BASIC INFORMATION ABOUT PLANT TISSUE CULTURE

AND ESTABLISHMENT OF PLANT TISSUE CULTURE LAB:

REPORT PREPARED BY: IQRA JUNEJO

SUPERVISED BY: MEHBOOB UL HAQ , CEO, SEDF

PREPARED FOR: SINDH ENTERPRISE DEVELOPMENT FUND

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ACKNOWLEDGEMENT:

I am thankful to almighty ALLAH who made me capable enough to get opportunity to work as

internee in Sindh Enterprise Development Fund.

I would like to thank to MR MEHBOOB UL HAQ, CEO, Sindh Enterprise Development Fund

who provided me opportunity to work as summer internee under his super vision and to make

report on such an important topic of PLANT TISSUE CULTURE & PLANT TISSUE

CULTURE LABORATORY. He cooperated and appreciated me in every step which gave me

potential through which i became able to make this report.

I would like to thanks MR NIAZ MUHAMMAD NIZAMANI, PLANT TISSUE CULTURE

LAB OWNER,TANDO ALLAH YAR, for his cooperation.

I would like to thanks PROF DR SAIF ULLAH, INCHARE BIOTECHNOLOGY WING,HEJ

RESEARCH INSTITUE,UNIVERSITY OF KARACH for his cooperation.

I have tried my level best to keep this report simple yet technically correct .I hope I succeed in

my attempt.

IQRA JUNEJO

MANAGEMENT TRAINEE

SEDF

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TABLE OF CONTENT

S no CONTENT PAGE #

1 ACKNOWLEDGMENT 2

2 PREFACE 4

3 HISTORY OF PLANT TISSUE CULTURE 5

4 PLANT TISSUE CULTURE 5

5 PROCEDURE OF PLANT TISSUE CULTURE 6

6 PLANT PROPAGATION METHODS 6

7 THE TISSUE CULTURE PROCESS 7

8 CONTAMINANT FREE ENIVRONMENT 7

9 TISSUE CULTURE MEDIA 7

10 PROPAGATION RATE 7

11 ADVANTAGES&DISADVANTAGES 8-9

12 COMMONLY USED TERMS IN TISSUE CULTURE 9-12

13 REQUIRED FACILITIES FOR PLANT TISSUE CULTURE LAB 13

14 WASHING ROOM 13

15 MEDIA PREPARATION ROOM 14

16 GLASS WARES AND CULTURE ROOM 14

17 PRECUATIONS OF PROCESS 15

18 QUESTIONNAIR 16-18

19 QUESTIONNAIR AND MEETING REPORT 19

20 MEETING REPORT 20-22

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PREFACE

This report is about plant tissue culture and plant tissue culture laboratory.

This report provides all the useful and basic information about plant tissue procedure and

establishment of plant tissue culture laboratory.

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HISTORY OF PLANT TISSUE CULTURE

Plants have a remarkable regenerative power, as evidenced by the relative ease with which they

can be rooted, transplanted and grafted. Early attempts at growing plants in vitro from isolated

parts were unsuccessful, because knowledge of plant nutrition and physiology was inadequate.

With the discovery of essential plant hormones, some progress was made starting in the 1920s

and 30s. A major advancement was made by Philip R. White in 1939 with his report of

continuous culture of carrot and tobacco done completely in vitro. Further progress was made by

Folke Skoog, who discovered new and important properties of the hormone auxin. Skoog, along

with Toshio Murashige, went on to develop the still widely used standard plant nutrient

solution---Murashige-Skoog (MS) medium. The work begun by Kenneth Vivian Thimann in the

late 1950s, which demonstrated that kinetin broke the dormancy of lateral buds, allowing them to

develop as if they were at the tips of plants, paved the way for rapid advancements. From then

on, new and important results were announced almost every year. Today almost any plant can be

grown under laboratory control from a wide range of starting tissues.

PLANT TISSUE CULTURE

Plant tissue culture is a method or techniques to isolate parts of plants (protoplasm,cells,tissues

and organs)and grow them on artificial media in aseptic conditions in a controlled space so that

parts of these plants can grow and develop into complete plants.

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PROCEDURE OF PLANT TISSUE CULTURE

Plant tissue culture processes were initially developed and practiced in university and government-based laboratories.

In recent years, however, the process has moved beyond these research facilities to widespread use among

commercial enterprises as a cost-effective tool for plant propagation, new variety introductions and research.

