Hi Sara:

This shouldn't be difficult to find. For example, .http://hortsci.ashspublications.org/content/28/2/155.full.pdf

but I'm sure there are many others....

In particular, for Salvia spp

http://www.ncbi.nlm.nih.gov/pubmed/17285979

http://treephys.oxfordjournals.org/content/27/7/993.full.pdf

Hope it helps

Pablo

Thanks Pablo!!!!

Thanks a lot Flor!!!!

Protocol: Feulgen reaction for plants chromosomes 1.- Germinated seeds 2.- Cut roots of germinated seeds (approximately 20 mm) and then immersing for 12 hours in a 0.002 M solution of 8-hydroxyquinoline at a temperature of 8-10 ºC or immersing in 0.05% solution of colchicine for 3 hours at room temperature. 3.- Fix the roots in 3:1 ethanol:acetic acid for 24-48 hours and stored in 70% ethanol.. 4.- Stain the fixed roots with cold Feulgen reaction: - Hydrolyzed in 5N HCl at 20 ºC for 60 min - rinse in distilled water and stained with Schiff reagent for 1 hour at room temperature. - rinse in bisulphite solution x 3 washes - 2 mins each (Bisulphite solution: 10% potassium metablsuphite 5ml, M HCL 5ml, Distilled water 90ml) 5.- Make the chromosome preparations by squash the root tips.

I'm agree, Feulgen technique is the best, but I prefer to use ice-water pre-treatment for 24-26 hours to accumulate metaphases.  

You may not have a straight protocol for Salvia, but you can try protocols that work on other plants to see which one that can work on your plant. If you don't find anyone that works perfectly, you may have to modify anyone that is close to what you want. You may alter the concentration or duration of exposure to acid if the protocol involves acid-facilitated maceration of cell wall. You may also employ heat to enhance stainability of the chromosomes. You may look for Osuji, Okoli & Ortiz, 1995; Ekanem and Osuji 2006; Osuji, Edeoga and Okoli 2005; Osuji, Okoli and Heslop-Harrison 2006 for help. The papers contain reliable protocols for bananas and plantains, taro and tannia, Bambara groundnuts and cucurbits.

Hi Sara, 

I have only a few months experience, but finally am having good luck with a midday harvest, 24-hour pretreament in prechilled dH2O, fixation in Farmer's (3:1 95% ethanol to glacial acetic acid), then staining in Wittman's iron-aceto-hematoxylin and squashing in 50% Hoyer's.

To all the above suggestions I would add that you first need to be sure that your plants are very happy and that root tips are growing actively! I am growing seedlings and field-harvested (montane to subalpine Ranunculaceae) plants in a growth chamber with 18 hour days at 20C and they are happy. You will learn (if you have not already) to recognize healthy root tips (white tip, rounded or pointy depending on the taxon) that are full of actively dividing cells. I have experience only with Solidago (Asteraceae) and Caltha (Ranunculaceae), but attached my protocol for Wittman's iron-aceto-hematoxylin adapted from those given to me from Neil Luebke (Milwaukee Public Museum) and Carl Taylor (Smithsonian, US), and a longer document full of notes, resources, troubleshooting, "recipes", etc. Good luck!

Best, Keir

I am also having difficulties in finding chromosomes in a fynbos species Cyclopia. Its a species endemic only to South Africa. Your comments to Sara are very helpful. For me i am not even seeing the cells.   One day i have luck spotting the cells the next day its a different scenario. 

Regards,

Mlamuli

Thanks all. I finally managed to get the chromosomes with a bit of protocol modification. However, still struggling a bit to get them nicely spread in order to count them easily especially with species that have more than 90 chromosomes.. Does anyone have an idea what I can do/ improve on?

Dear Motsa

if you work on landrace and endemic plants difference in results are predictable because of genetic variation unlike cultivars. For chromosome counting you can take pictures via CCD or Camera on light microscopy then it is easier to counting on monitor but absoloutly direct observation method is time consuming and hard.

Best wishes