Doubled Haploid Plants Research Articles

Enhancement of microspore embryogenesis induction and plantlet regeneration of sweet pepper (Capsicum annuum L.) using putrescine and ascorbic acid.

Production of doubled haploid (DH) plants is an efficient tool in genetic and plant breeding programs; however, sweet pepper (Capsicum annuum L.) is recalcitrant to microspore embryogenesis and DH production. Trying to break the barrier of DH production, three independent experiments were conducted on microspore embryogenesis of sweet pepper. In the first experiment, the effect of cold (4°C) and heat (32°C) pretreatments were investigated on microspore embryogenesis of three genotypes of sweet pepper including "Inspiration F1," "Maratus F1," and "Magno F1" cultivars in a factorial design with three replications. Heat shock (32°C for 7days), applied to mannitol-starved anthers of "Inspiration F1," showed higher multinuclear microspore percent, number of multicellular structures, total embryos, cotyledonary embryos, and regenerants. In the second experiment, the effect of different concentrations of putrescine (0, 0.5, 1, 2, and 5mgl-1) was evaluated on microspore embryogenesis of the three aforementioned cultivars of sweet pepper. The highest mean number of multicellular structures, cotyledonary embryos, and regenerants were achieved by applying 0.5-1mgl-1 putrescine during the mannitol starvation and heat shock (32°C) treatments of isolated microspore culture of "Inspiration F1" cultivar. Significant decrease in microspore embryogenesis efficiency was observed when high levels of putrescine (2 and 5mgl-1) were used. Microspore embryogenesis was prevented completely at 5.0mgl-1 putrescine. In the third experiment, the effect of different concentrations of ascorbic acid (0, 20, 50, 100, and 200mgl-1) was investigated and the results showed that the application of ascorbic acid (20 and 50mgl-1) during mannitol starvation and heat shock treatment (32°C) caused remarkable improvement in the number of produced cotyledonary embryos and their regeneration ability compared to control treatment. However, the application of higher levels of ascorbic acid (100 and 200mgl-1) inhibited microspore cell divisions and embryogenesis. In conclusion, the results indicated that both putrescine and ascorbic acid have significant effect on microspore embryogenesis efficiency of sweet pepper when they are used in appropriate concentrations.

Targeted mutagenesis in wheat microspores using CRISPR/Cas9

CRISPR/Cas9 genome editing is a transformative technology that will facilitate the development of crops to meet future demands. However, application of gene editing is hindered by the long life cycle of many crop species and because desired genotypes generally require multiple generations to achieve. Single-celled microspores are haploid cells that can develop into double haploid plants and have been widely used as a breeding tool to generate homozygous plants within a generation. In this study, we combined the CRISPR/Cas9 system with microspore technology and developed an optimized haploid mutagenesis system to induce genetic modifications in the wheat genome. We investigated a number of factors that may affect the delivery of CRISPR/Cas9 reagents into microspores and found that electroporation of a minimum of 75,000 cells using 10–20 µg DNA and a pulsing voltage of 500 V is optimal for microspore transfection using the Neon transfection system. Using multiple Cas9 and sgRNA constructs, we present evidence for the seamless introduction of targeted modifications in an exogenous DsRed gene and two endogenous wheat genes, including TaLox2 and TaUbiL1. This study demonstrates the value and feasibility of combining microspore technology and CRISPR/Cas9-based gene editing for trait discovery and improvement in plants.

Antimitotic and hormone effects on green double haploid plant production through anther culture of Mediterranean japonica rice

Rice double haploid (DH) plants are produced mainly through anther culture. In order to improve the anther culture protocol, microspores of two japonica rice genotypes (NRVC980385 and H28) were subjected to three growth regulator combinations and four colchicine treatments on induction medium. In addition, a post anther culture procedure using colchicine or oryzalin was tested to induce double haploid plantlets from haploid plantlets. A cold pre-treatment of microspores for 9 days at 10 °C increased callus induction 50-fold in the NRCV980385 genotype. For both genotypes, 2 mg L−1 2,4-D and 1 mg L−1 kinetin on colchicine-free induction medium gave the best culture responses. The culturability of both genotypes changed on colchicine-supplemented induction media. A high genotype dependency was recorded for callus induction, callus regenerating green plantlets and regeneration of green double haploid plantlets. Colchicine at 300 mg L−1 for 48 h enhanced callus induction 100-fold in H28. Colchicine-supplemented media clearly improved green double haploid plantlet regeneration. We showed that the post-anther culture treatment of haploid plantlets at 500 mg L−1 of colchicine permitted fertile double haploid plantlets to be generated. Finally, an enhanced medium-throughput flow cytometry protocol for rice was tested to analyse all the plantlets from anther and post anther culture.