Plant tissue culture has revolutionized the flower and nursery markets by making valuable new hybrid clones

available in commercial quantities comparatively soon after their first discovery. It is possible to start with a single

plantlet and, in the space of 10-12 months, create in excess of 250,000 identical copies or clones.

Plant tissue culture processes:

Plant propagation methods

The tissue culture process

Contaminant-free environment

Tissue culture media

Propagation rate

PLANT PROPAGATION METHODS

Plants produced by vegetative propagation including top cutting, root divisions, pseudobulbs, offshoots and plantlets,

are genetically identical to the mother plant and thus members of a single clone. Plant tissue culture is a laboratory

based extension of these plant propagation techniques.

Through tissue culture, very large numbers of identical plantlets can be derived from one mother plantlet. This

technology and the resulting plantlets now form the basis of many plant nursery and flower trade industries.

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THE TISSUE CULTURE PROCESS

The mother plant selected should be healthy and free from all micro-organisms. A piece of live tissue is removed

from a part of the plant and placed into culturing flasks containing appropriate nutrient media under aseptic

conditions.

In successful culture, these cells will divide, multiply and differentiate into thousands of plantlets having the same

characteristics as the parent plants. In tissue culture, the presence of bacteria and fungi will prevent the growth of the

plant tissue. It is important that the plant material used is thoroughly sterilized and the procedure is carried out in an

aseptic environment

.CONTAMINANT-FREE ENVIRONMENT

Laboratory techniques and specialized equipment such as a laminar flow cabinet combine to present an area for the

manipulation of sterile plant tissues. A working environment that has virtually all of the bacteria and fungal spores

removed is a requirement for successful plant tissue culture.  A sterile environment is prepared, now we prepare

sterile instruments to remove an apical shoot from a plant

.

TISSUE CULTURE MEDIA

The excised bud is transferred into a tube containing a sterile nutrient medium. The success of tissue culture depends

very much on the stage of explant selected, the sterilization period and the type of culture media used; different types

of plants require different sets of culture media. The rich tissue culture media provides a good food source for bacteria

and fungi, therefore precautions against microbial contamination must be taken in all in vitro procedures.

PROPAGATION RATE

Once a plant has been successfully entered to in vitro culture and is growing, a multiplication program may be carried

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out.  

Careful manipulation of the culture media and the plant tissues will yield a 4-10 fold increase in plant numbers every

18-40 days.

When sufficient plant numbers have been developed, a process to move the plantlets from the confines of the culture

vessel to a greenhouse is undertaken.  The following table compares a conventional propagation scheme on the left

with a plant tissue culture production system on the right.  Note the number of days and the number of resulting

plants:

  Cutting Propagation     Tissue Culture Propagation  

  Root 50 cuttings   Day 1 One plant in tissue culture media  

  Roots first appear   Day 40 5 plantlets cut & transplanted in media  

  Good rooting observed   Day 80 25 plantlets  

  Cold Storage   Day 120 125 plantlets  

  Cold Storage   Day 160 625 plantlets  

  Cold Storage   Day 200 3,125 plantlets  

  Cold Storage   Day 240 15,625 plantlets  

 Transplant 50 to

Greenhouse  Day 280 Transplant 15,625 plants to Greenhouse 

  Transplant 50 to Field   Day 320 Transplant 15,625 plants to Field 

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ADVANTAGES:

1. To produce many copies of the same plants then which may be used to produce plants with

better flowers, odors, fruits or any other properties of the plants that are beneficial to the human

beings.

2. To produce plants anytime we want although the climates are not appropriate to produce a

plant. Moreover, if seed is not available, it is possible to produce a plant with this method.

3. If there is plant with partially infected tissue, it is possible to produce a new plant without

infection.

4. Very helpful in the genetically modified organism studies.

5. Very useful solution for the prevention of starvation in third world countries since the process

is highly efficient, by using only one plant, it is possible to produce more than one thousand of

the same plant with higher productive if its genome changed.

6. The equipments are cheaper when compared to the animal cell culture.

DISADVANTAGES:

1. If large scale production is being thinking, the costs of the equipments are very expensive.

2. The procedure is very variable and it depends on the type of the species so sometimes it needs

trial-and-error type of experiments if there is not any review about that species.

