1. 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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2. 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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3. TABLE OF CONTENT
S no
CONTENT
PAGE #
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
1
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4. 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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5. 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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6. 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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7. 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
out.
Careful manipulation of the culture media and the plant tissues will yield a 4-10 fold increase in plant numbers every
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8. 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
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.
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9. 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
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
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10. 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.
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.
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11. 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.
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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12. PLANT TISSUE CULTURE LABORATORY
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13. 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
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14. MEDIA PREPARATION ROOM:
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
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15. 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.
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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16. 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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17. 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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18. ANS: Over irrigation.
Q15: Are there any special techniques applied to harvest fruit produced by tissue culture
plantlets?
ANS: No.
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19. 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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20. 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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21. 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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22. 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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