GardenGenetics(R) blog

In late September, GardenGenetics will be hosting its first private showings of our proprietary plant genetics products.  As of 12 September 2011, most of the available viewing dates have already been booked.

Many of these novel plant introductions are available immediately for both exclusive and non-exclusive licensing.  We anticipate that G2's new genetics will be extensively trialed by potential licensees in 2012, with the earliest licensed introductions entering the global horticultural market in the 2013 season. 

Our vegetative-annual products are G2's first ready-to-release products.  We are actively breeding in the following vegetative-annual classes, and will have more than 400 new varieties on display and available for licensing in September 2011 or 2012:

Abutilon (immediate)

Angelonia (2012)

Bacopa (2012)

Begonia boliviensis (immediate)

Buddleia (immediate)

Calibrachoa (2012)

Canna (immediate)

Dusty miller (2012)

Lantana (2012)

Pelargonium (immediate)

Pentas (immediate)

Petunia (immediate)

Phlox (immediate)

Salvia (2012)

Viola (immediate)

Zinnia (immediate)

G2 has a broad range of herbaceous perennial breeding projects underway.  We have herbaceous perennial products in the following classes available for immediate licensing:

Hibiscus moscheutos

Perovskia atriplicifolia

Rudbeckia fulgida

Tanacetum niveum

As a start-up plant genetics company, we initiated breeding projects in many genera simultaneously.  Some of these convert into products quickly --- vegetative-annuals is a good example.  Some projects take much longer, simply because of the life-cycles of the plant.  Woody ornamentals take many years to develop, and G2 does have a broad range of woody projects in development.  However, we currently only have these woody perennial products available for licensing:

Callicarpa (immediate)

Diervilla (immediate)

Syringa microphylla (immediate)

Syringa vulgaris (2012)

G2 is actively breeding in a broad range of vegetable and culinary herbs.  We currently have the following vegetable products in development, available for 2012 release into 2013 trials:

Cucumbers

Peas (snap)

Peppers

Swiss chard

Tomatoes (parallel trials available for 2012)

If you have not yet visited G2's research facilities, and you are responsible for in-licensing new plant genetics, this might be a good time to schedule a visit. 

Those of you who have been following G2 for awhile might be interested in this photo.

We've changed a lot since we built the greenhouses in 2007/8.  The farm is essentially fully in use except for a three-quarter acre piece which has been maintained as an organic plot since 1996.  We are currently keeping G2's organic plot in rotating cover crops, as we attempt to bring the perennial weeds under control. 

Total area of the farm is just over 21 A (about 8.5 ha).  Buildings, roadways, and non-tillable spaces take up about half of that area, so we've got about 10 A in research plots available in any given year.  2011 turned out to be the first year that we are "out of field space". 

If you are planning on doing research trials with G2 in 2012, please be sure to book them early.  Field space is becoming a modest constraint. 

If you have been actively following the G2 blog, please recognize that it's spring.  I have the remainder of the polyploid induction series outlined and in rough draft form.  That said, finishing up each piece is likely to take a backseat to the current workload in the greenhouses.  Please be patient. 

Mike and I did a very quick run through the Spring Trials in California, and then headed back home to keep working.  This is the year that G2 will begin having extensive trials in the field, as well as with our prospective licensees.

We are also looking ahead to the fall, and are planning an early fall by-invitation-only open house for prospective licensees.  We've got more than 100 potential new varieties ready to release, and will have these on display for our prospective licensees. 

It will be a busy spring and summer! 

For my sake, I need to keep this simple.  I am not a cytogeneticist.  Neither am I a cell cycle molecular biologist.  I am a plant breeder, attempting to apply both modern and classical tools to the process of inducing polyploids in plants, as we attempt to develop new edible and ornamental varieties.  There.  I've stated my limitations.  You are getting my perspectives and understandings.  These drive my processes here at G2.  If you disagree --- good for you. 

Polyploid induction simply takes a diploid plant, and converts it to a higher ploidy level, usually tetraploid.  Words rapidly become confusing.  Many plant species are already polyploid --- zonal geraniums, for instance.  Doubling a tetraploid zonal would in theory give you an octoploid --- 4+4=8.  However, many polyploid species breed as though they are diploids.  Many do not.  There are examples (called segmental allopolyploids) where some loci segregate in a polyploid fashion, while others do so as diploids.  Fun, eh?

