When ordering media we find a baffling number of options in the
One of the
most complete media is in the Sigma catalog: Murashige and Skoog shoot
medium B (MSMB) (Sigma Catalog No. M7149). At the appropriate time,
pretransplant medium (Murashige syngonium stage III Pretransplant Medium
with sucrose: Sigma
You will also need a gelling agent, preferably a blend of agar and agar
substitute, such as Agargel.
Purchase sterile distilled water from a local grocery store. For 1 liter
use a 2 liter container (because the medium boils up). Add the powdered
the water and stir. (Don't add the agar (gelling agent) at this stage
gums things up when adjusting the pH).
Adjust the pH to pH 5.7 using 1N NaOH or 1N HCl (carefully, by the drop)
paper (3.5 - 6.8), or a pH meter. (If you don't have a pH meter, the
might.) Now add the agar. Use about 5 grams per liter of medium . Heat
stir until the the medium is clear. (The clarity tells you that the agar
Dispense into test tubes.
Sterilizing the Medium
Pour about one and a half inches of water into the pressure cooker.
upright in the cooker. To hold them upright place them in a wide mouth
a wire or wooden rack, or tie them with string in bundles of ten. They
over. Process at 15 pounds for 15 minutes according to the instructions
which come with
An explant is the part of a plant that you put in culture. The example
a strawberry runner tip. Select a young runner where the bud on the end
opened. Cut it off the plant one inch or so from the end. Transport the
classroom in a plastic bag in which you have placed a wet, but not
towel. If the runner tips are not going to be processed immediately,
held in a refrigerator for a day or two.
Fill a pint jar half full of sterile water (boiled, pressure cooked tap
water or the
store-bought bottled distilled water. Add 2 or 3 drops of liquid dish
(or Tween 20, a wetting agent). Place the runner tips in the jar and
screw-on lid. Vigorously shake the jar by hand for one minute. Pour off
two or three times with fresh sterile water. Repeat this operation (or
alcohol for a few seconds and rinse. In another container add 30 ml of
bleach to 270 ml of sterile water(10% bleach). To this add 2 drops of
detergent. Add the
explants and shake intermittently for 10 minutes. Quickly drain and add
cover and shake. The explants are now ready to take to the transfer
Starting the Explants
The transfer chamber should be ready with the walls and workspace wiped
with 10% bleach/sterile water solution or 70% isopropyl alcohol (not if
using a Bunsen
burner). There should be a container of 10% bleach/sterile water and a
of 1% bleach/sterile water to sterilize and rinse the instruments and
hands of the
Immerse the forceps and knife for 30 seconds or more in the 10% bleach
in the 1% bleach and rest them on a sterile holder or paper towel to dry
seconds. With the forceps place a sterile paper towel on the workspace.
forceps place a runner tip on the towel. Place the forceps in the other
to hold the tip
while the first hand uses the knife or scalpel to cut off 1 cm of the
the knife in the 1/10 bleach, move the forceps to that hand. Grab a
tube of medium with the other hand, hold it by the base. With the little
finger of the hand
holding the forceps, remove the cap and cradle it there while you use
to firmly lay the bud on the medium. Recap the test tube and seal with a
Scotch tape (or Parafilm).
Place the test tubes in the planter tray (or other appropriate holder
and place the
tray on a shelf under fluorescent light which is 8-10 inches above the
tubes. Room temperature is o.k. Continuous light is acceptable, but 16
8 hours darkness is standard.
Check daily for contaminants. If any are found, sterilize tube and
discarding the contents.
Transfer the explant every two weeks or so until it is actively growing.
two months you should be able to divide the culture into two pieces,
is about 0.5 cm in diameter. Continue to divide and transfer until you
plantlets. The plantlets should be singulated as you transfer to
medium which has no hormones (or only IAA).
When the plantlets begin to root, perhaps two to four weeks, transplant
them to a
light artificial soil mix, such as peat/pearlite, in a seedling tray.
clear plastic and place on a lighted shelf or in a shaded greenhouse.
three weeks begin leaving the plastic off for a period of time each day.
time the plantlets
are left uncovered should get longer each day, until after about a week,
can be left off completely. (Tissue cultured plantlets are more delicate
than seedlings as the stomates remain open until they slowly adjust to
normal humidity and light.
After having successfully tried plant tissue culture, you will wonder
every plant you see, asking yourself, "I wonder how that plant will
respond in tissue
preparation | background