II. 31. Experiments on chromosome doubling of barley haploids with colchicine.
J. Thiebaut and K. J. Kasha, Crop Science Dept., University of Guelph, Guelph, Ont., Canada N1G 2Wl.
The interspecific cross Hordeum vulgare L. x H. bulbosum L. results in the production of H. vulgare haploids (Kasha & Kao, 1972). These haploids treated by a colchicine solution yield completely homozygous doubled haploids. Although the percentage of doubled haploids obtained has already been improved by the addition of dimethyl sulfoxide (DMSO) used as a adjuvant to the colchicine solution (Subrahmanyam and Kasha, 1975) it is still low. The results of 3 additional experiments to increase this percentage of doubling will be presented here.
In all 3 experiments the haploid seedlings were treated at the 2- or 3-leaf stage when still in the culture vial. The seedling crown was submerged with a solution of colchicine for 5 hours. The plants were treated in a growth room under artificially controlled conditions (Stoskopf et al., 1970) at 22°C with lights on. After treatment they were rinsed twice with distilled water and placed in 8 cm clay pots containing a mixture of turface and peat moss. When the plants reached the 5-leaf stage (12-15 cm high), they were transplanted into 14 cm plastic pots containing soil.
The percentage of haploids doubled (% doubling) was calculated on the basis of the total number of haploid plants included in the treatment minus the number of plants which died. However, in some cases there was a differential mortality among the treatments and a more realistic estimation of treatment differences is the adjusted percentage of doubling, calculated on the basis of the total number of plants. The haploid seedlings used were obtained from a number of genotypes. To reduce genotype effects, where possible, equal numbers of haploids from one genotype were randomly placed in each treatment. In the first experiment (see Table 1), four different concentrations of colchicine were used: 0.05, 0.1, 0.2, and 0.4% w/v together with 2% v/v dimethyl sulfoxide (DMSO) and 1 drop of Tween 20 per 100 ml of distilled water. Based on these data, the percentage of doubling increased from 58 to 100% as the colchicine concentration increased. However, the percentage mortality following colchicine treatment also increased, so that the adjusted percentages of doubling are similar for each of the four concentrations. Consequently, a concentration of 0.05 or 0.1% is recommended, since it is as effective as the higher concentrations and results in less mortality.
In the second experiment (Table 2), Tween 20 was added to the colchicine solution as a wetting agent at a concentration of 1 drop per 100 m. Tween 20 increased the percentage of doubling from 33 to 54%, which is significant at the 1% level. However, there was also a significant (1% level) increase in plant mortality from 7 to 27%. The adjusted percentage of doubling still favors the use of Tween 20, although the difference is not significant statistically.
Table 2. Chromosome doubling and haploid plant mortality when using a wetting agent (Tween 20).
These data in Table 2 come from the averages of the 2 treatments through several different experiments, but reflect the trend seen in the particular experiment we did to test the effect of Tween 20 where the levels of significance were somewhat lower (10 and 5% respectively for the percentage of doubling and the mortality). This was probably due to the smaller number of plants involved (74 in each treatment). No difference between the 2 treatments was found concerning the average number of seeds per plant from those with doubled sectors.
Gibberellins are known to enhance stem elongation among other effects. Whether the gibberellin effects are due to a stimulation of cell division or of cell enlargement has been extensively debated. In the case of stem elongation, however, there is now little doubt that an early and pronounced effect of gibberellins on responsive plants is a heightened rate of cell division in the subapical meristem (Steward, 1972), although this does not exclude some cell elongation effect. Iyer and Randhawa (1965) found that the addition of GA3 to the colchicine solution considerably increased the number of mixoploid shoots obtained in grapes. They attributed the favorable effect of gibberellic acid to the accelerated rate of cell division and to the cell elongation which might have made the cells more permeable to the colchicine solution.
In our third experiment (see Table 3), GA added to the colchicine solution at a concentration of 10 parts per million (ppm) increased the percentage of doubling from 58 to 76% on a total number of 60 plants in each treatment. Although this is not significant statistically, it is close to the limit of significance when testing at the 10% level. When considering these 2 treatments through several experiments (based on totals of 181 and 113 plants respectively), the difference becomes significant at the 5% level. The average number of seeds per plant was not increased by using GA3.
Table 3. Chromosome doubling of barley haploids when GA3 (10 ppm) is added to the treatment solution
Treating the plants for 5 hr. with GA3 before colchicine treatment, as opposed to mixing it in the colchicine solution, resulted in no difference in the percentage of doubling.
References:
Kasha, K. J. and K. N. Kao. 1970. High frequency haploid production
in barley, (Hordeum vulgare L.). Nature (Lond.) 225:874-876.
Lyer, C. P. A. and G. S. Randhawa. 1965. Increasing colchicine effectiveness in woody plants with special reference to fruit crops. Euphytica 14:203-205.
Steward, F. C. ed. 1972. Plant Physiology - a treatise. Vol. VIB: Physiology of Development: The Hormones. Academic Press, New York and London.
Stoskopf, N. C., Hume, D. J., Gamble, E. E. and W. E. Tossell. 1970. Glasshouse replacement rooms for growing plants. Can. J. Plant Sci. 50:125-127.
Subrahmanyam, N. C. and K. J. Kasha. 1975. Chomosome doubling of barley haploids by nitrous oxide and colchicines treatments. Can. J. Genet. Cytol. 17:573-583.
