So You Want to Try Your Hand at Hybridization?

Part I - The Easy Part

Marilyn HS Light
Copyright November 2005

You have wanted to take the plunge, to try your hand at making your very own hybrid orchid but wonder just how to go about it. The topic is divided into three parts.

• Part I - The Easy Stuff, deals with doing it: flower structure, pollen removal, storage, and pollination techniques. Also covered are some aspects of parent selection.
• Part II - The Challenging Stuff, deals with the consequences of doing it: choice of seed and pollen parents, speculative crosses, line breeding, novel breeding lines and seed-borne diseases.
• Part III - I have a million babies! Will address the reality of the first time experience including what to do with millions of seeds, thousands of protocorms, hundreds of compots, and how to realize success from the adventure.

Part I - The Easy Part - Flower Structure

Anybody with basic knowledge of orchid floral anatomy can locate the anther and the stigma. They are both located on the column, a structure found in all orchid blooms. Flowers of Cypripedium, Paphiopedilum and Phragmipedium: There are two anthers located at the base of the column, one on each side. The pollen may be sticky or mealy. The pollen-receptive structure is the stigma which is located behind the staminode and faces inwards toward the back of the pouch. Its surface may be smooth or rough to the touch.

Here is a section cut through a Cypripedium flower to show the structures.

The stigma is white, the staminode yellow, and the one visible anther, golden brown. Close examination will reveal tiny papillae on the stigmatic surface which capture the pollen as it is deposited by an exiting insect.

Here we can see yellow pollen on a Cypripedium stigma. The lefthand anther is normal but the righthand one (arrow) shows fungal infection which can happen when a flower ages and under humid conditions. Infected pollen does not germinate and cannot be used to sire seeds.

Other Orchid Flowers: In other orchids, a single anther is found at or near to the tip of the column.

Here is the column structure of Sobralia xantholeuca.

The pollen-containing structure called a pollinarium is usually fully or partly covered by an anther cap which protects the pollen mass until it is dislodged by a visiting pollinator or your toothpick. Loss of the anther cap can lead to premature flower fading.

In some orchids such as Podangis dactyloceras, the anther cap is a contrasting color like green.

The mass of pollen (pollinium) may have a stalk attaching it to the sticky disk (viscidium). We can see the viscidium and part of the stalk protruding from beneath the anther cap in Gongora pleiochroma. The sticky viscidium is the part which attaches the pollinarium to a pollinator's body.

We can likewise remove pollinaria by touching a toothpick to the viscidium if it is present. In Sobralia xantholeuca, the viscidium is present but inconspicuous. When we dislodged this anther cap, the four oval pollinia were still enclosed. They had to be removed before using them for pollination.

Sometimes flowers will self pollinate as we see here with Bletia rosea. Flowers which self-pollinate before they open are not appropriate for breeding but in some cases, self-pollination will happen only as the flower ages or under very humid conditions.   In this case, the yellow pollinium to the left has come in contact with stigmatic fluid and a swollen pearly mass of pollen tubes can be seen.

In some orchids such as Platanthera and Disa, the pollen is arranged in wedge-shaped packets of tetrads (massula) which are in turn gathered together into pollinia. The massulae are connected by an elastic material so that only a few massulae detach as the pollinium is dragged across a stigma. One pollinium can be used to pollinate several flowers.In other orchids such as Dendrobium, Cymbidium and Phalaenopsis, pollen is tightly compressed into firm to hard pollinia. There are usually two or four pollinia per pollinarium. Each pollinium can be removed and used separately to pollinate blooms. In Cattleya and some relatives, the pollinia are composed of flattened plate-like structures and each piece can be used separately to pollinate a bloom.

