Nitrous oxide (N₂O) is effective in chromosome doubling of maize seedlings
--Kato, A

Chromosome doubling of maize is important for subsequent haploid breeding and production of polyploid series. Recently successful chromosome doubling of maize calli has been reported using colchicine or other antimicrotubule herbicides in maize anther culture, though chromosome doubling of maize seedlings still remains difficult. The problems to address in the seedling treatment include the permeability of the leaf tissues of maize to the colchicine solution and/or prolonged effect of colchicine on maize growing tissues. The author proposes here the use of a gaseous substance, nitrous oxide (N₂O), for chromosome doubling of maize seedlings.

A diploid inbred line Oh43 (2n=20) was used in this experiment. Ten seedlings with a shoot length of 2 cm were treated with a gaseous mixture (1 atm air and 8 atm N₂O) for one to four days in an iron container (15 cm diameter, 20 cm height) at 28 C. The shoot meristem tissues of the treated seedlings were cut and fixed in an ethanol acetic acid solution (3:1) just after the treatments, and the effect of nitrous oxide on cell mitosis and ploidy was examined microscopically for each of four preparations.

N₂O gas affected significantly the mitosis of maize cells. Normal anaphase cells were completely replaced by N₂O-anaphase cells (Figure 1, Table 1). There was a reduction in the proportion of prophase cells and an increase in the number of metaphase cells. In the three- and four-day treatment, mitotic indexes decreased. Tetraploid cells were observed in all the treatments (1-4 days) (Figure 2, Table 2). In the four-day treatment, thirty-five percent of dividing cells were tetraploid and one octoploid cell was observed.

The seedlings subjected to the four-day treatment recovered from N₂O injury within two weeks. The ploidy level of three root tips and one shoot meristem of one of the seedlings (four-day treatment) was analyzed. The shoot and one root tip were diploid and one of the remaining two root tips was tetraploid, while the third root tip showed a mixture of diploid and octoploid cells. Further analysis on the remaining seedlings is currently underway.

Table 1. Mitotic index and incidence of cells in prophase, metaphase and anaphase in the N₂O-treated maize shoot meristem.
 

Days of treatment	Mitotic index (%)	Prophase (%)	Metaphase (%)	Anaphase (%)	N2O-anaphase (%)	No. of dividing cells observed
1	3.2	4.8	88.1	0.0	7.1	378
2	4.1	11.6	80.7	0.0	7.7	259
3	0.7	15.3	77.6	0.0	7.1	98
4	1.4	7.1	85.0	0.0	7.9	140
Control	3.7	30.4	36.0	33.6	0.0	289

Table 2. Incidence of diploid, tetraploid and octoploid cells in N₂O-treated maize shoot meristem.
 

Days of treatment	Diploid cells (%)	Tetraploid cells (%)	Octoploid cells (%)	No. of dividing cells observed
1	99.4	0.6	0.0	317
2	83.9	16.1	0.0	242
3	73.2	26.8	0.0	82
4	64.0	35.4	0.6	178
Control	100.0	0.0	0.0	172

Figure 1. Anaphase of normal mitosis (left) and N₂O-induced abnormal anaphase (right).

Figure 2. N₂O-induced tetraploid cell (left) and octoploid cell (right).

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