Discrimination of haploid and diploid maize kernels via multispectral imaging

The use of doubled haploids (DHs) in maize has become ubiquitous in maize breeding programmes as it allows breeders to go from cross to evaluation in as little as 2 years. Two important aspects of the in vivo DH system used in maize are as follows: (i) the identification of haploid progeny and (ii) doubling of the haploid genome to produce fertile inbred lines. This study is focused on the first step. Currently, identification of maize haploid progeny is performed manually using the R1‐nj seed colour marker. This is a labour‐intensive and time‐consuming process; a method for automated sorting of haploids would increase the efficiency of DH line development. In this study, six inbred lines were crossed with the maternal haploid inducer ‘RWS/RWK‐76’ and a sample of seed was sorted manually for each line. Using the VideometerLab 3 system, spectral imaging techniques were applied to discriminate between haploids and hybrids. Using DNA markers to confirm the haploid/diploid state of the tested seed, for the majority of genotypes haploid identification was possible with over 50% accuracy.     

Chromosome doubling of haploid maize seedlings using nitrous oxide gas at the flower primordial stage

In maize, inbred lines are used for the production of hybrid varieties. Corn breeders and researchers have considered using haploids to develop inbred lines; however, this procedure has not been practically applied because of the inefficiency of chromosome doubling of maize haploid seedlings. In this report, a procedure has been developed to overcome this difficulty. Maize haploid seedlings obtained from eight different genotypes were treated with nitrous oxide gas (2 days at 600 kPa). Treatment at the six‐leaf stage (flower primordia formation stage) significantly increased the occurrence of fertile sectors on both tassels and ears so that approximately half (44%) of the treated haploids produced kernels after self‐pollination. In the control, only 11% of haploids produced selfed kernels owing to spontaneous chromosome doubling. A strong genotypic effect on the occurrence of fertile sectors after the treatment was observed. This procedure can be used for inbred line development in maize breeding programmes.     

Molecular identification of Pm12-carrying introgression lines in wheat using genomic and EST-SSR markers

The traditional process of obtaining maize hybrids involves the generation of inbred lines through successive generations of selfing and subsequent testcrosses in order to identify the best combining ability by allelic complementation. A fast alternative to obtain inbred lines is to induce the formation of haploids followed by chromosome doubling. However, even with the aid of haploid-inducing genetic sources, this strategy has not been widely used in maize breeding programs, partly due to difficulties inherent to haploid generation and identification. In order to evaluate the possibility of using dihaploids to generate homozygous maize tropical lines, we used the androgenetic haploid inducer line W23 as a female parent in crosses with the tropical single-cross hybrid BRS1010. Within the progeny of these crosses, 462 seeds were phenotypically selected as putative haploids by the purple-colored endosperm and colorless embryo conditioned by the R1-nj gene. Among these, only four individuals were confirmed as being haploids using SSR markers, chromosome counting and flow cytometry, showing that the phenotypic marker was not efficient in detecting haploids in the tropical maize genotype used. All four haploids as well as some diploid plants presented reduced size, corroborating the difficulties for haploid identification by phenotypic evaluation. Genetic diversity analysis revealed by SSR markers divided the haploids in two groups represented by flint and dent maize inbred lines, which could be helpful in identifying complementary dihaploid lines. The present article demonstrates that a combination of haploid production and SSR fingerprinting is a feasible strategy for maize hybrid development in tropical germplasm.     

Maternal induced haploids in rye (Secale cereale L.)

Summary An inbred line of rye named P8 was found to possess haploid plants in their diploid stock. Phenomenon of spontaneous haploidy was examined in this line. Fifty families derived from this inbred line were screened to determine their ploidy level by mitotic chromosome count taking root tips. Seven families were found to contain haploid plants. Among these seven families, plants from two families which were diploid were crossed reciprocally with the standard out breeding cultivar Conrah. As all of the tested plants of P8 were found aa allele for 6-Phosphogluconic dehydrogenase (6-PGD) isozyme, therefore, only those Conrah plants which have bb alleles for the same isozyme were used in crossing programme.F1 seedlings were screened with the same isozyme marker and out of 1741 F1 seedlings tested, 51 were found to be non-hybrid phenotype for 6-PGD enzyme. Among these, only five plants were found haploid and the rest were diploid. Haploid plants were obtained only from those crosses where P8 was female which indicates that haploids in this line was maternally induced.     

