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From Pekka.Olin@lut.fi Fri Jun 14 04:56:19 1996
Subject: The Asymbiotic Method for Orchid Seed Sowing
To: ahicks@mailhost.nmt.edu (Aaron Hicks)
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Hi Aaron,
here's the file. Please feel free to correct my English; lord
knows there's a lot to be fixed!
All the Best,Pekka.
- cut here -
_______________________________________________________________________ _______
Source:
"Harrastajan Suuri Orkideakirja" ("Hobbyist's Great
Orchid Book") by Esko Puupponen. 2nd edition, 1968.
page 127:
Asymbiotic sowing method
The sowing method explained here does not require the addition of
purebred fungus into the nutrition-jelly-mix, but the germination and
the initial growing are started by the chemical substances in the
nutrient solution. Only later, during the so called compot stage, the
plants receive the fungus in their growing medium, and their growth
is then greatly enhanced. The symbiotic method is very complicated,
and it has been almost totally given up with the introduction of the
easier asymbiotic method which is an equally sure method.
This method has been seen to be a fairly easy one, and another of its
advantages is that only few apparatuses are needed. If the task is
carried out carefully, the rate of success can be quite high, even so
that only very few bottles are contaminated with mold spores.
Performing the sowing goes as follows: the needed amount of 200
cu. cm Erlenmayer bottles (Jena or Pyrex) is washed profoundly
and placed in the oven. (14 to 16 bottles are needed for each
litre of solution.) The temperature of the oven is adjusted to 150
degrees [N.B. the Finns use centigrades. 150 degs. centigrade
is equal to 302 degs. Fahrenheit]. It is an advantage if the bottles
are sufficiently hot when the nutrient solution is poured into them.
When preparing the solution, the needed amount of distilled water is
measured and poured into a kettle, and 0.1 litres of basic solution
is added per each litre of water. Sugar, laevulose and dextrose
acquired at the pharmacist's are added: 10 grams per each litre of
water. If these are not available, the usual household sugar can be
used, and its amount per litre of solution is 20 grams.
The recipe of the solution given here is one that the German orchid
grower Hans Thomalen has used for germinating Paphiopedilum
seeds, but in many experiments it has been proven to be very good
for Laelia and Cattleya. It is recommended that one has the basic
solution made at the pharmacist's so that all the chemical substances
mentioned are added with tenfold ratio in one litre of water. This way,
only 0.1 litres of the solution is needed when making one litre of
the final jelly.
Ammonium sulphate 0.06 grams
Ammonium nitrate 0.37 grams
Potassium nitrate 0.40 grams
Monosodium phosphate 0.30 grams
Magnesium nitrate 0.11 grams
Ferric sulphate (FeSO4 . 7H2O) 0.02 grams
Agar-agar about 16.0 grams
Laevulose and dextrose (or sugar) 10.0 + 10.0 grams
Distilled water 1000.0 grams
Nicotine acid amide
Nicotine acid amide, mentioned in the end of the recipe, has been
seen to be beneficial. 0.1 grams of it is solved in 100 grams of water,
and 10 cu. cm of that solution is per each litre of final solution.
Agar-agar is sold by the kilogram as straplike pieces; it is very
light so that the amount needed for a litre of solution is a good-sized
pile. As the quality of the agar is greatly variable, it is difficult to
state in weight units the exact amount of the agar needed. Even a
few extra grams of agar can make the jelly excessively hard; seeds
can not adhere to it and when the surface of the jelly dries up and
becomes leathery, there's no hope of germination. On the other hand,
if the jelly, due to inadequate amount of agar, is too viscose, seeds
sink in it and can even pile up, unable to spread evenly on the surface
of the jelly. The jelly cake, even if whole, moves easily in the bottle,
and is thus apt to be attacked by mold. One must pay special attention
not to ruin the jelly-forming ability of the agar by keeping it boiling
for too long. It is of great advantage to cut the dry agar with scissors
into rather small pieces and add them into the solution only when it has
warmed up. If the agar is dirty or dusty, it is good to rinse it once,
using a vigorous shower of water and a common kitchen sieve. The
water thus added to the agar by no means changes the ratios of the
solution, because some amount of water evaporates from the solution.
