Viçosa, Brazil
Universidade Federal de Viçosa
Maize root tip cell cycle synchronization -- Carvalho, CR, Saraiva, LS, Otoni, WC Accumulation of the cells in metaphase implies the use of some cytogenetic strategies. The well-known spindle mitotic inhibitors action, on normal meristematic cycle treatment, leads to metaphasic chromosome numbers appropriated for several cytogenetic approaches. However, this chromosome amount is not suitable for running flow cytometry analysis. This way, chromosome accumulation procedures, previously to the metaphase blocking, must be improved by means of an additional synchronization step. Treatments were optimized in our laboratory to synchronize meristematic root tip cell cycles of germinating maize seeds (test line L-869). Seeds were germinated in Petri dishes containing a film of distilled water, and incubated at 29 C in the dark. Seedlings (1.5 to 2 cm root length) were carefully transferred to a plastic mesh adapted inside a 6 cm diameter plastic vessels containing 100 ml of either 0, 1, 2, 4, and 6 µM hydroxyurea (HU). After two cycles, approximately 18 h, the roots were recovered by washing for 15 min (running tap water) and incubated in distilled water under the same conditions as before. Thereafter, samples of root tips were taken from 0 to 10 h, at 1 h intervals, and fixed in a fresh ice-cold methanol:acetic acid solution (3:1), and kept in a freezer for at least 24 h. Next, root tips were excised at 0.1 cm and macerated with 200 µM of freshly prepared Flaxzyme (NOVO) enzymatic solution plus 1.6 ml distilled water, and incubated at 35 C for 2h 30 min. The macerated cells were dissociated in a clean slide with a freshly fixative solution, air-dried and stained with a Giemsa solution. Meiotic figures were photomicrographed and digitized directly by a microscope-coupled CCD video camera to a computer. Under the experimental conditions, 2 µM HU enabled higher cell synchronization indexes (Figure 1) as compared to higher HU concentrations. By using only the synchronization step, at 2 µM HU, combined with 6-7 h recovering time, an average of 28% of metaphasic cells (Figure 2) was obtained in comparison to 0 µM HU control treatment (Figure 3), that normally displayed less than 1% of metaphasic cells. It was also noted that at 6 µM HU the cells remained in interphase, therefore losing the reversibility. This technique proved to be reproducible, being applied not only to cytogenetic and cytometric purposes, but to a wide range of cell cycle studies.

Figure 1. Highly synchronized interphasic maize root tip cells after 18 h at 2 µM HU. Bar = 20 µm.

Figure 2. Highly synchronized metaphasic maize root tip cells obtained after 2 µM HU treatment and 6-7 h recovering time without HU. Bar = 20 µm.

Figure 3. Typical pattern of maize root tip cell cycle from control treatment (0 µM HU). Bar = 20 µm.
 
 


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