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 Laboratory Requirements in Plant Tissue Culture

Lesson Prepared Under MHRD project “National Mission on

Education Through ICT”

Discipline: Botany

Paper: Plant Biotechnology

National Coordinator: Prof. S.C. Bhatla

Lesson: Laboratory Requirements in Plant Tissue Culture

Lesson Developer: Dr. Ashima Khurana

Department of Botany, Zakir Hussain Delhi College,

University of Delhi and

Dr. Rahul Kumar

RTGR, Department of Plant Sciences, University of

Hyderabad

Lesson Reviewer: Dr Rita Sharma

School of Life Sciences, Jawaharlal Nehru University

Language Editor: Namrata Dhaka

Department/College: Department of Genetics, University

of Delhi South Campus

Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL

Institute of Lifelong Learning, University of Delhi 1

 Laboratory Requirements in Plant Tissue Culture

Learning Outcomes

The chapter is aimed to enable the readers to learn about the following:

 Basic facilities required to start a plant tissue culture laboratory.

 Function of all these facilities and their importance in context of successful tissue
culture practices.

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 Laboratory Requirements in Plant Tissue Culture

Chapter: Laboratory Requirements in Plant Tissue Culture

Table of Contents

 Introduction
 Basic facilities required in a tissue culture laboratory
 Washing area
 Glassware/Plastic ware
 Area for media preparation
 Transfer area
 Culture room and incubators
 Greenhouse
 Basic laboratory equipment and apparatus
 Summary
 Exercise/ Practice
 Glossary
 References/ Bibliography/ Further Reading

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 Laboratory Requirements in Plant Tissue Culture

Introduction
Plant tissue culture techniques have played a pivotal role in addressing many questions of
basic and applied fields of plant science. Initially, this technique was used for studying the
roles of hormones in cytodifferentiation and organogenesis. It is now also used for the
functional characterization of genes, elucidation of underlying regulatory mechanisms by
developing genetically modified (transgenic) plants. Due to their intensive use in various
fields of applied plant science, plant tissue culture techniques have also revolutionized the
agriculture sector in modern times. For example, selected plants tissues/cells are cultured
as suspended cells to produce plant products. Additionally, transgenic plants with enhanced
agronomic traits such as high carotenoids (golden rice: Ye et al., 2000) or longer shelf life
(Flavr Savr tomatoes: Redenbough et al., 1992) have been produced. Further, tissue culture
methods also help in developing the somatic haploid embryos. Such somatic haploid
embryos can subsequently be used for the production of homozygous plants. Hence, tissue
culture techniques have played an important role in the progression of academic
and applied plant science.

The major applications of plant tissue culture techniques are:

 The large scale production of ornamental plants.

 Conservation of endangered plant species.

 Production of plant-derived secondary metabolites and recombinant proteins on a large-

scale in liquid culture of plant cells in bioreactors.

 Production of novel hybrids by either fusing protoplasts of distantly-related species or

embryo-rescue technique.

 Molecular, pharmacological and biochemical investigations of different aspects of plant

growth and development such as in vitro flowering.

 For the induction of polyploidy, by treating plants with of antimitotic agents such
as colchicine.

 To produce virus free plants from virus-infected stock, such as potatoes by culturing
meristem/tip culture.

 Production of genetically modified crops with improved agronomic traits.

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 Laboratory Requirements in Plant Tissue Culture

Brief overview of the techniques involved in Plant tissue culture

Figure: Steps involved in tissue culture. A number of tissues can be used as explants
and used for in vitro culture to regenerate whole plants by various methods of tissue
culture.

Source: ILLL Inhouse

Figure: A general scheme of plant tissue culture. The figure shows the different steps

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 Laboratory Requirements in Plant Tissue Culture

involved in plant tissue culture starting from culture of explants on nutrient media, then
callus regeneration, followed by differentiation of callus and formation of a complete
plantlet.

Source: ILLL inhouse

Figure: Different steps of tomato tissue culture on MS-agar (solid) medium.