The solution does not need to be kept boiling for a long time; slight
churning and bubbling is sufficient, and at once when the agar is
dissolved the kettle can be taken away from the hot plate. While
excessive boiling can ruin the agar's ability to form jelly, it must
be kept in mind that the jelly can become too hard if too much water
evaporates out of the solution. The solution must be mixed well,
as only this way one can obtain certainty that the agar has been
dissolved properly.
If one is to perform germination tests repeatedly, it is wise to
buy all the agar for these tests at the same time, as the properties
of agar in the following batch can be very different.
While the solution is heating up, it is time to place a heat tolerant
0.1 litre beaker into the oven, among the bottles which have already
been heated up. Even prior to that, cotton wads of suitable size for
closing the bottles are made. Sterile cotton is nowadays sold in
handy packs from which it can be drawn as narrow strap, and one
needs not open a whole big packet. Cotton is torn (not cut) into
suitable pieces which, depending on their thickness, should be 6-8
cm in diameter. They are then rolled into wads so that their thickness
is the same as a sturdy thumb's, and it is then tested how these wads
fit a cold bottle's mouth. If the wad stays in its place without
dropping into the bottle when pushed lightly, it is of good size.
This task must be practiced well, because a wad that has become loose
can be the reason for the later attack of mold.
Filling the bottles is hot work and one needs a pair of woollen
gloves for protection of one's fingers. Rubber gloves won't do,
because fingers can easily burn to blisters in them. When boiling
steam is blowing out of the hot bottle, one must act calmly and even
let the bottles cool down for a few seconds, because after boiling
in the bottle the amount of the solution is by no means the same
as it was when poured. It is anyway usual that, after filling a couple
of bottles, the person performing this task takes off the glove of
his right hand and starts using a small piece of thick cloth instead.
That way, it is much easier to handle thew one decilitre measuring
cup. On the other hand, when placing the cotton wad, one can not
cover one's both hands with gloves, or one needs to have an assistant
for that task.
Furthermore, while filling the bottles, one needs to have a sturdy
wooden forceps. A handy man can make it himself, using thin pieces
of wood and a piece of leather for the hinge. A shallow notch is
carved near the end of each piece of wood so that it is easier to
grab the bottles.
When filling the bottles, a kitchen bench is put in front of the stove,
and covered with a piece of paper. The kettle is been kept on the hot
plate, but the solution in it needs not boil any longer. The temperature
in the oven is kept at 120 - 130 °C (248 - 266 °F). A bottle is taken
out of the oven with one's left hand, using the wooden forceps, and the
1 dl measuring cup is used to take about half a decilitre or slightly
more of the solution from the kettle. The solution is poured swiftly
into the bottle so that the bottle's neck and mouth are not stained with
the solution. Pouring the solution is done properly if hot steam can
be seen blowing out of the bottle. Of course, the bottle must not be so
hot that the solution goes on boiling in it or, worse still, foams out
of the bottle. It is fine if the cotton wad can be placed in the
bottle neck while the steam is still blowing out; thus no mold spores
can end up on the jelly. The cotton wad must placed so that it fits
tightly in the bottle's mouth in order to avoid its becoming loose
while the bottles are moved. All the bottles are filled this way, and
they are then allowed to cool down in room temperature.
It is useful to find out the acidity of the solution, if one has the
possibility for that. Usually, it is automatically suitable.
The solution should be slightly acidic, the best pH range is considered
to be from 4.8 to 5.1.