3. The procedure needs special attention and diligently done observation.

4. There may be error in the identity of the organisms after culture.

5. Infection may continue through generations easily if possible precautions are not taken.

6. Decrease genetic variability.

COMMONLY USED TERMS IN TISSUE CULTURE

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Adventitious: Development of organs such as buds, leaves, roots, shoots and somatic embryos

from shoot and root tissues and callus.

Agar: Natural gelling agent made from algae

Aseptic technique: Procedures used to prevent the introduction of microorganisms such as

fungi, bacteria, virus and phytoplasma into cell, tissue and organ cultures, and cross

contamination of cultures.

Autoclave: A machine capable of sterilizing by steam under pressure

Axenic culture: A culture without foreign or undesired life forms but may include the deliberate

co-culture with different types of cells, tissues or organisms.

Callus: An unorganized mass of differentiated plant cells.

Cell culture: Culture of cells or their maintenance in vitro including the culture of single cells.

Chemically defined medium A nutritive solution or substrate for culturing cells in which each

component is specified.

Clonal propagation: Asexual multiplication of plants from a single individual or explant.

Clones: A group of plants propagated from vegetative parts, which have been derived by

repeated propagation from a single individual. Clones are considered to be genetically uniform.

Contamination: Infected by unwanted microorganisms in controlled environment

Cryopreservation: Ultra-low temperature storage of cells, tissues, embryos and seeds.

Culture: A plant growing in vitro in a sterile environment

Differentiated: Cultured cells that maintain all or much of the specialized structure and function

typical of the cell type in vivo.

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Embryo culture: In vitro culture of isolated mature or immature embryos.

Explant: An excised piece or part of a plant used to initiate a tissue culture.

Ex vitro: Organisms removed from tissue culture and transplanted; generally plants to soil or

potting mixture.

Hormone: Generally naturally occurring chemicals that strongly affect plant growth

In Vitro: To be grown in glass

In Vivo: To be grown naturally

Laminar Flow Hood: An enclosed work area where the air is cleaned using HEPA filters

Medium: A solid or liquid nutritive solution used for culturing cells

Meristem: A group of undifferentiated cells situated at the tips of shoots, buds and roots, which

divide actively and give rise to tissue and organs.

Micropropagation: Multiplication of plants from vegetative parts by using tissue culture

nutrient medium.

Propagule: A portion of an organism (shoot, leaf, callus, etc.) used for propagation.

Somatic embryos: Non-zygotic bipolar embryo-like structures obtained from somatic cells.

Subculture: The aseptic division and transfer of a culture or portion of that culture to a fresh

synthetic media.

Tissue culture: In vitro culture of cells, tissues, organs and plants under aseptic conditions on

synthetic media.

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Totipotency: Capacity of plant cells to regenerate whole plants when cultured on appropriate

media.

Transgenic: Plants that have a piece of foreign DNA.

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PLANT TISSUE CULTURE LABORATORY

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REQUIRED FACILITIES FOR PLANT TISSUE CULTURE LAB:

In each laboratory where plant tissue culture techniques are used must have a number of

facilities that include among others.

Outdoor media preparation, sterilization and storage

Washing room

Media preparation room

Water(distilled water)

Transfer space (laminar air flow)

Culture room

Glass wares

Microscope

Shaker

Autoclave chamber

Tools dissection (spatula, scalpels,(tweezers)forceps, scissors)

Shade or green houses

Computer for record keeping

WASHING ROOM:

Washing room should have a sink.

A desk made up of material resistant to acids and akalis

Drying ranks and channels for deminerallisi or distilled water

Space for the oven drying

Equipments /washing machines and dryers

Storage racks or equipment cabinets

Disinfectants

Oven

MEDIA PREPARATION ROOM:

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In the media preparation room should be available space for storage of chemicals,

glass culture and the lid

Sturdy table or bench for storage of hot plate magnet stirrer

Ph meter, scales and dispenser must be available

Vacum devices

Distilling units

Bunsen burner

Refrigerator

Freezer for storage of stocks, solutions and chemicals ,microwave ,gas stove

Glass ware and other equipments

All the chemicals utilized in the manufacturing of culture media

Split AC

GLASS WARES:

Glass ware used in lab of tissue culture generally made up of pyrex Erlenmeyer of various

sizes(50,125,250,500,1000,2000 ml)

Glass tubes

Petri dishes

Bottle of jam jam

Glass cup

Measuring pipettes

CULTURE ROOM:

Suspension cells, protoplast cells, anther cells aRe the most sensitive to environment

Room temperature for the growth of culture generally range between 15c to30c

The range of temperature greater may be required for the purpose of the experiment

Temperature and light must be programmed for 24hrs.