Haploid plant regeneration from hardy kiwifruit (Actinidia arguta Planch.) anther culture

Haploids are important for studying plant breeding and genome sequencing. Regeneration from in vitro anther culture under aseptic conditions has achieved success in many higher plants. This study focused on anther culture of hardy kiwifruit (Actinidia arguta Planch.) to obtain haploid or double haploid plants that originated from microspores. Using the anthers of hardy kiwifruit as the test materials, we report the relationship between the microspore development stage and the bud morphological characteristics, some factors influenced the callus induction and the differentiation of the adventitious buds. The results showed that microspore development correlated with the bud diameter and morphological characteristics during development. The microspore development stage, pretreatment at low temperature (4 °C), basic medium, plant growth regulators and genotype all affect hardy kiwifruit anther callus induction, and the type and concentration of plant growth regulators significantly affect the regeneration of adventitious buds. The ploidy of 119 regenerated plants was verified using flow cytometry. The results showed that the ploidy of the plantlets obtained from the vitro anther culture of hardy kiwifruit was complex, being 3.36% haploid, 56.3% tetraploid, 4.20% hexaploid, and 36.13% octoploid.

PRODUCTION OF DH-PLANTS IN CULTURE OF ISOLATED MICROSPORE IN CARROT

The main goals of the research were to study the factors affecting on the process of embryogenesis in culture of isolated microspores, optimize the existing protocols, and finally produce the doubled haploid plants in carrot (DH-plants). Out of 10 carrot accessions tested in 2017 the embyoids were obtained in 8 carrot accessions with the use of 5 different media. The highest yield of embyoids was obtained in accessions 7kt (84 embryoids per Petri dish). It was shown that optimal explants were buds containing microspores at late vacuolated uninucleate stage. The first divisions in microspores were seen in 3 days of cultivation. After 40 days, the welldeveloped embryoids can be observed by naked eye. It was also shown that further cultivation of these embryoids on medium with 13% agarose over 60 days provoked the active secondary embryoid formation. Owing to this, it is recommended to place the embryoids at heart-shaped stages on another regeneration medium supplied with 2 % of agarose, enabling to register correctly the number of well-formed embryoids. We carried out the study on influence of such factors as genotype and composition of medium, and their combination on embryoid formation in 8 genotypes on 5 various media. ANOVA analysis showed that the plant genotype was the main factor causing the embryoid formation, whereas the effect of both factors had an impact on the number of embryoids developed. The counting the chromosome number in meristem cells and also observation of chloroplast number in stoma guard cells enabled to reveal that most of the plants produced were the doubled haploids

Coupling Seq-BSA and RNA-Seq Analyses Reveal the Molecular Pathway and Genes Associated with Heading Type in Chinese Cabbage.

In Chinese cabbage, heading type is a key agricultural trait of significant economic importance. Using a natural microspore-derived doubled haploid plant, we generated self-crossed progeny with overlapping or outward curling head morphotypes. Sequencing-based bulked segregant analysis (Seq-BSA) revealed a candidate region of 0.52 Mb (A06: 1,824,886~2,347,097 bp) containing genes enriched for plant hormone signal transduction. RNA Sequencing (RNA-Seq) analysis supported the hormone pathway enrichment leading to the identification of two key candidate genes, BrGH3.12 and BrABF1. The regulated homologous genes and the relationship between genes in this pathway were also revealed. Expression of BrGH3.12 varied significantly in the apical portion of the leaf, consistent with the morphological differences between overlapping and outward curling leaves. Transcript levels of BrABF1 in the top, middle and basal segments of the leaf were significantly different between the two types. The two morphotypes contained different concentrations of IAA in the apical portion of their leaves while levels of ABA differed significantly between plant types in the top, middle, and basal leaf segments. Results from Seq-BSA, RNA-Seq and metabolite analyses all support a role for IAA and ABA in heading type formation. These findings increase our understanding of the molecular basis for pattern formation of the leafy head in Chinese cabbage and will contribute to future work developing more desirable leafy head patterns.

