I am doing karyomorphological study of the plant, sarcostemmma. I did not get good slide preparation yet. Roots were usually pretreated with PDB and traces of Aesculin for 1 hour for spreading of cells. But no result of it. I couldnt observe chromosomes too.
Please give me suggestions
https://www.researchgate.net/publication/230319241_Deviating_chromosome_numbers_in_Asclepiadaceae
You may want to try this technique.
Article Deviating chromosome numbers in Asclepiadaceae
Well, what you need to do depends on what the problem was. If the cells did not separate well, you need to look at the hydrolyzing agent. You may try hydrolysing the root tips with various agents. You can try enzyme combinations like cellulose, pectinase cytohelicase etc. You may also try dilute HCl of various strengths: 5, 10, 12 15% etc. If the cytoplasm stained deeply and made chromosomal observation difficult, you may increase the acid concentration during cell wall maceration. If the chromosomes did not stain at all when you macerate with acid, you need to reduce the acid concentration and probably the time of exposure also. You may also consider applying heat after quashing in stain drops or after staining of microtometric section. You may search for some of my papers online to see some of the methods I have used in some of my works. I have also attached a few publications for your attention.
Article Cytology and Molecular Cytogenetics of Cucumeropsis mannii N...
Article Karyological Studies on Vigna subterranea (L.) Verdc. from Nigeria
Article Karyotypes of the A and B Genomes of Musa L
Article An improved procedure for mitotic studies of the Eumusa sect...
YOU CAN FOLLOW THIS SIMPLE TECHNIQUE. TAKE THE ROOT TIPS AND SLIGHTLY WARM IN 1 N HCL. ALLOW IT TO COOL. THEN WASH THE ROOT TIPS IN WATER. DRY THE ROOT TIPS AND HEAT THEM IN ACETO-CARMINE STAIN FOR 3-4 MINUTES AND KEEP THE ROOT TIPS IN THE STAIN FOR 30 MINUTES. SQUASH THE TIPS IN 45% ACETIC ACID.
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