Efficient production of callus-derived doubled haploids through isolated microspore culture in eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

Production of doubled haploids (DHs) through androgenesis induction is an important biotechnological tool for plant breeding. In some species, DHs are efficiently obtained through embryogenesis from isolated microspore cultures. In eggplant, however, this process is still at its infancy, despite the economic relevance of this important agricultural crop. To date, only two studies have focused previously on this process, suggesting that in eggplant microspore cultures, the only morphogenic response is callus formation. Given the notable lack of studies on eggplant microspore cultures, in this work we explored this process with different experimental approaches. We studied the response of different cultivars and characterized the development of microspores induced to divide and proliferate. We demonstrated that microspore-derived embryos (MDEs) can be produced in eggplant; however, MDEs stopped at the globular stage, to turn into euploid and principally mixoploid calli. From these calli, 60 % of DH plants could be regenerated. In order to promote microspore induction we evaluated the effect of polyethylene glycol (PEG) and mannitol. PEG, but not mannitol, significantly increased induction of microspore embryogenesis. We also tested the ability of eight different media compositions to promote efficient plant regeneration from calli. In order to test it in a genotype-independent manner, we previously developed a method to generate clonal callus populations derived from single microspore-derived calli. Together, the results presented hereby constitute an efficient way to produce eggplant DHs through microspore culture. In addition, they contribute significant insights into the knowledge of the particularities of androgenesis induction in this species.     

茄子花培后代的观察与应用

对从长茄杂交种“牟尼卡”（编号06-1001）进行花药培养获得的花培品系二代（DH2）和三代（DH3）的主要植物学性状进行了观察与比较。结果表明,从同一接种材料可以获得不同类型的花培品系,其始花节位、果形指数和单果重变异幅度很大,15个二代品系中,有46.7%的品系始花节位与接种材料达到显著或极显著差异标准,有40.0%的品系果形指数与接种材料达到显著或极显著差异标准,有33.3%的品系单果重与接种材料达到显著或极显著差异标准。同一品系不同世代间始花节位、果实横径、果实纵径、果形指数和单果重能稳定遗传;品系内个体间始花节位、果实横径、果实纵径、果形指数和单果重变异系数较小(<10%),系内整齐一致。此外,初步选育的花培品系“06-139”和以花培品系为亲本的杂交组合“05-203”开始在农业生产中应用。     

Genetic variation of seedling traits responded to brassinosteroid and gibberellin inhibitors in maize (Zea mays) doubled haploid lines

The purpose of this study was to examine the genotypic variation in maize doubled haploid (DH) lines response to brassinosteroid and gibberellin inhibitors. Plant responses to hormone inhibitors were determined in growth chamber experiments using germination paper for three different seedling treatments: application of propiconazole (Pcz), uniconazole (Ucz) or water (control). Mesocotyl length (ML) was more sensitive to hormone inhibitors, especially to the Ucz treatment, than other seedling traits. ML was significantly correlated with other traits in the Ucz treatment. All the seedling traits showed moderate-to-high broad sense heritability values, ranging from 0.39 to 0.82. The Euclidian genetic distances of inbred line pairs ranged from 1.27 to 19.94, indicating there was a high level of variability across the maize DH lines used in this study. DH lines with extreme MLs were identified, which can provide valuable breeding resources for improving abiotic stress tolerance, and for further genetic studies.     

Evaluation of androgenic competence through anther culture in common eggplant and related species

Anther culture is a convenient technique to obtain androgenic haploid and doubled haploid (DH) plants. In common eggplant (Solanum melongena), this technique has been used to develop DH pure lines for producing uniform F1 hybrid seed of some commercial varieties. However, a comprehensive study of the variation of this useful trait among different materials of common eggplant and related species is still lacking. In this work, we studied the androgenic response of 12 accessions of common eggplant and related materials from the primary (eggplant complex) and secondary genepools. We cultured anthers of all the accessions under the same experimental conditions, and studied their competence to produce calli, embryos and plants, as well as the quality and origin of the embryos produced. In our conditions, anthers of 11 out of the 12 accessions produced somatic calli, whereas only 5 also produced microspore-derived embryos, with variable results in terms of embryo quality and of frequency of embryo induction and plant germination. Embryos of responding accessions were initially haploid, and reached the DH status, verified with SSR markers, after a defined period of culture. In addition to other aspects common to many androgenesis-responsive species, our results allowed us to extract conclusions particular to common eggplant and relatives, including the difficulty for finding sources of androgenic competence out of S. melongena, the reduced impact of calli in the production of non-DH individuals, and the need to avoid the occurrence of severe anatomical and functional problems in the apex of most embryos, which seriously reduces their germinative success.     

