On the number and size of cross combinations in a breeding programme of self-fertilizing crops

Summary The number and size of crosses in a breeding programme were discussed to the conclusion that the number of crosses rather than the size of a cross should be increased with a given total population size. The advantage due to this manipulation, however, is relatively small when such genotypes as improved for many (twenty-five or more) loci are to be obtained, or when a high proportion (around 0.1 or more) of available combinations have the potentiality of releasing the objective genotypes. In such a case, the ease with which crossing and cultivation can be carried out becomes the decisive factor.     

Production of salt tolerant rice breeding line via doubled haploid

Summary A haploid breeding program was initiated to develop doubled haploid salt tolerant rice breeding line via anther culture. Two sensitive breeding lines BR4608-R₁-R₂ and BR4909-R₁-R₂ were crossed with a salt tolerant line IR13146-13-3-3 to transfer its salt tolerant character to the doubled haploids.Anther from confirmed F₁s of the two crosses were cultured in defined medium for callus induction and eventual plant regeneration. Fifteen doubled haploid (DH) lines were obtained from two crosses. Test for salt tolerance were done in vitro. Five out of 15 lines were found tolerant at the level of 8–10 decisiemens/m (ds/m) while the rests were sensitive to that level of salinity.Field experiment was conducted to evaluate the doubled haploids under saline and non saline soil. Five salt tolerant lines produced comparable yield with the resistant control (BR 23) under saline condition, whereas these lines yielded even higher in non saline soil under irrigated condition when evaluated with other 10 sensitive DH linesAbbreviations LSD Least Significant Difference - NAA Napthalene Acetic Acid     

A comparison of bulk testing and direct-line testing in self-fertilizing crops

Summary Computer simulation techniques were used to compare the efficiency of bulk-yield testing with direct-line yield testing. Bulk testing was almost always more efficient than direct-line testing for a wide range of values for population parameters. The differences between the two methods were smaller when the among-bulk heritability was low, the within-bulk genetic variance was high relative to the among-bulk variance, the variance of the within-bulk genetic variance was high, and the correlation between bulk means and within-bulk genetic variances was highly negative.With the direct-line testing method, discarding lines with low means after the first year of testing was always more efficient than retaining all lines for 2 years. Selection of 25% of the lines was slightly more efficient than selection of 50%. In general, it was better to evaluate more crosses and less lines per cross with the direct-line testing procedure.     

Segregation distortion for agronomic traits in doubled haploid lines of barley

Four barley doubled haploid populations were produced by anther culture from the reciprocal crosses between two six‐row barley cultivars, ‘Plaisant’ and ‘Orria’; the doubled haploid lines (DHLs) derived from each cross were subsequently assigned to weak or vigorous populations according to the weak or vigorous nature of the originating embryos. Well‐formed embryos at day 25 on the induction medium were considered vigorous, whereas embryos maturing later were considered weak. The classification of vigorous and weak was closely associated with the ratio of green to albino plantlets regenerated. A random set of 25 DHLs from each of the four populations were selected for field testing in a replicated trial. Furthermore, a second set consisting of a total of 454 unreplicated DHLs from the four populations were also field assessed for grain yield. Distortion during in vitro culture may impede regeneration of a random array of microspores from a given cross, and may bias genetic estimates of specific trait/marker association in genetic studies. However, no significant differences were detected in this study among the four populations for days to heading, height, grain yield and thousand‐kernel‐weight when measured on the replicated trial of 100 DHLs, nor for grain yield in the second collection of 454 entries. This suggests that the likelihood of producing improved agronomic pure lines is independent of the direction of crossing and, more importantly, independent of the time when embryos matured in the induction media, at least for these particular six‐row cultivars and for the anther culture method used.     

Weighing in on a method to discriminate maize haploid from hybrid seed

The doubled haploid breeding method can produce maize inbred lines faster than traditional methods, but there are challenges associated with it. Sorting haploid from hybrid seed based on visual colour markers is time consuming and can be difficult due to colour inhibitors that obscure pigmentation needed to distinguish between haploid, hybrid and outcrossed seed. In this study, weight was evaluated as a method to sort haploid from hybrid seed. A first experiment utilized two families for analysis in a preliminary study. Eleven haploid and hybrid kernels from both families were weighed for a total of 44 experimental units. A second experiment was carried out using six families, using the same format as the previous, for 132 experimental units. Hybrid seed weighed significantly more than haploid seed in both experiments. However, the interaction between line and kernel type was significant in the second experiment. In conclusion, efficacy of sorting haploid from hybrid kernels based on weight depends on the genotypes involved.     