Developed by: Authors

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 Laboratory Requirements in Plant Tissue Culture

Basic facilities required in a tissue culture laboratory

A laboratory is a place where all sorts of scientific and research activities can be performed
under controlled conditions. Laboratories used for scientific research may work on different
scientific problems and therefore, differ in the requirements of specialists in the various
fields of science and engineering. For example, physics lab would contain a particle
accelerator or vacuum chamber, whereas, a plant tissue culture lab would have a laminar
hood and tissue culture room. Despite the significant differences among laboratories, some
facilities are common. These facilities include the use of workbenches for sitting and
ensuring comfortable working conditions, use of cabinets to store the laboratory equipments
and use of a laboratory notebook/computer workstation for recording an experiment's
progress data collection and analysis. In this chapter we have focused on the facilities which
are required for a basic tissue culture laboratory.

Plant tissue culture can be defined as a collection of techniques/methodologies that are

employed to grow or maintain plant cells, tissues or organs on a nutrient culture medium
under aseptic growth conditions. Micropropagation is a technique which is widely used for
the mass production of clones of a plant. Due to its diverse applications in
reviving/maintaining plants (refer to Chapter Tissue Culture Applications – Part I), this
technique has immensely benefitted our society. Therefore it becomes important to
understand the requirements to start a basic tissue culture laboratory.

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 Laboratory Requirements in Plant Tissue Culture

Figure: An example of a floor plan of a laboratory.

Source: http://nptel.ac.in/courses/102103016/2 (cc)

Some of the prerequisite basic facilities for any laboratory, which actively practices the plant
tissue culture methods to grow plants, are:

 Washing area
 Glassware
 Autoclave
 Areas for media preparation, sterilization, and storage

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 Laboratory Requirements in Plant Tissue Culture

 Area for transfer of cultures/medium in aseptic conditions

 Environmentally controlled incubators or culture rooms

Washing area

The washing area is one of the most important places in a laboratory as its use starts even
before the actual start of an experiment and continues till the experiment gets completely
over. This area is mainly used for cleaning and washing of glassware and other items used
in actual experiments, therefore maintenance of its cleanliness is equally important. The
washing area should consist of sinks and proper draining boards. It should also have
provision for production of deionized/double-distilled water. Moreover, it must also have a
specific place to dry the washed glassware/vessels and a storage cabinet to store cleaned
and dried culture vessels.

Glassware/Plastic ware

The glassware is mainly used in media preparation, storage and setting up of the
experiments for growing the cultures. To prevent the contamination/infection in the growing
cultures, the glassware used in tissue culture experiments should be restricted to such
experiments only. Due to their continuous use, sometimes toxic metal ions can also be
absorbed on glassware which can be troublesome in the repeatability of the experiments.
Therefore, glassware should be washed thoroughly with a good laboratory detergent and
rinsed with tap water several times after use. Finally, the washed glassware should be nicely
rinsed with highly purified water to prevent any metal ion absorption. The glassware,
particularly the culture vessels, should be made of Pyrex or borosilicate glass which is
resistant to heat and also is less prone to breakage and scratching. Whereas the large
volume Erlenmeyer flasks are used in the preparation of culture media, the small capacity
flasks (50-, 125-, 250-ml capacity) are mostly used to grow cultures. Additionally, tissue
culture glass jar with poly propylene cap (commonly called jam bottles), test tubes, petri
plates, and magenta boxes etc. can also be used in tissue culture experiments. A number of
presterilized disposable polystyrene culture containers ( Falcon, Corning, Nalgene and Nalge
Co.) are available.

Before the use of any new glassware, it is highly recommended that it is properly washed
with detergent and autoclaved, thereafter. This practice prevents contamination of culture
media. Beakers, volumetric flasks, pipettes, and graduated cylinders are the other
glassware which is commonly required in tissue culture laboratories. Altogether, washing
and cleanliness of glassware determines the fate of a tissue culture experiment as dirty

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 Laboratory Requirements in Plant Tissue Culture

glassware often lead to contamination of growing cultures when incubated for longer
periods.

Figure: Photograph of an Erlenmeyer flask.

Source:
http://en.wiktionary.org/wiki/Erlenmeyer_flask#mediaviewer/File:Erlenmeyer_flask_hg.jpg(
cc)

Figure: Photograph of Jam bottles with caps containing tissue culture explants.

Source: http://honorsgenetictechnology2012-1.wikispaces.com/Logen+Casella(cc)

Washing of glassware

1. Immediate cleaning of reusable glassware used in plant tissue culture. Do not allow
media or agar to dry inside the glassware.

