Contents
Plant Tissue Culture
1) Introduction
2) Experimental Design
3) Week 1 Method: Preparation of media
4) Week 2 Method Sub-culturing
5) Week 4 Method Quantitative Assessment of Growth
6) Table 1(sterilisation methods)
7) Table 2 (MS media composition)
8) Concentrations
9) Dilutions
PART1. PLANT TISSUE CULTURE
The aims of this element of the practical work are:
* to investigate the influence of culture media on the development of
plants grown in tissue culture.
Learning Outcomes
 Plan and carry out a piece of investigative experimental work.
 Be able to make up defined nutrient medium from original materials,
including performing accurate calculations for diluting stock solutions.
 Understand the effect of plant growth regulators (PGRs) on plant
development.
Introduction
Plant tissue culture is central to plant biotechnology, as this group of techniques
allows the regeneration of entire plants from either a single cell or a small group of
cells (in vitro cultures or tissues).
There are several important applications of this technology for plant production
and breeding.
 The production of plants, which are identical to the parent material using
techniques such as micropropagation.
 The introduction of variation through regeneration of material with different
characteristics to the parent material.
e.g. through somaclonal variation or somatic embryogenesis.
 The regeneration of entire plants following genetic transformation techniques.
 In vitro cell culture techniques can be used to produce industrial quantities of
plant products.
The basic considerations and procedures in achieving a particular type of culture
are:
 choice of plant material



preparation of material (see Table 1)
preparation of media (see Table 2, concentrations and dilutions)
culture environment
1
To introduce new material into culture, sections of tissue are sterilized (to
prevent contamination) and then inoculated onto media with or without relevant
plant growth substances, with a view to producing adventitious or axillary buds.
N.B. The plant material, once it has been introduced into tissue culture is referred
to as an explant.
Callus is a mass of undifferentiated cells, usually cultured on a nutrient medium
solidified with agar. Inclusion of the synthetic plant growth regulator 2,4-D
encourages development of callus. If the callus is then transferred to medium with
different plant growth regulator(s), this medium can encourage the development of
roots or shoots (organogenesis). Callus can be initiated from various sources of
explant including; leaves, stems, seeds, although undifferentiated cells provide
the best source, e.g. the zygotic embryo, or undeveloped inflorescence.
Once cultures have been established, they must be regularly divided and
transferred to fresh media. This process is known as sub-culturing and involves
dividing the plantlets, either by splitting, or taking nodal sections. Appropriate plant
growth regulators in the media determine the type of growth which occurs.
Experimental Design.
As a class group, you will collectively design an experiment to examine the effect
of different concentrations of plant growth regulators (and possibly) different
carbon sources on plant growth. This practical investigation covers several weeks.
In the first week you will prepare agar-solidified media; in the second week you
will sub-culture plants on to the pre-prepared agar, and four or five weeks later
you will harvest the plant material and assess the effect of the media on growth
and development. The growth data obtained will be used as the basis for
Assignment 2 (Poster).
When sub-culturing plant material, different combinations of auxin and cytokinin
will give different types and rates of growth and development.
For this exercise it is suggested that the media used should have either no growth
substances or a combination of cytokinin: (BAP) and auxin (NAA) at different
concentrations.
For the plants you will be sub-culturing the usual concentrations are BAP 0.8 mg l1, NAA 0.1 mg l-1. You may wish to try some treatments with half the concentration
of BAP or NAA and combinations thereof.
The source of carbon may also influence growth therefore different carbon
sources, either glucose, fructose or sucrose may also be tested.
Each group will test a different ‘set’ of media (treatment), however each treatment
will be replicated twice within each group. The class group results will be collated
so it is extremely important that you keep a record of the composition of your
media and all measurements/observations that you make.
Treatment notes:
Code:
Description of treatment:
2
METHODS
Do Tissue cultured plants follow the rules of proportions of cytokinin (BAP to auxin
(NAA) in media regarding shoot, root or callus production – Hypothesis (other types of
hormones)
If doing it in other plants do you need to take into account the internal hormone levels of
that plant
Looking at growth (dry mass) and development (what it grows into – root, shoot, callus)
What should we measure?