Agronomic performance of anther‐derived doubled haploid and single seed descent lines in crosses between primary and secondary winter triticale

Octoploid (8x) and hexaploid (6x) primary triticales (xTriticosecale Wittm.) can be used as crossing parents with secondary 6x triticales to enlarge the genetic basis of a breeding programme or introgress traits. Doubled haploid (DH) production permits to develop homozygous lines more rapidly from a segregating generation than other breeding methods such as single seed descent (SSD). Both anther‐derived DH and SSD lines were produced from reciprocal cross‐combinations between 8x and 6x primary and 6x secondary triticales. Field experiments of DH and SSD lines were conducted in three environments as two‐replicate lattices to measure seven agronomic traits. A tendency for higher grain yield, taller plants and a higher 1000‐kernel weight of SSD lines compared with DH lines was found. Significant genetic variation for all traits in both breeding methods was revealed, indicating their suitability to select superior genotypes. Hexaploid and even more so 8x primary triticales can profitably be included as crossing parents in a commercial breeding programme. In such crosses, the primary triticales should be used as the male parents if followed by DH method.     

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.     

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.     

3个辣椒花药培养再生株群体主要农艺性状及苗期抗病性研究

对3个辣椒花药培养再生株DH-R2群体的主要农艺性状,包括株高、株幅、首花节位、叶色、单果质量、商品果横径、商品果纵径、果形指数、老熟果色等进行了较详细的测量和统计分析。结果表明:辣椒花药培养再生株群体的农艺性状存在基因型偏性;再生株群体的单果质量、商品果纵径、果形指数这3个性状都表现为低于供体,株高、株幅、首花节位、商品果横径这4个性状都表现为近似于供体;通过单倍体培养途径可以得到不同颜色层次的老熟果色的辣椒纯系,证明了该途径是多基因控制性状育种的有效途径;对耐TMV和CMV的亲本与感病亲本的杂种进行花药培养,获得了兼具TMV和CMV抗性的株系,证明了单倍体培养途径可以创造新的超亲抗病种质。     

Molecular and agronomic evaluation of wheat doubled haploid lines obtained through maize pollination and anther culture methods

Although maize pollination (MP) and anther culture (AC) are alternative techniques widely used for wheat doubled haploid (DH) production, there is only limited information on the attributes of the plant materials produced through both methods. This study was conducted to evaluate genetic fidelity, transmission of parental gametes, and to compare field performance of DH populations produced by the MP and AC methods from the F1s of two crosses between spring bread wheat cultivars. The DH populations were compared to single seed descent (SSD) lines created from the same crosses. In total, 76 MP and 122 AC lines of the cross between cultivars of divergent origin were subjected to RAPD and AFLP analysis. Only changes in AFLP banding patterns, at similarly low frequencies, 0.18% (MP) and 0.21% (AC), were detected. The frequency of the DH lines affected by the variation, 14.5% (MP) and 14.8% (AC), was similar in both populations. For most of the DH lines, variation in 1‐2 loci only, out of several hundreds scored, was observed. A total of 14.3% (MP) and 22.2% (AC) marker loci showed the significant segregation distortion from the expected 1 : 1 ratio, but in at least one polymorphic locus the within‐cultivar variation was responsible for the skewed segregation. The field performance of the corresponding MP and AC lines derived from two crosses confirmed the equivalency of both DH populations. In most of the traits analyzed, the MP and AC lines performed the same as the SSD populations created from the same crosses. No, or very small differences in means and ranges, were observed when the best 10% of the lines from all three methods were compared. Moreover, the best 10 % of the lines of the cross between Polish wheat cultivars adapted to the local environment performed significantly better for some traits than different groups of checks used in the study.     