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 Laboratory Requirements in Plant Tissue Culture

2. The conventional method of cleaning glassware is to soak it for 4 hrs in sodium

dichromate- sulphuric acid and then washed vigorously in tap water. An alternative
is to soak the glassware in detergent solution for 16-20hrs and the wased in tap
water followed by rinse in distilled water.

3. Glassware used for culturing microorganisms should be decontaminated and

autoclaved.

4. All labels and marking should be removed from culture vessels prior to soaking.

5. Glassware soaked in 5% detergent solution for a minimum of 1 hr.

6. Personnel cleaning the glassware should wear facemask and acid resistant apron and
gloves.

7. Agar remaining in the culture vessel should be melted and collected in sieve and
discarded.

8. Washed glassware should be rinsed with distilled water and dried and stored in dust
proof cabinets.

Area for media preparation

The area dedicated to media preparation should comprise of sufficient storage space for the
chemicals, culture glassware/vessels etc. Additionally, proper bench space for small
equipment for example: hot plates/stirrers, pH meter, balances, water baths, and media
dispensing equipment should be available. Other necessary equipments which are needed in
media preparation and decontamination include two autoclaves including one for sterilizing
the media before initiation of the experiment and second for sterilization of waste and left-
over materials/cultures/infected media after the experiment, refrigerators and freezers to
store stock solutions, and a convection oven for heating the solidified stored medium.

The preparation of media requires careful weighing of all the components. Even if a
commercially prepared medium is used, care must be taken in preparing it and the stock
solutions that are required.

Only analytical grade (AG) chemicals should be used for preparing culture media. To
minimize the media-originated variations among the biological and technical replicates of
the same experiments, good laboratory practices, including accurate weighing of chemicals
and precise measurement of pH should always be practiced. To ensure accuracy, a step-by-

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 Laboratory Requirements in Plant Tissue Culture

step procedure should be followed for media preparation. The water used to prepare media
should be highly purified. Use of tap water should be avoided as it is a rich source of
undesirable salts, particulate matter (silt, organic matter, etc.), and microorganisms (algae,
fungi, bacteria). Instead either Reverse Osmosis or double distilled water should be used.
Overall, all the instruments used in media preparation should be checked time-to-time for
their proper functionality. This practice will prevent variations which might be induced due
to improper working of any instrument. Moreover, if more than one person is using the
same media for their experiments, common stock solutions for all the lab members should
be prepared. This practice is helpful in minimizing the media and/or user-based variations
among the tissue culture experiments.

Figure: Photograph of an area for media preparation in ILRI's forage genebank on the
ILRI Addis Ababa campus where a lab technician, prepares media for plant tissue culture in
the tissue culture lab (photo credit: ILRI/Stevie Mann).

Source: http://commons.wikimedia.org/wiki/File:Preparing_media_for_tissue_culture_-
_ILRI_Forage_Genebank_on_the_ILRI_Addis_Abeba_campus.jpg(cc)

Transfer area

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 Laboratory Requirements in Plant Tissue Culture

Transfer area is a central place in a tissue culture laboratory. It is possible to perform the
tissue culture techniques on an open working table, however, in that case the laboratory
must be perfectly clean and dry conditions should be maintained throughout. The most
commonly used instrument for making transfers in tissue culture laboratories is a laminar
flow hood. Due to its importance in tissue culture, transfer area should be well equipped.
The basic equipments include a source each of electricity, ultraviolet light, compressed air,
vacuum and a positive pressure ventilation unit. The ventilation should have a high-
efficiency particulate air (HEPA) filter. The most common type of transfer area is a laminar
flow hood. In the laminar hood, air passes through a dust filter followed by a HEPA filter.

Figure: Photograph of a Laminar flow hood with HEPA filters.

Source: Namrata Dhaka, Department of Genetics, University of Delhi, South Campus

Thus constant flow of both dust- and microbe-free filtered air prevents settling of dust or
microbes, respectively, on the working surface. It is recommended that before the transfer
of cultures, laminar hood should be sterilized by an ultraviolet light on daily basis. Further,
its surface should be wiped periodically with 70% alcohol during the transfers of culture
materials. Removal of any contaminated/infected culture should be preferably performed
once transfer of all the healthy cultures is completed. It is a good strategy to prevent
spread of infection during plant tissue culture.