Haploid differentiation in maize kernels based on fluorescence imaging

A new fluorescence‐based method for inbred haploid differentiation in maize kernels was developed by utilizing the R1‐nj colour marker in combination with fluorescence microspectroscopy and imaging. Seven inbred lines with varying R1‐nj expression were used in this study. The fluorescence response of the diploid kernels at the embryonic dye spot was shown to simultaneously exhibit lower intensity and occur at a higher wavelength than the fluorescence of the dye‐lacking haploid embryos. Intensity and area thresholds were applied to fluorescence images to sort the haploids from mixed sample populations, and sorting efficiencies of greater than 80% were achieved in all seven inbred lines (with values greater than 90% for five lines). The potential for high‐throughput sorting when fluorescence imaging is combined with existing technologies for seed handling as well as high sorting efficiency may make fluorescence a viable and promising alternative to current sorting methods for some inbred lines.     

Regular segregation of four recessive marker genes among maternal haploids in maize

The objective of the study was to investigate whether a population of maternal haploid plants represents a random gametic array. Four inbred lines of maize (A619, MK01, 092 and 19‐3‐3) were used in the present study. These were crossed with line TO carrying four recessive genes: brown midrib (bm2), liguleless (Ig1), white endosperm (g1) and golden plant (g1). Maternal haploids were produced from the F ¹ hybrids, using a haploid‐inducing pollinator line. Segregation of haploids for the marker genes was estimated under field conditions. The observed segregation data agreed with the expected 1:1 ratio. It is concluded that no segregation distortion occurred during the production of maternal haploids.     

Vigor and variation expressed by anther-derived doubled haploids of burley tobacco (Nicotiana tabacum L.). I. Comparison of sexual and doubled-haploid populations

Summary The genetic consequences of anther culture and chromosome-doubling techniques on burley tobacco (Nicotiana tabacum L.) were examined in this study. Three diploid populations, obtained from a burley tobacco inbred by conventional and anther-culture techniques, were compared. The first population consisted of 50 conventionally-selfed lines; the second population was made up of 35 doubled-haploid lines obtained from individual haploid plants by in vitro techniques (IVDH); and the third population consisted of 20 doubled-haploid lines whose chromosome complements had been doubled with colchicine (CDH). Comparisons of doubled-haploid lines with sexually-derived lines revealed significant differences for yield, maturity, leaf length, and alkaloid content. Yield reductions in the doubled-haploid populations averaged 8.5%. Significant differences observed between the IVDH and CDH populations indicate that the reported deleterious effects of colchicine contributed to the vigor reduction of doubled haploids. The anther derived lines in this study exhibited greater variation than did the sexual materials. This variation could provide useful variation for a breeding program. Variation exhibited by the sexual progeny of the highly inbred line, Kentucky 16, suggests that the differences among anther-derived materials are at least partially due to natural phenomena.Contribution from the department of Agronomy, University of Kentucky, Lexington, KY 40546-0091. This research was supported, in part, by a grant from R. J. Reynolds Tobacco Co. This paper (no 85-3-5) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.     

Culture of unpollinated ovules,ovaries, and flower buds in some species of the genus Allium and haploid induction via gynogenesis in onion (Allium cepa L.)

Summary Induction of haploid plants is of great importance for breeding purposes because of the possibility to obtain from haploids homozygous material by artificial chromosome doubling in relatively short times. The present study reports the first evidence of successfull haploid induction in onion. Isolated ovules, ovaries, or whole flower buds of different Allium species were cultured on BDS agar medium. Testa browning in the ovules and an extensive growth of the latter were observed. In cultures of ovaries and flower buds, development of callus and subsequent regeneration of plantlets from the region of the nectaries were observed. In leek, sometimes supernumerary flower organs like ovules were formed in this callus. In onion (Allium cepa L.), plantlets developed from the ovules in all culture methods. Chromosome numbers of these plantlets were counted in root tip squash preparations. They were found to be haploid. Haploid plants were significantly smaller than diploid ones. They were transferred to soil and developed until bulb formation. Because of their importance for breeding, haploid plants obtained by gynogenesis are further stored in vitro.     