辣椒DH群体果实性状的分离及与F2群体的比较

为分析DH群体的遗传稳定性及其DH群体内性状分离与同来源的F2群体之间异同，本研究利用辣椒花药培养技术，构建一个由“羊角椒(97403)×方灯笼甜椒(97410)”的杂交而成的牛角椒组合作为供体的且由103个DH系组成的DH群体，对该DH群体进行单果重等5个主要果实性状的遗传表现进行分析，并与同来源的F2群体进行比较。结果表明：原供体及其双亲的5个果实性状的变异系数均较小，三者之间的各性状均存在显著差异，说明原供体及其双亲的整齐一致性；对DH群体和F2群体各性状的平均值、变异系数及其性状分离区间的分析和对比结果表明，5个果实性状均是受多基因控制的数量性状，且由于基因重组无论是DH群体还是F2群体均能产生正向和负向两个方向的超亲基因型。但DH群体与F2群体比较各性状的分离区间明显增大，超亲分离类型明显增加，且获得的各DH系均为稳定遗传的自交系，提高了花培育种的选择效率。     

Characterization of wheat-triticale doubled haploid lines by cytological and biochemical markers

Five wheat-triticale doubled haploid (DH) lines— M08, V209, DH220-14-2, DH696-3-4 and M16 —derived from anther culture of F₁s resulting from crosses involving hexaploid or octoploid triticale × hexaploid wheat, were characterized by cytological and biochemical markers. Cytological evidence from genomic in situ hybridization and C-banding indicated that DH lines M08 and V209 (2n= 42) each contained a pair of 1BL/1RS translocation chromosomes. DH220-14-2 (2n= 42) was also a translocated line with two pairs of chromosomes containing small fragments of rye. One of the translocation fragments carried the Sec-1R gene originating from the satellite region of 1RS; the origin of the other one remains unknown. DH696-3-4 (2n= 42) contained a 3D(3R) substitution. In M16 (2n= 44), three pairs of rye chromosomes, 3R, 4R and 6R, were present, 4R as an addition and 3D(3R) and 6D(6R) as substitutions. Biochemical, isozyme and storage protein markers confirmed the cytological conclusions. The advantages of transferring alien chromosomes or chromosome fragments into wheat and creating alien aneuploid lines by anther culture of hybrid F¹s are discussed.     

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

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

Androgenesis from a Lolium multiflorum × Festuca arundinacea hybrid to generate extreme variation for freezing-tolerance

New opportunities for plant breeding using androgenesis in Lolium × Festuca hybrids have been identified. Plants derived by anther culture from a Lolium multiflorum × Festuca arundinacea (5x) hybrid were screened for freezing-tolerance, and their post-freezing recovery compared. The androgenic population showed extreme diversity in freezing-tolerance. While the majority of androgenic plants had inferior freezing-tolerance compared with the freezing-sensitive L. multiflorum parent, 6% of the population were more freezing resistant than the freezing-tolerant Festuca parent. Novel Lolium and Festuca gene combinations resulting from rare meiotic events were recovered within the androgenic population. The two most freezing-tolerant androgenic plants carried virtually the entire F. pratensis subgenome of F. arundinacea. F. pratensis is known to carry genes for freezing-tolerance and would be expected to be the primary source of genes governing this trait within the F. arundinacea genome. The most freezing-tolerant androgenic plants were more freezing-tolerant than the hybrid plant from which they were derived. Consequently, androgenesis was also effective in removing factor(s) reducing the expression of freezing-tolerance within the L. multiflorum × F. arundinacea (5x) hybrid.     

