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.