Weight of roots
Weight of shoots
Weight of callus
No. of leaves
No. of shoots >2mm
WEEK 1. PREPARATION OF MEDIA
The same basic media is used for many types of plant tissue culture, and is based
on the medium developed by Murashige and Skoog in 1962 (MS medium). It
contains macro and minor nutrients, with a source of iron and vitamins (see
Table 2). The medium is sold commercially in powdered form, and only requires
addition of a carbon source, growth substances and agar.
To prepare 500ml of medium

ml/500ml
BAP
NAA
0
0
8
0.5
2
0.5
2
2
0.5
2
0.5
4
dilution factor =
initial concentration/final conc
100mgl-1/0.8 =125
volume of stock = 1/df x volume of final solution
1/125 x 500 = 4
Ratio
0:0
10 pots with 10 replicates per pot
8:1
4:1
4:4
1:4
1:8
3
Dissolve 2.27 g MS powder in ~ 500ml distilled water

Add:
appropriate amount of carbon source (either sucrose of glucose
15g)
appropriate volumes of growth regulator stocks
(Please refer to your experimental design treatment data for details of
what you need to add at this stage).

Adjust pH to 6 and make up to 500ml litre.
Weigh out 4.5 g agar and transfer direct to glass bottle.
NB: Usual bottle size is 300 or 500 ml, so divide up volumes/agar
accordingly.
Pour the 1 litre of medium in, shake, and screw top on, but not completely tight.
Note: for information only
For some plant material, alternative media are used, such as McCowan’s woody
plant media, This has less nitrogen than MS.
 For initiation of cultures of dicotyledenous species it is usual to have
cytokinin and auxin present, with more of the cytokinin than auxin. For this
exercise media used should have either no growth substances or a
combination of cytokinin: (BAP) at 1 M and auxin (NAA) at 0.1 M
 Likewise, when sub-culturing material different combinations of auxin and
cytokinin will give different types and rates of growth. For these plants, the
usual concentrations are BAP 0.8 mg l-1, NAA 0.1 mg l-1


Autoclave at 15 psi for 15 min.
Allow to cool (about 40 mins), then pour into containers:
- about 12 - 15 ml of medium into the small plastic vials,
- about 50 ml into the large tub containers
Pouring should be carried out in a sterile laminar flow cabinet, but may be carried
out on the bench if care is taken. Place lids on top but do not press on.
WEEK 2: SUB CULTURING PLANT MATERIAL
Each pair should use a different culture media
(refer back to your treatment notes, page 3).
1) Collect the media you prepared the previous week and check for
contamination.
2) Record the weight of the pots containing media.
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3) Retain at least one pot (do not add plant material to this pot!).
4) Sub-culture the plants provided, working carefully in the tissue culture suite. It
should be possible to avoid contamination by using good sterile technique, and
avoiding passing your hands over the culture pots.
Divide the plants provided by splitting the basal clump into individual shoots.
Cut tissue into relevant size pieces, and transfer to the containers prepared the
previous week.
5) Clearly mark the cultures, with a treatment code (e.g. A) and a number (1-6)
6) Weigh the pots containing the plant material
7) Incubate at 25 oC in the growth cabinet.
8) Observe at weekly intervals.
WEEK 4: QUANTATIVE ASSESSMENT OF GROWTH
As a group you will decide which measurements and data to collect for analysis.
You will need to compare the effects of the various treatments on the rate and
type of growth.
You may wish to consider the following;
1) The weight of the pots, the wet and dry weights of the plant material.
2) Differences in plant morphology (you may wish to take photographs)
Data collection.
Growth?
Development?
5
Table 1
Summary of Sterilisation Methods
Tissue
Pre-sterilisation*
Sterilisation
Storage
organs
Scrub clean under
running tap water.
Submerge in NaOCl (1-1.4%
available Cl2) for 30 mins.
Stems and
Roots
Scrub clean under
running tap water.
Submerge for 10 secs in
absolute EtOH.
Submerge in NaOCl (1-1.4%
available Cl2) for 20 mins.