Grain dormancy QTL identified in a doubled haploid barley population derived from two non-dormant parents

Grain dormancy provides protection against pre-harvest sprouting (PHS) in cereals. Composite interval mapping and association analyses were performed to identify quantitative trait loci (QTL) contributing grain dormancy in a doubled haploid (DH) barley population (ND24260?×?Flagship) consisting of 321 lines genotyped with DArT markers. Harvest-ripe grain collected from three field experiments was germinated over a 7-day period to determine a weighted germination index for each line. DH lines displaying moderate to high levels of grain dormancy were identified; however, both parental lines were non-dormant and displayed rapid germination within the first two?days of testing. Genetic analysis identified two QTL on chromosome 5H that were expressed consistently in each of the three environments. One QTL (donated by Flagship) was located close to the centromeric region of chromosome 5H (qSDFlag), accounting for up to 15% of the phenotypic variation. A second QTL with a larger effect (from ND24260) was detected on chromosome 5HL (qSDND), accounting for up to 35% of the phenotypic variation. qSDFlag and qSDND displayed an epistatic interaction and DH lines that had the highest levels of grain dormancy carried both genes. We demonstrate that qSDND in the ND24260?×?Flagship DH population is positioned proximal and independent to the well-characterised SD2 region that is associated with both high levels of dormancy and inferior malt quality. This indicates that it should be possible to develop cultivars that combine acceptable malting quality and adequate levels of grain dormancy for protection against PHS by utilizing these alternate QTL.     

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.     

QTL mapping of three ear traits using a doubled haploid population of maize

Ear shape substantially correlates to grain yield, so understanding their genetic architecture is of great significance in maize breeding. Ear length (EL), ear diameter (ED), length of barren tip (LBT) and cob diameter (CD) were determined for 240 doubled haploid maize lines, and all four traits showed a relatively high broad sense heritability around 77%. Using this DH population consisting of 240 lines and a genetic map constructed from 964 SNPs, a total of five, four and three QTLs were identified for EL, ED and CD, respectively, in three various growing conditions. Among these, qEL1‐1, qED1 and qCD1 were consistently mapped at an overlapping location on Chr1, which contributed 15.7, 28.3 and 22.6% of the phenotypic variation in EL, ED and CD, respectively. All other QTLs exhibited minor effect with the phenotypic variation explained ranging from 4.7% to 7.8%. Because most of the QTLs were detected in at least two different planting environments, they appear to be potential loci for gene isolation and marker development in maize molecular breeding.     

Mapping QTLs for seedling root traits in a doubled haploid wheat population under different water regimes

A deep and thick root system has a positive effect on wheat yield, particularly in drought environments. A doubled haploid (DH) population of 150 lines derived from the cross Hanxuan 10?×?Lumai 14 was used to map QTLs for seedling root characteristics. The DH lines were cultivated in an agarose gel-chamber under well-watered (WW) and water-stressed (WS) regimes. Water stress was simulated by adding mannitol to the agarose gel. The seminal root traits, including maximum root length (MRL), seminal root number, total root length, project root area, root surface area, and seminal root angle were measured after 6?days of seedling development. Grain yields (GY) were measured in a field experiment. A total of 29 QTLs were identified for seedlings cultured under WW regimes, and 23 QTLs under WS regimes. Individual QTL accounted for phenotypic variations ranging from 4.98 to 24.31?%. The QTLs were distributed on 17 chromosomes, except 1D, 4D, 6B and 6D. Seven consistently expressed QTLs were detected for all the traits tested except MRL under both water regimes. The QTLs for root traits were unevenly distributed among chromosomes, and clustered in eight loci on seven chromosomes, showing pleiotropic effects on target traits. One region in the interval Xgwm644.2?CP6901.2 on chromosome 3B contained 9 QTLs affecting most root traits. The present data provide an insight into the genetic basis of seedling root development under different water regimes and may benefit breeding programs using marker-assisted selection (MAS) for root traits.     

Assessment of different anther culture approaches to produce doubled haploids in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Cucumber is one of the most important vegetable crops worldwide, which makes it a good candidate to produce doubled haploid (DH) lines to accelerate plant breeding. Traditionally, these approaches involved induction of gynogenesis or parthenogenesis with irradiated pollen, which carries some disadvantages compared to androgenesis. Despite this, studies on anther/microspore cultures in cucumber are surprisingly scarce. Furthermore, most of them failed to unambiguously demonstrate the haploid origin of the individuals obtained. In this work we focused on anther cultures using two cucumber genotypes, different previously published protocols for anther culture, different in vitro culture variants to make it more efficient, and most importantly, a combination of flow cytometry and microsatellite molecular markers to evaluate the real androgenic potential and the impact of anther wall tissue proliferation. We developed a method to produce DH plants involving a bud pretreatment at 4 °C, a 35 °C treatment to anthers, culture with BAP and 2,4-D, and induction of callus morphogenesis by an additional 35 °C treatment and sequential culture first in liquid medium in darkness and second in solid medium with light. We also found that factors such as genotype, proliferation of anther wall tissues, orientation of anthers in the culture medium and growth regulator composition of the initial anther culture medium have a remarkable impact. Our rate of chromosome doubling (81%) was high enough to exclude additional chromosome doubling steps. Together, our results present androgenesis as an improvable but yet more convenient alternative to traditional gynogenesis and parthenogenesis-based approaches.     

