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.     

Production of haploids of potato (Solanum tuberosum subsp. tuberosum) and their identification with electrophoretic analysis

Summary The frequency of haploid production following the interspecific pollination of eight tetraploid potato cultivars (Solanum tuberosum subsp. tuberosum) with Solanum phureja clone 1.22 was investigated. A total of 185 haploids were produced with an overall haploid frequency of 3.9 haploids/100 fruits. The haploid frequency was affected by the genotype of the maternal parent. Atlantic, ND860-2, Superior, Saginaw Gold, Spartan Pearl, Nooksack and Onaway had frequencies of 6.2, 5.1, 4.7, 3.9, 2.3 and 0.7 haploids/100 fruits, respectively. There were 60 and 57 haploids produced from Atlantic and Saginaw Gold, respectively, and no haploids were extracted from fruits of Lemhi Russet. Isozyme analysis and visual examination were performed independently to compare the efficiency of discriminating hybrids from haploids. Approximately 80% of total hybrids could be identified by electrophoretic analysis, while 77% were distinguished through visual examination. Pgm-2 ¹, which is unique in the clone 1.22 and absent from all seed parents, was found to be the most useful locus in hybrid identification and 50% of total hybrids could be distinguished by this allele. With similar rationale, Mdh-1 ¹ allozyme, which was absent in six of the eight parents, identified 37% of total hybrids. A combination of both visual and electrophoretic methods made hybrid identification even more efficient, with an average identification efficiency of 91%. A scheme was proposed to develop a new haploid inducer which would be homozygous for both Pgm-2 ¹ and embryo spot.     

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.     

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.     

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.     

AFLP analysis indicates no introgression of maize DNA in wheat x maize crosses

Amplified fragment length polymorphisms (AFLPs) were used to follow the possible introgression of maize DNA into haploids of wheat as a side‐effect of exploiting wheat x maize hybridization for haploid production. AFLPs were generated with 64 MseI/ EcoRI and 64 MseI/ PstI primer combinations, and the AFLP profiles of haploids were tested against those of maize and of the regular wheat varieties involved in the crosses. On average, 45.1 and 110.7 fragments were produced per assay with the MseI/EcoRI and MseI/PstI combinations, respectively. Different numbers of fragments were produced for wheat and maize: an average of 81 in the haploid, 80 in the wheat parent, and only 67.1 in maize. No evidence was found for introgression of maize into the wheat genome. Three unique AFLP fragments were detected in haploids, which were not present in the parental wheat genotypes. These ‘novel’ AFLP bands in the haploids could be caused by nucleo‐cytoplasmic interaction in the hybrid zygote. Such instability in the wheat genome is defined as temporal, as it was not detected in further generations when colchicine‐doubled progeny of the haploids was tested for the presence of polymorphic fragments.     

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.     

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.     

不同除草剂加倍玉米单倍体的效率

通过比较3种除草剂加倍玉米单倍体的效率,提出了利用除草剂加倍玉米单倍体的新方法。以先玉335、中农大4号和8607×8609三个基因型诱导的单倍体籽粒为材料,利用20、40、80和160 μmol L^-1浓度的甲基胺草磷、炔苯酰草胺和氟乐灵作为加倍药剂,在单倍体植株生长到三叶期和五叶期时,用滴心法处理幼苗,选择有花粉的单株自交,收获后调查果穗加倍率;采用细胞学方法观察单倍体的染色体数目和花粉的活性。结果表明,20~160 μmol L^-1的3种除草剂对玉米单倍体加倍均有效果,加倍率在3.42%~26.32%之间。甲基胺草磷、炔苯酰草胺和氟乐灵的加倍率分别为4.29%~26.32%、3.85%~20.81%和3.42%~17.61%;其中80 μmol L^-1甲基胺草磷的加倍效果最佳,使用80 μmol L^-1甲基胺草磷处理3个杂交种的单倍体,平均加倍率分别为25.02%、20.13%和14.99%。方差分析表明,3个基因型间的单倍体加倍率均呈极显著差异,可见使用甲基胺草磷、炔苯酰草胺、氟乐灵可以提高玉米单倍体的加倍频率,但不同基因型单倍体对除草剂的敏感性存在差异。     

