A protocol for rapid, measurable
plant tissue culture using stem disc
meristem micropropagation of garlic
(Allium sativum L.)
Gerry Peat and Meriel Jones
ABSTRACT Plant tissue culture is becoming an important technique for the mass propagation of
plants. Problems with existing techniques, such as slow growth and contamination, have restricted
the practical work in plant tissue culture carried out in schools. The new protocol using garlic
meristematic stem discs explained in this article addresses many of these problems and aims to
produce measurable growth over two weeks. These methods could be developed in a range of
further investigations.
Plant tissue culture and micropropagation
laboratory techniques are becoming economically
and ecologically important across the world. In
the commercial horticultural world, plant tissue
culture techniques enable rapid production of
disease-free plants such as Phalaenopsis orchids
and Gerbera produced in the Far East, the
Netherlands and Madeira, and palm oil palms
used in tropical countries to establish biofuelproducing plantations. The benefits of such
culture include rapid reproduction, identical
flower colour or oil production due to the cloning,
and high-density, virus-free plants. In the UK,
specialist companies provide contract propagation
services to wholesale nurseries, growers and
public bodies. From the customer providing
a stockplant to the delivery of thousands of
rooted shoots takes, on average, 18 months. The
techniques are used for conservation purposes,
either to grow seeds to maintain genetic diversity,
or to bulk up numbers of rare or endangered
plants for re-introduction back into the wild.
On a Cheadle Hulme School group visit to
Micropropagation Services of East Leake,
Loughborough, we were shown how cowberry
(lingonberry) plants (Vaccinium vitis-idaea) are
being propagated using these methods for the
reclamation of eroded moorland in the Peak
District. The techniques are also being used in
industrial and academic research for a range of
purposes, for example to clone varieties and to
produce genetically modified plants.
The techniques for plant tissue culture were
developed in universities in the 1960s and 1970s
and at that time it was often used as a research
tool, for example to bioassay hormone levels.
Methods that could be used in school laboratories
were designed and published by Tony Storr
(1985), who was working as a teaching fellow
for the Standing Conference on Schools’ Science
and Technology (SCSST) project presenting
industry-related science practicals for schools.
He worked in partnership with Unilever plc in
Bedfordshire and produced protocols for the
culture of cauliflower, carrot callus, Nicotiana
and Saintpaulia. From his experience, Storr
suggested a success rate of about 80% for these
cultures. However, a survey taken at a recent
teachers’ conference by the author (GP) shows a
different picture, with about 5% uncontaminated
measurable growth of cultures. Of the 20 schools
represented, 18 indicated that they tried tissue
culture on a regular basis because they thought
that the techniques were important, but few had
any success or expected good uncontaminated
growth of cultures. For three decades, this poor
success rate has deterred schools from tackling
plant tissue culture. All schools in the survey
were using versions of the carrot or cauliflower
protocol, and all stated that they thought it was
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Plant tissue culture using stem disc meristem micropropagation of garlic
important that students were exposed to the
techniques involved in tissue culture.
Problems with existing protocols
The methods of culture outlined in the SCSST
project and those published elsewhere were trialled
in class sessions over the course of two years
and practical problems identified. The cultures
were set up using aseptic techniques performed
on a school laboratory bench rather than using
transfer chambers. Medium was produced using
recipes given in the SCSST project protocols and
sterilised by autoclave. The key problems that
were identified are discussed below.
Contamination
The major factor in the failure of cultures was
contamination by both bacteria and fungi. This
resulted in decay and failure of 95% of cultures.
The plant material was difficult to sterilise
because of surface bacterial and fungal spores
in crevices and folds of tissue. Increasing the
strength of sterilising fluid or the duration of
immersion resulted in damage or death of the
plant material. Starting from material grown in
greenhouses has been recognised as being more
successful because of a reduced bacterial load
compared with plants grown in open fields. Fuller
and Pizzey (2001) tried to address the problem
when culturing cauliflower by adding a biocide
called Plant Preservative Mixture™ (PPM™) to
the medium at the concentration of 1 cm3 dm−3.
Slow growth
Slow growth rates were a major problem because
groups that had set up the cultures could only
see measurable results after a month or, in many
cases, several months. By this time, they had
moved on to different topics in class and there
was a loss in continuity so they had forgotten the
ideas and methods. Insight gained from visits
to commercial micropropagation nurseries and
research departments would suggest that, to bring
a new plant variety into routine use, trial and error
methods over 18 months were required to gain
significant growth and established cultures.
Difficult to measure
The cultures that grew were difficult to measure,
especially the cauliflower propagation and the
carrot callus culture. Any measurement required
the removal of the plant material from the
culture tube and consequently an increased risk
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Peat and Jones
of contamination. Because the growth occurred
over months, it was also a problem to arrange
appropriate measuring intervals.