Genetic analyses of agronomic and seed quality traits of doubled haploid population in Brassica napus through microspore culture

Summary The results showed that the F1 genotype from the cross (Brassica napus cv. Zheshuang 758 × cv. Z-4115) had good response to embryogenesis, and their embryo yield and rate of plant regeneration reached 69.8 embryo/bud and 46.9%, respectively. Characters from the doubled haploid (DH) populations in B. napus were analyzed and it was showed that the means of each agronomic trait were between their parents, but they were nearer to the paternal in 6 agronomic traits (plant height, branch position, number of pods in the main raceme, length of pod, number of pods/plant and number of seeds/pod). The number of genes controlling each agronomic trait was analyzed based on the DH populations. The results showed that the number of genes controlling number of pods in the main raceme was the highest (15.6), and the least number of genes was involved for stem width (only 7.9). According to estimated coefficients of skewness and kurtosis of the traits tested, gene interaction was found to be absent for stem width, plant height, length of main raceme, number of primary and secondary branches, pod density in the main raceme and seed weight/plant. Complementary interaction was also observed in five agronomic traits (number of pods in the main raceme, length of pod, number of pods/plant, number of seeds/pod and 1000-seed weight). A significantly positive correlation was observed between seed yield/plant and four agronomic traits (length of main raceme, length of pod, number of pods/plant and 1000-seed weight). The experiment also showed that the erucic acid, glucosinolate, oil and protein contents of DH populations were 34.23%, 87.09 μmol/g, 44.09% and 42.67%, respectively. The numbers of genes controlling each quality trait were 7.8, 9.7, 9.4 and 8.7, respectively. Partial correlations between the seed quality traits and the agronomic characters of DH populations were analyzed. In this experiment, the partial correlations among seed quality traits were also analyzed and it was found that the oil content had a negative correlation with the other three seed quality traits.     

Genetic distance among doubled haploid maize lines and their testcross performance under drought stress and non-stress conditions

In contrast to conventional inbreeding that takes up to seven generations to develop inbred lines, the doubled haploid (DH) technology allows production of inbred lines in two generations. The objectives of the present study were to: (a) evaluate testcross performance of 45 doubled haploid lines under drought stress and non-stress conditions (b) estimate heritabilities for grain yield and other traits and (c) to assess the genetic distance and relationship among the DH lines using 163,080 SNPs generated using genotyping-by-sequencing (GBS). The 45 hybrid and five checks were evaluated using a 10 × 5 alpha lattice in six drought stress and nine well-watered environments in Kenya, Uganda, and Tanzania. Differences in trait means between the drought stress and well-watered conditions were significant for all measured traits except for anthesis date. Genetic variances for grain yield, grain moisture, plant height and ear height were high under well-watered environments while genetic variance for anthesis date, root lodging and stalk lodging were high under drought stress environments. Combined analyses across drought stress and well-watered environments showed that ten top hybrids produced 1.6–2.2 t/ha grain yield under well-watered condition and 1–1.4 t/ha under drought stress condition higher than the mean of the commercial checks. Genetic distance between pairwise comparisons of the 38 of the 45 DH lines ranged from 0.07 to 0.48, and the overall average distance was 0.36. Both cluster and principal coordinate analysis using the genetic distance matrix calculated from 163,080 SNPs showed two major groups and the patterns of group was in agreement with their pedigree. Thirteen (13) of the best hybrids are currently in National Performance Trials testing, an important step towards commercialization in Kenya, Tanzania and Uganda.