Identification of BABY BOOM homolog in bread wheat

Modern breeding practice of small grain cereals necessitates the development of an efficient system for the large scale and reproducible production of the doubled haploid (DH) lines. It is believed that among the available DH generation techniques, only isolated microspore culture (IMC) can satisfy the demand of public and private breeding programs. Unfortunately, the IMC method is prone to several challenges that jeopardizes its large scale adoption. One of the approaches to limit the variation in DH plant production and increase the efficiency of the method is manipulation of embryogenesis-related genes. Here we set up a study to map BABY BOOM in a bread wheat genome. The gene is one of the morphogenic regulators of somatic embryogenesis in plants. To achieve this task, we used amino acid sequences of Zea mays BBM-like proteins. TaBBM homoeologs were mapped to chromosomes 6AL, 6BL and 6DL. Amino acid sequence analysis revealed the presence of two AP2 domains and bbm-1 motif in the A and D copies and only one AP2 domain and bbm-1 motif in the B copy. This, along with the absence of both gene expression and predictable TATA-box, suggests that TaBBM-gB is a pseudogene. The expression pattern of the identified A and D homoeologs was similar to that for the BBM-like genes in other species and presence of the transcript was detected in an embryogenic microspore population. Identification of the TaBBM homolog can have application in elevating the efficiency of DH production, tissue culture, plant transformation and genome editing for wheat improvement.

Production and conversion of haploid embryos in chickpea (Cicer arietinum L.) anther cultures using high 2,4-D and silver nitrate containing media

Due to recalcitrant nature of chickpea (Cicer arietinum L.) to androgenesis, the production of double haploid plants has been only reported by Grewal et al. (Plant Cell Rep 28:1289–1299, 2009) using some physical stresses such as anther centrifugation and electrical shock. In the present study, we successfully obtained haploid plants from cultured anthers of two chickpea cultivars, Bivanij and Arman, using high 2,4-D and silver nitrate containing media without applying of these time and labor consuming stresses. For induction of androgenesis, different concentrations of 2, 4-D (0, 2, 5 and 10 mg/l) and silver nitrate (0, 5, 10, 15, 25 and 50 mg/l) were used in embryo development medium. In Bivanij cultivar, anther induction medium containing 10 mg/l 2,4-D and 15 mg/l silver nitrate produced the highest number of embryos (0.188) and regenerated plants (0.1) per each cultured anther, while the highest frequencies of embryos (0.1) and regenerated plants (0.075 and 0.063) were obtained from Arman cultivar when 10 mg/l 2,4-D was combined with 15 and 50 mg/l silver nitrate in anther culture medium, respectively. In second part of this study, different cold (4 °C for 4 and 7 days) and heat (30 °C for 10 days, 32 °C for 2 days and 35 °C for 8 h) pretreatments were applied on cultured anthers of Bivanij cultivar. Incubation of cultured anthers at 32 °C for 2 days significantly enhanced the rate of embryo formation up to 0.222 embryos per each anther, while the highest number of regenerated plants/anther (0.0332) was obtained when cold treated anthers at 4 °C for 7 days incubated at 30 °C for 10 days. Taken together, these results provide a good basis for large-scale generation of DH plants in this important legume species.

Inheritance patterns of the response to in vitro doubled haploid induction in perennial ryegrass (Lolium perenne L.)

The ability to produce doubled haploid (DH) plants has found broad application in research and breeding. For major crop species such as maize (Zea mays L.) and barley (Hordeum vulgare L.), routine large-scale production of DHs has enabled the acceleration of breeding processes, for example through efficient generation of homozygous lines. However, in forage crops such as perennial ryegrass (Lolium perenne L.), low and genotype-specific responses to in vitro anther culture (AC) still limit wide-spread use of DHs. Here, we report the responses of nine bi-parental populations, segregating for microspore embryogenesis and plant regeneration capacity, to an effective AC protocol. Genotypes of exceptionally high androgenic ability, producing over 200 green plants per 100 anthers cultured, could be selected. Continuous and distinctly shaped distributions for the evaluated traits were indicative of quantitative polygenic control and the presence of different alleles in each population. An insignificant association of embryo production with plant regeneration, as well as a low correlation between green and albino plant yield (ρ = 0.20), suggested that different genes influence these traits. The populations evaluated here provide a rich source of alleles needed for the introgression of high levels of androgenic capacity into recalcitrant material. Moreover, this germplasm is ideally suited for use in future genotyping and mapping studies so that the genetic control of androgenic capacity in perennial ryegrass can be elucidated. Ultimately, our results will help to realize the potential of DH induction in one of the world’s most important forage crop species.

