Why use
the meristem?
Tissue culture can be done with two
different sets of tissue from the Hosta plant: the meristem or the very young scape
(flower stalk). To replicate a variegated Hosta, the meristem MUST be used, otherwise the
resulting explant will be a single color - green or blue. This phenomena is based on the
way color is genetically transmitted in Hosta. (See numerous articles in the American
Hosta Society's Journal. Also see:
www.aggie-horticulture.tamu.edu/tisscult/chimeras/chimeralec/chimeras.html
The newly placed explant on media
sometimes takes a while before it becomes "established" or
"acclimated" and starts to grow vigorously. Don't give up. One professional
tissue culture lab reports this period can last up to eight months.
Scape Tissue
If you use the scape tissue, it must be just
emerging and rapidly growing from the center of the crown. Cut the tissue in lengths of
about 2-4 cm and insert the tissue upside down, after thorough sterilization, in the cell
proliferation media.
Preparation of meristem
and explant initiation on media
Dig out crown and
wash off dirt, or remove dirt from around the crown in the ground. Lateral bud or offshoot
should be well developed and growing but just short of unfurling first leaves. (See
photo) |
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Cut/remove offshoot
from crown so that there is a small portion of the crown material at the base of the
offshoot.
Slowly peel layers of petioles and leaves from the
offshoot, until you get to the center growth stem.
Note: if you leave a layer or two extra, the
world will not end and you can still have success; however, the fewer the layers the less
chance for contamination. As an analogy, you have a miniature crown with all but the
center growth point left. In many cases, the base material will be about .2-.3" thick
and .25" in diameter; the meristem itself will be about .5" long. The sizes, of
course, vary with the size of the plant itself. (See photo) |
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- Make sure that the "base material" is blemish free. No
"blackheads" or other spots allowed. These spots contain bacteria and will
contaminate your explant.
- Place prepared meristem in tea strainer under a stream of lukewarm
water for about one hour to wash off bacteria and dirt.
- Place meristem in a baby food jar with 70% isopropyl alcohol, stir
rapidly, for 30 seconds. Rinse in sterile water.
- Place in 10% bleach solution, add a drop or two of detergent, stir
rapidly, for 10 minutes.
- Rinse in sterile water (3 stages and 3 containers) 5, 3, and 1
minutes each, stirring rapidly. Note: always use sterile forceps for the transfer
process.
- Place last jar from rinsing process containing the meristem inside
your sterile "hood" Using sterile conditions, transfer meristem on to cell
proliferation media (you do this by "poking" the explant into the media so that
the base/crown part is submerged in the media and the "leaf part" is above the
media) and replace cap.
- Put jar inside of plastic bag (spray inside with pyrethrum based
insecticide) about 3"- 5" under flourescent lights with a 17/7 hours on/off
cycle. Adjust height of lights to maintain temp around 75 F.
- Now wait 4+ weeks.
Other Tidbits
This is a young
plant with two shoots ready for tc. Crown dug out of soil, washed off, and two shoots
harvested. After the two shoots were taken and placed in tc, the crown was soaked 30 min
in 3,000 ppm of BAP-10. |
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| This is the result 10 days later. The miracles of BAP-10. The younger shots were induced by using BAP-10(TM) - A real bonus to the Hosta
tc'er.
Each shoot shown here would be OK to use in tissue culture. They are
growing rapidly, but no leaves are yet unfurled.
NOTE: BAP-10(TM) is a plant hormone that overcomes apical
dominance so that lateral buds (at the base of each petiole) can develop more quickly than
they would in nature.
Extensive research has been done at Auburn Univ by Dr. Gary Keever and by Dr. Ed Elslager,
a retired researcher and a Hosta enthusiast. A number of articles reporting their research
have been printed in recent issues of the American Hosta Society's Journal.
There is much to be learned about the uses of BAP-10(TM) and early research and practices
indicate a wide range of uses and responses. Many anecdotal reports also have been
reported in a number of local Hosta societies' newsletters. |
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Text and photos for this article
were produced by Charles Tuttle
and are copyright protected. No reproductions of this can be
made without written permission of Mr. Tuttle. |