The stigma is usually located behind the anther and on the undersurface of the column but the position can vary with the genus. The stigma is usually a simple sticky depression. The stickiness causes the pollinia to remain in place. The fluid is also an ideal germination medium for pollen grains. Stigmas are usually easy to spot but in some orchids like Gongora and Promenaea, there are slit-like openings that can be challenging to manoeuvre when it comes to placing pollen. Try to imagine how an insect enters and leaves the flower to position the pollen accordingly. Pollinia are always perfectly shaped and sized to enter the stigma of their species. When making hybrids, there often serious disparity in size and shape which is when a bit of imagination is needed to be successful.

Choice of seed and pollen parents is important. Each parent carries traits which contribute to various plant and flower characteristics. The seed parent can be most influential because extra-nuclear DNA is transmitted only by the seed parent via cell organelles called plastids. If either parent is polyploid, that is, has more than the typical two sets of chromosomes, it may be more challenging to breed. Crossing a diploid (2n) with a tetraploid (4n) may yield seeds and seedlings but many may be triploid (3n) and sterile.

Timing of Pollination: Sometimes a stigmatic surface is appropriate to pollen deposition only while the bloom is fresh or when it is most sticky. Receptivity may coincide with fragrance peaks which may vary with flower age. Flowers may be less receptive when a plant is young, has been recently disturbed/repotted or if the plant lacks vigor. A healthy mature plant is always a better choice as a seed parent.

Flower Position: In a multi-flowered inflorescence, pollen can be removed from almost any flower but flowers destined to be hand pollinated should be those toward the base/bottom of the inflorescence.

Pollen Storage: Orchid pollen is very short-lived if stored under in humid conditions. Fresh Cypripedium pollen can be stored in a sealed vial in a freezer for at least one year without noticeable loss of viability. Hard pollinia can be stored dry in a refrigerator at 4C for many years. Remove pollinia from fresh blooms. Clean off any extraneous material such as anther caps. Place the pollen in a fold of waxed or parchment paper, seal and label with at least the name of the plant and the date. Record this information in a database, propagation book, stud book or similar reference. Pour a layer of dry silica gel into the bottom of a bottle. Cover the silica gel with waxed paper and place the sealed packets on top. Seal the bottle, label, and place in the refrigerator.

Additional Skills Required: Some skill will be needed to artfully remove pollen either as sticky, powdery or hard pollinia and to place this on a stigma. Pollinia do come in all shapes and sizes, colors too, but even the tiniest structures such as those found in pleurothallid flowers can be successfully transferred provided one does not sneeze! Always work over a piece of white paper!

A simple toothpick is an ideal tool for pollination. The flat end is suited to removing sticky pollen then smearing it on a stigma. The pointed end can be used to pick up pollinaria. Use as is or further whittle to create a fine tip. Use one toothpick per pollination to minimize transmission of disease. Discard used toothpicks.

• Step 1 - Locate the anther. Use a toothpick to gently dislodge the cap. If the pollinia are not apparent, check within the anther cap. Gently lever the pollinium out of the cap but be careful not to press too hard otherwise you see your pollen flying through the air, never to be seen again! Pollen may be used immediately or stored until needed.
• Step 2 - Locate the stigma. With a toothpick, transfer all or part of a pollinium to the stigmatic surface. Label the flower/plant with a code or words to describe the date and the pollen parent. Additional information can be kept in a Propagation Notebook. The act of pollination does not guarantee fertilization or the production of viable seeds. Your speculative cross may never get past the starting gate. The parents may be genetically incompatible. Either or both parents may be functionally sterile.
• Step 3 - Measure and wait. The process of pollination is separated from fertilization by a matter of days to months. Unless the pollen never germinates on the stigma, hormones will be produced readying the ovary tissues for the reproductive cycle. The ovary will begin to enlarge in length and width.

To follow the process, measure and record the length and width of the ovary at pollination and at the same point weekly or until the fruit aborts development and yellows or withers. If pollination leads to fertilization and seed development, the fruit will increase in girth for the first few weeks then pause for about a week before resuming growth. This pause is when fertilization is happening. When the fruit ceases growth in girth, seeds are matured.

Once we know How To, should we?

