Hello, I am trying to use the root squash method to observe mitosis in almond (Prunus) shoot apical meristems, but I'm not getting clear staining of chromosomes. This is the protocol I'm using so far:
1) Collect apical buds from the tree and preserve in a cooler on ice.
2) Dissect an almond meristem and inner-most leaf primordia from an actively growing apical bud.
3) place meristem on a microscope slide. Treat with an even mixture of HCl (I've tried 2M to 0.5M) and aceto orcein dye (I've tried 1% to 0.025%).
4) Place slide on a hot plate on low heat to evaporate the mixture. Don't dry completely.
5) Resaturate meristem with aceto-orcein dye and place a coverslip on top.
6) Cover slide with a paper towel and "squash" the meristem into a single cell layer by pressing down with an Erlenmeyer flask.
7) view slide under microscope starting at 4X and going to 1000X (oil immersion)
I'm very confident in my ability to excise meristems. I believe the HCl is properly dissolving the pectin from between the cell walls and "squash" step are correct since the meristem appears to be in a single layer of cells when viewed under the microscope.
There doesn't seem to be anything in the literature about shoot apical meristem squashes. Is there a good reason for that? Does anyone have a suggestion to improve the result? the first 2 photos are the shoot apical meristems (using different percentages of dye). The second photo is from a colleague who applied the same method to rice roots and clearly shows the chromosomes.
There doesn't seem to be anything in the literature about shoot apical meristem squashes. Is there a good reason for that? Does anyone have a suggestion to improve the result? Thanks in advance!
Maybe an alternate DNA stain would help? You could do the light imaging you currently have to visualize cell outlines and morphology then try a DAPI stain for the chromosomes.
Brian Allen
May be you should try treating the tissue with a fixative (Carnoy's) before proceeding with the squash procedure. Treatment with a prefixative can also be tried.
Try to collect samples at the morning and fix it in carnoy's solution.
Hi Amy, Pendyala, and Surendhar
Thank you for your suggestions!
This protocol was originally designed to visualize mitosis in onion roots, so I have repeated that experiment to ensure my reagents work correctly. I treated them with HCl in a 60°C oven, then stained with 1% aceto-orcein for 5 minutes. This was successful (see image below).
Returning to the almond shoot meristems, I followed your recommendation and tried fixing the tissue with a Carnoy's solution I made from 3:1 absolute ethanol and glacial acetic acid. I then treated the meristems with HCL in a 60°C oven, and stained with aceto-orcein at these treatment times and concentrations:
- 1% for 5 minutes
- 1% for 12 hours
- 0.05% for 12 hours
None of the treatments showed the same staining seen in onion roots. My conclusion is that this particular dye cannot penetrate the cell membrane in almond meristems.
My next step will be to find an alternative dye that can penetrate the cell membrane and, ideally, can be visualized with a light microscope.
- Badges
- Science topic
- Similar topics
- Agricultural Science
- Horticulture