Early diagnosis of ploidy status in doubled haploid production of maize by stomata length and flow cytometry measurements

Reliable discrimination of haploid (H), doubled haploid (DH) and crossing (C) plants in early growth stages could streamline DH production in maize. By detecting in early growth stages undesirable sterile H plants and undesirable heterozygous C plants, a large proportion of resources required for DH production could be saved. The goal of this study was to evaluate the effectiveness of early classification of plants in growth stages V3‐V4 as H, DH or C in the context of DH production using flow cytometry and stomata length. As the reference classification, we used a field score based on plant phenotype commonly applied in DH production and research. Our results show that identification of misclassified C seeds is possible because the overlap in distributions of stomata length between H&DH and C plants is small and the association between flow cytometry and the reference field score is high. In contrast, overlap between H and DH distributions is substantial. Consequently, the main application we see for these classification methods in early growth stages is the identification of C seedlings.     

Efficient haploid and doubled haploid production from unfertilized ovule culture of gentians (Gentiana spp.)

Factors affecting reliable plant regeneration from unfertilized ovule culture of gentians (Gentiana spp.) were examined. Cold pretreatment (4°C) of flower buds enhanced or maintained production of embryo-like structure (ELS). When 43 genotypes were surveyed in two different labs, 40 of them produced ELSs ranging from 0.01 to 26.5 ELSs per flower bud. No ELSs could be obtained in three genotypes. A significant correlation (r = 0.64) was observed between the number of ELS per flower and the frequency of responding flower buds. Eight genotypes of G. triflora, which were used as common materials in two different labs, produced ELSs in both labs. The ploidy levels of a total of 1,515 regenerated plantlets were determined, revealing that the majority of these plants consisted of haploids (57.9%) and diploids (34.3%). However, the frequency of haploids and diploids was different between G. triflora and G. scabra, and G. triflora showed higher frequencies of haploids than G. scabra. When haploids were treated with oryzalin for chromosome doubling, diploids and tetraploids were obtained. These results demonstrate that the unfertilized ovule culture technique of gentians is a powerful tool for obtaining haploids and DHs because of its reproducible and reliable nature and application to a wide range of genotypes.     

Dicamba and growth condition effects on doubled haploid production in durum wheat crossed with maize

Doubled haploid plants are useful in genetic studies and plant breeding, but a consistent and satisfactory frequency of production has been difficult to achieve in durum wheat. Triticum turgidum L., using the maize pollen method. The objective of this study was to develop an objective method of producing doubled haploids in durum wheat. Plant growing and handling conditions, aspects of hormone treatments, wheat genotype and pollen source were considered. The number of caryopses, embryos, haploids, doubled plants and doubled plants that set seed were measured. Although growth conditions, pollen source, method of handling plants and wheat genotype are important considerations, the type of hormone was found to be most significant in the production of doubled haploid plants. When 50mg/l dicamba was substituted for 100 mg/l 2,4‐D the number of doubled haploids per spike increased from 0.2 for the best 2,4‐D treatment to 1.3 for the dicamba treatment. This increased frequency was largely attributed to an increase in the number of caryopses generated for each spike emasculated and from an increased frequency of germination of embryos to haploid plantlets. The best production of caryopses was 0.41 caryopses per florest with 2,4‐D. The best production of haploids per 100 florets was 12 with dicamba and 1.65 with 2,4‐D. The frequency of one doubled haploid per emasculated spike through the use of dicamba is a practical level for generating populations for genetic studies.     

Difference between USA and French isolates of tobacco etch virus and pepper mottle virus displayed by doubled haploid line analysis

Summary Doubled haploid peppers resistant in France to specific strains of tobacco etch virus (TEV) and pepper mottle virus (PeMV) were indexed for resistance to USA strains of TEV and PeMV. The doubled haploids were inoculated when four to six true leaves had developed. The peppers were indexed after an incubation period, and a disease index was calculated. Plants without viral symptoms were reinoculated. Plants challenged to TEV were inoculated four times. Doubled haploids, TEV-resistant in France, were susceptible to the USA strain by the fourth screening. All doubled haploid lines showed TEV symptoms after the fourth inoculation. Two doubled haploids lines, no. 4 and no. 8, had about 6% resistance to TEV after the fourth inoculation. Doubled haploid line no. 5 was 100% resistant to PeMV. The results confirm that the USA PeMV and the USA TEV isolates are different pathotypes than the French ones used originally to screen the doubled haploids. In addition, the use of doubled haploids to identify pathotypes of PeMV and TEV is demonstrated.     

How to develop from three parents a new variety of a self-fertilizing crop?