The next day, or as well the day after that, the sterilization of the
bottles is carried out. If the bottles happen to contain any mold spores,
these will have germinated during the two days, and they must be killed
by heating the bottles. A wooden trellis, where the required amount
of bottles can be stored at an angle, almost in a lying position, is
built in advance. Of course, each bottle will have its own niche in
this trellis so that the bottles can not roll - this could result in
the jelly becoming loose inside the bottles. It is useful to design
these trellises so that several of them can be piled up. Precious
space can be thus saved, and piled up trellises will not gather
dust easily.
Sterilizing the bottles is a rather simple task. I have used for that
purpose a common pressure cooker that has room for four bottles in
standing position. A slightly bigger model might have room for seven
bottles and the task would then go remarkably faster.
A sufficient amount of water is poured into the pressure cooker so
that the water level reaches the bottom plate with holes. After placing
the bottles into the vessel, the pressure cooker is closed and put onto
a hot plate to warm up. When steam starts blowing out of the pressure
cooker, the pressure controlling unit is installed and this starting time
is verified with a clock. There is no reason to use high pressure, and
the temperature of the hot plate as well as the location of the pressure
cooker on the plate must be adjusted so that the water inside is boiling
properly but steam is not blowing forcefully out. The pressure control
unit must not, thus, be allowed to clank restlessly. If the temperature
of the hot plate can be controlled, it should be adjusted to rather low
as the task is advancing. If the only available hot plate is a
portable one with a tendency of becoming too hot, one can place a
knitting needle between the plate and the cooker in order to avoid too
vigorous boiling. Sufficient time for sterilizing the bottles is 13 to
15 minutes, after which the kettle is lifted away from the hot plate
and allowed to cool down slowly. Letting the steam out or cooling the
pressure cooker with cold water is not wise, because the cotton wads
might then not stay in their places, and the heating up should be
renewed. It is best to let the pressure cooker to cool down to the
point where the lid can be opened without any danger. The bottles,
still hot to almost boiling, are then placed on their sides in the
trellis, and the position to which the jelly is to take is checked.
It would be fine if the jelly forms a thick layer in the bottle, even
near the neck, because a thin layer of jelly easily dries out at its
edges and is prone to be infected by mold near the mouth of the bottle.
Successful sowing is especially difficult if the jelly reaches near
the mouth of the bottle. There is, therefore, no reason to try getting
the surface of the jelly as large as possible.
The final closing of the bottles with rubber corks carried out only
after the jelly has been completely coagulated. Usually it is done
the day after sterilizing the bottles. It might, of course, be wise
to wait and see that no mold is appearing in the bottles, but as
mold invades the bottles only when the sowing is carried out, there
is not always much reason in waiting.
When closing the bottles with rubber corks, sturdy forceps (so called
operation room forceps) and a spirit lamp or a gas torch (the one with
trademark Kosan is very handy) are needed. Furthermore, one needs to
have juice bottle corks, made of rubber. According to the size of the
bottles' mouth, cork sizes 2 and 3 are used.
Closing the bottles with corks must be carried out swiftly but still
with calm. Shaking the bottles vigorously must be avoided, because
this might result in the jelly cakes to become loose. A bottle is
held in the left hand so that its bottom is firmly against the palm.
