David... root tip squash question

David, thanks for sending me your root tip squash protocol a while back. I’ve been playing with it without too much success yet but will keep at it. I have a whole bunch of cuttings atm (see pic) that are two weeks old (of “Temple Bells”) and they are callousing strongly already. Because they were done in moist newpaper they are very clean and I was wondering whether this callous material would be suitable to perform a squash to determine ploidy? It makes sense as they are made up of actively dividing cells but I don’t think there would be much organisation… if that makes sense. So what do you think? Do you think it might be suitable material to prepare?

Hi Simon!

It took me awhile to get the procedure working smoothly. Part of it is that the contrast with the chromosomes, etc. can take awhile for ones eye to pick up. The root tip is especially great tissue because we can isolate just the region of dividing cells and separate away the older cells with greater cell wall development which which make it harder to physically squash the cells and spread their contents for counting. With all the secondary cell wall development it is harder to get just a single cell layer think and a very flat plane to count chromosomes. I suspect the callus would have a lot of cells with too much cell wall development and it will be challenging. I would go ahead and give it try tough and I don’t want to discourage you. It looks like you should get some nice root tips soon though.

After you have a slide with some cells you see chromosomes in and your eye becomes more familiar with what to look for, I bet the cells will start popping out easily. The main thing above all is to have actively growing root tips. Don’t even bother with tips that seem like they are slowing down their growth, etc. Don’t leave them on ice more than 24 hours because that affects things too. Using the Farmers fixative (3/4 ethanol and 1/4 glacial acetic acid) to kill fast is important. If you use just ethanal there is problems in finding the cells you are looking for too.

Take Care,

David

I tried it quickly at work (I only had an hour off between classes), just with methylene blue and found the cells are really really small. I could see chromosomes but they were too small at x400 to see properly. I tried with the x1000 oil immersion lens without too much more success. There seemed to be a lot of tissue in there that made finding cells to look at difficult. It was also difficult to prepare without destroying the actual cells (maybe I’m just too heavy handed). Will try with your protocol too to see if I can improve my results. I have my lab tech on the job now too and she’s brilliant so I’m sure between the two of us we can work it out :slight_smile:

Simon, I love your picture of the callused cuttings - very nice.

Jim Sproul