Limited student skills
Many of the contamination problems were down
to the lack of skills shown by the students. Despite
clear demonstrations, students are slower, leave
the tissue and tubes exposed for longer, and fail
to perform the aseptic technique adequately.
Commercial micropropagation nurseries take
three months to train up their laboratory staff to
use the same aseptic techniques efficiently; it is
therefore perhaps unrealistic to expect school
students to perform to the required standard in one
or two practical sessions.
Lack of facilities
Many schools lack the ideal equipment for plant
tissue culture, such as transfer chambers and
sterile culture tubes and bottles.
Proposed new student protocol
Our research has tried to address these issues and
produce a method that is:
l fast growing;
lmeasurable;
l reliable, with a high success rate and a low
rate of contamination problems;
l easy for students to learn adequate skills;
l possible to set up on a normal laboratory
bench without bacterial or fungal contamination;
l visually exciting to students.
Preparing the medium
The medium was prepared using a modified
method developed at the Micropropagation Unit,
Royal Botanic Gardens, Kew, and available
on the Science and Plants for Schools (SAPS)
website. This recipe, originally designed for
cauliflower tissue culture, has several advantages.
It does not require an autoclave because the
sterilising effect of the Milton solution (active
chemical dichloroisocyanurate) remains in
the medium to provide continuing protection
against microbial contamination infection during
incubation of the cultures. In this method, the
chemicals are dissolved and then the agar is
melted in a microwave before the addition of
the Milton solution. Because some tenacious
fungi continued to cause problems, the method
was modified with the addition of 1% Nipagin
(methyl p-hydroxybenzoate, also known as
Peat and Jones
Plant tissue culture using stem disc meristem micropropagation of garlic
methylparaben) to inhibit their growth. CLEAPSS
and other safety data sheets suggest Nipagin poses
no significant hazard, but may be an irritant to
skin and eyes. It is recommended that technicians
use eye protection when preparing the medium
from powder, and that students avoid skin contact
with the prepared medium.
Suitable containers
Small jars or tubes with screw lids are most
suitable. They are easy to place tissue into and
they retain moisture for long periods of time.
Most school laboratory suppliers have a suitable
container in their catalogues. We used 70 cm3 clear
plastic containers with screw-on lids (catalogue
number BO 03091) from Timstar Laboratory
Supplies, Crewe, Cheshire. Purpose-designed
tissue culture jars with vented lids can be bought
but good growth has been achieved with unvented
jars. Containers could be bought ready-sterilised or
glass jars could be autoclaved (plastic containers
can be microwaved for 30 seconds). The jars need
to be filled to a depth of 1 cm with the growth
medium. If the jar is unvented the agar will not dry
out within the time required for this experiment.
We have not seen any adverse effects on growth
from the inevitable changes to oxygen and carbon
dioxide concentrations over time, although this is
something that future experiments need to consider.
Petri dishes could be used but they are not ideal
because they dry out quickly and need careful
sealing, such as with electrical insulating tape.
Plant tissue
Garlic meristematic stem disc has proven to be
both reliable and fast growing. This follows the
work of Ayabe and Sumi (1998; 2001), who
produced a technique called the ‘stem-disc dome
culture method’ for commercial production of
virus-free cultivars. Older and slightly drier
garlic bulbs are used in preference to fresh ones.
Ayabe and Sumi found that pretreatment by
storing the garlic bulbs in a refrigerator at 4 °C
for approximately eight weeks enhanced growth
and especially shoot development. We have
found that even one week of cold storage in a
refrigerator can be quite critical: batches of garlic
cloves bought fresh from the supermarket showed
a low percentage of growth, but the same batch
after storage for a week all showed growth after
just one day. Zheng et al. (2002) found that many
cultivars responded in similar ways to a range
of culture techniques and we have experienced
similar success from a wide range of suppliers.
The dry scales were removed from a single
clove of garlic and a 2 mm thick slice was cut
from the basal part of the clove which is the stem
disc. The upper part of the clove was discarded.
The 2 mm thickness is quite critical as the first
millimetre may only consist of dried root tissue
from the previous year’s growth but the second
millimetre contains the meristematic cells.
The cloves within the garlic bulb have each
developed from an axillary bud and therefore
each represents a condensed shoot. Garlic is a
monocotyledonous species and therefore the
basal stem disc is meristematic tissue waiting to
develop in appropriate conditions. The cells on the
basal surface are likely to develop into root tissue
and those on the apical surface into shoot tissue.
