Colchicine is known to be an unsurpassed chemical agent used for the polyploidization of plants. Simultaneously, it has a strong mutagenetic property. Possibly this is the cause of its insufficient efficiency during doubling of maize haploids. The haploid chromosome set is known to be more sensitive to mutagenic effect in comparison with the diploid one (Tyrnov, 1970). The other possible reason is an asynchronous division of meristem cells, which causes their varying sensitivity to the polyploidizing action of colchicine. Prophase, metaphase, and anaphase cells duplicate, but the mutagenic action of colchicine results in a significant number of chromosome aberrations, blocking of cell division, death of a part of them. Simultaneously, interphase cells are less sensitive to colchicine and, therefore, they maintain the former level of ploidy and the ability of a normal division (Davoyan, 1972).

It is known that chemical mutagens are characterized by the capacity to influence directly not only the DNA molecule, but also its precursors. This specific type of mutagenesis is characteristic of the presynthetic (G1) and synthetic (S) phases of the cell cycle, when modified bases or nucleotides may be inserted into the forming DNA molecule (Dubinin, 1968).

In order to overcome the barriers impeding effective chromosome duplication in haploids, we propose the utilization of a method contributing to the increase in the cell number at the phase of mitotic division during the treatment with colchicine.

Seedlings with a root length of about 1.5cm were used for colchicine treatment, as mitosis in the stem meristem proceeds just at this stage of maize development (Gulyaev, 1958; Berlin, 1972). It has been established that the rootlets reach an appropriate length after 3-day germination of seeds at 26 C. Then the seedlings were placed in a refrigerating chamber and kept at the low temperature (2-4 C) for 72 hours. Further, the seedlings were placed into a thermostat at the same temperature (26 C) and the meristem tissue was examined using a cytological method with equal time intervals. It had been established that all the cells were proceeding with the interphase stage after they were kept in the refrigerating chamber. This condition was maintained for a few hours and the mitotic activity of cells appeared only after seven hours of their storage at the temperature of 26 C. In this case, the mitotic activity was ample in comparison with the meristem tissue of the seedlings which were not exposed to low temperatures.

The effect of mass mitotic activity was studied on three genotypes showing no significant difference among them. The findings of the cytological examination had led us to the conclusion that it was the synchronization of cell cycles we were observing in maize at low temperatures.

The synchronization was employed as a possible means of enhancing the efficiency of haploid diploidization, as well as a possiblity to reduce the mutagenic action of colchicine.

Colchicine treatment was conducted seven hours after the seedlings had been placed into the thermostat. The seedlings were soaked in colchicine solutions at the concentration of 0.02%, 0.04%, and 0.06% supplemented with DMSO(0.5%).The treatment was carried out at the temperature of 18 C and 26 C for 12 hours. The colchicine treated seedlings were planted into sand filled tubs and kept in dark for 24 hours at the temperature of 26 C Further, the seedlings continued to grow for 48 hours at sun light then were planted in the field.

The experimental findings on the duplication of the chromosome number were verified under field conditions. The number of haploids plants producing pollen was estimated.

Table. The method of treatment and results obtained
 

Treatment conditions			Results
Temperature	Concentration	Number of plant	Haploids	Flowering haploids	%
-	-	404	404	0	0
18 C	0.02%	34	32	2	5.8%
	0.04%	35	25	10	28.5%
	0.06%	45	36	9	21%
Total		114	93	21	18.4%
26 C	0.02%	55	48	7	12.7%
	0.04%	57	38	19	33.3%
	0.06%	31	25	6	19.3%
Total		143	111	32	22.3%

The effect of the cell cycle synchronization was more apparent in the treatments with the colchicine concentration of 0.02% while flowering plants were absent in the treatment under similar conditions and concentration but without synchronization.

The percentage of flowering haploids in the treatments with the conlchicine concentration of 0.06% was close to that without synchronization. The null influence of different temperatures and synchronization on the duplication result with an increased colchicine dose is likely to be associated with the fact that its diploidizing action, to a larger extent, occurs after soaking, i.e. colchicine, as an alkalyzing chemical mutagen, is characterized by the capacity to remain in tissue for some time. The colchicine action in these cases is likely to take place during at least two cell cycles. This supposition is also linked to the fact that plants supposedly having tetraploid traits were found among the haploids.

The seedlings treated with the concentration of 0.06% colchicine showed a suppressed growth and strong thickening at initial stages.

The highest percentage of flowering haploids was obtained after the treatment with the 0.04% concentration plus synchronization 28.5% at the treatment temperature of 18 C, and 33.3% at 26 C.

A significant difference in the percentage of flowering plants was recorded between the treatments at different temperatures, especially at the concentrations of 0.02% and 0.04%. This is, probably, connected with the highest efficiency of colchicine at high temperatures.

Flowering panicles were not found in the untreated control comprising 404 haploid plants.

Thus, synchronization of cell cycles in combination with low colchicine concentrations and elevated temperature can significantly increase the efficiency of diploidization of maize haploids.
 
 

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