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Improvements of the doubled haploid technology in maize
(2019) Molenaar, Willem; Melchinger, Albrecht E.
The in vivo doubled haploid (DH) technology in maize carries many advantages over traditional line development by recurrent selfing and has played an integral role in numerous breeding programs since the early 21st century. A bottleneck in the DH technology is still the success rate of chromosome doubling treatment, which has a strong influence on the costs of DH production. Currently, only a minority (~10%) of treated D0 haploid plants result in DH lines. Improvement in the chromosome doubling step of DH production would not only make DH lines cheaper, but could also change the optimum allocation of resources in hybrid breeding. In addition, the development of treatments using alternative doubling agents to colchicine, which is toxic to humans, would improve worker safety and simplify waste disposal issues for developing countries to benefit from the DH technology. Initiating such developments is the goal of this thesis. In a first step, we evaluated anti-mitotic herbicides with different modes of action as alternatives to colchicine for reducing the toxicity of chromosome-doubling treatment and for potentially increasing the success rates. In a series of experiments, we evaluated anti-mitotic herbicides with different modes of action in different concentrations and combinations. Based on the results of the initial experiments, we chose a specific concentration of amiprophos-methyl for evaluation in combination with varying concentrations of pronamide in a further experiment. This revealed the optimal concentration of pronamide in combination with the chosen concentration of amiprophos-methyl. However, this less-toxic treatment showed slightly lower success rates and slightly higher costs per DH line as compared to the standard colchicine treatment. In a second step after evaluating anti-mitotic herbicides for seedling treatment, we evaluated gaseous treatments using nitrous oxide (N2O), an anti-mitotic gas, in varying concentrations and combinations with air and pure oxygen. In two years of evaluation, we found an N2O treatment which had similar success rates as colchicine. The major benefit of such treatment is that this gas can simply be released into the atmosphere, eliminating the difficulty of proper chemical waste disposal, which is difficult to secure in developing countries. The only requirement is a treatment chamber, in contrast to the laboratory facilities required for handling colchicine. In a third step, we evaluated the potential of spontaneous chromosome doubling (SCD) as an alternative to chemical treatment-based chromosome doubling. Although previous studies found significant genetic variation and high heritability for SCD, a classical quantitative genetic analysis, elucidating the type of gene action governing this trait, and a selection experiment for improving SCD was missing in the literature. We found a predominance of additive genetic effects compared to epistatic effects, and a large selection gain after three cycles of recurrent selection for SCD to levels far beyond those reached by standard colchicine treatment. This indicates the great potential of SCD to improve the DH technology. The approximately ten-fold increase in spontaneous chromosome doubling rate (SDR) reached in our recurrent selection experiment marks a paradigm shift in the chromosome doubling step of DH production in maize. DH production efficiency can be greatly increased by the vast improvement in SDR, and production can be further simplified to enable even higher throughput. Instead of chromosome doubling treatment, which involves much handling of seedlings, haploid seeds from germplasm with a high innate ability to produce seed set without chemical treatment can be simply seeded in the DH nursery, eliminating the most costly production steps. Thus, this thesis has provided new opportunities to increase worker safety and reduce toxic waste in DH production, and further provided a proof of concept for genetic improvement of spontaneous chromosome doubling, which has great prospects for increasing the efficiency of DH production in maize.