Adsorption of cell-penetrating peptide Tat2 and polycation luviquat FC-370 to triticale microspore exine

Microspores are the pre-gametophyte stage of pollen, and have proven to be a successful tissue culture material for the production of doubled haploid plants. Microspore culture has also been used as a platform for the production of transgenic plants. The use of cell-penetrating peptides (CPPs) as transfection agents in microspores has been previously demonstrated, but at low efficiencies. Here, the pH dependent adsorption of the cationic CPP Tat2 to the sporopollenin surface of the microspore (the exine) has been explored using electrophoretic light scattering (ELS). Furthermore the adsorption of a commercially available polycationic polymer; Luviquat FC-370 (polyquaternium D-16, PQ-D16) was similarly measured using ELS. It was found that a suspension media with a pH less than 7.0 showed an approximately ten fold decrease in the amount of Tat2 that was required before apparent surface neutralization. This data suggests that a change in the surface chemistry of the microspore occurs in acidic pH conditions, that modulates the binding affinity of the CPP Tat2 in a non-trivially complex manner.

Production of doubled haploid plants from anther cultures of borage (Borago officinalis L.) by the application of chemical and physical stress

Anther culture can be used as a powerful tool to produce doubled haploid (DH) lines in medicinal plants, thus accelerating breeding programs. In the particular case of borage (Borago officinalis L.), a method to produce DH plants has not been yet published. In this work we evaluated the effect of different culture media and of different chemical (colchicine and n-butanol) and physical stresses (centrifugation and electroporation) on androgenesis induction and plant regeneration in borage anther cultures. We found that the highest response can be obtained with culture medium containing B5 salts and NLN vitamins, the addition of 200 mg/l colchicine during 4 days, a pretreatment of anthers with 0.2% n-butanol for 5 h, or the application to anthers of single physical stresses (either centrifugation at 300×g or a 100 V electrical shock, but not combined). This is the first report on the production of DH plants in borage. Together, the results presented hereby can be used as a basic framework for large-scale generation of DH plants in this important medicinal species.

Reduced ascorbate and reduced glutathione improve embryogenesis in broccoli microspore culture

Isolated microspore culture (IMC) is an effective alternative technique for the production of doubled haploid (DH) homozygous lines in plant breeding. However, death of most of the cultured cells during culture is a critical obstacle that affects embryo production in the practical application of IMC technology. In this present paper, we evaluated the effects of two antioxidants of both reduced ascorbate (ASC) and reduced glutathione (GSH) on microspore viability and the induction of embryogenesis in the IMC of broccoli (Brassica oleracea L. var. italica). The results showed that cell death occurred soon after stress treatment (at 32.5°C for 24h), and the number of dead cells reached about 80% after 2d. The addition of 10mgl−1 ASC and 20mgl−1 GSH significantly decreased microspore mortality, and had a strong effect on the number of embryos produced. In the genotype ‘B415’, embryo yields increased approximately 1.2-fold and 2.5-fold after the addition of the 10mgl−1 ASC and 20mgl−1 GSH, respectively. Moreover, the addition of ASC and GSH also influenced positively the rate of plant regeneration, which respectively increased by 4.2% and 9.7% for ‘B415’. Taken together, our results show that the induction of embryogenesis in broccoli is strongly affected by the use of ASC or GSH. The effects on embryogenesis induction could confirm the importance of the redox environmental regulation in IMC, specifically at the early phase of IMC. The obtained knowledge may accelerate and guide the application of microspore-derived DH plants as a breeding tool in broccoli.

Kultur Antera untuk Mendapatkan Galur Padi Toleran Salinitas

<p>ABSTRACT<br /><br />Haploid breeding through anther culture allows shortening of the breeding cycle and production of homozygous lines from a segregating population in the immediate generation. This technique has been used for crop improvement especially in rice. The objective of this research was to determine regeneration ability of twelve F1s, derived from reciprocal crossing between high yielding rice variety and rice tolerance to salinity, through anther culture. Completely randomized design with 20 replications was used in this research. Medium for callus induction was based on N6 medium + 2.0 mg NAA L-1 + 0.5 mg kinetin L-1 + 1 mM putrescine, while regeneration medium was based on MS + 0.5 mg NAA L-1 + 2.0 mg kinetin L-1 + 1 mM putrescine. Rooting were done in MS medium + 0.5 mg IBA L-1 + 1 mM putrescine. The result indicated that F1 derived from IR77674/Inpari 29 (3.1% green plants/total anther) was the most responsive genotypes in rice anther culture (high anther culture ability). After greenhouse grow out 125 putative double haploid plants were obtained (41.5% from total acclimated green plantlets). <br /><br />Keywords: double haploid, green planlets, indica rice, salt tolerance <br /><br /></p>

RNA-Guided Cas9-Induced Mutagenesis in Tobacco Followed by Efficient Genetic Fixation in Doubled Haploid Plants.