Relative Efficiency of Anther Culture and Chromosome Elimination Techniques for Haploid Induction in Triticale × Wheat and Triticale × Triticale Hybrids

Summary The study was undertaken to evaluate the relative efficiency of anther culture and chromosome elimination (by crosses with maize) techniques of haploid induction in intergenotypic triticale and triticale × wheat hybrids. For this, 15 triticale × wheat and 8 triticale × triticale F₁ hybrids were subjected to anther culture and were also simultaneously crossed with the `Madgran Local' genotype of maize (Zea mays L.) to induce haploids through the chromosome elimination technique. The haploid embryo formation frequency through the chromosome elimination technique was significantly higher in both, triticale × wheat (20.4%) and triticale × triticale (17.0%) F₁ genotypes, as compared to the calli induction frequencies through anther culture (1.6 and 1.4%, respectively). Further, four triticale × wheat and three triticale × triticale F₁ genotypes failed to respond to anther culture, whereas, all the F₁ genotypes formed sufficient number of haploid embryos through the chromosome elimination technique with no recovery of albino plantlets. The haploid plantlet regeneration frequencies were also significantly higher through the latter technique in both triticale × wheat (42.7%) and triticale × triticale (49.4%) F₁s as compared to anther culture (8.2 and 4.0%, respectively), where the efficiency was drastically reduced by several constraints like, high genotypic specificity, low regeneration frequency and albinism. The overall success rates of obtaining doubled haploids per 100 pollinated florets/anthers cultured were also significantly higher through the chromosome elimination technique (1.1% in triticale × wheat and 1.5% in triticale × triticale hybrids), proving it to be a highly efficient and economically more viable technique of haploid induction as compared to anther culture, where the success rates were only 0.2% and 0.1%, respectively.     

Properties of maternal haploid maize plants and potential application to maize breeding

Summary Presented are the results of a two-year study of haploid maize plants in the field. The haploids were produced with the aid of inducer line ZMS. In total, 604 and 1030 haploids were obtained and studied in the first and second years, respectively. Tassels of haploid plants were found to be almost completley sterile. Fertility of ears was studied by pollinating them with the pollen from diploid inbred lines, the cross resulting in almost all of the haploid ears carrying kernels. On average 27.4 kernels per ear of haploid plant were obtained in the first year of study and 26.3 in the second. These gave rise to normal diploid plants. This property allows genotypes selected at the level of haploid plants to be involved in breeding process. Unusual plants were found among haploids, phenotypically resembling homozygous lines. It was assumed that the plants had resulted from spontaneous chromosome doubling in haploids. The results of comparative studies of progenies of unusual plants and inbred lines derived from the same synthetic population are presented.     

Genetic dissection of haploid male fertility in maize (Zea mays L.)

Haploid genome doubling is a key limiting step of haploid breeding in maize. Spontaneous restoration of haploid male fertility (HMF) provides a more promising method than the artificial doubling process. To reveal the genetic basis of HMF, haploids were obtained from the offspring of 285 F_2:3 families, derived from the cross Zheng58 × K22. The F_2:3 families were used as the female donor and Yu high inducer No. 1 (YHI‐1) as the male inducer line. The rates of HMF from each family line were evaluated at two field sites over two planting seasons. HMF displayed incomplete dominance. Transgressive segregation of haploids from F_2:3 families was observed relative to haploids derived from the two parents of the mapping population. A total of nine quantitative trait loci (QTL) were detected, which were distributed on chromosomes 1, 3, 4, 7 and 8. Three major QTL, qHMF3b, qHMF7a and qHMF7b were detected in both locations, respectively. These QTL could be useful to predict the ability of spontaneous haploid genome doubling, and to accelerate the haploid breeding process by introgression or aggregation of those QTL.     

