Maize Doubled Haploid Research Articles

Improving in vivo maize doubled haploid production efficiency through early detection of false positives

With 1 figure and 2 tablesAbstractIn vivo doubled haploid (DH) technology provides a means of creating new maize inbred lines relatively quickly; however, productivity is limited by false‐positive (FP) plants for haploidy and for dihaploidy, which consume resources of space and labour until detected. This work examines the potential for using stomata guard cell length measurement as a means for early detection of FP plants. We found that the true haploid and DH plants could be differentiated from FP and untreated diploid controls as early as Leaf 2 stage by stomata guard cell length measurement. Furthermore, DH plants were distinguishable from haploid and other diploid plants by the Leaf 7 growth stage. Results suggest that, when used together with screening through the anthocyanin colour marker system and flower fertility, stomata guard cell measurement is an easy, non‐destructive, early screening method that may lead to a greater efficiency in DH production systems and optimization of resource allocation for space and labour.

Variation and covariation for Gibberella ear rot resistance and agronomic traits in testcrosses of doubled haploid maize lines

Gibberella ear rot (GER) of maize caused by Fusarium graminearum reduces grain yield and leads to contamination of the grains with deoxynivalenol (DON), a mycotoxin that adversely affects the health of humans and animals. The objectives of this study were to (1) analyze means and genotypic variances for line per se performance (LP) and testcross performance (TP) of doubled haploid (DH) lines for GER severity and DON concentration as well as for some agronomic traits, (2) examine correlations among these traits, (3) validate QTL for resistance detected in previous studies for LP and their effect on TP and (4) investigate the relative efficiency of indirect selection (RE) for LP to improve TP. Testcross progenies of 94 DH lines originating from four flint populations were developed using a susceptible dent tester as pollinator. Artificial inoculations with F. graminearum led to appreciable disease development. Average TP for GER severity and DON concentration were lower than the mean mid-parent values of the tester and DH lines, indicating mid-parent heterosis for resistance. Genotypic variation for resistance was significant for LP and TP. Genotypic correlations between LP and TP were low and resistance QTL for LP had no significant effects on TP. Accordingly, RE for resistance was low, suggesting to allocate resources mostly to the evaluation of testcrosses. Correlations of resistance to GER and DON contamination with grain yield (measured under non-inoculated conditions) were not significant, indicating that selection for resistance and higher grain yield can be carried out simultaneously.

In vitro selection of microspores for resistance to oxidative stress resulted in chilling tolerance in doubled haploid maize lines

The chilling tolerance of doubled haploid (DH) maize plants selected and regenerated from microspores exposed to prooxidants, paraquat or tert-butyl hydroperoxide was determined by monitoring cold-induced changes in the photosynthetic electron transport, CO2 assimilation processes and chlorophyll breakdown in young leaves after cold treatment (8°C for 5 days). The results were compared to those of the non-selected DH line and the original hybrid plants. Chilling stress caused a great reduction in the Fv/Fm, qP and ΔF/Fm’ fluorescence parameters, related to the photosynthetic electron transport processes, and in carbon assimilation, and resulted in chlorophyll breakdown. These changes were less extensive in the selected DH plants, which showed elevated antioxidant capacity both at ambient and at low temperature. Among the antioxidant enzymes tested, the activity of GR and GST was induced by chilling stress to the greatest extent. Correlations between cold-induced changes in the photosynthetic apparatus and the antioxidant capacity of the plants suggested that the better protection against oxidative stress induced by the elevated antioxidant capacity of the plants contributed to protecting the photosynthetic apparatus from cold.

Improved Cold and Drought Tolerance of Doubled Haploid Maize Plants Selected for Resistance to Prooxidant tert-Butyl Hydroperoxide

To improve the abiotic stress tolerance of maize (Zea mays L.), doubled haploid (DH) plants were produced by in vitro selection of microspores exposed to tert-butyl hydroperoxide (t-BuOOH) as a powerful prooxidant This study investigated the tolerance of the progenies of t-BuOOH-selected DH lines to oxidative stress, cold and drought in controlled environment pot experiments by analyses of photosynthetic electron transport and CO2 assimilation processes, chlorophyll bleaching and lipid peroxidation of leaves. Our results demonstrated that the t-BuOOH-selected DH plants exhibited enhanced tolerance not only to oxidative stress-induced by t-BuOOH but also to cold and drought stresses. In addition, they showed elevated activities of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, catalase, glutathione reductase and glutathione S-transferase when compared with the DH lines derived from microspores that were not exposed to t-BuOOH and to the original hybrid plants. The results suggest that the simultaneous up-regulation of several antioxidant enzymes may contribute to the oxidative and cold stress tolerance of the t-BuOOH-selected DH lines, and that the in vitro microspore selection represents a potential way to improve abiotic stress tolerance in maize.