Callus will develop if a suitable growth regulator
regimen is supplied. The stem disc was cut in
half, sterilised and finally placed on the growth
medium. One half-disc was placed with the basal
surface down and this tended to encourage shoot
and callus growth, which may be a function of
the kinetin in the medium stimulating the shoot
and inhibiting root growth. The other disc was
placed upside down and this showed good root
growth. Placing the stem disc halves in this way
will therefore demonstrate the full regeneration
potential of the tissue.
Sterilisation and aseptic technique
The practical was carried out on normal open
laboratory benches that had been wiped down
with a household antibacterial cleaner. Aseptic
technique was used, with scalpel and forceps
dipped into a beaker of 70% ethanol to sterilise
them before flaming and use. Material was cut
inside sterile plastic Petri dishes and lids opened
as little as possible to reduce contamination
from airborne microbes. The slices of stem disc
were sterilised by immersion in 70% ethanol
for 2 minutes and then transferred aseptically
to a Universal bottle of sterile distilled water
containing 1% Nipagin and agitated for 1 minute.
After this initial wash to remove the ethanol, the
tissue was transferred aseptically to a second
identical Universal bottle of sterile distilled
water plus Nipagin for a further wash for at
least 3 minutes. The tissue was then removed to
a sterile Petri dish, ready for placement in the
culture jar.
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Plant tissue culture using stem disc meristem micropropagation of garlic
Placement in culture jar
Two pieces of stem disc were placed in each
culture jar, one with the basal side upwards
and the other with it downwards. The jars were
placed in a growth chamber with fluorescent tube
lighting providing 10 000 lux, and a photoperiod
of 16 hours. The lights provided some heating
such that the temperature was maintained at
approximately 20 °C. We have achieved good
growth on a laboratory windowsill with less light,
but night-time temperatures below 10 °C are likely
to affect growth adversely. Trying to find the
optimum growth conditions is a suitable area for
further research but we have found that it is not
critical to the success of the practical.
Measurement of growth
Our experience suggests that in a class set of
cultures there will be both root and shoot growth.
For quantitative results, the number of roots and
shoots can be counted, and their total length could
be estimated since the culture jar must remain
closed to maintain sterile conditions. This could
be done from photographs. Growth is obvious
from day 3 and should give good results after
10 days (as shown in Figure 1). After 7 days, some
cultures could show signs of callus. Development
can continue for at least 50 days, producing
shoots, roots and bulbils (small bulbs) (Figure 2).
Health and safety issues
l Wear eye protection when sterilising the discs
of garlic.
l The major hazard with carrying out the aseptic
technique is the risk of an alcohol fire. The ethanol
must be kept well away, at least 1 metre, from the
Bunsen flame at all times. Keeping the ethanol in
a covered container except when briefly dipping
forceps or scalpel into it will reduce the risk.
l A sharp scalpel is needed to cut the stem disc
and students should be alerted to the need to cut
away from hands.
l Technicians should be aware that dissolving
the Milton tablet, and adding the solution to the
medium, generates chlorine, so this must be done
in a well-ventilated area.
Figure 1 Stem disc growth after 10 days
Figure 2 Shoot, bulbil and callus development after 25, 30 and 50 days
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Plant tissue culture using stem disc meristem micropropagation of garlic
Student protocol
Micropropagation of garlic (Allium sativum L.) using stem disc tissue derived from cloves
Garlic is an important crop plant that originated in
Central Asia but is now used worldwide for both
culinary and medicinal purposes. Most varieties
of garlic are sterile and cannot be reproduced by
seed. Therefore it is propagated vegetatively by
growing individual cloves from the bulb separately.
This method has the disadvantages of increasing
numbers slowly and passing on viral diseases.
New methods, including tissue culture and
micropropagation, have been used to solve this
problem. In commercial systems, the garlic tissue
is cultured to form callus tissue. Callus is a mass
of mostly undifferentiated cells that can be grown
in laboratory media. This is subdivided into many
fragments, each of which can grow back into
a new plant using a culture medium containing
appropriate levels of plant hormones. The method
below is used to establish the initial cultures and
has been found capable of producing stem, root
and callus tissue from the pieces of meristem cut
out of the clove.
Materials
l cloves of garlic that have been stored at low
temperature (4–6 °C) for 2 weeks;
l culture jar containing sterile growth medium
(Murashige and Skoog basal medium
supplemented with sucrose and kinetin);
l two sterile plastic Petri dishes;
l 100 cm3 beaker containing 70% ethanol for
aseptic technique;
l sharp scalpel and blunt forceps;
l two Universal bottles of sterile distilled water
containing 1% Nipagin antifungal chemical;
l one specimen tube containing 70% ethanol;
l waterproof marker pen.