Exercise 1 (3 parts for discussion):

We have two plants blooming which could be potential seed or pollen parents. 1 - What characteristics might make either one a suitable parent? 2 - What additional information should we have before making a speculative cross? 3 - How could we learn more with the plants in hand?

Dor. pulcherrima alba

Dor. pulcherrima

Please refer to the plants as alba or pink when discussing them.

1 - What characteristics might make either one a suitable parent? Color, shape, number of blooms, branching, vigor, reliability of blooming... Any other possibilities?

N_Calif_Kathy
I'd say vigor. After that I'd ask if there's something about the parent I liked that I thought may pass along, like if I liked the lip (for example). In terms of the alba parent here I always wonder about the tendency for albas to wash out colors as well as their small flowers

MarilyninOttawa
Lip shape is a good point. Also, Doritis tends to produce more erect spikes in its progeny. Albas may wash out colors but you might wish to breed white progeny.

N_Calif_Kathy
But even with white progeny might their vigor not be compromised by using alba genes?

MarilyninOttawa
Look for a vigorous seed parent. Genes are transmitted by the seed parent including mitochondrial ones which influence vigor.

MarilyninOttawa
2 - What additional information should we have before making a speculative cross? Has this clone been used previously with success (proven breeder)? Can the plant carry a capsule to maturity without suffering loss of vigor? What traits does this plant have that I want to transmit to progeny?

N_Calif_Kathy
Something that I'd forgotten about was the prevalence or tendency of the person I bought my parental stock from to use 4N plants in his breeding program, so now I'm wondering about whether my crosses - if they take - will be 3N... so knowing the source material and their ploidy is a factor I hadn't even considered prior to picking up the toothpick.

MarilyninOttawa
Ploidy is important when working with line bred plants, even species, that have been selected over and over again. If you want to do serious breeding work, it is worthwhile investing in proven seed and pollen parents of known ploidy levels. Expensive but worthwhile if you want good results.

N_Calif_Kathy
So many breeders are using 4N parents that to cross them back to 2N species...well the one time I did that the cross didn;t take, but that was probably operator error.

MarilyninOttawa
3 - How could we learn more with the plants in hand? Self and outcross to see what happens. Try the pollen on a range of plants to see if capsules form. Young plants may have viable pollen but may not set fruits until they have attained a critical mass. (NB Doritis requires 60 days from pollination to fertilization but if pollen does not germinate then flowers fade and drop quickly). Speculative capsules can be removed before maturity. Have the ploidy levels analysed by a specialist.

Ken_in_Traverse_City_MI
In regards to color, would the offspring be a combination of the two parent's color? in this example the white and the purple would create a "blush"? Is it possible/probable that the offspring would be white or purple?

N_Calif_Kathy
Good question... would a blush be the result? Or would they all be purple? Is this the same as crossing to a white (which I think would produce a pink color) versus crossing with an alba that has no color genes such that the purple would be the only color expressed?

MarilyninOttawa
Ken, a lot depends on what you don't see. In Cattleya, albas are the result of mutations in the pigment production pathway but not all albas result from the same mutation. It is possible to breed two different albas and have purple-flowered progeny. Breeding two having the same mutation will produce albas. Subsequent generations can yield some white, mauve and purple blooms.

Kathy, as I said, a lot depends upon what you don't see. Is an alba a true alba or is it the result of a mutation which can be countered when the 'alba' is bred with a plant without the pigment mutation? You cannot know unless you try.

N_Calif_Kathy
I know there are lists of proven alba clones of various species. I think this month's Orchid Digest magazine publishes one such list for paphs. I have been impressed with a hybridizer's knowledge of parental stock, not just knowing the species/hybrids but the individual plants (clones) that have the traits they want. I guess one needs to have friends that have done crosses in the past and know how traits are passed, in their experience, too.

MarilyninOttawa
Breeders get to know the traits of their stud plants through hands on experience. I know the traits of individuals within one of my primary crosses by doing the experiment. Not all are seed parents and sometimes the ones with the best flowers do not produce seeds.