I will attempt to use polyploid to only indicate tetraploid or higher ploidy levels.  When I mean tetraploid, I will use tetraploid.  When I mean triploid, I will use triploid. 

The goal is to convert a diploid plant into a tetraploid plant.  2n=2X needs to become 2n=4X.  To do this means that somehow you have to force the plant to make an error in cell division.  There are many ways in which this can occur, some of them natural, others induced. 

In a plant, all cells are ultimately derived from a single meristematic cell.  If you can somehow double the number of chromosomes in that single cell, all subsequent cells will also have double the original number.  The problem is that that single meristematic cell only exists for a few hours after the ovule is fertilized as a seed begins to develop.  As the embryo develops in the ripening seed, cell division is occurring, and tissue differentiation begins.  By the time a seed is ripe, the embryo is fully differentiated. 

That said, the embryo within a seed is relatively accessible to external influences.  One of the typical ways in which you can induce polyploidy is to treat germinating seeds with an acute dose of a mitotic spindle inhibitor.  These chemicals --- typically, plant people use either colchicine or oryzalin, although there are others available --- stop cell division by stopping the cytoskeletal structures known as mitotic spindles from developing.  These spindle structures are contractile fibers that pull doubled chromosomes apart during cell division, so that each resultant cell gets one set of chromosomes.  Stop spindle formation, and the chromosomes remain doubled.  Restart cell division --- and the new cells will have twice the original chromosome number.  If you originally started with a diploid, you will have just created a tetraploid. 

But what if you can't restart cell division?  The embryo dies.  In fact, many mitotic spindle inhibitors are commercial herbicides at higher dose rates.  Oryzalin is the active ingredient in SurflanTM.  Other herbicides are known to work by this mechanism.   

To effectively use a mitotic spindle inhibitor, you must be able to apply it; stop cell division; remove it;  and restart cell division.  This uses an acute dose. 

The problem is that an acute dose only works when there are meristematic cells entering the right stage of the cell cycle --- that point in the cycle when the chromosomes are duplicated/doubled but not yet pulled apart by the spindle fibers.  That complicates things a bit.  Not only do you need to get the chemical to a very small number of meristematic cells, those cells have to be in just the right stage of the cell cycle in order to double the chromosome number. 

In many cases, the easiest way to get the most numbers of meristematic cells at just the right stage is to dose germinating seedlings.  That sounds simple, right?  But in practice, there is a fair amount of trial and error necessary to determine the when and how much for each species (and in some cases, for each variety).  You need to determine the optimal dose rate for each species with which you are working.  You need to determine the optimal pre-germination timing (hours in the germination chamber) for each species with which you are working.

More later, with a detailed explanation of our standard protocols. 

In addition to the potential end-user drawbacks discussed earlier, for the plant breeder, polyploid induction has its own set of potential problems. 

Poisons: Polyploid induction chemicals are mitotic spindle inhibitors.  As such, they are inherently cytotoxic, potentially mutagenic, and potentially carcinogenic.  That said, with the same care, safe use practices, and personal protective devices that you would use in any chemical laboratory, any breeder can induce polyploids. 

Conversion problems: For some species, inducing polyploids is simple and straight-forward.  For others, it can be quite frustrating.  You will find different species sensitivities to various polyploid induction chemicals.  You will find different species sensitivities to various additives such as surfactants used to enhance the penetration of the chemicals through the plant cell wall and membrane.  You will find different species responses to the stage at which you dose the plant.  Each of these issues will be addressed in a later post, one in which I describe my conversion protocols. 

Fertility or fecundity problems: Induced polyploids frequently display both decreased fertility and decreased fecundity, especially in early generations.  When you double the chromosome number of a plant, there become more opportunities for the chromosomes to pair up incorrectly during meiosis.  Until you go through enough seed generations to eliminate these pairing errors, fertility is going to be negatively affected.  The good news is: you can readily select for increased fertility.  However, decreased fecundity may or may not be so easy a problem to select against.  Why not?  Polyploid seeds are typically larger than diploid seeds, and as such, contain more biomass.  Under ideal conditions, partitioning of plant biomass into seeds is likely to favor more and smaller seeds, rather than fewer and larger.   It is as though the plant can produce a finite amount of biomass for seed production, and if it is making larger seeds, it is also making fewer of them. 

