I am dealing with already fixed leaf tip meristem as root tips are not available, the plant is a berry crop cultivated at a botanical graden (quite far away) and it's roots are woody, seeds don't germinate, twigs don't root - a total disaster. I have tried flower buds, but unfortunatelly missed meiosis last spring. I am able to get splashed preparations from enzyme digested leaf, but my spreads are of very poor quality. I tried cellulase, pectinase at different %, temp, timing, ratio, cold-hot hypotonisation, various dropping techniques (mixing cold, hot with dry, wet slide) - still no improvement...
You could try contacting Professor Guijun Yan at University of Western Australia School of Plant Biology, as he may be able to help you with methodology.
Hello, Asta !
I use for making chromosome preparations shaking off-imprinting technique. Maybe in our article you can imagine how it's done. Recommend some other articles. Regards Antonina Natyaganova.
1) Cytologia 66: 11-17, 2001
A Combination of the "Squash" and "Splash" Techniques to
Obtain the Karyotype and Assess Meiotic Behavior of
Prosopis laevigata L. (Fabaceae: Mimosoideae)
F. Tapia-Pastrana, and P. Mercado-Ruaro
2) JAPAN. J. GENETICS Vol. 53, No. 4: 251-255 (1978)
KARYOTYPE ANALYSIS IN RICE.
METHOD FOR IDENTIFYING ALL CHROMOSOME PAIRS
NORI KURATA AND TAKESHI OMURA
dear Antonina, I'm ashamed to confess, but i can't get any of these articles you recommend, because of limited access as our library has accessibility to only few databases :/ could you please share your article with the shaking off-imprinting technique? i found only: G- AND C-BANDING OF CHROMOSOMES IN BAICALASELLUS ANGARENSIS (CRUSTACEA, ISOPODA), but i also can't get it :/ other articles with your name is not about chromosomes, by the way, i read in russian :)
Hi. All papers written by Maluszynska et al. and Hasterok et al. There are details which need to be studied before you will do any chromosome spreads. We use enzymatic digested roots for squashing (based on Heslop-Harrison` protocolls) and the results are very good, especially for karyotyping. You can also come to us. Regards, Tomasz.
@Tomasz, I am really good at squashed root tip preparations, but the main problem is i can't get roots from that plant - blueberry, so i have to deal with the leaf tip meristem and even though I am able to get splashed preparations from enzyme digested leaf, but my spreads are bad, not suitable even for counting what to say about karyotyping, I tried to adjust every step in protocol and now i'm stuck with still the same bad chromosome plates. Thank you for the comment, will study papers you recommend, hoping it will help.
A big thanks, Antonina! i'm anxious to investigate the new information you've provided :)
I have a question, more than a suggestion: Could other methods, like flow cytometry, be used in this case?
@Anne-Kari, flow cytometry can be used in this case, if only you have it :)
There is a lab in Netherlands that provide flow cytometry services! You can read about it at http://www.plantcytometry.nl/
Try different timing for different strengths of antimitotics, try different hypotonics for variable periods, To do cytogenetics is to try infinite number of combiinations - fiddling around.
I did that for 6 months, made few hundred slides - no improval :(
The quality of cytologic preparations depend directly on the following combination: the type of anti-mitotic drugs, time and temperature adopted in the pretreatment of the tissue to be used for cytological preparations (the most common is the root tip meristem) . However you can try various combinations until you find the ideal. Tip drug 8 hydroxyquinoline (300 ppm), cycloheximide (3 to 6 ppm) (can be used together), saturated solution of para-dichlorobenzene and then colchicine. Chilled water also works but for large chromosomes. The temperature is 15 C and the time varies from 2 to 4 hours on average.
Hydrolysis is also very important to get clean and preparations with extended chromosomes. The choice of an enzyme or a mixture of several of them (pectinase, cellulase, etc.) can be decisive. The hydrolysis may also be done with 5N HCl (1 to 8 minutes)
Try this method: Mol Cytogenet. 2014 Mar 6;7(1):21. doi: 10.1186/1755-8166-7-21. An easy "SteamDrop" method for high quality plant chromosome preparation. Kirov I, Divashuk M, Van Laere K, Soloviev A, Khrustaleva L1.
Try this method:
An improved method for examining meiotic chromosomes in Musa L.
HortScience 37(6):959-961. 2002.
This method is for meiotic chromosomes, but maybe it will be useful for you
If you are dealing with material that is already fixed, then that is that, it's fixed.
Dear Asta
pretreatment of ice water (0 centigrade) could be effective for small chromosomes. You can examin for 2 to 24 hours but often it is enough for 7 hours. I never suggest colchicine for small chromosomes because it compact them and absolutely you will not be successful in observation. Sometimes simple and cheap material works better!
Good luck
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