Genetic,quantitative and microscopic evidence for fusion of haploid nuclei and growth of somatic calli in cultured <Emphasis Type="Italic">ms10</Emphasis><Superscript><Emphasis Type="Italic">35</Emphasis></Superscript> tomato anthers

In plant breeding, androgenic doubled haploids represent powerful tools to save time and resources for pure line generation. While in many species efficient protocols are known, in tomato (Solanum lycopersicum), the knowledge on the induction of androgenesis is still very scarce, and little is known about the particularities of this highly recalcitrant species. The only known method capable of yielding haploid/doubled haploid tomato plants is anther culture. However, this method has important limitations, including low efficiency of haploid induction and a low proportion of spontaneously doubled haploids. To understand these limitations better, we have analyzed the process of callus formation in anthers of tomato lines carrying the ms10 ³⁵ gene for male-sterility, using light and electron microscopy, flow cytometry and genetic analysis with morphological and molecular markers. Our results demonstrate that haploid, doubled haploid and diploid calli occur in tomato anthers, although at different frequencies. Diploid calli derived either from somatic cells or from the fusion of two genetically different haploid nuclei account for more than 90% of the total of calli produced. Somatic calli are derived from the stubs of connective tissue present in the interlocular septa of anthers. This growth is markedly increased in the ms10 ³⁵ mutants, which explains their higher callogenic rates than standard tomato lines. Together, our results reveal serious drawbacks that explain the low efficiency of anther-derived, doubled haploid production in tomato, and stress the need for alternatives towards doubled haploidy.     

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.     

Improved Techniques for Haploid Production in Wheat using Chromosome Elimination

Wheat × maize and wheat × pearl millet crosses have proved efficient for haploid production using various genotypes of wheat; 22 and 27 % of florets produced embryos. In favourable conditions 6—9 haploid plants per spike were produced. The following simplifications or improvements in technique are recommended: 1. Only a single treatment with an aqueous solution of dicamba or 2,4-D (50–100 ppm) for embryo stimulation in vivo; 2. Application by spraying or dipping the spikes; 3. Application time two to four days after pollination; 4. Embryo rescue 15 to 18 days after pollination; 5. Crosses without emasculation are possible if pollination occurs 1–2 days before anthesis. More than 450 haploids and some doubled haploid (DH) lines (after colchicine treatment in vitro) were produced using these methods. No hybrid plants, chromosome additions or substitutions were found.     

Relative efficiency of maize and Imperata cylindrica for haploid induction in Triticum durum following chromosome elimination‐mediated approach of doubled haploid breeding

Intergeneric hybridization in seven diverse durum wheat genotypes was carried out using two composite varieties of Himalayan maize, viz., Bajaura Makka and Early Composite, and a wild grass, Imperata cylindrica, as pollen sources. Observations related to various haploid induction parameters put forth I. cylindrica as significantly better pollen source for haploid induction in durum wheat over maize in terms of pseudoseed formation (46.93%), embryo formation (38.06%), haploid regeneration (40.42%) and haploid formation efficiency (7.44%). The line x tester analysis revealed that both male and female genotypes had significant effects on all haploid induction parameters except haploid formation frequency in later. Among the pollen sources, I. cylindrica emerged as best combiner based on GCA values when compared with the two Himalayan maize composites. Durum wheat genotype, A‐9‐30‐1 was recognized as the best general combiner followed by PDW 314. The present investigation proposed durum wheat × I. cylindrica as a superior technique over maize‐mediated system, and its large‐scale use can open a new horizon in the sphere of durum wheat doubled haploidy breeding programme.     