General and specific combining ability of in vitro doubled haploid maize lines in the field

The breeding of hybrid maize now has a history of over 100 years. In 1908, George H. Shull was the first to report on the high yields, great uniformity and homogeneity of hybrids derived from a cross between two inbred lines. Following this discovery, consistent self-fertilisation over a period of six to eight generations was found to be an extremely efficient method for developing maize lines. From the mid-1970s, however, with the elaboration of the monoploid ( in vivo ) and microspore culture ( in vitro ) techniques, it became possible to develop homozygous lines within a year.With the help of an efficient plant regeneration system based on anther culture, large numbers of doubled haploid (DH) lines can be produced. In the course of the experiments the seed of DH plants selected over several years was multiplied and crossed with Martonvásár testers, after which the hybrids were included in field performance trials in three consecutive years (2005–2007). The aim was to determine whether the field performance of hybrids developed in this way equalled the mean yield of standards with commercial value. The data also made it possible to calculate the general (GCA) and specific (SCA) combining ability of the parental lines, indicating the usefulness of the parental components in hybrid combinations and expressing the extent to which a given line contributes to yield surpluses in its progeny.A total of 52 maize hybrids were evaluated in the experiments in terms of yield and grain moisture content at harvest. The combinations, resulting from crosses between 12 DH lines, one control line developed by conventional inbreeding and four testers, were found to include hybrids capable of equalling the performance of the standards, and four DH lines were identified as improving the yield level of their progeny. As the experiment was carried out on a very small number of genotypes, the results are extremely promising and suggest that, if the range of genotypes used to develop DH lines is broadened and the sample number is increased, it will be possible in the future to find maize hybrids, developed with in vitro DH parental components, that surpass the performance of commercial hybrids.

Enhanced Tolerance to Oxidative Stress with Elevated Antioxidant Capacity in Doubled Haploid Maize Derived from Microspores Exposed to Paraquat

To improve the oxidative stress tolerance of maize (Zea mays L.), doubled‐haploid (DH) plants were regenerated from in vitro selected microspores exposed to paraquat. The efficiency of in vitro selection was tested by physiological and biochemical investigations, in which the tolerance to paraquat‐mediated oxidative damage and antioxidant capacity of the second generation of fertile microspore‐selected lines (DH2) were determined. In the leaves of paraquat‐selected DH2 lines, reduced accumulation of reactive oxygen species, lower rates of chlorophyll bleaching, protein breakdown and membrane damage, but higher photosynthetic activity were detected on exposure to paraquat stress than either in the leaves of nonselected DH2 lines or in the original hybrid used as controls. The tolerant DH2 plants showed elevated activities of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase, glutathione S‐transferase, and catalase as compared to both controls. These results suggest that elevated antioxidant capacity may contribute to the paraquat tolerance of the paraquat‐selected DH2 lines and that in vitro microspore selection represents a potential way to improve oxidative stress tolerance in maize.

Combining ability of doubled haploid maize parental lines

Combining ability of doubled haploid maize parental lines

Field performance of hybrids developed from doubled haploid maize inbred lines

Field performance of hybrids developed from doubled haploid maize inbred lines

Rapid attainment of a doubled haploid line from transgenic maize (Zea mays L.) plants by means of anther culture

We present a strategy for establishing a transgenic doubled haploid maize line from heterozygous transgenic material by means of anther culture. Compared to conventional inbreeding, the in vitro androgenesis technique enables a faster generation of virtually fully homozygous lines. Since the androgenic response is highly genotype-dependent, we crossed transgenic, non-androgenic plants carrying a herbicide resistance marker gene (pat, encoding for phosphinothricin acetyl transferase) with a highly androgenic genotype. The transgenic progenies were used as donor plants for anther culture. One transgenic and three non-transgenic doubled haploid lines have been established within approximately 1 yr. The homozygosity of all four doubled haploid lines was tested by analysis of simple sequence repeat (SSR) markers at 19 different loci. Polymorphisms were found between the lines but not within the lines indicating the homozygous nature of the entire plant genome gained by anther culture. Southern blot analysis revealed that the transgenic donor plants and their doubled haploid progeny exhibited the same integration pattern of the pat gene. No segregation of the herbicide resistance trait has been observed among the progeny of the transgenic doubled haploid line.