Root tip squash ?

General Discussion
1

Could anyone tell me what the minimum required magnification would be to perform a chromosome count on a root tip squash?

I would also appreciate any opinions on the best stain to use.

Thanks.

Jinks.

2

I suggest 400x- 10x eyepiece and 40x objective.

Acetocarmine is a great stain and what I use most commonly. Acetoorcein is fine and Shiffs reagent okay. The main thing is to have tissue at the right stage. The stain just gives greater contrast. The truth is with practice you can see the shadows of chromosomes without stain if need be. People, in my opinion, seem to get carried away with more minor details like stains and some other things when they would be making far more progress admitting and working on collecting and preparing their samples at the right stage. If you don’t have nice metaphase cells, varying your stains won’t help to find what’s not there anyway.

Sincerely,

David

3

David,

I have access to microscopes with 10X eyepiece and three objectives (4, 10, and 20). Do you think I’d be wasting my time trying to use the 10X eyepiece and 20X objective, or would I have a decent shot at seeing rose and/or iris chromosomes at this power of magnification?

Thanks, Tom

4

Hi Tom,

Great question. I usually scan a slide at 100x, 10x eyepiece and 10x objective to find cells that look promising (flat, spread well…) and then go to the 40x to be able to count chromosomes. For roses and at least my eyes I think I can get a pretty good idea of general ploidy if I look at multiple cells with a 20x objective, especially if the chromosomes are spread well, but I’m so used to the 40x. The chromosomes of roses still seem small with a 40x objective. If Iris chromosomes are like lily (I suspect they should be large like lily since they are somewhat closely related), it would be easier to see them than roses and your 20x ojective should work fine. Perhaps if you are able to buy a 40x objective and replace one of your other ones that would be great. Perhaps if you start looking at root tips using the scopes you have access to you can get an idea if it would be worth it for you to get a 40x objective. Perhaps if you ask around there may be one you can borrow.

I really enjoy cytology. I love the peacefulness of being in the dim microscope room listening to a peaceful radio station and finding chromosomes at different stages of mitosis or meiosis.

Please let us know how it goes Tom.

Sincerely,

David

5

Thanks David,

I’ll have to give the 20X a try for now, since I’m on a very limited budget. And I don’t have any stains yet, so I’m especially encouraged by your previous statement that “you can see the shadows of chromosomes without stain if need be.” Also, I’m not necessarily looking for absolute counts right now; so, the “idea of general ploidy” would be all I’d need.

Specifically, I have an iris hybrid that I’d like to know the general ploidy of. And I have a trifluralin treated version of a sterile diploid hybrid rose, that I’d like to confirm ploidy of. I may have more difficulty obtaining roots of this one, since it’s been relatively slow to grow at all.

I’ll let you know how it goes.

Thanks again, Tom

6

How important do you suppose the quality of the microscope is? I also need to stay within a fairly tight budget, and I’ve seen advertised microscopes that are quite affordable (less than $100, even less than $50) and would do the job - if, that is, they are all they claim to be. I also have some plants of various genera for which I really need to know chromosome numbers before commencing breeding activities. It really would be a boon to be able to do this, but I would hate to waste any money!

Stefan

7

Hi David,

Thanks for your reply.

I should have guessed that it would take more than having a microscope to perform a chromosome count.

I have looked at root tips at 400X and not made anything out with any degree of certainty. Though I have to admit that I

8

A couple RHA issues or so ago I wrote a step by step article on the process of root tip squashes highlighting what has been working well for me. I included pictures of most steps and what to look for from root tip collection to isolating the cells which are most amenable. If you don’t have the issue, E-mail me and I’ll try to send it as an attachment.

David



zlesak@rocketmail.com

9

I was just curious if anyone knew of the specific procedures for doing a chromosome count with buds rather than root tips, as this is what i have available. any information anyone can give on the matter, including info i might find on the web is much appreciated! thanks

Emily

10

Hi Emily,

Yes there is a paper by Ma et al. (1990’s) in HortScience where they do just that. THey use some enzymes to soften the tissue a bit and allow the cells to spread more. I’ve just tried the same technique on shoot tips as root tips with difficult roses and have found some mitotic cells which could be counted. I didn’t have the enzymes on hand and for this side project and few roses that I had trouble rooting cuttings of it wasn’t worth buying them. It takes a bit more patience to find the right cells, but I’ve done it.

Sincerely,

David