Culture room should be well ventilated with 20-98% humidity range.

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PRECUATIONS:

Distilled water should be used.

Glass wares should be washed with hot water(.>70c)+soap.

Glass ware should be dried in an oven at 150c and wrapped in aluminum foil, then

stored in closed cabinet.

Culture room should be cleaned once a week with disinfectants and soap.

Dissection equipments should be sterilized by sterilization combustion techniques,

because these equipments may be responsible for blast of autoclave chamber as

equipments are made of metals...

Several compounds belonging to the group of proteins, vitamins, amino acids ,

extracts ,carbohydrates that are thermo labile, there may be decomposition when

sterilized by autoclave ,so should be sterilized by filter.

Millipore filter with porosity +-0.2 micron (um) is one of the filters that should be

used for sterilization of materials that are thermo labile.

Parts of plants should be soaked with dis infectant.

Don’t wash herbaceous plant.

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QUESTIONNAIR

Q1: What kind of soil is ideal for tissue culture planlets?

ANS: Ideal media must have 5.75 PH.

Q2: we sterilize equipments , tissue of plants is there any need to sterilize soil as well? When we

plant tissue culture plantlets in the field?

ANS: No.

Q3: We wash ex plant with detergent before performing the practical, are plantlets produced as a

result of this practical immune to disease, virus after planting in the field?

ANS: NO.

Q4: What kind of manure or fertilizer will be ideal for tissue culture plantlets after planting in the

field?

ANS: NPK.

Q5: What kind of effect will be on plant tissue culture if we couldn’t sterilize soil before planting

in the field?

ANS: Nothing.

Q6: How long I can keep my plantlets under shade in case of no buyers?

ANS: One or two months

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Q7: What are major challenges associated with performance of practical?

ANS: Electrical power

Unskilled labor

Improper weighing of plant materials

Improper measuring of chemicals utilized in manufacturing of media

Improper operation of Equipments

Q8: What are the major challenges associated with transportation of tissue culture plantlets?

ANS: High transportation cost

Q9: What will be the effect of improper transportation on plant tissue plantlets?

ANS: Plants may be damaged

Q10: Why the price of tissue plantlets is so high as compared to ordinary plantlets?

ANS: Conventional plants are free and production cost of the TC plant is high.

Q11: Do tissue culture plantlets need extra attention or special management after planting in the

field?

ANS: Yes initial care is required that is a disadvantage of TC plant.

Q12: What will be the effect of improper or poor management on growth rate of tissue culture

plantlets after planting in field?

ANS: Proper management shall give uniform crop and early yield.

Q13: What are the sources of contamination during performance of practical?

ANS: Improper handling and autoclaving, frequent visits of people.

Q14: What are the chances of wilting, because of improper management under shade house?

And after planting in the field?

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ANS: Over irrigation.

Q15: Are there any special techniques applied to harvest fruit produced by tissue culture

plantlets?

ANS: No.

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QUESTIONNAIR

Q1: WHAT IS PLANT TISSUE CULTURE??WHAT IS THE IMPORTANCE OF IT??

Q2: HOW MUCH AMOUNT OR FUND IS REQUIRED TO SET UP A LAB?

Q3 : HOW MUCH MAN POWER IS REQUIRED TO SET UP  A LAB??

Q4: WHERE IN PAKISTAN WORK ON PALNT TISSUE CULTURE IS BEING DONE ON

PLANT TISSUE CULTURE??

Q5: WHAT BASIC EQUIPMENTS ARE REQUIRED FOR PLANT TISSUE CULTURE LAB?

Q6: HOW MUCH TIME IT WILL APPROXIMATELY TAKE TO PROPERLY SET UP  

PLANT TISSUE CUTURE LAB?

Q7: WHAT ARE THE IDEAL LAB CONDITIONS FOR PLANT TISSU CULTURE??

Q8: WHAT IS PROCEDURE OF PLANT TSSUE CULTURE??

Q9: HOW PLANT TISUE CULTURE IS BENEFICIAL FOR OUR BUSINESS?(SMALL AND

LARGE SCALE) , WE ARE GETTING GENETICAL IDENTICAL OFF SPRINGS OR

PLANTS FROM THIS TECHNIQUE

Q10: CAN WE GET DISEASE RESISTANCE VARIETIES FROM PLANT TISSUE

CULTURE??