Jade_in_GR
Good reason to keep really accurate records of progeny.

Jade_in_GR
So the seed parent is similar to female genetic transfer in humans?

MarilyninOttawa
The egg contains organelles that contain extra-nuclear DNA. In plants, this includes chloroplasts, mitochondria and other plastids including those producing carotenoid pigment (yellow/orange/red). In animals, organelles in the egg include mitochondria.

MarilyninOttawa
We have two Paphiopedilum species blooming which could be potential seed or pollen parents. 1 - What characteristics might make either one a suitable parent? 2 - What additional information should we have before making a speculative cross? 3 - How could we learn more with the plants in hand?

Paph. hookerae B

Paph. hookerae N

Steve_in_the_Adirondacks
I say, forget the cross and just make divisions of B ;-)

MarilyninOttawa
1 - What characteristics might make either one a suitable parent? Color, shape, vigor, reliability of blooming...
We can test pollen germinability in cyps, paphs and phrags by taking a tiny amount of the pollen and germinating it in a drop of sugar solution. Even examining some pollen under the microscope will reveal healthy grains or shrunken ones.

N_Calif_Kathy
Is B poliploid?

MarilyninOttawa
I don't think so Kathy but I cannot be certain. This plant is not too many generations from the wild. It was exhibited at the Ottawa show in 1998. It is likely that the flower form would be seen in at least some of the progeny. I personally would want to get more species seed from it as well as trying hybridization.

What additional information should we have before making a speculative cross? Has this clone been used previously with success (proven breeder)? Can the plant carry a capsule to maturity without suffering loss of vigor? Check the culture as the plant must be in tiptop condition to mature a fruit. You do not want to jeopardize the safety of a superior clone by taxing its strength. As Steve suggested, dividing the plant, if it is large enough, might ensure that in the future, there are two instead of just one.

Ken_in_Traverse_City_MI
How do you choose which one should be the pollen parent and which one the seed parent? Wouldn't the end result be close to the same? Wouldn't breeding the broad to the narrow produce a "medium"? and the same the other way around?

MarilyninOttawa
First, we can only speculate on the dominance of the broad petalled trait. You might get only narrow-petalled progeny but chances are that at least some of the progeny of the broad-petalled plant as a seed parent would also have broad petals. The strength of the plant to carry a capsule is probably the most important consideration. The pollen quality is the next consideration and this can be assessed shortly after the flower opens. Then it is wait and see. Learn from experience.

N_Calif_Kathy
And, When putting pollen on the pod parent flower does one *have* to remove the anthercap and pollinia of the pod parent? Is there some sort of biofeedback to the flower if its pollen *hasn't* been removed? I removed the pollen from the pod pernt flower anyway, and so I had pollen for the reverse cross.

MarilyninOttawa
It is always best practice to remove the pollen from the flower to be pollinated before pollinating it. You can use pollen from the same flower or another on the same inflorescence to produce a SELF or pollen from another plant of the same species or grex to produce a SIB CROS, or you can use pollen from a different species or hybrid grex to produce a new hybrid.

MarilyninOttawa
We have three plants blooming which could be potential seed or pollen parents. 1 - What characteristics might make either one a suitable parent? 2 - What additional information should we have before making a speculative cross? 3 - How could we learn more with the plants in hand?

C. labiata alba

C. labiata rosata

C. labiata

1 - What characteristics might make either one a suitable parent? Color, shape, number of blooms, fragrance, vigor, reliability of blooming...

2 - What additional information should we have before making a speculative cross? Has this clone been used previously with success (proven breeder)? Can the plant carry a capsule to maturity without suffering loss of vigor? What traits does this plant have that I want to transmit to progeny?

3 - How could we learn more with the plants in hand? Cattleyas can take quite a few years to bloom and before they do, they get big!!!

N_Calif_Kathy
How about selfing the alba to be sure its a true alba? Same with the semi alba.

