Re: CROSS: tri/tetraploid

Martin,

I just joined this group and therefore answer a question from 
some months ago.

For chromosome counting one need to have mitotic cells  , 
because only in his phase the condensed chromosomes are 
visable. Tissue in which cell division takes place are the 
meristems, normally the root meristems are used. One has to 
soften the tissue, stain the chromosomes, press the root tip in 
order to get a layer of one cell thick and then look under the 
microscope to see if cells are present in the right stage and then 
count the number.

I have only experience with polyploidization of bulbous Irisses, I. 
xiphium, I. tingitana, I. reticulata. I do polyploidization in tissue 
culture because for the breeding work I need to convert sterile 
interspecific hybrids which produce no seeds. With this tissue 
culture material I used also apical meristems for chromosome 
counting.

Nowadays I use for ploidy determination flow cytometry . Nuclei 
are released from tissue by chopping it with a razor blade in a 
buffer. The suspension with released nuclei are stained with a 
DNA stain. The staining intensity of nuclei is proportional with the 
DNA content. With a flow cytometer the staining intensity of 
individual nuclei can be measured. By comparing the intensities 
with that of a plant with known chromosome number, the 
chromosome number can be determined.  

The advantages of this technique is that you can use any tissue 
leaf, root etc, one has to use only a small piece of tissue. Since 
there are differences in the DNA content between species, the 
technique can also be used for assing interspecific nature of a 
hybrid: Hybrids have intermediair DNA content of the parents. 
The disadvantage of the technique is that the equipement is very 
expensive.

Beside chromosome counting and flow cytometry there are 
some other techniques for ploidy determination:  The amount of 
chloroplasts in the guard cells of stomata in the leaves and also 
the size of the stomata is often correlated with the ploidy level. 
The ways of analyses are however ofen not very accurate.

If you are interested I can look up the protocol for chromsome 
staining and send it to you. I could also mail you a copy of an 
small article on polyploidization and flow cytometric analyses in 
Irisses. 

Regards,  

Eloy Boon, The Netherlands

--- In iris-talk@y..., Foley Martin <emjfoley@y...> wrote:
> 
>  Happy Freedom Day,
> How does one determine the chromosome count? Is there a 
procedure to do this?
> Regards, Martin, Birmingham MI, Zone 5
>  
>   craigiris@i... wrote: Hi Martin,
> 
> The only way to be sure what ploidy an iris is, is to get a 
chromosome
> count.
> 
> Vicki
> 
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Service. 
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