Summary The aim is to combine the genes for attractive levels of expression for three different characters. These genes occur in three different lines of an autogamous crop. The 12 breeding procedures evaluated comprise each combination of three different crossing programs with selection among the obtained F2 plants (or not) and with random mating among the (selected) plants (or not). For independent segregating loci random mating has no effect or only an unrewarding small effect, depending on the crossing program.In case of selection the crossing programs differing from the conventional scheme for a three-way cross deserve attention. With selection in F2 the frequency of plants with the desired genotype is increased by 20% with different crossing programs.     

Rye doubled haploids as a research and breeding tool – a practical point of view

Doubled haploid (DH) plants were produced using anther culture from out‐crossing rye, including breeders’ lines, cultivars and F₁ plants with DH parents, to examine the feasibility of using the DH technique for breeding and specifically for developing mapping populations. Only 10–36% of green regenerants produced via anther culture were suitable for research or breeding purposes because of low survival rate or low fertility. Spontaneously arising DH regenerants were more often fertile compared with the colchicine‐treated ones. The fertility of spontaneous DHs varied from sterile to half that found in a normal rye population, which has implications for the design of a crossing scheme and subsequent anther culture. In the reciprocal crosses within one DH population, fertility was the lowest observed, probably because of self‐incompatibility factors, whereas in the DH crosses with normal heterozygous cultivars fertility was the highest. Two mapping populations using DHs were established, the first for out‐crossing rye it would seem. These populations will be used for mapping two important traits, the semi‐dwarf growth habit and preharvest sprouting resistance in rye.     

Potential use of doubled haploid lines for the screening of resistance to yellow rust (Puccinia striiformis) in hexaploid wheat

Doubled haploid lines derived from anther culture of two Iranian spring wheat genotypes‘Ghods’susceptible and‘9106’resistant to yellow rust in Iranian field conditions, and their F₁ hybrids were used in this study. Seedlings of 36 doubled haploid lines, selected out of 96 according to their agronomic traits and the two parental genotypes were inoculated with eight races of yellow rust. The parental genotypes (‘Ghods’and‘9106’) were segregating for some of the races but their doubled haploid lines were either resistant or susceptible to them.‘Ghods’was susceptible to three of the races studied but three doubled haploid lines derived from it were resistant to them. Five selected doubled haploids from the‘9106’genotype and six from F₁ hybrid plants were resistant to all eight races tested. After further investigations in Iranian field conditions it was found that some of these lines can be used as donor genotypes for resistance to yellow rust in wheat breeding programmes. Use of these genotypes should be possible if the French yellow rust races used for selection also represent the dominant races in Iran. It can be concluded that anther culture provides an efficient method for fixing genes of resistance to yellow rust and desirable doubled haploids from F₁ plants can be derived.     

Review of doubled haploid production in durum and common wheat through wheat × maize hybridization

Production of doubled haploids (DHs) is an important methodology to speed the process of breeding and development of mapping populations in crops. The procedure for DH production includes two major steps: haploid induction and chromosome doubling. In recent years, wide hybridization between wheat and maize has become a main approach for haploid production in wheat. In this method, the maize chromosomes are completely eliminated during the early development of the hybrid seeds after wheat spikes were pollinated with maize pollen. Numerous wheat cultivars and mapping populations have been developed using wheat–maize hybridization. In this study, we review the procedures of DH production of durum and common wheat via wide hybridization with maize, the factors which affect the efficiency of DH production, and the mechanism of selective elimination of the maize genome during the early development of the hybrid embryos. We also report a highly efficient protocol for DH production in durum and common wheat, which was established based on the optimal conditions for each of the factors that affect the efficiency of DH production.     

Analysis of QTL responsible for grain iron and zinc content in doubled haploid lines of rice (Oryza sativa) derived from an intra-japonica cross

Quantitative trait loci (QTL) related to the grain iron and zinc contents of brown rice were mapped by using a doubled haploid population derived from an intra-japonica cross between 'Hwaseonchal' and 'Goami 2'. QTL–QTL, background–QTL, and background–background interactions and candidate genes that affect grain iron and zinc contents were preliminarily identified. Twenty-one iron- and zinc-related QTL were found. The major-effect QTL qFe7 and qZn7 provided the highest contribution to phenotypic variance for grain iron and zinc contents. The colocation of zinc- and iron-related QTL on chromosomes 1, 4, 7 and 11 may account for the strong correlation between iron and zinc contents. A region on chromosome 7 and epistatic interaction between loci on chromosomes 2 and 10 affected iron content. qZn7 and qZn11.3 exerted additive effects on zinc content. Eleven iron- and zinc-related candidate genes colocated with qFe7, qZn7 and the region on chromosome 7 with an additive effect on iron content. The major-effect QTL identified here may be useful for breeding biofortified rice.     

