Seed Sowing Manual
                         by
                  Barry L. Cohen
                  G&B Orchid Laboratory
                  2426 Cherimoya Dr.
                  Vista, CA 92084
                  619-727-2611

      Reprinted, with permission, by Aaron J. Hicks, for the Orchid FAQs

      The purpose of this manual is to provide a guide to the first time
tissue culture practitioner. However, many of the suggestions may be of
use to the old-timer as well. It should be pointed out that there are many
ways to do the same thing; each lab person may find an easier way to
accompish the same task. Until you have become acquainted with sterile
procedures, I would suggest that you stick closely to the procedures
outlined in this manual.

      Let's assume you have made your first cross, and are prepared to sow
this seed.

      Chapter 1
      Making a flasking case

      Depending upon the intent of the user, a glove box can be made very
easily and inexpensively. My first glove box was made out of an old
jewelry display case from a pharmacy. I used the three clear sides of the
plastic case as the box, and covered the ends with some greenhouse plastic.
I cut holes into the ends, and attached some plastic kitchen gloves with
duct tape. Then I would use the kitchen table top or some other hard table
top as a work surface. In such a device you can seed sow or replate about half
a dozen flasks at a time! The cost of the box is less than $10. You can make
one out of plexiglass, or other materials which you may have some preference
towards, or make a more expensive laminar flow clean air station. This is
much more expensive, but gives one lots of freedom of movement as well as
efficiency. Either way, you will still be able to get the job done.

      Now that you have a flasking box (or for replate), we should discuss
procedure on cleaning the green pod. I suggest attempting to sow green pods
the first time around, as success rates are highest when you start with a
sterile capsule. You may overstay with a pod, and have the thing open up quite
unexpectedly, and be faced with a lot of loose contaminated seed. Understanding
the principles of green pod sowing is important, so we'll start with that,
and move on to cleaning contaminated seed.

      Chapter 2
      Green pod seed sowing

      Let's prepare our seed pods with some record keeping system. It is
critical to do this, and is best done with any kinds of system that you
are comfortable with, and will allow for proper record-keeping. The system
that I like best is to obtain a ledger book with the cross names and
an assigned numbering system. You may like to use some date code or other
system of numbers and letters in combinations. But it is VERY important
that you use at least 4 numbers or numbers and letters in combination
to avoid duplication or confusion. In your ledger book, write the name of the
cross and the number assigned, and record the date, the cross that was made,
and leave room to record information about the results of the cross and
so forth. This log book will become a valuable tool as a reference for
future crosses, and to evaluate the success of your stud plants as parents.

      Now let's mark our pods with an indelible pen (Sharpie, etc.) or
laundry marker with a fine tip with your assigned number. Of course, this
number will be placed on the seed sowing flasks as well. I find it quite
easy to group no more than half a dozen pods at a time in our glove box.
Next, we will need some tools: disposible scalpels, a tooth brush, 2 pint sized
Tupperware containers with lid, and a gallon of laundary bleach. If you are
working in a laminar flow hood, you might want to wear some kind of kitchen
gloves with the cuffs rolled up as protection against dripping bleach.
Scalpels may be had at many hospital supply stores; hardware stores and
hobby shops may have X-acto knives, which are less desirable, but still
usable for our purposes. Straight blades are superior to hooked blades
for what we will be doing.

      Since the pods are marked with an indelible ink (you tested it in
bleach and alcohol, right?), we can soak them in a solution of laundry bleach
(30% in tap water) in the Tupperware container (eds note: some people do a
pre-soak in 70%+ ethanol for 3 minutes) for 10-15 minutes. Now take
the toothbrush, scrub the pod, and return it to the bleach. During this
time, the scalpels should be soaking in the bleach as well. Now grab the
stem end of the seed pod with the left hand, and prepare to cut the seed
pod with the scalpel in your right hand- hold it so that the bleach will
run back DOWN your hand, away from the pod. Now you can cut the pod as if
slicing a water melon, exposing the seed.
      Taking a step backward, you should have prepared the flasks such that
they are covered with foil caps (4 or more layers of aluminum foil). Just
before attemping to open the pod, you will remove the foil cap and stopper
in one piece, and place them as a single unit on a clean ledge, out of the
way. If you are using a glove box, the interior should have been cleaned
using a spray bottle full of 30% bleach using a fine mist. If you are in a
laminar flow hood, none of this is necessary, except to keep the bench
sterilized.
      With the scalpel blade, scrape out a portion of the seed pod, and drop
it into the flask. I don't trust myself, and place another portion into
a second seed flask. In case I might contaminate the flask, and thus lose the
contents, I will still have another flask available. Restopper the flask,
and keep your fingers crossed for 2-6 weeks. With luck, it should green
up.

      Chapter 3

      Dry seed sowing technique

      Dry seed sowing is another story. Once a seed pod has split open,
the contents must be assumed to have been contaminated with bacteria or
fungal spores, which will contaminate your flask. The biggest problem
is fungal spores. Spores possess an impervious coat that makes them
difficult to kill with chemicals or heat. In order to sterilize the seeds of
orchids, you must expose the seeds to a chemical solution capable of
destroying the spores without damaging the seeds! There are a number of
chemicals available for this purpose, and most of them are some type of
chlorine salt with sodium or potassium. I have found excellent success with
common household bleach. The trick is to expose the seed and villains to
the solution long enough to kill the enemies, but not the seeds. I have found
that 5% volume/volume of household bleach made with sterilized distilled
water, when exposed to the contaminated seeds for a total time of 10
minutes and followed with a double rinse of sterile distilled water works
in 70 to 80% of all contaminated seed sowing cases.

