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Full grown plants from tiny bits of
leaves
By Sameera Ali
Tissue culture, in simple terms, is the science of growing
plant cells, tissues or organs isolated from the mother plant,
on artificial media.
Generally such cultures are axenic, which means there is no
other life form present on the culture medium than the plant
tissue of interest. This plant tissue is maintained on the
culture medium for a specified period of time and may be
transferred to fresh medium periodically, or to a different
medium to alter the path of development. This technology is
based on the concept of Totipotency - "the ability of a single
cell to express the full genome in the cells to which it gives
rise by cell division."
In Karachi, Husein Ebrahim Jamal (HEJ) Research Institute of
Chemistry is making remarkable headway into the field of
tissue culture technology.
Commercial production of plants through micropropagation
techniques has several advantages over the traditional methods
of propagation. For instance: plant propagation is faster
(more than ten times in some instances); it is independent of
of climatic changes; no need to worry about fungus, pestsand
virus;it can be accurately programmed. Production of
virus-free varieties from grossly contaminated plants is
possible. Plantlets require less space and can be stored in
refrigerator for several years. And, above all, identical
clones of plant varieites are esaily generated through this
method. Let's see how it's done.
Explant preparation
The tissue or organ that is removed from a plant and placed
into culture is called the explant. Before the explant is
placed onto a sterile culture medium, any contaminating
organism that might be present must be removed or otherwise
eliminated. This process of cleaning the explant is referred
to as surface sterilization or surface disinfestation. The
first step may be a simple rinse with soapy water to remove
any insects, loose spores and bacteria, and other debris that
might be harboring germs.
Following the initial wash, explants are then transferred to a
home bleach solution (generally 10 per cent bleach solution),
or to an alcohol solution (usually 70 per cent ethanol - not
methanol which is toxic). The explant is submerged in the
bleach solution for various times, but 10 minutes often works
well for many plants. If you use ethanol, the time is
generally shorter, with many tissues ready after one minute of
exposure.
When the explant has been "surface sterilized", it is usually
removed from the sterilizing solution and rinsed several times
in sterile, distilled water. This last step is performed
inside a laminar flow hood to maintain the axenic condition of
the explant and to prevent the re-introduction of
contaminating microbes. The explant is then ready to be
trimmed if necessary and placed onto a tissue culture medium.
Tissue culture media
Tissue culture media provides nutrition to the explant.
Explants may be immersed in a liquid medium as might occur in
root-culture, or positioned above the medium, but in contact
with the medium, which may be gelled by the addition of agar
(powdered china grass). This will support the explant and keep
it from submerging in the medium, yet allow diffusion of the
medium ingredients into the plant tissues.
Environmental conditions
After placing the explant on a nutritive medium, the culture
vessel containing the explant is placed in a controlled
environment. The amount of light, the spectral quality, the
periodicity, the temperature all vary with the plant tissue
involved and nature of the medium. There are few
generalizations that are worthwhile when it comes to
environmental conditions, except to say that the environment
should not be too hot or cold. Some developmental events
require light; for others to occur, the tissue should be kept
in the dark.
After the plant starts growing, it is shifted to the nursery
where is kept for several weeks - under close observation.
This is done to make the baby plants ready to face the
climatic conditions out in the open.
The local scenario
When these plants are grown up enough, they are shifted to the
field outside and in this way thousands of plants are produced
from a single meristem tip.
In Pakistan, the Plant Tissue Culture and Biotechnology
Division was established at the HEJ Institute of Chemistry,
University of Karachi, under the supervision of Dr Saifullah
Khan (see his article "Molecular farming to mark the next
decade" in Dawn Sciencedotcom, May 25 issue - Editor).
Techniques have been developed here to propagate virus-free
sugarcane and potato plants; efforts have been made to
initiate work on the production of secondary metabolites from
cell and tissue cultures, use of plant cells for
biotransformation, and use of tissue culture techniques for
the micropropagation of important medicinal, and disease-free,
plants.
Currently, in the Plant Tissue Culture lab, micropropagation
has been applied to ornamentals (ixora and crotons), fruit
trees (banana; "Amrit Sagar"), and a variety of other plants.
Ixora
Commonly known as the "flame of the forest", ixora belongs to
the family of Rubiaceae. This beautiful flower is very
commonly found in south-east Asia. Its long tubular flowers in
clusters (as many as 60 at the end of one branch) are found in
a wide range of colours (yellow, orange, scarlet, pink and
white). Commercialization of ixora will accelerate the
economic activity in the agricultural sector.