The effect of spikelet position on the success frequencies of wheat haploid production using the maize cross system

Barbela is an old Portuguese landrace of wheat that is highly genetically heterogeneous. Different Barbela populations when subjected to aluminium stress show variable levels of tolerance. In order to study the inheritance of this character, doubled haploid (DH) lines were developed. These DH were obtained by intergeneric crosses of 14 different lines of Barbela with maize. During this process the efficiency of the technique was evaluated and suggestions for its improvement were obtained. Several parameters were studied in the crosses: % of crossability, % of embryos per florets pollinated and % of embryos per seed set. The different genotypes of Barbela showed significant variation for the parameters analysed. When the reciprocal crosses were analysed, no differences were found, indicating that cytoplasm differences do not influence the parameters of DH production. However, different spikelet positions (lower, middle and upper) gave highly significant differences in all parameters analysed. Highest success frequencies were obtained for pollinated spikelets in the middle of the spike. This can indicate that concentrating on the middle part of the spike can increase the frequency of DHs obtained using inter generic crosses of wheat with maize. This revised version was published online in August 2006 with corrections to the Cover Date.     

Efficient application of in vitro anther culture for different European winter wheat (Triticum aestivum L.) breeding programmes

The efficiency of our anther culture protocol was tested with high‐ and low‐responding genotypes, ‘Svilena’ and ‘Berengar’, and 93 F₁ winter wheat crosses in 2010 and 2011. Based on data for these genotypes, the effect of genotype influenced the number of embryo‐like structures, regenerated plantlets and green plantlets, while the number of albino plantlets was affected by genotype, year and environmental factors. Although genotype also influenced the production of green plantlets from breeding crosses, with green plantlets per 100 anthers ranging from 0.04 to 28.67, the average regeneration rate over all crosses was 5.3 green plantlets/100 anthers, which resulted in a total of 11 416 well‐rooted green plantlets. The survival rate of green plantlets following acclimatization was 97.21% in 2010 and 96.34% in 2011. In this study, the phenomenon of albinism and genotype dependency did not hinder the production of more than five thousand green plantlets each year. In our experiments, anther culture proved to be an efficient method in winter wheat breeding programmes with lower costs than alternative technologies.     

Selection for yield in early generations of self-fertilizing crops

Summary If a breeder is to apply selection to a cross-population of a self-fertilizing crop and wishes to maximize his chances of finding a productive genotype, he will have to introduce yield tests in the F₃ lines. Should he not do so because of his intention to work on many populations simultaneously, his tests would be considerably less accurate and the finding of a favourable combination more or less a matter of mere chance.Bulk breeding will result in genetic drift or numbers of plants too large to manage, or in a combination of both.Application of the Single Seed Descent for quantitative characteristics causes such a genetic drift, that the method is not to be recommended.Multiple crosses (double-crosses) to improve quantitative characteristics require populations of a size, that cannot be realized and/or selected. This type of crosses is more suited for cases in which qualitative characteristics form the breeding goals and when it is impossible to obtain the favourable combination using only two varieties.Genic recessive male sterility may be a means to raise large progenies. In the F₃ it confuses the issue of yield of fruits or seeds, which is highly vulnerable in any case. In the F₂ its significance for finding the proper recombinant is very low.     

High frequency spontaneous production of doubled haploid plants in microspore cultures of Brassica rapa ssp. chinensis

Brassica rapa (syn. Brassica campestris) ssp. chinensis is an important vegetable crop, but it is relatively recalcitrant to microspore culture. One genotype each of B. rapa ssp. chinensis var. communisand var. utilis were used formicrospore culture. Embryo production of3.8–42.4 embryos/bud was obtained. A high rate of plant regeneration directly from microspore-derived embryos without subculture was achieved by an improved protocol involving replacement of culture media and reduction of sucrose concentrations after 48 h of induction,among other modifications. More than 70%of regenerated plants were spontaneous diploids. Some spontaneous tetraploid plants were also obtained from isolated microspores of both genotypes tested. These tetraploids may be directly exploited a snew varieties in a Brassica rapabreeding programme. This revised version was published online in July 2006 with corrections to the Cover Date.     

Segregation of genetic markers among wheat doubled haploid lines derived from wheat x maize crosses

Summary Wheat doubled haploid (DH) lines were produced from the F₁ hybrid, Fukudo-komugi x Oligo Culm, through intergeneric crosses between wheat and maize. F₂ plants and 203 DH lines were analyzed for the segregation of the eight genetic markers, namely, grain proteins, grain esterases, GA-insensitivity and glume traits. The segregation in the F₂ plants fitted to the expected ratios. No deviation was observed among the DH lines, either, except for the glume pubescence. The result indicates the absence of correlation between the markers investigated and the efficiency of embryo formation in the DH lines.