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.     

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.     

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.     

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.     

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.     

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.     

Double haploids,markers and QTL analysis in vegetable brassicas

Double haploid (DH) plants of Brassica spp. can be produced via anther culture or culture of microspores. This paper reviews the uses of double haploids in crop improvement research in vegetable brassicas (B. oleracea). Applications of DH lines are described for breeding; construction of linkage maps; genetic analysis of quantitative traits and capturing genetic variation. The advantages and disadvantages of DH lines are discussed     

Efficient doubled haploid production in microspore culture of loose-curd cauliflower (Brassica oleracea var. botrytis)

We present an improved protocol for highly efficient production of doubled haploid loose-curd cauliflower plants (Brassica oleracea var. botrytis) via microspore culture. Our experiment explored factors such as donor plant treatment, flower bud pretreatment, embryo germination medium, and ploidy characterization of regenerated plants. Our technique efficiently produced embryos from both tight- and loose-curd donor plants, although the embryo yields were genotype dependent. We achieved a germination rate of around 30 % by employing a hormone combination of zeatin, indole-3-acetic acid, and 6-benzylaminopurine pretreatment culture. We also used 1–4 days of cold pretreatment of the flower buds, which were submerged into NLN-13 medium, to induce microspore embryogenesis. Analysis using an FCM Ploidy Analyzer showed that more than 50 % of regenerated plants were spontaneously doubled haploids, more than 25 % were tetraploids, and fewer than 7 % were haploid. Visual examination of plants in the field revealed that they had distinct phenotypic characteristics relating to their ploidy level. The efficient production of double haploids using our improved microspore culture technique is a promising approach that can be applied in loose-curd cauliflower breeding programmes and genetic research.     

Comparison of fertility between the F1, F2 and anther derived lines in the crosses of indica/japonica and japonica/indica in rice (Oryza sativa L.)

Summary Response of anthers in in vitro culture was examined in the indica-japonica hybrids of rice (Oryza sativa L.). Significant genotypic differences were observed for callus induction and regeneration among the different interracial hybrids of indica-japonica races. Induction frequency of haploids ranged from 57.7 to 72.9 per cent and doubled haploid androgenic lines ranged from 27.1 to 42.3 per cent in the anther culture of the different hybrids. The indica-japonica hybrids recorded partial pollen grain and spikelet fertility in F1 (29.9 to 41.5% and 19.4 to 48.7% respectively) as well as in F2 (42.7 to 50.6% and 37.1 to 54.4% respectively). In contrast, the androgenic doubled haploid lines recorded significant increase and the pollen grain and spikelet fertility was 76.3 and 78.6 per cent respecitively. The results suggested that the sterility barriers for realising genetic recombinants and fixation of fertile homozygous lines in indica-japonica hybridization programme could be overcome through F1 anther culture technique.Abbreviations BAP Benzyl Amino Purine - 2,4-D 2,4-Dichlorophenoxy acetic acid - MS Murashige & Skoog medium - IAA Indole Acetic Acid     

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     

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.     

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.     

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.     

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.     

Analyses of phenotype and ARGOS and ASY1 expression in a ploidy Chinese cabbage series derived from one haploid

The aim of this research was to improve our understanding of how ploidy level influences phenotype and gene expression in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Haploid plants (2n = 10) was induced by 0.2% colchicine to produce diploid (2n = 20) and tetraploid plants (2n = 40). The aneuploid (2n = 24) was also obtained by hybridization between diploid plants as the female and tetraploid plants. The ploidy levels of all plants were identified through chromosome counts and flow cytometry. Leaves and petals became larger as the ploidy level increased from haploid to diploid, and from aneuploid to tetraploid. Similarly, expression of ARGOS was regulated by genome size, increasing in parallel with the level of ploidy. Among the four ploidy types, expression was stronger in the floral buds than in the leaves. Expression by ASY1 also differed according to ploidy level, being highest in diploid plants, followed in order by tetraploids. Expression was similar between haploids and aneuploids at two stages—prior to and after meiosis—but was higher in the haploids during meiosis. When buds were compared within the same ploidy type at different stages, ASY1 expression was obviously higher during meiosis than either before or after. Our study demonstrated the generation and phenotype of a ploidy Chinese cabbage series derived from one haploid. Expression of genes ARGOS and ASY1 were modulated by genome size in this ploidy series, and the regulated patterns of the two genes was different.     

Microspore mutagenesis of Brassica species for fatty acid modifications: a preliminary evaluation

A microspore mutagenesis protocol was developed for Brassica rapa, Brassica napus and Brassica juncea for the production of double haploid lines with novel fatty acid profiles in the seed oil. Freshly isolated Brassica microspores were first cultured with ethyl methane sulphonate (EMS) for 1.5 h. The EMS was removed and the microspores were then cultured according to the standard Brassica microspore culture protocol. This protocol was used to generate over 80 000 Brassica haploid/double haploid plants. Field evaluation of B. napus and B. juncea double haploids was conducted between 2000 and 2003. Fatty acid analysis of the B. napus double haploid lines showed that saturated fatty acid proportions ranged from 5.0% to 7.7%. For B. juncea, saturate proportions ranged from 5.4% to 9.5%. Of the 7000 B. rapa lines that were analysed, 197 lines had elevated oleic acid (>55%), 69 lines had reduced α‐linolenic acid (<8%) and 157 lines had low saturated fatty acid proportions (<5%), when compared with the parental lines.     

Shoot Regeneration from Anther Culture of Sunflower (Helianthus annuus) and Some Interspecific Hybrids as Affected by Genotype and Culture Procedure

Anther culture has been demonstrated to be an applicable technique for the development of doubled haploid, i. e. homozygous lines of many crop species. In some species, androgenetic doubled haploids have already been shown to be a useful tool for breeding. However, anther culture results in sunflower have been rather unsatisfactory up to now. As in other species, anther culture response of sunflower (Helianthus sp.) is strongly affected by physical, nutritional, physiological and genetical factors. By testing a number of different culture parameters, i. e. donor plant stages, culture media and conditions, and appropriate schedule could be worked out for the successful regeneration of shoots – at least for a number of sunflower lines and interspecific hybrids.     

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。     

Genomic structure of androgenic progeny of pentaploid hybrids, Festuca arundinacea × Lolium multiflorum

To assess the usefulness of the doubled haploid (DH) method in the breeding of forage grasses, a sample of anther-derived progeny of pentaploid F₁ hybrids of Festuca arundinacea × Lolium multifiorum was karyotyped using genomic in situ hybridization (GISH). The technique allowed scoring of the total number of chromosomes, the number of chromosomes contributed by each parent, and the number and positions of the Festuca-Lolium translocation breakpoints. Among 27 plants analysed, 13 belonged to three clones, effectively reducing the number of different progeny karyotyped to 17. These included 10 haploids, five doubled haploids and two plants for which the origins could not be explained. In all plants analysed, a mixture of chromosomes of both parents was present, including an average of 1.88 intergeneric translocations per plant. The translocation breakpoints were distributed along almost the entire length of the chromosome arms. Chromosome variation among androgenic progeny appeared much wider than that in the conventional backcross but low vigour and high mortality suggest that this additional variation may be difficult to exploit directly in breeding. However, a change in the pattern of recombination makes the entire genome accessible to manipulation.