Remove cut ends (where
cells have been killed) and
cut into small sections with
sterile scalpel.
Wash three times in
sterile distilled H2O.
Dry between pieces
of sterile filter paper.
Seeds
(a) Submerge in
absolute EtOH for 10
secs or
(b) Submerge in NaOCl
solution (1-1.4%
available Cl2) for 15
mins. Rinse in sterile
distilled H2O. [Soak
overnight in sterile
distilled H2O (for
removal of radicle etc.)]
Select seeds with intact
testas. Submerge in NaOCl
(1-1.4% available Cl2) for 20
mins. Alternative sterilising
agents include 10% (w/v)
calcium hypochlorite or 1%
(w/v) bromine water.
Inclusion of 0.5% detergent
(eg. Teepol) is
advantageous, especially for
seeds with rough surfaces.
Wash three times in
sterile distilled H2O.
* May be
omitted
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Procedure
Post-sterilisation
Wash three times in
sterile distilled H2O.
Planting out and callus initiation
Cut off area of skin with sterile scalpel.
Remove small discs of tissue with a sterile
cork borer and scalpel. Plate out face down
on agar medium for callus induction.
Plant vertically in agar medium for callus
induction.
(a) For very small seeds, whole seeds may
be plated out on agar medium for callus
induction or germination.
(b) Seeds may be germinated in sterile
H2O, on damp sterile filter paper or on
culture medium lacking growth regulators.
This gives a good source of sterile root,
shoot or leaf tissue for callus initiation.
(c) With larger seeds, radicle and plumule
tips can be dissected out and transferred
directly to callus induction medium.
Table 2
Murashige and Skoog (MS) Medium
mg 1-1
Molarity
1650
1900
4440
370
170
20 mM
19 mM
3 mM
1.5 mM
1.25 mM
0.83
6.2
22.3
8.6
0.25
0.025
0.025
5 M
100 M
100 M
30 M
1 M
0.1 M
0.1 M
27.8
36.7
100 M
100 M
Macro salts
NH NO3
KNO3
CaCl2. 2H2O
MgS04. 7H2O
KH2PO4
Micro salts
KI
H3BO3
MnSO4. 4H2O
ZnSO4. 7H2O
Na2M10O4. 2H2O
CuSO4. 5H2O
CoCl2. 6H2O
Iron source
FeSO4. 7H2O
Na2 EDTA. 2H2O
Vitamins
Myo-Inositol
Nictonic acid
Pyridoxine-HCl
Thiamine-HC1
Glycine
100
0.5
0.5
0.1
2.0
Carbon source – needs to be added
3 x 104
Sucrose (or glucose)
7
8
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PART 2: MOLECULAR BIOLOGY
Modern molecular biology techniques use an understanding of the structure of
DNA to identify genetic characteristics, which can be exploited by plant
breeders to enhance plant performance.
1) EXTRACTION OF DNA
The aim of this session is to provide an introduction to the basic technical aspects
of DNA extraction.
Learning Outcomes.
 Understand the principles of the methods used for the extraction of
DNA from plant material
 Demonstrate technical ability to perform laboratory techniques of
DNA extraction
Introduction
Many different methods are now available, but all methods appear to involve
three stages;
• Disruption and lysis of the starting material
• Removal of proteins and other cellular fragment contaminants
• Recovery of the DNA
Disruption and lysis of the starting material
Disruption is often achieved by drying or freezing in liquid nitrogen and
grinding in a pestle and mortar. This can be followed by lysis, which is the
disruption of the cellular membranes, usually utilizing a detergent or soap
material to breakdown the structures and release the chromosomal and DNA
materials
Removal of proteins and other cellular fragment contaminants
This typically involves the digestion of proteins (e.g. histones) with proteinase
K (an enzyme isolated from a fungus). This will also degrade any nuclease
enzymes, which would otherwise breakdown our isolated DNA/RNA.
The process of ‘salting out’ is then undertaken, which is a method to
precipitate the proteins and other contaminates out of solution, often using a
high concentration of salts such as potassium acetate or ammonium acetate.
The precipitates are then removed by centrifugation.