MarilyninOttawa
That is a possibility. Albas are always in demand. I would look for any coloration of seedling foliage. Anthocyanins are sometimes expressed in the foliage of purple-flowered plants but not in plants that will have white or near white blooms. If this indicator is present, you could select seedlings at replating time. I would also look for seedling vigor, not so much as an indicator of alba but as a marker for future growth and ease of cultivation.

MarilyninOttawa
A first for Orchidsafari HOMEWORK! - Exercise 2 : The next time you view an orchid, try to identify the column and if you are uncertain, ask a fellow member to assist. If you can, remove a flower of the same type to be bred and identify the column, pollinaria and stigma. Note how the parts are arranged and see if there is a sticky viscidium present. Try removing the pollinarium by touching a toothpick to the viscidium or remove the anther cap and examine the pollinarium. Find the stigma and touch it gently to see if the surface is sticky. Using a toothpick, pick up a pollinarium and deposit the pollen mass on the stigma. If it adheres, you have succeeded in pollinating an orchid flower.

Anyone up to homework? I hope that you will take a close look at especially those unusual flowers like Stanhopeas. There was a hybrid Stanhopea at the Montreal show so somebody had to know where the essential parts were and how to remove and place the pollen.

Jade_in_GR
I have a stanhopea in bloom right now. Hopefully it will be accessible tomorrow am. I did find the pollen area. so far so good.

N_Calif_Kathy
Ok, how hard do you have to press the pollen onto the stigmatous surface? I really pressed it in hard because I wanted full contact, but is that really necessary? Will laying it on the surface and depending on the surface stickiness be sufficient?

MarilyninOttawa
Good question with a range of possible answers. First, think of the pollinator and the force it must have to attach pollinia to a stigma. Not very much I would think. Stigmas vary in stickiness and are usually stickiest when fresh. That is when pollinia will adhere with the least force by yourself. You can gently press the pollinium into the stigmatic fluid layer if it is thick but no great force is required. All it has to do is touch the surface for the pollen to germinate.

N_Calif_Kathy
I also came to understand why people sacrifice floral lips when setting crosses. they just get in the way!

MarilyninOttawa
I usually remove the lip to gain access to the stigma but this is not necessary with all orchids, Phals for instance. Removing the lip at the time of pollination will not interfere with the reproductive process.

Ken_in_Traverse_City_MI
Homework question: is it a good idea to remove part of the flower to get to the column? I'm assuming that the column provides some sort of protection after pollination. Is that correct?

MarilyninOttawa
The column is essential to the reproductive process and should not be damaged during pollination. After pollen germinates on the stigma, tiny pollen tubes travel down the center of the column to the ovary where fertilization takes place. The column protects the delicate pollen tubes during their trip. You can either bend the lip to one side or gently remove it. It is not necessary to remove it.

Jade_in_GR
This was a whole new world for me, Marilyn. Really sounds challenging and i love a good challenge. Me who is a species nut! Will I stay in that room or head out to the hybrids. Stay tuned for further news.!!!!!:-))) Marilyn, you did it again!! I can't want for the next chapters!!!

MarilyninOttawa
Part III - I have a million babies! will address the reality of the first time experience including what to do with millions of seeds, thousands of protocorms, hundreds of compots, and how to realize success from the adventure.

MarilyninOttawa
Remember that fertilization takes place 20 to 60 days after pollination during which time nothing might happen. What you cannot see and what has likely happened by now, is a few pollen tubes have grown and this has induced enough hormonal feedback to keep the ovary green. But, there may be very few seeds from this cross or the fruit may cease development prematurely. Chalk it up to experience.