The HEJ Research Institue is propogating ixoras by tissue
culture and has produced about 4,000 plants which have been
trnasferred to soils and they are growing very well.
Crotons
Crotons are popular garden plants found in hot and humid
subtropical regions. In our cities most of the croton
varieties are imported from Sri Lanka and Singapore; its local
price ranging from Rs150 to Rs500 per plant, depending upon
the plant size and species.
At the HEJ, the tissue culture protocols have been optimized
and have started production in some varieties; the work on
other varieties are in progress.
Banana
Banana is propagated by means of suckers but the rate of
multiplication of a clone is slow and suckers may get infected
with various systemic soil-born and viral diseases.
In the recent past due to an epidemic of viral disease, the
Banana Bunchy Top Virus Disease" (BBTD), there was a decrease
in the yield of bananas. This disease was first observed in
the coastal areas of Thatta district in 1988 and had decreased
the productivity to a staggering 50 per cent. The only
solution to this problem is rapid multiplication of clean
material using tissue culture technology. At the HEJ, this
technology is beign routinely used for the production of
disease and virus free banana plants.
Tissue culture for home
Whilst in a research laboratory such as that in the HEJ
Research Institute ofChemistry, high-tech equipment is used
for plant production through tissue culture. It is important
to note that many home gardeners and hobbyists could
substitute the high-tech equipment and instruments with
ordinary household items such as:
1. A sterile air cabinet used to transfer plants. A fish tank
on its side makes an ideal transfer cabinet. Any glass chamber
about 50cm long, 40cm high and 40cm deep could easily be made
into a transfer cabinet.
2. A pressure cooker to sterilize media, instruments, water,
paper towel (or white copy paper), etc.
3. Small glass jars (baby food jars are excellent; see
picture) and take-away food containers with lids which can
withstand the heat inside a pressure cooker are ideal.
4. Scalpel and forceps.
5. Tissue paper ot white copy paper, cut to size, can be
sterilized and used for a sterile cutting surface.
6. A spirit lamp for flaming the instruments.
7. Handheld spray bottle containing 70% ethyl alcohol solution
to spray the transfer chamber and other surfaces. (Never use
methyl spirit for this purpose as it si toxic.)
8. Dilute chlorine solution; for example 1/4 dilution of the
household bleach for use in surface sterilization of plant
material.
9. Any skin disinfectant obtainable from any chemist shop.
10. Media (see below)
Media preparation
All the ingredients given below can be purchased at a
supermarket or chemist shop.
1. Two cups of distilled water
2. A quarter cup of sugar
3. Fertilizer stock: 1/2 tablespoon all-purpose 10:10:10
(nitrogen/potassium/phophorus) water-soluble fertilizer in one
litre of water: use just one cup of this stock for this
recipe.
4. Inositol tablet (500mg): 1/2 tablet
5. Vitamin tablet with thiamine: 1/2 tablet (any multivitamin
tablet will do).
6. Agar flakes (china grass): four tablespoons
This is the basic medium. For preparation of multiplication
and rooting media add 1/2 cup of coconut milk and 1/2 teaspoon
of malt. Replacing the coconut milk with 1/2 cup of green
tomato puree or 1/2 cup of freshly squeezed orange juice may
produce different responses. Ensure that the pH of medium is
always between 5 and 6 (using narrow range pH indicator tape).
Adjust pH if necessary, with acid (citric acid) or alkali
(cooking soda).
Mix the ingredients in a saucepan and gently boil until the
agar (china grass) has dissolved, stirring continuously to
stop the agar from sticking or burning at the bottom of the
pan. Dispense into glass jars, using a deep spoon so that the
medium is about 2cm deep. Cover and cook in a pressure cooker
for 15 minutes after full pressure is reached (this will be
achieved when the pressure valve starts letting steam out).
Sterilizing
Forceps and scalpels can be sterilized after wrapping them in
aluminium foil and cooking in the pressure cooker for 15
minutes. These items can also be sterilized by being washed in
chlorine solution or being dipped in alcohol and flamed.