If your crop is vegetatively-propagated, this is probably not an issue.  If your crop is seed-produced, however, decreased fecundity could be a serious limitation.  Especially if your crop is being hand-pollinated as an F1 hybrid. 

Genetic instability: Forgive the anthropomorphism, but not all plant species seem to "like" being polyploid.  More than just taking a few seed generations to clean up the genetic junk, these species tend to be very unstable when doubled, and during subsequent seed generations continue to eliminate the additional chromosomes.  Unfortunately, as this occurs, extra chromosomes (aneuploidy) or extra pieces of chromosomes are likely to be working their way through your breeding project.  You may think that you are selecting a particular phenotype, only to discover that your desired phenotype only occurs in a particular aneuploid form, and that that form is neither seed-stable nor vegetatively-stable. 

 "Mixoploid production":  One of the problems with inducing polyploids is that plants are comprised of multiple layers of cells, and that you can convert one layer while leaving the rest at a diploid level.  People often (and informally) refer to these incomplete conversions as "mixoploids". 

Technically, a mixoploid is a cytochimera.  Imagine that you are wearing a glove on your hand.  In a plant chimera, the glove may be of one genotype (or one ploidy) while your hand is a different genotype.  This is often the physical explanation for plant variegation.  But it is also very possible to have the outer tissue layer (or layers) of a plant be at a different ploidy level than the inner tissue layer(s). 

Imagine that you are attempting to convert a vegetative bud.  You apply the chemical to the outside of the bud.  If the chemical does not penetrate deep into the bud and convert the actual meristematic cells, you may convert only the outer layers, leaving the innermost layers diploid.  In my hand-in-glove example, the glove would be tetraploid, while your hand --- the central tissue --- remains diploid. 

Why is this a problem?  Germ cells --- ovules and pollen --- are produced by the inner cell layers.  If your goal is to breed with your induced polyploid, you need the tissues producing germ cells to be converted.  Otherwise you have a plant that looks like a tetraploid, but breeds like a diploid --- and does not transmit the tetraploid condition to the next generation.  Let me repeat that statement: a ploidy cytochimera can look like a tetraploid but breed like a diploid. 

Please realize that unless you can determine the actual ploidy of these inner cell layers (and there will be posts on ploidy verification coming), you cannot identify a cytochimera from a "solid" conversion.  A solid conversion is one in which all cell layers are converted --- the ideal result.  Unfortunately, cytochimerae are not stable, and if a plant is not stable, it should neither be protected nor released to market.  Cytochimerae can sport to solid diploid forms; they can sport to solid polyploid forms; they can sport to a form with is outside-diploid, inside-tetraploid.  And as a breeder, you may or may not be able to distinguish these forms. 

Polyploid genetics are more difficult: Polyploids are generally more challenging to work with as a breeder.  In the simplest consideration, there are now 4 alleles segregating at a tetraploid level compared to 2 at the diploid.  More possibilities for recombination are good, but finding those possibilities in a segregating population requires growing out much larger populations, and thus, much more bench space or field space, and much more labor for the observation of the increased population size.  More, in order to find the desired phenotypes, the phenotypes resulting from additive effects of the additional alleles available in a polyploid are very likely to require more generations of recombination.  More space, more labor, more time.  Polyploid breeding is inherently slower than diploid breeding. 

It is common for laypersons to get extremely excited about the possibilities of induced polyploids in plants, especially ornamental plants.  Many think of the chemicals used to induce polyploidy as a "magic sauce", and attempt to use them carelessly, and in so doing, put themselves at significant risk.  Polyploid induction chemicals can be extremely dangerous to use!

But even when done properly, safely and with appropriate caution, there are frequently problems --- true horticultural problems --- with induced polyploid plants. 

Delayed germination: It is extremely common for seed germination in induced polyploids to be delayed when compared to the parental diploid lines.  This can be true for many generations following conversion, although time-to-germination is generally a selectable trait.  For example, diploid Pelargoniums typically take 5 to 7 days to germinate, while tetraploid pels typically take 10 to 12 days. 

Delayed flowering:  Induced polyploids frequently take longer to come into flower than comparable diploid lines.  This can be an advantage if slower flowering (i.e., non- or late-bolting) is desirable, but for flowering ornamentals, this delay would generally be perceived as a flaw. 

