The genotypes from which the haploids were produced are listed in Table 1. All material was grown in a growth room under a 16/8 h day/night, photoperiod and 19/16C temperature. Haploids were produced using the wheat x maize hybridization technique described earlier (Sadasivaiah et al. 1999). Haploid seedlings with 5-6 tillers (about 8 weeks old) were washed free of soil, and the roots trimmed to about 4 - 6 cm. The seedlings were then placed in a beaker with the crowns immersed in 0.2% colchicine solution for 5 h at ambient laboratory temperature under low intensity light. After the treatment, the seedlings were thoroughly rinsed in running tap water. The shoots were then trimmed to about 10-15 cm before transplanting into 2.2 in (6.3 cm) square pots containing Cornell mix (Boodley and Sheldrake 1977) and returned to the growth room for further development. The presence of viable seed at maturity was used as the criterion of chromosome doubling due to the colchicine treatment. The percentage of plants with doubled sectors was calculated as the number of plants that produced seed, divided by the number of plants that survived the colchicine treatment and developed spikes. The chi-square test was used to compare frequencies of chromosome doubling. Pearson correlation coefficient was calculated to evaluate the relationship between the number of haploids treated and percent survived and fertile.


Results and discussion

The haploids varied with regard to vigor and tillering habit both within and among genotypes. Data on the number of seedlings treated, post-treatment survival and chromosome doubling frequencies (based on seed set) are presented in Table 2. Only 14.1% of the plants failed to survive colchicine treatment, with a range of 0 to 34.8% among the genotypes. In most cases the mortality appeared to be due to poor seedling vigor resulting in an inability to overcome the toxic effect of colchicine (Jensen 1974; Hansen et al. 1988; Mentewab and Sarrafi 1997; Hansen and Anderson 1998). Of the 85.9% that survived the treatment, only 10.1% were completely sterile, with a range of 0 to 53.3% among the genotypes.

In most cases the 89.9% of plants that survived the colchicine treatment and subsequently produced seeds showed only partial fertility, although occasional heads were fully fertile. There was a very low correlation between the number of haploids treated and percent survived (r = 0.03) and the number of haploids treated and percent fertile (r = 0.31). Similarly, the correlation coefficient for the percent survived and the percent fertile was also low (r = 0.37). This suggests that there is no relationship between the number of haploids treated and the rate of doubling. The frequency of surviving plants setting seed varied with the genotype, ranging from 46.7 to 100%, with a differential response that was highly significant (P<0.01). In a study of the effect of colchicine treatment in two winter wheat lines, Metz et al. (1988) found that 98% of the treated plants of one line, Centurk, produced seeds, whereas only 43% of another line, NB88, did. A similar genotypic difference in response to colchicine treatment was observed in the wheat haploids studied by Mentewab and Sarrafi (1997).

The seed harvested from. colchicine-treated plants was viable and produced phenotypically normal, fully fertile progeny. The colchicine treatment technique used in this study is simple and effective in producing a high frequency of doubled haploids from a diversity of genotypes and facilitates the attainment of instant homozygosity thereby enhancing the efficiency of selection in breeding programs.


Acknowledgments

This work was supported in part by the Farming for the Future Program (Projec #950648) of the Alberta Agricultural Research Institute. The authors wish to thank Dr. J. R. Byers for valuable suggestions and Mr. T. Entz for statistical analysis. LRC Contribution No.3870066.