Method
1. Remove all papery scales from the surface of
the clove, and, if dry scaly material covers the
meristem, break this away leaving firm white
tissue.
2. Sterilise a sharp scalpel and cut a 2 mm slice
off the base of the clove. This is the stem disc
l Any contaminated cultures should be
autoclaved unopened before disposal. A pressure
cooker operating at 15 psi could be used for this
purpose. Many modern pressure cookers do not
that includes all the meristematic tissue.
3. Place the stem disc into one of the sterile Petri
dishes and cut it into equal halves.
4. Place both pieces of tissue into the specimen
tube containing 70% ethanol for 2 minutes.
Shake the tube to ensure the surface is fully
sterilised.
Note: Keep the beaker of ethanol covered and
well away from the Bunsen burner.
5. From now on use careful aseptic technique
to ensure the tissue remains free of microbial
contamination.
6. Transfer the pieces aseptically into the first
Universal bottle of sterile distilled water and
Nipagin and leave for 1 minute. Shake the
bottle to ensure all ethanol is washed off the
garlic pieces.
7. Transfer the pieces aseptically into the second
Universal bottle of sterile distilled water and
Nipagin and leave for at least 3 minutes.
Remove the garlic stem disc pieces with a
sterile pair of blunt forceps and place inside
the second sterile Petri dish. Ensure the Petri
dish lid is replaced to prevent contamination
from airborne microbes.
8. Aseptically place one piece of stem disc onto
the surface of the medium in the culture jar
with the stem side down. Place the second
piece in the same jar but in the reverse
orientation; i.e. with the basal root side down.
Ensure the lid of the jar is raised as little as
possible, in line with good aseptic technique.
9. Label the lid of the jar: NAME, DATE, GARLIC,
2 min ethanol.
10.On the body of the jar draw a small reference
line so that any repeated photographs can be
lined up correctly.
11.Place the completed jars in the growth chamber
(10‌ 000 lux, 16 hour photoperiod, 20 °C).
12.Observe and record growth changes every
2 days for 3 weeks.
have interchangeable weights but they all operate
at a similar pressure of 15 psi, which is equivalent
to 2 atmospheres of internal pressure.
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Peat and Jones
Conclusion
Acknowledgements
The key aim of investigating tissue culture
methods and micropropagation in schools is for
students to observe development of callus tissue
and whole new plants. As with all practicals
involving plant tissue culture, uncontaminated
rapid growth is not guaranteed, but experience
has shown that starting with garlic stem discs and
taking the precautions explained in this article
significantly improves the chances that students
will achieve a good percentage of growth. The
growth is so vigorous that some contamination
by fungi will not significantly interfere with the
results in the first 10 days.
This work was supported by a Partnership
Grant from the Royal Society. Cheadle Hulme
School provided a sabbatical half term to allow
the author (GP) to start this work. Our thanks
to Helen Bradley and Grace Barker of Cheadle
Hulme School for running initial trials, Barbara
Wright of Micropropagation Services, East Leake,
Loughborough, for discussions on commercial
applications, and Margaret Ramsey of Kew
Gardens for a tour of facilities and discussion on
possible lines of research.
References
Ayabe, M. and Sumi, S. (1998) Establishment of a novel
tissue culture method, stem-disc culture, and its practical
application to micropropagation of garlic (Allium sativum
L.). Plant Cell Reports, 17, 773–779.
Ayabe, M. and Sumi, S. (2001) A novel and efficient
tissue culture method – “stem-disc dome culture” – for
producing virus free garlic (Allium sativum L.). Plant Cell
Reports, 20, 503–507.
Fuller, M. P. and Pizzey, T. (2001) Teaching fast and
reliable tissue culture using PPM and brassicas. ISHS
Acta Horticulturae, 560, 567–570.
Storr, T. (1985) Experimenting with Industry. 13.
Plant Tissue Culture. Hatfield: Association for
Science Education. Available at: www.docstoc.com/
docs/56346770/Experimenting-with-Industry-PlantTissue-Culture.
Zheng, S., Henken, B., Krens, F. and Kik, C. (2003) The
development of an efficient cultivar-independent plant
regeneration system from callus derived from both
apical and non-apical root segments of garlic (Allium
sativum L.). In Vitro Cellular & Developmental Biology –
Plant, 39, 288–292.
Website
Science and Plants for Schools (SAPS) – Tissue Culture
and Micropropagation – Cloning Cauliflowers: www.
saps.org.uk/secondary/teaching-resources/706.
Gerry Peat is the head of biology at Cheadle Hulme School, Cheshire. Email: [email protected]
Meriel Jones is a senior lecturer in the School of Biological Sciences at the University of Liverpool.
Email: [email protected]
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