Rubber gloves must be worn to protect the hands, although they can
sometimes feel, especially after warming up, uncomfortable. The cotton
wad in the mouth of the bottle is lit with the flame of the spirit
lamp or gas torch, and after letting it burn for a few seconds, it is
extinguished by blowing strongly. The bottle is still kept in a
horizontal position above the flame and the cotton wad is removed with
forceps from the mouth of the bottle. The distance from the flame is
at this stage about 10 - 20 centimetres, depending on the size of the
flame. If the filling of the bottle was carried out skillfully, the
cotton wad can be removed easily; bottles whose mouths were stained
with jelly during the filling are not so easy to deal with as the wad
is tightly glued to the mouth of the bottle. The upward flow of hot air
efficiently stops the mold spores from getting in the bottles. (If steam
from boiling water is used instead of real flame, the risk of
contamination is higher.) The forceps is put aside and a rubber cork,
which has been kept in boiling water, is taken using one's right hand,
the inside of the cork is, for safety's sake, once still allowed to heat
up in the flame for an instant, and it is then, while still hot, pushed
on the bottle's mouth. If the cork is moist and sufficiently hot, it can
be placed easily, without the risk of shaking the jelly inside the bottle
to pieces. In this last stage one is of course not to keep the mouth of
the bottle or one's hands in the hot flame, but once the bottle has been
closed it can be lifted away. The procedure is went on with until all
the bottles are ready for sowing. The cotton wad can be removed even when
aflame, but then there must be a bucket of water nearby into which the
flaming wad can quickly be thrown. In addition to hydrogen peroxide
solution, a syringe (2 millilitres) and a couple of injection needles
are required for sowing the seed. One of the needles must be with as
large bore as possible so that the solution with the seed can easily flow
through it, the other should be thin and fairly long. Furthermore, it
would be good to have a small 10 millilitres measuring beaker and a
measuring pipette as well as a few 4 to 6 centimetres long test tubes and
their caps. These should have a slightly greater volume than the syringe,
but also should they not be of greater length than the injection needle
with the larger bore. In some stages of the task a flame might be needed,
too. If a clock, equipped with a second hand, is added to this
paraphernalia, one can start with the sowing.
In this method, 2 % hydrogen peroxide is used. The same solution does
well for cleaning the needles and syringes. Many instructions published
abroad state that the only possible disinfectant is chlorine chalk, but
if it is used, the seeds must be rinsed after their disinfection. The
disinfecting effect of hydrogen peroxide quickly disappears after the
seeds and the solution have been injected into the bottle, so that no
rinsing is needed.
If one is determined to sow seeds more than once, it is wise to purchase
the disinfection solution as strong storage solution. It is known to be
available at 40 % strength. It is then easy to dilute to 2 % strength
with distilled water for overall purposes. That kind of solution is
required not only for disinfection of the seeds but keeping the syringe
and the needles clean. The solution intended for disinfection of the
seeds can be kept on one's working table in a bottle which can be easily
closed with a glass stopper, while that for other purposes can be put
in an ordinary glass where the syringes and their needles can be kept
in an upright position so that no air can enter them.
There's no need for pre-soaking the seeds. Only in cases where a
remarkably low amount of viable seeds among husks has been detected
with a microscope, it is useful to let the seeds soak in distilled water
in a glass tube, so that the viable seeds sink to the bottom while the
husks keep floating. Distinguishing the seeds from the husks with this
method is not always successful, especially with certain Paphiopedilum
seeds that are covered with such a well sealed, airtight sheath that
neither water nor hydrogen peroxide can soak it thoroughly, so that both
the seeds and the husks keep floating on the surface and are thus
impossible to get into the syringe. The same applies to Cymbidium seeds,
which, even if completely mature, might go on floating. In these cases,
one can of course add some surfage tension reducing agent, like spirit or
saponine, but rinsing them off, especially the latter, may be difficult.
It is important to remember that repeated rinsings, soakings and subsequent
disinfectings increase the risk of infection. In my opinion, if just a
few percent of the seeds seem to be viable, it is best to sow the whole
batch, husks included, but here at least some of the seeds must be
disinfected using a stronger solution (2.5 - 3 %) and also the disinfecting
time must be increased to 3, even 4 or 5 minutes. There's no use hoping for
a complete success at the first time. It is easy, by using a sufficiently
strong solution, to clean the seeds perfectly so no mold is left, but then
they won't germinate. Experience is needed in order to be able to find the
proper solution strength and soaking time for each batch of seeds. One must
keep in mind that once air enters the syringe or the needles or even the
bottles, there is the risk of contamination. The most careful disinfection
is not always a guarantee of success, but mold can surprisingly appear in
several months old jelly regardless of a completely airtight rubber cap.