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 Laboratory Requirements in Plant Tissue Culture

Autoclave

The purpose of aseptic technique in plant tissue cultures is the elimination of

microorganisms during experimental procedures. If sterile tissues are available, then the
eradication of microorganisms is done by using sterile instruments and culture media. Media
and other apparatus are made sterile by autoclaving them at 15 lbs/square inch (121°C) for
15 minutes. The use of disposable sterile plastic ware reduces the need for autoclaving,
however, augments the total cost of the experiment. Alternative sterilization techniques
such as filter sterilization (FS) must be used for heat-labile substances like plant growth
regulators (e.g. cytokinins, auxins etc.) and antibiotics (such as kanamycin, ampicillin etc.)

Figure: Photograph of an autoclave.

Source: http://nptel.ac.in/courses/102103016/2

Culture room and incubators

Culture room is another important area required for plant tissue culture. Since cultured
explants/tissues initially need to be incubated/grown in more controlled conditions
(temperature, humidity, and light quality), culture room should also be well equipped with
basic instrumentations. Typically, the ambient temperature required for the optimum
growth of plant tissues during culture generally ranges between 20°C and 30°C. Therefore,

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 Laboratory Requirements in Plant Tissue Culture

temperature of culture room should also be maintained within this range; however, a wider
range in temperature may be required for specific experiments. Further, the culture room
should have an alarm system to indicate any large fluctuations in the temperature which is
beyond the permissible limits. The culture room should have enough fluorescent lighting to
reach the 10,000 lux. Additionally, specific experiments may require growing a culture in a
specific light condition such as either blue or red and far-red etc. Therefore, electrical
circuits of culture racks should also be amenable for installation of such specific light
sources. Further, both light and temperature should be programmable for a 24-hr period.
Light: dark photoperiod should be maintained automatically by using the timers. Sometimes
cultures are to be incubated in continuous dark, therefore, provision of black curtains in a
few culture racks should be there. The culture room should be fitted with double doors so
that it can be kept dust free and temperature can also be maintained effectively. The culture
room should have uniform forced-air ventilation, and a humidity range of 20-98%
controllable to ±3%. To regulate temperature and humidity, air conditioners in connection
with their regulators and with prefixed maximum and minimum thresholds for these factors
are used in tissue culture room.

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 Laboratory Requirements in Plant Tissue Culture

Figure: A tissue culture room. In the picture, shelves are arranged in parallel and some
walking space is left between the two shelves. The cultures plates are placed on these
shelves and to avoid carrying any contamination from outside, users are entering in the
culture room with lab coats and proper masks. On the left side, a cupboard can be seen that
carries a temperature controller microprocessor and a photoperiod programmable timer.

Source: Namrata Dhaka, Department of Genetics, University of Delhi, South Campus

Figure: Petriplates containing tissue culture explants, kept in a rack in tissue culture room.

Source: Namrata Dhaka, Department of Genetics, University of Delhi, South Campus

Figure: Photograph of an incubator shaker used for incubation of cultures.

Source: Namrata Dhaka, Department of Genetics, University of Delhi, South Campus

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 Laboratory Requirements in Plant Tissue Culture

Greenhouse

Greenhouse is used to grow plants which are used to obtain explants. It is also required to
grow in vitro raised plants for acclimatization. Greenhouse requires facilities for maintaining
humidity and temperature.

Figure: Photograph of a greenhouse.

Source:http://earthguide.ucsd.edu/eoc/special_topics/teach/sp_climate_change/p_greenho
use.html(cc)

Basic laboratory equipments and apparatus

Since in vitro propagation is a very labor-intensive process and regeneration potential of
explants depends on number of factors, the basic set up of laboratory and standardization
of the specific protocols for different starting materials are important determinants of the
final success of this process in a laboratory. The plant tissue culture techniques for most

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 Laboratory Requirements in Plant Tissue Culture

plant systems often require similar basic laboratory equipments. The following table enlists
the items that are commonly required in a laboratory for in vitro propagation of plant
materials:

