4. Evaluation5. Multiplication6. Distribution

However there are some limitations to it. One of the chief limitations is limited genetic

variability. Plant breeding methods enable the use of genes only from species that are

sexually compatible which places a limit on genes accessible for plant breeding.

Another technical difficulty is the production of hybrid variety of seeds. Hybrid seed

production requires emasculation. Hand emasculation is tedious, and a satisfactory

system of genetic emasculation is not possible in all plants. Plant biotechnology is offers

useful solutions to all such problems.

Plant biotechnology helps to generate plants from isolated cells, tissues and organs on

one hand and genetic manipulation on the other. These can be continuously maintained

under controlled nutritional and environmental conditions to enable regeneration ofcomplete plantlets. that are finally transferred to the field. Other important areas where

plant biotechnology is of utmost importance include production of synthetic seeds,

secondary metabolites, rapid clonal multiplication, development of dihaploid

homozygous lines etc. Plant biotechnology can be exploited for both crop improvement

and crop protection.


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Thus plant tissue culture techniques form an integral part of plant biotechnology and are

of great interest. Keeping this in mind work was initiated in this field. The main stresswas to skill ourselves with the various culture techniques. As a part of the project we

established protocols for micropropagation ofChlorophytum borivilianum, somatic

embryogenesis in Vigna unguiculata, and androgenesis in Citrus sp.


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Lay out of a Plant Tissue Culture Laboratory

Any laboratory, in which tissue culture techniques are performed, regardless of thespecific purpose, must contain a number of basic facilities. These usually include thefollowing:

A washing area

A media preparation area

An aseptic transfer area

Maintenance area

An observation/data collection area

Washing Area

The washing area should contain large sinks, some lead-lined to resist acids andalkalis, draining boards, and racks, and have access to demineralized or distilled water,and double-distilled water. Space for drying ovens or racks, automated dishwashers,acid baths, pipette washers and driers, and storage cabinets should also be available inthe washing area.

Media Preparation Area

The media preparation area should have ample storage space for the chemicals, culture

vessels and closures, and glassware required for media preparation and dispensing.

Equipments like hot plates/stirrers, pH meters, balances, water bath(s), distilled and

double-distilled water system, bunsen burners with a gas source, refrigerators and deep

freezers, a microwave or a convection oven, and an autoclave for sterilizing media,

glassware, and instruments.

Transfer Area

A laminar flow hood or sterile transfer room should be utilized for making transfers.Within the transfer area there should be a source of electricity, gas, compressed air,and vacuum. The most desirable arrangement is a small dust-free room with an


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overhead ultraviolet light and a positive-pressure ventilation unit equipped with a high-

efficiency particulate air (HEPA) filter (0.3-m) of 99.97-99.99% efficiency.

Another type of transfer area is a laminar flow hood. Air is forced into the unit through a

dust filter then passed through a HEPA filter. The air is then either directed downward

(vertical flow unit) or outward (horizontal flow unit) over the working surface. The

constant flow of bacteria-free filtered air prevents non-filtered air and particulate matter

from settling on the working surface.

Culture maintenance area

All types of tissue cultures should be incubated under conditions of well-controlled

temperature, humidity, air circulation, and light quality and duration. Culture room for

growth of plant tissue cultures should have a temperature between 25 and 30C, with a

temperature fluctuation of less than 0.5C.The temperature should be constant

throughout the entire culture room (i.e., no hot or cold spots). The culture room shouldhave enough fluorescent lighting to reach the 10,000 lux; the lighting should be

adjustable in terms of quantity and photoperiod duration. Both light and temperature

should be programmable for a 24-hr period. and a humidity range of 20-98%

controllable to 3 percent.


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STERILE TECHNIQUES

Aseptic technique is necessary for the successful establishment and maintenance of

plant cell, tissue and organ cultures. The in vitro environment in which the plant material

is cultured is also ideal for the proliferation of microorganisms. In most cases the

microorganisms outgrow the plant tissues, resulting in their death. Contamination can

also spread from culture to culture. The purpose of aseptic technique is to minimize the

growth of microorganisms in cultures.

Effective sterilization involves:

Sterilization of media

Sterilization of explants

Sterilization of working area

Sterilization of instruments

Sterilizing tools, media, vessels, etc.

Moist heat sterilizationAutoclaving is the method most often used for sterilizing heat-resistant items. In order to

be sterilized, the item must be held at 121C, 15 psi, for at least 15 minutes. It isimportant that items reach this temperature before timing begins. Therefore time in theautoclave will vary, depending on volume in individual vessels and number of vessels inthe autoclave.

Sterilizing tools, vessels, etc.

Empty vessels, beakers, graduated cylinders, etc., should be closed with a cap oraluminum foil and never autoclaved empty. Tools should also be wrapped in foil orpaper or put in a covered sterilization tray. It is critical that the steam penetrate theitems in order for sterilization to be successful.

Sterilizing Media and Other Liquids (moist heat & flame sterilization)

Culture media, distilled water, and other heat stable mixtures can be autoclaved in glasscontainers that are sealed with cotton plugs, aluminum foil, or plastic closures.