      The exact procedure is simple. First label 1 or 2 mother flasks
(seed sowing flasks) with the code name of the seed cross you wish to
decontaminate. Next, label a sterile vial or tissue culture tube which
has been autoclaved (ed- same as pressure cooked at 15 PSI for 15 minutes)
and the mouth and cap, down about 2-3 inches onto the neck covered with a
4-layer thick foil cover. Into the sterile vial, place a half portion of
the collected seed, and add a 5% solution of bleach in water, agitating
intermittently. In the last 2 minutes, let the seed settle or float to the top
without agitation. Then decant off the excess solution about a half minute
from the ten minute mark. At the last moment, add the first sterile distilled
water rinse, and agitate for about a minute. Decant the liquid, and repeat,
but this time do not decant all the water. Open the seed sowing flask(s),
and agitate the remaining seed and water, and pour the solution into the
seed sowing (mother) flasks and restopper them along with the foil caps.
This way, the seed will be in suspension when you pour it into the flasks,
and may be distributed on the surface of the agar as you desire.
      If all goes well, the seed should germinate within a few weeks.
If you fail in your first attempt, you still have the seed you saved from the
pod. If that fails, you will have to remake your cross next year, so record
the results carefully.
      Once you have successfully made your seed sowing flaks, you must find
a warm (70-80 degrees F) and well-lit place to grow them. Artificial light
is very good for this, either incandescent or fluorescent, for a period of
about 8 hours; daylight also works, if it is well-filtered. Do not try to mimic
the natural sunlight period with fluorescent lighting in tissue culture, as
this will only slow growth, and cause problems with phenol production, a sign
of stress, and toxicity to the developing protocorms.

      Chapter 4

      Transplanting

      After a few weeks, your seed should be germinating; this does
not mean you should rush to replate! Your protocorms should be large enough
to be handled with replating tools and allow for easy handling and
separation. Do not overcrowd replate flasks! Most plants, including orchids,
do not do well if they are in competition with other, closely packed
plants. So, when you begin transplanting into your replate flasks, consider
the density that will grow well in replate. If in doubt, underplant! Also
consider the number of plants that you would like to have when they grow
up. Remember that when the plants are small, they take up MUCH less space
than when they grow up!

      We will need to make some tools for the job known as replate forks.
I like to make my own out of stainless steel welding rod cut to length
and flattened with a hammer into a knife or fork or hook end (like a
long thin spatula). You might like to heat the end with a torch, or just
hammer it cold. You will need a mister bottle with the same 30% bleach
solution. You will need rectangular Tupperware containers or glass dishes
to soak your replate forks in prior to working with your mother flasks
(ed- the old, old ice cube trays with no divisions in them, the ones that
had the levers, are EXCELLENT for sterilizing these long, thin tools!),
gloves, a quart bottle with sterile distilled water, capped with a four layer
thick aluminum foil cover.
      One tip that I like to use when the mother flask is really packed
with protocorms that are competing heavily and are unlikely to grow evenly
is to make a spread flask. A spread flask is an intermediate replate flask
with a smaller number of protocorms than the mother flask, but is not a
replate. This will allow the protocorms to grow and mature a bit, and make
them "more survivable" than they are right now. When a spread flask is done,
this shortens the time needed to replate, and may save several weeks in
growing time.
      Begin by selecting your mother flask an dgathering the number of
replates you will want to transfer to, along with the tools into your glove
box or flow hood. Place your replate forks into a solution of 30% bleach,
allowing them to sterilize for 30 minutes as you label your flasks. Label
the flasks carefully, and sterilize the inside of your box with the sprayer,
using 30% bleach solution. Spray the tops of the bottles you will be opening,
the bottles of distilled water, the forks, and your gloves, Allow the mist to
settle and kill any little critters that might be hanging around.

      Now open the mother flasks or spread and the desired number of replates
that are ready to be filled with protocorms. Place the foil-covered stoppers
on a ledge, away from where the work will be done, on a clean, dry surface.
It is now important to think of where your hands go and what they are doing
inside of the box; this is part of the technique that you must develop in
order to minimize the chances of contamination. As mentioned earlier, I like
to use two containers to sterilize forks- in the first container all the forks
start, and are moved to the second container once they have been used.
When they have all been used, there is a time out for 20 minutes to let the
last fork soak in the first container. Next, I remove a fork from the first
container into my working hand and open a bottle of distilled sterile water,
and wash the fork by erecting the fork so the wash water drains towards the
hand. I then insert the fork into the mother flask or spread flask, and start
working the protocorms apart, and carrying them to the replate flask. I like
to work the protocorms against the side of the flask, rather than the media
itself, which is soft and unable to support any pressure at all. It is good
to count the number of protocorms removed and planted, as it is easy to
overplant and cause the transplant to fail to grow properly due to crowding.
I usually plant between 25 and 35 plants in a 500 ml replate flask, depending
upon the surface area of the vessel one is using. When you are done planting
2-4 flasks, it is a good idea to exchange forks before going back into the
mother flask again. It is also best to limit the number of entries into the
mother flask, to keep down the chances of contaminating more than one flask
from the plants you pull out: one fork to move protocorms from the
mother flask to the replates, and one or more forks to do the actual replating,
etc. If there is an accidental contact between the fork and something
unclean, exchange forks.

      Now that the replating is done, you can enjoy watching the plants
grow up, same as the seeds. 8 hours per day of artificial light in the
vicinity of 100 footcandles is suggested, or around 300 footcandles of
natural light as measured during noon on a clear day. Do not try to mimic
the duration of timewith artificial light, as there are some important
differences. I will not go into this now, but trust me for a moment.

      I hope that you will find my comments and suggestios helpful, and
that you will not have many difficulties in attempting your first seed
dowing and replate work. Keep in mind that practice perfects technique,
and that you may try some variations in what has been suggested here- you
may well find better ways to perform some of the things described.
Good luck!