Origin of haploids and semigamy in Theobroma cacao L.

Summary The origin of haploid plants of cacao was studied using morphological and enzyme markers. Analysis of 250 haploids arising either from controlled crosses or from open pollination in a biclonal seed-garden showed the existence of several types. They could be either or in origin, or chimaeric haploids or diploid/haploid chimaeras. In the majority of cases these chimaeras, visible at the cotyledon stage, were not maintained at the level of foliage, where only one genotype was observed. Studies of the haploid/diploid chimaeras show that the diploid part could be heterozygous and thus correspond to the fusion of the two parental gametes occurring after gametic cell divisions have produced haploid tissue. This phenomenon of semigamy could be facilitated in cacao by its particular system of self-incompatibility.     

Classification approaches for sorting maize (Zea mays subsp. mays) haploids using single-kernel near-infrared spectroscopy

Doubled haploids (DHs) are an important breeding tool for creating maize inbred lines. One bottleneck in the DH process is the manual separation of haploids from among the much larger pool of hybrid siblings in a haploid induction cross. Here, we demonstrate the ability of single-kernel near-infrared reflectance spectroscopy (skNIR) to identify haploid kernels. The skNIR is a high-throughput device that acquires an NIR spectrum to predict individual kernel traits. We collected skNIR data from haploid and hybrid kernels in 15 haploid induction crosses and found significant differences in multiple traits such as percent oil, seed weight, or volume, within each cross. The two kernel classes were separated by their NIR profile using Partial Least Squares Linear Discriminant Analysis (PLS-LDA). A general classification model, in which all induction crosses were used in the discrimination model, and a specific model, in which only kernels within a specific induction cross, were compared. Specific models outperformed the general model and were able to enrich a haploid selection pool to above 50% haploids. Applications for the instrument are discussed.     

Effect of pollinator on haploid production in durum wheat crossed with maize and pearl millet

The aim of this work was to analyse the influence of the male parent on the production of embryos and haploid plants in durum wheat crossed with maize and pearl millet, to find a proper trait to identify the most efficient pollinators and to evaluate the mixtures of pollen. Two genotypes of durum wheat, low and high responding, were crossed with eight pollen samples: (i) three maize hybrids, (ii) three pearl millet inbred lines, (iii) a mixture of maize pollen and (iv) another mixture of pearl millet pollen. No significant differences on embryos and haploid plant production were observed among the four samples of maize pollen, but there were clear genotypic differences for the production of haploids between genotypes of pearl millet. The best pearl millet genotype produced significantly more haploid plants than the other two and the mixture of pollen. There was no correlation between the production of embryos and haploid plants. Therefore, the production of haploid plants must be the criterion to identify superior pollinators. In addition, a mixture of pollen is inappropriate except when using genotypes previously identified as good pollinators.     

玉米单倍体加倍(DH)系籽粒性状表现的研究

玉米单倍体育种是快速选育纯合自交系的方法.研究利用单倍体诱导系,对来自BSSS杂种优势群的两个自交系BX08和BX19的杂交F1进行诱导获得单倍体,又经加倍获得33个加倍纯合系(DH),分析单倍体加倍纯合系与两个亲本及F1杂交种的果穗及籽粒性状表现.结果表明,单倍体加倍系的籽粒容积和质量比亲本籽粒都有所提高,子代的农艺性状获得显著改善,说明发生单倍体不是基因随机分离的结果,单倍体育种存在选择压力,有利于优良基因的累积,淘汰不良基因.     

Genetic and environmental effects on chromosome doubling of sugarbeet (Beta vulgaris L.) haploids

Summary Chromosome doubling has been a limiting factor for production of doubled-haploids, a means of obtaining fruly homozygous individuals, and a time-saving alternative to repeated selfing for the creation of inbred lines. The existence of genetic, environmental and genotype × environment interaction effects on chromosome doubling ability of sugarbeet (Beta vulgaris L.) haploids was investigated. Haploids were derived from four distinct, highly heterozygous diploid populations through in vitro culture of unpollinated ovules. Ovule-derived plants were treated with colchicine to double their chromosome complement. Environmental effects were determined from replication of the experiment at seven dates. Both genetic and environmental factors were found to affect chromosome doubling ability of sugarbeet haploids. No significant interaction between genotype and environment was identified. The presence of genetic effects on chromosome doubling ability is discussed with regard to its implications on breeding programs.     