Q11: WHICH VARIETIES OF BANANA ARE BEING DEVELOPED FROM PLANT

TISSUE CULTURE??? IN PAKISTAN

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Q12: FROM WHERE I CAN GET PROPER TRAINING ABOUT PLANT TISSUE CULTURE

IN PAKISTAN??

Q13: HARMFULL EFECTS OF PLANT TISSUE CULTURE ?IF ANY ??

Q14: BASIC REQUIREMENTS FOR INFRA STRUCTURE OF PLANT TISSUE CULTURE

LAB??

Q15: WHICH PARTS OF PLANTS ARE BEING USED FOR PLANT TISSUE CULTURE??

Q16: ARE THERE ANY SPECIFIC RULES AND REGULATIONS FOR PLACE WHERE

LAB HAS BEEN SET UP??OR ABOUT SIZE OF LAND, SIZE OF LABORATORY??

(NATIONALOR INTERNATIONAL)

Q17: WHAT IS THE LEGAL PROCESS REQUIRED TO SET UP A PLANT TISSUE

CULTURE LABORATORY? (IF ANY) MEANS NOC FROM ANY GOVERNMENT OR

PRIVATE ORGANIZATIONS, AND OTHER SPECIFIC DOCUMENTS)?

Q18: CAN PLANT TISSUE CULTURE CREATE PROBLEM?? IF YES THEN HOW??

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MEETING REPORT WITH DR SAIF ULLAH KHAN

MEETING DATE: Friday, June 28th

 MEETING TIME: 11:00 am to 12:00 am 

                                12:45 pm to 1:00 pm

ATTENDEE: IQRA JUNEJO

PLACES OF MEETINGS : (1) Biotechnology wing ,Room#1 

                                                H.E.J Research Institute, University Of Karachi

                                               (2) Centralized Science Lab, University Of Karachi

PURPOSE OF MEETINGS: Research in Establishment of Plant Tissue Culture Lab

THINGS DISCUSSED WITH DR SAIF ULLAH KHAN:

Sir dr saif is doing incredibly nice work in plant tissue culture, he appreciated me and

wished me good luck for my project, Sir as you know that we had already sent the

questionnaire to him, He answered all of my questions in a very polite way.

Following things were discussed:

Only 2-3 persons are required to run the lab , But they must be technically well informed

and trained they must be capable enough to handle all the situations.

Work on Plant Tissue Culture is being done in National Agriculture Research Centre

(NARC) (ISL), NIAZ Tissue culture lab tando allahyar, Commercial Lab (isl), H.E.J (k.u),

Botonical Garden(k.u),DHA Lab(khi), Ayub Agriculture (faisalabad).

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2-3 years are required for technical back up from lab.

Matric pass student can also work in lab, No special or specific qualification is required to

run the lab.

There are no specific rules and regulations are required to run the LAB, Only concerned

or authorized persons should be, Allowed to enter in the lab in order to maintain the

aseptic environment of lab., but lab should be near to road in order to minimize the

transport expenses.

Yes plant produced by plant tissue culture is of good quality but some time variations

come after one year, otherwise it is genetically identical to mother plant.

NO , we can’t produce disease resistance varieties, Only virus and disease free varieties

are produced..

NO varieties of different plant are not developed, Only varieties are being introduced in

Pakistan, People take small parts of plants from foreign countries and practice tissue

culture techniques on these parts..

You OR anybody can take training from NARC (isl) and HEJ research institute, university

of karachi, but HEJ is expensive then NARC.

NO, There is no harmful effect of this technique only the plant produced by it expensive.

NO there are no legal requirements for establisment of lab.

NO, plant tissue culture products do not create any problem.

No specific rules and regulations are required about size of lab,its depends how much

amount you want to invest on it ,or on what scale or capacity  you want to establish a lab..

HE provided me list of chemicals and equipments require to establish a lab. Which is

attached with this report.

Another fruitful  meeting was held with MR WAJID ,who works at centralized science lab

university of karachi, He assured me that he will give me  necessary assistance  for my this

research project...he also asked me to meet with DR EHTASHAM at Botanical

Garden ,university of karachi.. for further assistance as DR EHTASHAM is also working on

plant tissue culture..

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