Nitrous oxide (N2O) is effective for chromosome counting in maize
Nitrous oxide is a gaseous substance and has been used for chromosome doubling in red clover. Application of nitrous oxide to the pretreatment of chromosome counting in maize gives excellent preparations with many dispersed countable chromosome figures even in tetraploid maize (Figure 1).
Maize kernels were germinated in wet vermiculite at 25 - 30 C for 2 - 3 days. The germinating seeds with root length less than 1 - 2 cm were used. The excised roots were placed on wet vermiculite in a petri dish and were placed into an air-sealed ion container (10 cm inner diameter, 10 cm depth), and were treated with nitrous oxide gas (8 atm N2O + 1 atm air) for 3 hr at room temperature (21-23 C). The pretreated roots were fixed in ice cold 90% acetic acid for ten minutes and washed with tap water for five minutes. Root tips (1-2 mm) were excised with a razor blade and hydrolyzed with enzymatic solution (1% pectolyase Y-23, Kikkoman Co., and 2% cellulase Onozuka R-10, Yakult Honsha Co. LTD., dissolved in distilled water) for 90 min at 37 C. Hydrolyzed root tips were extended on glass slide according to the method described by Kato (Biotech. Histochem. 72:249-252,1997).
Preparations with 100 - 350 countable chromosome figures were obtained in diploid maize, and about 50 countable chromosome figures are expected in tetraploid maize according to this method.
Figure 1. Chromosome figures of a tetraploid maize stock Q-28-1(4n=40).
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