Table: Items needed to start a plant cell/tissue culture laboratory

Item Minimum Function Description/Specification

quantity/
number/
volume
Non-consumable
Water purification One Provides purified water Capable to purify water to a
system to make stock solutions resistivity of at least 200,000 ohms/
and other media cm and a conductivity 5.0
micromhos/cm level
Weighing balance One Measurement of At least 0.001 g readability with a
(electronic) biochemicals and media minimum 200 g weighing capacity
components
pH meter One For measurement and Range 0-14 +/- 0.01
adjustment of pH of the
prepared medium
Hot plate/stirrer One For the heating and 7” x 7” ceramic top; variable heating
mixing of the media and range (upto 400ºC) and stirring
stock solutions, speed (50-150 rpm); chemically
respectively resistant
Refrigerator and One each For the storage of stock Refrigerator: Capable to maintain a
freezer solutions, hormones and temperature of 2-6 ºC; Freezer:
prepared media Capable to maintain temperature at
approximately –20 ºC
Laminar flow One Provides a sterile Incoming air should be HEPA filtered
transfer hood atmosphere during to remove 99.99% of particles larger
transfer of the cultures than 0.3μm. It should also have a
UV-source for the sterilization of the
space inside the laminar hood
Glassware/ 250 ml (5-10), Mixing of stock solutions Made up of borosilicate glass
Beakers 500 ml (5-10), during media
1000 ml (5-10), preparation and its
2000 (5-8) and subsequent storage
4000 (5-8) ml
Wash bottles 5-10 with 500 Rinsing instruments, Pre-labeled, separate bottles for
ml capacity beakers, transplants dispensing water and isopropyl
from tissue culture alcohol
Bottles 100 ml (10-20), To store stock solutions, Made of borosilicate glass; black
500 ml (10-20) sterile distilled water polypropylene cap with rubber liner
and autoclaved media
Brushes 5-10 To clean glassware, Brushes of different length
flasks or bottles
Culture tubes One case For starting cultures in 25 x 150 mm, made of borosilicate
Stage I glass
Culture tube One case Holding culture tubes Able to hold 25 mm 24-40 test
racks tubes, able to withstand
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 Laboratory Requirements in Plant Tissue Culture

temperatures up to 121 ºC
Closures 500 Sealing culture tubes For 25 mm culture tubes
Culture vessel One case Culture vessel to Uses Magenta B Cap (C903) as
maintain plant cultures closure
Magenta B Caps One case Closure for food culture Closure for culture vessels; clear
vessel polypropylene closure; 100/ case.
Culture vessels Two cases Culture vessel for Autoclavable culture vessel and lid
maintaining plant made from clear polypropylene;
cultures round vessel measures
Erlenmeyer flasks 1000 ml(5), Mixing media Wide mouth, 1000 ml
2000 ml (5),
4000 ml (5),
6000 ml (5)
Filtration system One dozen For filter sterilization of Disposable, plastic, 0.22 μm pore
heat labile stock size nylon membrane
solutions
Forceps 8’ (5), bayonet Transferring tissue Serrated/nonserrated, stainless steel
(5), 5.5’ with
very fine point
(5)
Graduated 10 ml (5), 100 Preparing stock Glass or plastic
cylinders ml(5), 1000 ml solutions
(5),
Glass pipettes 1 ml (10-20), 5 Measuring out stock Graduated
ml (10-20), 10 solutions
ml (15-25), 25
ml (10-20)
Scalpel handles 5” length (10- Cutting explants Stainless steel
20), 8” length
(10-20)
Scalpel blades One box Cutting explants Stainless steel; individually
wrapped; sterile
Scoops 15 Measuring large Large, made up of plastic
volumes of biochemicals
Scoops One pkg Measuring small to Medium, 30 cm length, 2/pkg
medium amounts of
biochemicals
Spatula 20 To measure small to Stainless steel blade
medium amounts of
biochemicals
Sterilizers, Two Sterilizing media and Operates between 116- 126 ºC; 10-
pressure cooker instruments 20 psi; electric (for small
operations)
Sterilizers, Two For sterilizing media and operates at 121ºC with dial for fast
autoclave instruments or slow exhaust; 0-60 minute timer;
for large operations
Sterilizer Two Sterilizes instruments in dry heat with glass beads 120 V
hood between transfers (S636) or 240 V (S637)
Stir bars One pkg Used to mix stock Magnetic; teflon covered
solutions and during
media preparation
Stir bar retriever One Retrieving stir bars from With a magnet sealed in
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 Laboratory Requirements in Plant Tissue Culture