Slower growth: Induced polyploids invariably grow slowly in the first generation, the generation which was converted.  However, slower-than-diploid growth rates are not at all uncommon in induced polyploids, regardless of how many generations have passed post-conversion.  This is not a universal characteristic of induced polyploids, but it is quite common.  This slower growth rate is likely to be the same effect that one observes when the induced polyploid is considered to be more compact or more stocky than the diploid.

Decreased cutting or seed yield: Another potential drawback with induced polyploids which is also likely related to a slower growth rate is that polyploids frequently have decreased seed yields (fewer but larger seeds), or decreased cutting yields (slower growth rates lead to slower production of cuttings).  As a breeder, you want your developments --- your products --- to be produced and sold in high volumes.  As such, your products need to be produce-able. 

Decreased flower production: One of the benefits of induced polyploids is larger flowers.  The flip-side of larger flowers is that larger flowers in polyploids may have a cost: fewer flowers.  As an ornamental breeder, you frequently want BIG flowers, but BIG must always be balanced by flower number.  Selecting for total floral display may cause you to select away from the largest flowers possible. 

Changed plant architecture (more coarse, less refined):  Polyploids frequently have a distinctly different plant architecture than the comparable diploids.  Polyploids can appear to be coarse, open, and less refined.  However, this can also be viewed as an advantage, especially if the less-refined habit provides a better display for larger flowers.  Bigger leaves, thicker leaves, reduced branching --- all of these structural changes may create very different plant architectures with induced polyploids.  That said, leaf size and branching are highly selectable characteristics, whether at the diploid or tetraploid levels.  You can breed tetraploids to have the more refined tetraploid appearance. 

More vigorous growth: Induced tetraploids may also demonstrate more vigorous growth than comparable diploids.  Again, this becomes a matter of trial-and-error.  And again, if more vigorous growth occurs, it is generally not in the generation which was converted, but in future generations.

On the left (below) is a tetraploid Bacopa seedling.  On the right is a diploid seedling (Pan American's Snowtopia).  Please be aware that increased vigor like this does not always occur with induced polyploids.

And no, the tetraploid seedling on the left has not yet flowered.  It may have large flowers; it may not.  Not all tetraploid bacopas have large flowers. 

Larger leaves:  Many induced polyploids have larger leaves than their comparable diploids.  You can see this in the bacopas in the image to the left.  This may or not be beneficial.  However, large foliage can often appear "cabbage-y" and this is not generally considered to be a positive if you are not breeding vegetables. 

Genetic junk:  Last, one of the important considerations to remember is that when you induce polyploidy, you create more opportunities for things to go wrong during cell division and during gamete production.  Slow growth rates, reduced fertility or fecundity --- these may simply be reflections of chromosomal pairing abnormalities because there are extra sets of chromosomes in the dividing cells.  For this reason, as a breeder you should probably go through at least two seed generations post-conversion before making final selections.  You may find that what may have been potential problems simply disappear.  More, the new combinations of alleles that you are hoping for will ONLY occur in the advanced generations, as recombination begins to work for you, and the genetic debris from the initial conversion gets sifted out by meiosis. 

One of the practices in which G2 invests a significant amount of time is polyploid induction --- taking diploid genetics and creating tetraploids (and then potentially triploids or even higher ploidy levels). 

Let's review some basic terminology.  Every non-reproductive cell in a typical multicellular plant or animal has TWO sets of chromosomes.  This condition is called diploid (di = two), and is the normal state for most living things.  Cell division mechanisms work well when there are only two sets of chromosomes migrating at each division. 

When reproductive cells form, a diploid cell (e.g., a pollen mother cell) goes through a unique series of divisions called meiosis, and produces up to 4 microspores which end up as pollen grains.  Each microspore contains only ONE set of chromosomes.  This is called the haploid state. 

There is a standard abbreviation for the diploid condition: 2n. The abbreviation for haploid is 1n (or n --- lower case n, usually). 

Breeders and geneticists frequently include the number of chromosomes when describing the ploidy of a species.  For example, most seed-propagated Pelargonium xhortorum varieties are diploid, and have 18 chromosomes in each cell, or 2n=18.  Pollen and egg cells in these varieties would be haploid, and then be n=9.  Vegetatively-propagated zonal geraniums are tetraploid (tetra- meaning four, or four sets of chromosomes).  For zonals then, 2n=36. 