Be sure to take note of these References: We can find the Writings of Charles Darwin "On the various contrivances by which British and foreign orchids are fertilised by insects." London, John Murray (1862) on the web. button

Royal Botanic Garden (Kew) provides some excellent information on the subject. I read that they are putting the entire works of Darwin online

Ken_in_Traverse_City_MI
Thank you so much for the presentation. I'm very inspired now. Now off to find a couple to experiment with ... no pun intended. :)

N_Calif_Kathy
Ken, at the top of the page here is a link to the OrchiSafari archives, where I think we have a article called "the trembling toothpick". It may be on the link to 'full archives' at the top of that page (sorry the archives are all over the place. one of these days I'll get them all in one place and life will be easier.)

MarilyninOttawa
Hybridization, etc isn't for everyone but it is nice to know about how these flowers are contrived. Fascinating in fact!
Goodnight everyone! It was fun as usual and served to drive the snows away for yet one more day. Take care and keep 'em bloomin'

Prechat Handout

So you want to try your hand at Hybridization?

Part I - The Easy Part

Marilyn HS Light
Copyright November 2005

Introduction
You have been wanting to take the plunge, to try your hand at making your very own hybrid orchid but wonder just how to go about it.

The topic is divided into three parts. Part I - The Easy Stuff, deals with doing it: flower structure, pollen removal, storage, and pollination techniques. Also covered are some aspects of parent selection. Part II - The Challenging Stuff, deals with the consequences of doing it: choice of seed and pollen parents, speculative crosses, line breeding, novel breeding lines and seed-borne diseases. Part III - I have a million babies! will address the reality of the first time experience including what to do with millions of seeds, thousands of protocorms, hundreds of compots, and how to realize success from the adventure.

Part I - The Easy Part

Flower Structure
Anybody with basic knowledge of orchid floral anatomy can locate the anther and the stigma. They are both located on the column, a structure found in all orchid blooms. Many orchid-related books and web pages deal with the subject in detail. We can find the Writings of Charles Darwin "On the various contrivances by which British and foreign orchids are fertilised by insects." London, John Murray (1862) on the web. http://pages.britishlibrary.net/charles.darwin3/orchids/orchids_fm.htm

Royal Botanic Garden (Kew) provides some excellent information on the subject.
http://www.rbgkew.org.uk/scihort/orchids/orchidstructure.html

Flowers of Cypripedium, Paphiopedilum and Phragmipedium

There are two anthers located at the base of the column, one on each side. The pollen may be sticky or mealy. The pollen-receptive structure is the stigma which is located behind the staminode and faces inwards toward the back of the pouch. Its surface may be smooth or rough to the touch. Close examination will reveal tiny papillae on the surface which capture the pollen as it is smeared by an exiting insect.

Other Orchid Flowers
In other orchids, a single anther is found at or near to the tip of the column. The pollen-containing structure called a pollinarium is usually covered by an anther cap which protects it until it is dislodged by a visiting pollinator or your toothpick. Loss of the anther cap can lead to premature flower fading. The mass of pollen (pollinium) often has a filament attaching it to a sticky disk (viscidium). It is the sticky disk which attaches the pollinarium to a pollinator's body. We can likewise remove pollinaria by touching a toothpick to the viscidium if it is present.

Anther, Pollen, Pollinium, Pollinarium - Pollen Parent
The pollen may be in wedge-shaped packets of tetrads (massula) which are in turn gathered together into pollinia as in Platanthera and Disa. The massulae are connected to each other by an elastic material so that only a few massulae detach as the pollinium is dragged across a stigma. One pollinium can therefore be used to pollinate several flowers. More often, pollen is tightly compressed into firm to hard pollinia as we see in Dendrobium, Cymbidium and Phalaenopsis. There are usually two or four pollinia per pollinarium but we may also find six or eight in some genera. Each pollinium can be removed and used separately to pollinate blooms. In Cattleya and some relatives, the pollinia are composed of flattened plate-like structures and each piece can be used separately to pollinate a bloom. In others, the pollinia are round or oval.