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However, solutions that contain heat-labile components must be filter-sterilized. Forsmall volumes of liquids (100 ml or less), the time required for autoclaving is 15-20 min,but for larger quantities (2-4 liter), 30-40 min is required to complete the cycle. Thepressure should not exceed 20 psi, as higher pressures may lead to the decompositionof carbohydrates and other components of a medium. Too high temperatures or too

long cycles can also result in changes in properties of the medium.Organic compounds such as some growth regulators, amino acids, and vitamins may

be degraded during autoclaving. These compounds require filter sterilization through a

0.22 m membrane. Filters used may be pre-sterilized or larger ones can be set over a

sterile flask and a vacuum is applied to pull the compound dissolved in liquid through

the membrane and into the sterile flask (filter assembly). Smaller membranes fit on the

end of a sterile syringe and liquid is pushed through by depressing the top of the

syringe. The size of the filter selected depends on the volume of the solution to be

sterilized and the components of the solution.

Nutrient media that contain thermo labile components are typically prepared in severalsteps. A solution of the heat-stable components is sterilized in the usual way byautoclaving and then cooled to 35-50 C under sterile conditions. Solutions of thethermo labile components are filter-sterilized. The sterilized solutions are then combinedunder aseptic conditions to give the complete medium.

In spite of possible degradation, however, some compounds that are thought to be heatlabile are generally autoclaved if results are found to be reliable and reproducible.These compounds include 2,4-D, NAA, kinetin, pyridoxine, 2-ip, thiamine, etc. areusually autoclaved.

UV Radiation

Use of germicidal lamps to sterilize items in the transfer hood when no one is workingthere is a routine practice. UV lamps should not be used when people are presentbecause the light is damaging to eyes and skin. Plants left under UV lamps will alsoshow mutations.


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Autoclave Filter assembly


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Laminar air flow Glass bead sterilizer

Additional Guidelines forWorking in the Transfer Hood

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 unnecessarythings are removed

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

Spray or wipe the inside of the transfer hood (bottom and sides) with 70% EtOH.

Spray everything placed in the sterile area with 70% ethanol.

Remove watches, etc., roll up long sleeves, and wash hands thoroughly with soapand water before working.

Wipe hands and lower arms with 70% EtOH

Make sure that materials in use are to the side of your work area, so that airflow

from the hood is not blocked. Dont 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 gasburner.


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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 cooledby dipping in sterile water or medium before handling plant tissues.

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

Sterilize culture tubes with lids or caps on. When you open a sterile tube, touch onlythe outside of the cap, and do not set the cap on any laboratory surface. Instead,hold the cap with one or two fingers while you complete the operation, and thenreplace it on the tube.

After you remove the cap from the test tube, pass the mouth of the tube through aflame. Put only sterile objects into the tube. Complete the operation as quickly asyou reasonably can, and then flame the mouth of the tube again. Replace the lid.

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

When finished in the hood, clean up after yourself. Remove all unnecessarymaterials and wipe the hood down with 70% EtOH.

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

Surface-sterilizing Plant Material

Preparation and use of Stock Plants (Cultures)

Use of stock plants to obtain starting material may lessen the amount of contamination

that is present in field obtained explants. Plants grown in the field are typically moredirty than those grown in a greenhouse or growth chamber, particularly in humidareas. Treatment of stock plants with fungicides and/or bactericides is sometimeshelpful initially but may be inhibitory at a later stage.

To avoid this, the best solution is to use pre-conditioned plants as source of explants.Seeds may be sterilized and germinated in vitro to provide clean material. In case offield grown material, explants from which starting material will be cut can be washed inrunning water for 1 to 2 hours, followed by sterilization with a suitable sterilant and amild detergent added to it. This is followed by thorough washing with sterile distilledwater.

Ethanol (or Isopropyl Alcohol)

Ethanol is a powerful sterilizing agent but also extremely phytotoxic. Therefore, plantmaterial is typically exposed to it for only seconds or minutes. The more tender thetissue, the more it will be damaged by alcohol. Tissues such as dormant buds, seeds, orunopened flower buds can be treated for longer periods of time since the tissue that willbe explanted or that will develop is actually within the structure that is being surface-


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sterilized. Generally, 70% ethanol is used prior to treatment with other compounds.

Sodium Hypochlorite

Sodium hypochlorite is the most frequent choice for surface sterilization. It is readily

available and can be diluted to proper concentrations. Commercially available bleach is5.25% sodium hypochlorite. It is usually diluted to 10% - 20% of the originalconcentration, resulting in a final concentration of 0.5 - 1.0% sodium hypochlorite. Plantmaterial is usually immersed in this solution for 10 - 20 minutes. A balance betweenconcentration and time must be determined empirically for each type of explant,because of phytotoxicity.

Calcium Hypochlorite

Calcium hypochlorite is obtained as a powder and must be dissolved in water. Theconcentration that is generally used is 3.25 %. The solution must be filtered prior to use

since not all the compound goes into solution. Calcium hypochlorite may be lessinjurious to plant tissues than sodium hypochlorite.

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Establish Protocols