In those plant families where multiple ploidy levels may exist --- as with Pelargoniums --- it is standard practice to include a reference to the ploidy as you describe the chromosome number.  Somatic cells (non-reproductive cells) are still defined as 2n (think of normal mitosis as working on a "diploid" level since cells divide in two), and the chromosome number is described using an upper case "X".  For seed-propagated P. xhortorum then, 2n=2X=18, while for zonals, 2n=4X=36.

Why is any of this important?  Look at the picture below: 

A pair of Pelargonium xhortorum (4X, 2X) flowers is on the left.  A pair of P. peltatum (4X, 2X) flowers is on the right.  Within each pair, the flower on the left is the tetraploid, while the flower on the right is the diploid.  One of the classic "advantages" that polyploids exhibit is that they may have a larger flower size.  The Pel flowers above are typical --- in each pair of flowers, the tetraploid on the left is visibly larger. 

Tetraploids also tend to exhibit larger fruit, larger seed, larger pollen grains and sometimes larger plants.  Please recognize that these potential benefits of polyploids do not always occur, and that variation for size may be equally possible within the range of diploid variation. 

Often, the cells of a polyploid are larger than those of a diploid.  Larger cells can frequently create leaves and flowers which are thicker and denser.  The "feel" of a plant leaf or flower is referred to as "substance" and polyploids often have more substance.  Leaves and flowers of polyploids will feel thicker.

To an ornamental breeder, flower color is always a consideration.  Polyploids often display novel flower colors.  The reasons for this are many but I find it useful to think about this as a gene dosage effect.  In a diploid plant, there are two alleles (two genes) present for every biochemical step (and yes, I am oversimplifying here).  If each allele contributes 10 units of flower color, a diploid plant might then make a total of 20 units.  In a tetraploid, there are four alleles present.  Theoretically, then, a tetraploid could make 40 units of flower color.  If so, then tetraploids should have more color and perhaps be darker-colored than their diploid counterpart.  Indeed, this is frequently what seems to occur. 

But novel colors also occur in tetraploids.  Keeping to my hypothetical model, if red=20 units of color, and deep red=40 units, what happens when you have 3 doses of red (30 units) and one dose (10 units) of some other color, perhaps blue.  Red + blue = purple.  3 doses of red plus 1 dose of blue might give you a deep burgundy flower color.  This color combination would probably NOT be possible in a diploid, since there would only be two doses available, and even though red+blue=purple might occur at the diploid level, it would only be purple (1 red + 1 blue).  3 red plus 1 blue would give you a very different shade of purple, and could possibly give you a rich burgundy color. 

Other potential benefits to inducing polyploids include improved vase/shelf-life of flowers; improved cold-tolerance of plants; and improved pathogen-tolerance of plants or flowers.  Most of these effects seem to be related to the change in cell size that occurs when a polyploid is induced, but not always.  Most of us think first of BIG, but there are many other potential benefits possible when you create a polyploid.

Next time, I'll write about potential benefits to the breeder.

Andrea Murphy-Faust runs G2's TC lab.  Andrea is a Penn State grad (BS Horticulture, MS Plant Genetics) with extensive experience at the USDA-Beltsville in plant tissue culture and molecular biology.  She is only available to us on a part-time basis right now because her other job --- being the Mom to two wonderfully energetic young boys ---  is her full-time commitment.  Which is as it should be.

With G2, Andrea has an interesting and challenging job.  Whenever we make a selection with potential for release, we immediately put it into culture in order to minimize the future risk of potential infection.  This process alone keeps the lab quite busy.  Andrea has spent the past few years methodically figuring out how to put each of our project-species into culture, and then taking them back out of culture.  We do this for two reasons.  One, we want to keep our material-for-release as clean as possible, but also (two), we want to make the release process easier and smoother for our potential licensees.  The cleaner our stock, the faster the licensee can ramp up production.  Andrea's species count is currently in excess of 20, and increasing on a monthly basis.

But production-style TC can be boring, like any production-style process.  We like keeping our staff intellectually involved in our processes, so we try to give Andrea more challenging tasks, too.  She is currently working on a number of embryo-rescue projects.  What's embryo-rescue?  Sometimes when you attempt a cross, especially a wide-cross between two different species, the immature seed fails to mature, and the seed aborts.  Sometimes, this occurs very early in the process, and there is little hope of recovering a viable embryo.  Sometimes, the seed begins to develop and only fails later in the process.  If you can take the immature seed off of the plant; dissect out the immature embryo; and then successfully raise the embryo in sterile culture, you can "rescue" the embryo and recover the desired hybrid seedling.  Andrea's success with ER is quite good, and we are beginning to use this technique more and more as a result. 