Stigma, Ovary, Ovule - Seed Parent
The stigma is usually located behind the anther and on the undersurface of the column but the position can vary with the genus. The stigma is usually a simple sticky depression. The stickiness causes the pollinia to remain in place. The fluid is also an ideal germination medium for pollen grains. Stigmas are usually easy to spot but in some orchids like Gongora and Promenaea, there are slit-like openings that can be challenging to manoeuvre when it comes to placing pollen. Try to imagine how an insect enters and leaves the flower to position the pollen accordingly. Pollinia are always perfectly shaped and sized to enter the stigma of their species. When making hybrids, there often serious disparity in size and shape which is when a bit of imagination is needed to be successful.

Exercise: The next time you view an orchid, try to identify the column and if you are uncertain, ask a fellow member to assist. If you can, remove a flower of the same type to be bred and identify the column, pollinaria and stigma. Note how the parts are arranged and see if there is a sticky viscidium present. Try removing the pollinarium by touching a toothpick to the viscidium or remove the anther cap and examine the pollinarium. Find the stigma and touch it gently to see if the surface is sticky. Using a toothpick, pick up a pollinarium and deposit the pollen mass on the stigma. If it adheres, you have succeeded in pollinating an orchid flower.

Timing of Pollination
Sometimes a stigmatic surface is appropriate to pollen deposition only while the bloom is fresh or when it is most sticky. Receptivity may coincide with fragrance peaks which may vary with flower age. Flowers may be less receptive when a plant is young, has been recently disturbed/repotted or if the plant lacks vigor. A healthy mature plant is always a better choice as a seed parent.

Flower Position
In a multi-flowered inflorescence, pollen can be removed from almost any flower but flowers destined to be hand pollinated should be those toward the base/bottom of the inflorescence.

Additional Skills Required
Some skill will be needed to artfully remove pollen either as sticky, powdery or hard pollinia and to place this on a stigma. Pollinia do come in all shapes and sizes, colors too, but even the tiniest structures such as those found in pleurothallid flowers can be successfully transferred provided one does not sneeze! Always work over a piece of white paper! A simple toothpick is an ideal tool for pollination. The flat end is suited to removing sticky pollen then smearing it on a stigma. The pointed end can be used to pick up pollinaria. Use as is or further whittle to create a fine tip. Use one toothpick per pollination to minimize transmission of disease. Discard used toothpicks.

Step 1 - Locate the anther. Use a toothpick to gently dislodge the cap. If the pollinia are not apparent, check within the anther cap. Gently lever the pollinium out of the cap but be careful not to press too hard otherwise you see your pollen flying through the air, never to be seen again!
Pollen may be used immediately or stored until needed. Pollen should be protected from humidity otherwise it could be attacked by fungi. I have stored Cypripedium pollen for six months to one year in the freezer whereas it spoils after just 30 days when stored humid in the refrigerator or at room temperature. Hard pollinia should be placed in a piece of waxed paper which is then folded securely and labelled with name and date. I store hard pollen over silica gel in a sealed glass bottle in the refrigerator. Always collect pollen from fresh flowers. Pollen from aged or fading blooms may already be supporting fungal growth or may no longer be viable.

Step 2 - Locate the stigma. With a toothpick, transfer all or part of a pollinium to the stigmatic surface. Label the flower/plant with a code or words to describe the date and the pollen parent. Additional information can be kept in a Propagation Notebook. The act of pollination does not guarantee fertilization or the production of viable seeds. Your speculative cross may never get past the starting gate. The parents may be genetically incompatible. Either or both parents may be functionally sterile.

Step 3 - Measure and wait. The process of pollination is separated from fertilization by a matter of days to months. Unless the pollen never germinates on the stigma, hormones will be produced readying the ovary tissues for the reproductive cycle. The ovary will begin to enlarge in length and width. To follow the process, measure and record the length and width of the ovary at pollination and at the same point weekly or until the fruit aborts development and yellows or withers. If pollination leads to fertilization and seed development, the fruit will increase in girth for the first few weeks then pause for about a week before resuming growth. This pause is when fertilization is happening. When the fruit ceases growth in girth, seeds are matured.