Low relative humidity increases haploid production in durum wheat X maize crosses

Haploidization is a useful tool for genetic analysis and plant breeding, but a consistent and satisfactory protocol for haploid production has been difficult to achieve in durum wheat. The objective of this study was to analyze the influence of the relative humidity of the environment, when culturing detached tillers during the production of haploids plants in durum wheat by the maize method. Thirty‐eight F3 lines from eight crosses of durum wheat were pollinated with bulked pollen from three commercial maize hybrids. A mixture of 2‐4D and dicamba was used as a hormone treatment. The numbers of caryopses, embryos and haploids plants were scored. When 65‐85% (light‐dark) humidity was substituted for 55‐65% the number of haploids per spike increased notably. This increased frequency was largely attributed to a rise in the production of generated caryopses. On average, 15.2 vs. 9.3 caryopses, 5.0 vs. 2.8 embryos, and 3.1 vs. 0.6 haploid plants, per spike, were produced under low and high humidity regimes, respectively.     

Main Factors Affecting Cucumber (Cucumis sativus L.) Haploid Embryo Development and Haploid Plant Characteristics

This report gives the first detailed analysis of cucumber haploid plants and the first evaluation of genotype and season effects on haploid embryo development. Female flowers of four F1 varieties and two inbred lines were pollinated with 300 Gy-treated pollen and three-week old embryos were cultured in vitro on E20A medium. The variety ‘Polan’ produced the greatest number of embryos (1.34 per 100 seeds), 51 % of which were capable of further development. The number of embryos able to develop was four times higher in summer than in spring. All the plants obtained, except one, were haploids or aneuhaploids. Haploid plants grew slowly and were sterile, although they occasionally set seedless fruits. The length of the stems, leaves and guard cells was smaller than those of diploids.     

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.     

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.     

Somatic embryogenesis from floral tissue of cassava (Manihot esculenta Crantz)

The aim of this study was to examine the embryogenic potential of floral material of the cassava cultivar MCOL 1505. Macerated immature inflorescences were found to be highly embryogenic, with almost 78% of the explants producing somatic embryos. Somatic embryos were also produced from whole male florets and half florets although at much lower rates. No regeneration was obtained from anther, microspore or floret wall tissue. Somatic embryos derived from immature inflorescences were regenerated via organogenesis and the plants derived from this process were assessed in terms of phenotype and ploidy level. If haploid plants could be produced by this method, this would have significant implications in assisting traditional cassava breeding, as this would allow homozygosity to be reached more rapidly. In a crop such as cassava, which is highly heterozygous in nature, the use of haploids in a breeding programme could considerably shorten the time taken to produce new desirable cultivars. This is the first report on plant regeneration through somatic embryogenesis from floral tissue of cassava. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Inducer line generated double haploid seeds for combined waxy and opaque 2 grain quality in subtropical maize (<Emphasis Type="Italic">Zea mays.</Emphasis> L.)

The implementation of modern inducer lines in maize breeding can substantially decrease the time required to create elite inbred lines. In industrialized countries, this method has already largely replaced conventional backcross methods. However, the application of in vivo gynogenesis for inducing doubled haploids is still limited to European and US maize germplasms and has still to be adapted for exotic plant material. The reliability of three modern European inducer lines from the University of Hohenheim (Germany) was investigated for the production of haploid progenies from subtropical specialty maize. Three Chinese hybrids heterozygous for waxy maize and opaque 2 were used as maternal donor material, as maize double recessive for waxy and opaque 2 may improve the nutrition of ethnic minorities in Southeast Asia. However, many false positives were detected by flow cytometry among putative haploid seeds based on anthocyanin pigmentation because the color expression was inhibited in almost 50% of the induced seeds from this maternal plant material. Based on flow cytometry, the haploid induction rates were high with 10.2–12.3%, and the chromosome doubling rates were around 50%; therefore the principal potential of producing DH was confirmed for subtropical maize. However inducer lines for the precise and fast recognition of truly induced haploid seeds still need to be developed.