Sterile water is needed for rinsing plant material and sterile
paper towel or white copy paperto be used as a clean surface
to work on. The towel can be discarded after using once, and a
fresh sterile paper towel used. The water can be sterilised in
glass jars. Place papers inside a paper bag and cook them in
the pressure cooker above the water level. The bag will be wet
on completion of sterilization. Transfer the bag to an oven
set at 80oC and allow the bag to dry inside the oven. Do not
unwrap the papers until needed.
All plant material can be sterilized in diluted domestic
bleach, and detergent or liquid soap. Put plant pieces in a
jar containing the bleach for 10-20 minutes. Stirfrequently.
Discard the chlorine solution (bleach). This process will kill
bacteria and fungi and sometimes some parts of the plant such
as outer bud scales and softer shoots. Rinse plant pieces
twice with sterile water.
Sterile cabinet
Great care should be taken to ensure that your cultures are
free from contamination. To achieve this do the followings:
1. Tie back your hair, roll your sleeves up and remove your
watch and other jewellery. Wash your hands thoroughly with the
disinfectant solution suitable for skin application. If
allergic to any disinfectant wash your hands with water and
wear a pair of surgical gloves.
2. Sterilize the inside of the cabinet by spraying with 70%
alcohol and wiping dry with sterile tissue.
3. Collect and organize all the items you will need close to
or inside the cabinet.
4. Working in the cabinet: take a sterilized piece of stem
from the jar with a pair of forceps (do not touch the plant
material with your hands). Also sterilize your instruments by
dipping them in alcohol between each manipulation and flaming
them. Small pieces, 2-3 cm long with a few leaves can be cut
and transferred to agar medium. If the leaves are too large,
either remove them or cut them to one-third or half the size.
Put one piece of the shoot into each jar (it is important to
have only one shoot per jar at this stage so that if the shoot
is contaminated it cannot spread to the others). Shut the lids
of the jars. Store the jars at room temperature away from
direct sunlight. Leave these shoots for one month.
One of these three things will happen during this time: some
of the shoots will be killed by the chlorine solution and or
the toxin produced by plants themselves; some will be
contaminated by fungi and bacteria; or new shoots will grow
very rapidly from the axils of the stems in the uncontaminated
containers. Discard the dead and infested cultures.
Shoot multiplication
The shoots from the previous stage may have elongated during
the past four weeks. They need to be transferred to the medium
containing the coconut milk (coconut milk has some growth
promotion properties which makes plant segments to produce
more shoots) for further multiplication.
1. Repeat the preparation and sterilization steps for the
medium, instruments and chamber as before. Sterilize your
hands as before too.
2. Transfer the containers of shoots to one side of the
cabinet and the sterilized medium at the other side.
3. Use sterile paper, scalpels and forceps as before.
4. With a pair of forceps, remove a stem from its container
(jar) and cut on the surface of the sterile paper moistened
with some sterile water. It is important to do all the
manipulation on a damp paper towel as these plants are very
soft and can dry up quickly. The separated shoots can be
transferred to the new jars. At this stage, up to five pieces
of plant may be put inside each container.
5. Store cultures as explained in the previous stage.
The multiplication stage may be repeated every four weeks
until enough plants have been obtained. As a general rule,
shoots can multiply 3-4 times every four weeks. The high rate
of contamination during this stage suggests that you are
getting airborne contaminants due to poor hygiene.
Root formation
Once you have established enough shoots, let them grow to at
least 2cm before beginning the rooting process. Transfer
shoots to the rooting medium containing coconut milk and malt.
Up to five shoots may be put in each culture vessel. Store the
jars in their usual place. Roots should form within two to
four weeks.
Potting (acclimatization)
The operation at this stage is carried out on the open bench.
The rooted cultures can be treated as follows:
1. Fill the pots with suitable potting mix without any
fertiliser and water well. Allow to drain.
2. Remove the rooted plants from agar medium using a pair of
forceps.
3. Wash off the agar thoroughly from the roots using lukewarm
water.
4. Insert a hole in the middle of the potting mix and gently
insert the roots in that hole.
5. Spray the foliage with a hand spray containing water. These
pots can be kept inside a larger plastic containers with a
glass cover, out of the direct sunlight. Gradually remove the
glass cover but watch for signs of drying up and, if needed,
use the hand spray to spray water on the foliage.
6. When the roots are well established and the plants are
acclimatized (this should take about 4-6 weeks), they can be
given fertilizer and be treated like any other plant. It is
advisable to gradually increase the light intensity for the
plants too.
The writer is a freelance contributor.
Courtesy Dawn
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