mixing vessel polyethylene

Thermometers 2-5 To measure In a range from 20 to 150 ºC
temperature of liquids
and culture room
Timer One For timing sterilization Electronic stopwatch
and general lab use
Consumables
Stocks of At least 10 liters It is used to sterilize AR grade is not required
isopropyl alcohol instruments and the
work areas
Detergent One gallon To clean glassware Big packing size should be preferred
Culture dishes One case Sterile, for incubation of Disposable, sterile, 100 x 15 mm
Stage I cultures and
cutting explants
Chlorine bleach One gallon Surface sterilize 4% stock
(Sodium explants
hypochlorite)
Roll tape One Labeling cultures, All purpose
storage bottles, media
vessels, etc.
Gloves One pkg Safely removing hot Provides protection up to 350 ºF
items from autoclave
Parafilm One roll Wrapping culture 4” x 250 ft size
closures
Lab markers One packet Labeling cultures Assorted colors

Towels One case When sterilized, it act General lab use

as sterile work surface
to cut explants
Developed by: Authors

Overall, the basic procedures of the in vitro propagation of plants can be easily learnt.
However, certain attributes such as focus on precision, hygiene, and strict adherence to the
protocol are the key ingredients for a successful in vitro tissue culture experiment.

Summary
Plant tissue culture techniques are indispensable for both basic as well as applied research
areas of plant sciences. However, it is recommended to check the basic facilities before the
start of any experiment. To summarize, the major basic facilities used in tissue culture
techniques that should be present in a standard tissue culture laboratory include area for
washing and storage of plastic ware and glassware, preparation, sterilization and storage of
nutrient media, aseptic manipulation of plant material, maintenance of cultures under
controlled environmental conditions in culture room, data collection and photographic facility
and an area dedicated for the acclimatization of in vitro developed plants.
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 Laboratory Requirements in Plant Tissue Culture

Glossary
Autoclave: It is a device which is used to sterilize equipment and culture media by
subjecting them to high pressure saturated steam at 121 °C for around 15–20 minutes.

Bioreactor: Any manufactured or engineered device or system that supports a biologically

active environment is called bioreactor.

Callus: A mass of unorganized undifferentiated cells.

Cytodifferentiation: Specialization of cells in eukaryotes during their development is called

cytodifferentiation.

Embryogenesis: It is the process of formation of embryo.

Explant: It can be defined as the tissue or plant part to be cultured.

In vitro: Investigations which are performed with biological molecules outside their normal
biological context such as in culture vials or glass vials.

Meristem: A meristem is the tissue in most plants containing undifferentiated cells. It is

associated with the growth zones in the plant.

Organogenesis: It is the process of formation of organs.

Totipotency: It is the ability of a single cell to divide and give rise to a complete organism.

Exercise/ Practice

 What are the applications of plant tissue culture techniques?

 Define an “explant”.

 What is the function of an autoclave?

 List a few basic requirements of plant tissue culture laboratory.

 Why is a separate culture room required for raising plant cultures?

 What is the use of a greenhouse in plant tissue culture?

References

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 Laboratory Requirements in Plant Tissue Culture

Murashige T and Skoog F (1962) A revised medium for rapid growth and bio-assays with
tobacco tissue cultures. Physiol Plant 15(3): 473-497.
Redenbaugh, Keith, Bill Hiatt, Belinda Martineau, Matthew Kramer, Ray Sheehy, Rick
Sanders, Cathy Houck and Don Emlay (1992). Safety Assessment of Genetically Engineered
Fruits and Vegetables: A Case Study of the Flavr Savr Tomato. CRC Press. p. 288.
Ye, X; Al-Babili, S; Klöti, A; Zhang, J; Lucca, P; Beyer, P; Potrykus, I (2000). "Engineering
the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice
endosperm". Science 287 (5451): 303–5.doi:10.1126/science.287.5451.303

Web Links
 http://edugreen.teri.res.in/explore/bio/tissue.htm

 http://aggie-horticulture.tamu.edu/tisscult/tcintro.html

 http://members.aol.com/MrDJReed/private/PTC.html

 http://www.tamu-commerce.edu/coas/agscience/clasnote/pls497/PlantTissueWeb/

 http://www.uni-bonn.de/~ulp50b/tissue_culture.htm

 http://www.orchidmall.com/CEVIE/cevtc-en.htm

 http://www.sigmaaldrich.com/content/dam/sigmaaldrich/product7/143/v8630.tif/jcr
content/renditions/medium.jpg
 https://www.labstockstore.com/v5Files/sfwlHpleNfygCJH2/148412/640/blstirretrieve
r.jpg

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