I am trying to determine the ploidy level of a legume plant specie that is only endemic to the Cape fynbos biome of South Africa. No cytological studies have been previously done, hence the ploidy level of the plant is not known. I have been trying with no success. Can anyone with a working protocol for ploidy level determination in indigenous plants species, where the chromosome number is not known assist. In addition, i would also appreciate a protocol of using pollen mother cells.
*NB. I have no genetics background. I am trying a new field and hence still familiarizing myself with genetics.
I have not done it myself but I guess you can find papers in PubMed (http://www.ncbi.nlm.nih.gov/pubmed) by inserting key words (e.g. ploidy level determination plants ) and searching. Then, by clicking "last" you will get the first papers published about it. You will need to read some "methods" sections on the papers that most fit to your question; it may not be strightforward. Bests.
These are non dividing somatic cells. If you want to determine the ploidy, please soak the seeds and when the length of root root attain 0.5-1 cm , excise them and fix. After fixing squash them properly and then locate the dividing cells.
Thanks Milagros and Anwar.
@Milagros, thanks will continue researching. i am currently trying the different methods but its proving to be a daunting task. @Anwar, i was using root tips from cuttings but have since germinated seeds to try and use them.
What you need to determine first is the chromosome number and the procedure is as Anwar said, after that you can continue. Review the book Investigating chromosomes for Adrian F. Dyer 1979.
And I suggest that control the technique using some other characterized specie (may be one related that you are studing) to be sure the technique is appropiated used.
Usual procedure to find out chromosome number in plants is by root tip squash methods. This will also determine ploidy level. Take 1 cm long young root germinated from soaked seeds. Use standard chromosome staing procedures and squash stained root tip only from this root on a glass slide under cover slip and observe chromosomes under the compound microscope.
Chromosome staining is the best approach. Acetocarmine /galc ac. acid based staining protocol commonly used. . Chromosome Techniques — Theory and Practice , a famous book by a legendary cytologist Prof. Aruna K. Sharma is a good reference.
Another reference on chromosome staining is "Handling of Chromosomes" by C.D.Darlington and Janaki Amaal. Chromosomes in dividing cells can be seen only by staining.
Did you use colchichine on your protocol? It will destroy spindel formation causing the chromosome to spread around within the cell. It will than be easier to be counted at the metaphase when a good preparate is obtained. Surely it is not easy when the ploidy level is high and the size of the chromosome is small.
@Samuel, thanks, no i hadn't use colchine but propionic carmine. yes it appears that all the species are of ploidy level. We are presently trying a number of stains to see which best works.
Propiono carmine is used for staining chromosomes like acetocarmine or acetoorcein, but for chromosome counting you look at mitotic metaphase spreads in root tip squashes for which colchicine treatment before staining is done. Read book on Chromosome techniques mentioned in earlier answers to your question for detailed procedure. All your difficulties will be solved. Wish you good luck.
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