Rice Meiotic Chromosome Spread Preparation of Pollen Mother Cells

Materials and Reagents

1. Young panicles containing meiocytes of rice (Oryza sativa ssp japonica cv. Zhonghua 11)
   
2. 70%, 90% and 100% ethanol (Analytical Reagents)
   
3. 60% (v/v) acetic acid (Analytical Reagents)
   
4. Carmine (Sigma-Aldrich, catalog number: C1022-25G )
   
5. Liquid nitrogen
   
6. Vectashield Mounting Medium with DAPI (Vector Laboratories, catalog number: H-1200 )
   
7. Carnoy’s fixative (see Recipes)
   
8. Aceto-carmine (see Recipes)
   

Equipment

1. Stereo microscope
   
2. Fluorescence microscope (Zeiss, model: AX10 )
   
3. Microscopic slides and cover slips
   
4. Dissection needles and fine forceps
   
5. Heating block
   
6. CCD camera (Hamamatsu Photonics K.K., model: ORCA-R2 C10600 )
   

Procedure

1. Fix young panicles by immersing whole young panicles (from 4 cm to 15 cm in length) at proper developmental stages into about 100 ml freshly prepared Carnoy’s solution at room temperature for 2-4 h and store the fixative materials in Carnoy's solution at 4 °C.
   
2. Dissect about 15 florets under stereo microscope and remove all parts except anthers on a microscopic slide.
   
3. Put the anthers into 20 μl Aceto-carmine stain on a slide, and keep cutting the anthers with a small scalpel until no obvious large debris were observed.
   
4. Add 30 μl 60% (v/v) acetic acid to chopped anther on the slide and put the slide on a heating block at 50 °C for 2 min.
   
5. Cover the slide with a piece of glass cover slip and put the slide and cover slip under a piece of filter paper, then press the cover slip down tightly with thumb for 10 sec to squash the meiocytes. Keep the slide at -20 °C for at least 30 min (keep the glass slides from cracking when it was placed into liquid nitrogen next step).
   
6. Put the squashed slide into liquid nitrogen for 5 min and remove the cover slip with a scalpel.
   
7. Dehydrate the slides for 2 min in 40 ml of 70% ethanol, 90% ethanol and 100% ethanol, sequentially.
   
8. Add 15 μl VECTASHIELD Mounting Medium with DAPI on the air-dried slide, and seal the slide with a glass cover slip.
   
9. Observe the chromosome spreads under fluorescence microscope and capture the images with CCD camera (Figure 1).
   

Representative data

Figure 1. Male meiotic chromosome spread by DAPI staining at pachytene (A) and diakinesis (B) in wild type. Bar= 5 μm

Recipes

1. Carnoy’s fixative
   Ethanol: Glacial acetic acid 3: 1 (v/v)
   Freshly prepared
   
2. Aceto-carmine
   0.5 g carmine was dissolved in 100 ml boiling 45% acetic acid
   

Acknowledgments

This protocol was adapted from the previously published paper Li et al. (2013), and was supported by the 863 Project Grant (2012AA10A303), the National Natural Science Foundation of China (31171441 and J1103510), the National Key Basic Research Developments Program (2013CB126900), the Fundamental Research Funds for the Central Universities, and the Program for New Century Excellent Talents in University.

References

1. Chelysheva, L., Vezon, D., Chambon, A., Gendrot, G., Pereira, L., Lemhemdi, A., Vrielynck, N., Le Guin, S., Novatchkova, M. and Grelon, M. (2012). The Arabidopsis HEI10 is a new ZMM protein related to Zip3. PLoS Genetics 8(7): e1002799.
   
2. Higgins, J. D., Armstrong, S. J., Franklin, F. C. and Jones, G. H. (2004). The Arabidopsis MutS homolog AtMSH4 functions at an early step in recombination: evidence for two classes of recombination in Arabidopsis. Genes Dev 18(20): 2557-2570.
   
3. Li, X., Chang, Y., Xin, X., Zhu, C., Li, X., Higgins, J. D. and Wu, C. (2013). Replication protein A2c coupled with replication protein A1c regulates crossover formation during meiosis in rice. Plant Cell 25(10): 3885-3899.
   
4. Wang, K., Tang, D., Wang, M., Lu, J., Yu, H., Liu, J., Qian, B., Gong, Z., Wang, X. and Chen, J. (2009). MER3 is required for normal meiotic crossover formation, but not for presynaptic alignment in rice. J Cell Sci 122(12): 2055-2063.