Alternatively an organic extraction method can be used, following lysis with
detergent. The organic substances used are typically phenol, chloroform and
isoamyl alcohol. The correct pH and salt concentration help to ensure the
contaminants enter the organic phase and the DNA remains in the aqueous
phase. Note that this method uses toxic compounds and generates toxic
waste, which need to be disposed of with care and in accordance with
hazardous waste guidelines.
Recovery of the DNA
DNA recovery is usually achieved by alcohol precipitation, following which, the
solution can be centrifuged again and the pellet of DNA recovered.
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Method: Isolation Of Plant Genomic DNA Using the Sigma
Extract-N-AMP Plant Kit ™
Materials
• Ethanol
• Cork Borer
• Heat block or water bath at 95°C
• Sigma Extraction Solution
• Sigma Dilution Solution
• Microfuge tubes (2ml)
• Forceps
• P200 pipette and tips
• Vortex
Procedure
We intend to use a kit to extract and purify the DNA from fresh leaf material
that you have collected (Extract-N-AmpTM Plant PCR kit -Sigma-Aldrich, UK).
http://www.sigmaaldrich.com/life-science/molecular-biology/dna-andrna-purification/extract-n-amp-plant.html#description
1. Collect plant material suitable for taking leaf disks the size of the hole from
a typical paper punch.. You could test neighboring plants of the same
species and try to determine if they are the same, or simply compare the
results from your species with other species collected by your colleagues.
2. Wash the plant material, first in tap water and then with distilled water.
3. Rinse a cork-hole-borer and forceps in 70% ethanol prior to use and
between handling different samples.
4. Punch a 0.5 to 0.7 cm disk of leaf tissue into a 2 ml collection tube
5. Add 100 µl of the Extraction Solution to the collection tube and vortex
briefly. Make sure the disk is covered by the Extraction Solution.
6. Incubate at 95 oC for 10 minutes. Note that leaf tissues usually do not
appear to be degraded after this treatment.
7. Add
100
µl
of
the
Dilution
Solution
and
vortex
8. Store the diluted leaf disk at 2 to 8 oC.
It is not necessary to remove the leaf disk before storage.
11
to
mix.
Extraction of DNA Activity
Identify the role of the components and the stage of isolation and purification at
which they act, for the Dellaporte genomic DNA extraction method shown on
the following page:
12
Dellaporte genomic DNA extraction procedure.
0.5-0.7g of fresh plant material was snap frozen in liquid nitrogen, ground to a
fine powder and transferred to a pre-cooled 30ml centrifugation tube. The
tissue was resuspended in 15 and transferred to a pre-cooled 30ml
centrifugation tube.
The tissue was resuspended in 15 ml of extraction buffer (100mM Tris. pH 8,
50mM EDTA pH 8, 0.5M NaCl with 10.5 l -mercaptoethanol, added
immediately before use). 2ml of 10% SDS was added, the mixture was
shaken vigorously and placed at 65 oC for ten minutes with regular agitation.
This was followed by the addition of 5ml of 3M Potassium 5M Acetate. The
samples were placed at 0 oC for twenty minutes prior to centrifugation at
25000 x g for 20 minutes at 4 oC.
The supernatent was carefully transferred to a fresh tube avoiding the carry
over of cellular debris, 10ml of iso-propanol added, mixed well and placed at –
20 oC for at least 30 minutes.
Samples were centrifuged for 15 minutes at 4 oC and the resultant pellet was
allowed to dry, before resuspension in 0.7 ml of TE (10mM Tris. 1mM EDTA).
Contaminating RNA was removed by the addition of 14 l RNase A (stock
concentration 20 mg/ml) and a 30 minute incubation at 37 oC.
Protein was removed by two phenol extractions, followed by a precipitation
step in 0.1x volume of NaOAc(sodium acetate) and 1x volume of isopropanol.
The pellet was washed in 70 % ethanol, the excess ethanol removed using a
pipette and the sample allowed to briefly air dry before being re-suspended in
500 l of water. The integrity and quantity of the sample was assessed using
both a spectrophotometer and agarose gel electrophoresis
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