Andrea recently reminded me that we are a long way from "production-style" TC.  Even her most routine projects are still under-going refinements: tweaking cleaning protocols; tweaking media for multiplication, rooting, and maintenance; working through the TC-to-bench protocols.  In addition, she's been thinking about additional processes such as cold storage, and photoautotrophic micropropagation.   

Andrea is also beginning to methodically hybridize hardy ferns in vitro.  Reproductive biology of the ferns is very different from that of flowering plants, and as a result, has its own set of challenges.  In order to make fern hybrids effectively, she is developing some very innovative techniques.  You will eventually see some of Andrea's techniques in G2's blog as she reduces these to routine practice. 

One of the more challenging breeding projects underway at G2 works with variegated Pelargoniums.  These can be absolutely gorgeous plants, and are routinely consumer favorites. 

Why, then, are there so few variegated Pels in the market?  Because they are not easy to produce, and they are not easy to breed.  If the plant is difficult to produce, most growers will not attempt to grow it more than once, regardless of how much demand there is.  If the plant has to be handled differently from other varieties in the same product class, most growers will not attempt to grow it more than once, regardless of demand.   

The picture above is an old variegated variety --- bred at Penn State by Dr. Richard Craig --- named 'Ben Franklin'.  I will use "Ben Franklin' to demonstrate some of the challenges which occur in both breeding and production. 

The variegated Pelargonium above is what a botanist would call a "chimera".  A chimera is a plant which contains tissues of different genotypes.  In this case, the white tissue is one genotype, while the green tissue is another.  The white tissue has NO chlorophyll (the green pigment in plants), while the green tissue has an abundance of chlorophyll.  Why is this important?  Because chlorophyll is the pigment in a plant which absorbs the energy from sunlight and converts it into sugars (the form of energy which the plant needs to grow). 

A variegated chimera is thus a mix of green- and white-colored tissue.  Only the green portions contain chlorophyll, and only the green tissue is generating energy for the plant.  The white portions may make the plant attractive, but to the plant, these non-green tissues use energy without producing energy.  If there are two plants side-by-side, one all green, and the other variegated, which one do you think will grow faster?  The all-green one.  To a grower, this means that the variegated plant takes longer to grow, to get ready for market, takes more time on the production bench, and has to be treated separately from the all-green forms.  For many growers, this is a serious problem.  Can they sell the variegated forms at a higher price?  If not, then they make less money on the variegateds, and growing them could be a bad business decision. 

Breeding variegated Pelargoniums is also a challenge.  When you make crosses using variegated Pels as the seed parent, you only recover 3 to 5% variegated seedlings.  When you make crosses using a variegated Pel as a pollen parent, you recover even fewer (ca. 1%) because there is not much cytoplasm transferred from pollen to embryo in Pels. 

And Pels are one of the few known species in which there is any transfer of cytoplasm during fertilization.  In most variegated chimeras, you would only use the variegated parent as the seed parent. 

Why does this matter?  Well, it simply makes the numbers of crosses you have to make, and the numbers of hybrid seed you need to produce for each experimental cross, increase dramatically.  If you would normally want to look at 100 progeny from a cross to have a good chance of finding a superior type, you would need to look at 100 / 0.03 progeny --- 3,333 progeny --- to find a similar superior type that was also variegated.  That's a lot of seed to make, and a lot of seedlings to grow out. 

Furthermore, it's actually not that easy.  If you recover 100 variegated seedlings, only about 10 of them will eventually turn into that nice white-margin or white-edge form (called a periclinal chimera).  There are lots of propagation tricks you can do to try and "encourage" a chimera to go periclinal, but in my experience, the harder it is to get to periclinal, the harder it is to stay periclinal.  Periclinal chimerae in Pels will invariably sport to either all-green, or all-white forms.  However, some are much more stable than others. 

In the end, it's all about probability --- how many seedlings do I need to look at in order to have a good chance of finding what I am looking for?  These numbers games make breeding variegated pelargoniums very difficult.