A small amount of dry seeds is placed in a short glass tube. It is difficult
to approximate the amount of the seeds, but almost without exception there
will be too much. If they germinate at all, even at a low percentage, there
will soon grow a jungle of seedlings in the bottle; greatly resembling a
spring-time lawn. If an end of a knitting-needle is hammered flat it is thus
formed to a suitable spoon to ration a portion of seeds to sow a bottle.
There are good grounds for sowing just one bottle with the seeds of each
tube, because otherwise the portion left in the tube would stay exposed to
hydrogen peroxide for too long and thus lose viability. 3 - 4 milliliters of
2 % hydrogen peroxide is poured on the seeds. A slightly stronger solution
may sometimes be required, but according to my experience, a solution
stronger than 3 % damages the seeds. One must also pay attention to the
disinfection time. Usually three minutes is enough, but the time can without
harm be extended to 5 minutes. The tube is closed tightly with a cork, and
it is shaken continuously so that the seeds won't rise to the surface.
If the seeds, or at least a part of them, sink to the bottom after a few
minutes, it's an OK sign. Then it is easy to suck up the seeds with a large
bore injection needle into a syringe. With experience, one soon grabs the
best ways to do that. The cork is slightly opened, the tube is still kept
gently shaken, and the syringe is filled up. One must take care that no air
is let into the syringe - it must be filled with 2 % disinfection solution.
Even when filling up the syringe, not even the smallest air bubble can be
allowed to enter it.
In normal cases, when the seeds are small, hairless and well ripened and
when they are evenly wetted and sink to the bottom slowly but steadily,
the task of sowing is easy. If the seeds sink like stones, the person
performing this task must really have quick fingers so that the seeds,
swimming in the solution, can be caught in the syringe before they reach
the bottom while the syringe and the tube are kept shaken. The ideal case
is the one where the seeds sink very slowly to the bottom, so that one
has abundantly of time to calmly take the solution and the seeds through
a large-bore needle into the syringe. If it can clearly be seen that good
seeds are piled on the bottom of the tube while a thick layer of husks is
floating on the surface of the solution, one can use a pipette to add
carefully the solution into the tube so that the husks flow over the rim
of the tube. One can also push the needle straight through the husk layer
to the bottom of the tube and take only the good seed into the syringe.
The sowing is carried out simply so that the needle is pushed through
the rubber cap and the contents of the syringe is injected on the surface
of the nutrient jelly. Just before injecting, the syringe is shaken
lightly in order to have the seeds evenly distributed in the solution, so
that they flow steadily with the solution. Ordinarily, the first
injectings have a low success rate. The needle is often aimed too high and
the liquid hits the bottom of the bottle and the main part of the seeds
are glued to the glass surface. They are extremely difficult to pick up
again without breaking the jelly cake. The seeds can also form a belt
between the jelly and the glass. A flow which is aimed straight onto the
surface of the jelly results in a more even distribution of seeds,
especially when the jelly has just the right hardness. By tilting the
bottle one can spread the seeds on the surface of the jelly so that they
are not in big lumps. If the amount of liquid in the bottle seems excessive,
the extra water can be removed using the same syringe or using a long,
thin needle. At this stage, it is easy to make some conclusions about the
amount of agar which was used while making of the jelly. If the jelly is
too hard, the seeds will not stay in their places but they flow with the
water to, and below, the edge of the jelly. If the jelly is too soft, the
stream of water from the syringe bores into the jelly and the seeds are
inevitably piled in great lumps. It is completely impossible to take them
apart from each other. If lots of seed is left on the bottom of the tube,
one can add the disinfection solution and carry out the sowing as quickly
as possible.
It is, all in all, comparably slow to sow the seeds. Sowing seeds into ten
bottles takes a bit more than one hour. Like it was mentioned above, one
can of course take only a part of the seeds in the disinfection solution
and sow several syringefuls one after the other. The rate of success, if
this method is used, depends on the swiftness of the performance.
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