The effects of parthenogenesis on wheat embryo formation and haploid production with and without maize pollination

Doubled haploid (DH) lines are important in wheat (Triticum aestivum L.) breeding, and haploids produced via maize pollination precede DH line development. Although maize pollination has proven reliable and broadly applicable to wheat, its success is determined by the wheat and maize genotypes employed. A wheat genotype consisting of nuclear and cytoplasm components predisposing it to parthenogenesis was compared with three other genotypes, each possessing only one or neither component necessary for parthenogenesis. In a glasshouse experiment, each genotype was pollinated with maize and subsequently treated with a2,4-Dichlorophenoxyacetic Acid (2,4-D) solution to determine if parthenogenesis affected embryo formation frequency (EFF)and haploid formation efficiency (HFE). Wheat genotypes were also treated with the2,4-D solution alone to determine if embryos and haploid plants could be produced in vivo without maize pollination. ‘Salmon(K)’, a parthenogenetic genotype consisting of a Salmon 1BL.1RS nucleus in a Ae. kotschyii cytoplasm, had a mean EFF of 32%; whereas, the non-parthenogenetic genotypes had mean EFF calculations ranging from 7 to 21%. Mean HFE for Salmon(K) was not significantly different than the mean HFE for non-parthenogenetic Salmon; however, EFF and HFE calculations for Salmon(K) and Salmon, each with a 1BL.1RS translocation, were generally higher than calculations for genotypes without the translocation. Salmon(K) was the only genotype to produce a 3% or higher EFF and HFE after treatment with 2,4-D alone. Parthenogenesis significantly affected the frequency at which embryos were produced after pollination with maize and the frequency at which embryos and haploid plants were produced after treatment with 2,4-D alone. This revised version was published online in July 2006 with corrections to the Cover Date.     

Improving androgenesis‐mediated doubled haploid production efficiency of FCV tobacco (Nicotiana tabacum L.) through in vitro colchicine application

Production of doubled haploid plants through androgenesis in flue‐cured Virginia (FCV) tobacco is a promising and convenient alternative to conventional selfing techniques for the generation of absolute homozygous lines. Here, we show a robust in vitro haploid and doubled haploid development protocol in FCV tobacco with major emphasis on improving the efficiency of chromosome doubling using in vitro colchicine treatment. We used five FCV tobacco hybrids for comparison of colchicine treatments. The anther culture response varied with developmental stages of the buds, and the highest response was observed in stage 2 buds. The effect of cold pretreatment was significant, and 4 days of pretreatment was optimum for gametic embryogenesis. Among the methods used for determining the ploidy status of plants, flow cytometry was found to be easy, fast and reliable for high‐throughput screening of haploids. Doubled haploids regeneration percentage varied from 6.77 to 11.95 in in vivo treatment, while the range of variation was 22.11% to 28.40% in in vitro colchicine treatment. We observed a pronounced increase in plant survival and the proportion of doubled haploid plants in in vitro treatment compared with the standard in vivo approach.     

Production of Haploid Sugar Beet (Beta vulgaris L.) by Culturing Unpollinated Ovules

The culture of unpollinated ovules is shown to be a suitable system for the production of haploid sugar beet (Beta vulgaris L.). The yield of haploids depended upon the genotype and varied between 0 and 13 % with a mean of 1.0 %. Haploid plants could be produced from approximately 50 % of all genotypes examined. The majority of the haploids isolated (about 90%) maintained the haploid genome level during the in vitro culture and propagation; 10% of the haploid clones showed a spontaneous doubling to the diploid genome level.     

Transfer of cytoplasmic male sterility by spontaneous androgenesis in tobacco (Nicotiana tabacum L.)

Summary Five cytoplasmic male sterile lines of Nicotiana tabacum homozygous for a dominant rootless mutation [Rac^-] were used as female parents in crosses with three male fertile varieties. Androgenetic haploids were selected by their ability to form plantlets with a normal root system, whereas hybrid plantlets failed to grow. High frequencies of androgenetic haploid plants were obtained with N. debneyi cytoplasm, and with one paternal genotype. Chromosome doubling was performed using three different methods (pollination, in vitro culture, acenaphthen) and cytoplasmic male sterile doubled haploid plants have been obtained. The results demonstrate the efficiency of this method for a rapid cytoplasm transfer in Nicotiana tabacum; the integrity of the cytoplasm is conserved. For the first time, we possess a counter selectable marker universally usable for Nicotiana tabacum and closely related species.