Customizable endonucleases are providing an effective tool for genome engineering. The resulting primary transgenic individuals (T0) are typically heterozygous and/or chimeric with respect to any mutations induced. To generate genetically fixed mutants, they are conventionally allowed to self-pollinate, a procedure which segregates individuals into mutant heterozygotes/homozygotes and wild types. The chances of recovering homozygous mutants among the progeny depend not only on meiotic segregation but also on the frequency of mutated germline cells in the chimeric mother plant. In Nicotiana species, the heritability of Cas9-induced mutations has not been demonstrated yet. RNA-guided Cas9 endonuclease-mediated mutagenesis was targeted to the green fluorescent protein (GFP) gene harbored by a transgenic tobacco line. Upon retransformation using a GFP-specific guide RNA/Cas9 construct, the T0 plants were allowed to either self-pollinate, or were propagated via regeneration from in vitro cultured embryogenic pollen which give rise to haploid/doubled haploid plants or from leaf explants that form plants vegetatively. Single or multiple mutations were detected in 80% of the T0 plants. About half of these mutations proved heritable via selfing. Regeneration from in vitro cultured embryogenic pollen allowed for homozygous mutants to be produced more efficiently than via sexual reproduction. Consequently, embryogenic pollen culture provides a convenient method to rapidly generate a variety of genetically fixed mutants following site-directed mutagenesis. The recovery of a mutation not found among sexually produced and analyzed progeny was shown to be achievable through vegetative plant propagation in vitro, which eventually resulted in heritability when the somatic clones were selfed. In addition, some in-frame mutations were associated with functional attenuation of the target gene rather than its full knock-out. The generation of mutants with compromised rather than abolished gene functionality holds promise for future approaches to the conclusive functional validation of genes which are indispensible for the plant.

Doubled Haploid Laboratory Protocol for Wheat Using Wheat-Maize Wide Hybridization.

In traditional wheat breeding, the uniformity of lines derived from a breeding population is obtained by repeated selfing from the F1 which takes several generations to reach homozygosity in loci controlling traits of interest. Using doubled haploid technology, however, it is possible to attain 100% homozygosity at all loci in a single generation and completely homogeneous breeding lines can be obtained in 1-2years. Thus, doubled haploid technology may significantly reduce cultivar development time. Two major methods for producing wheat doubled haploids are androgenesis (anther culture and microspore culture) and embryo culture using wheat-maize wide hybridization, the latter being the most effective and widely used method. The method of wide hybridization between wheat and maize is laborious but is widely successful for rapidly obtaining homozygous lines. This technique includes six major steps: emasculation of the wheat flower; pollination of the emasculated flower with maize pollen; hormone treatment; embryo rescue; haploid plant regeneration in tissue culture medium; and chromosome doubling. It has been observed that the efficiency of doubled haploid production depends on both maize and wheat genotypes, good plant health and proper greenhouse conditions (without disease, insects, or drought stress), and proper conduct of all procedures. Therefore, the procedures may need minor modification in order to produce higher numbers of embryos, haploid green plants, and doubled haploid plants.

High Frequency of Doubled Haploid Plant Production in Spelt Wheat

AbstractThis is the first study to report an efficient anther culture (AC) method for spelt wheat, which has an increasing importance not only in applied research but also in organic farming and changing nutritional standards. In this study, an efficient AC protocol has been described for ‘GK Fehér’ spelt wheat. The number of AC-derived embryo-like structures (ELS) was 62.2/100 anthers, from which we were able to regenerate 30.6 green plantlets per 100 anthers. The percentage of green plantlets production was 89.0% among the regenerated plantlets, while the phenomenon of albinism was restricted (3.8/100 anthers). Altogether, from AC of ‘GK Fehér’ 306 green plantlets were producedin vitroand 241 plants were acclimatized to the greenhouse conditions. Based on ploidy level analyses, 83 spontaneous doubled haploid (DH) plants were produced (8.3 DH plants/100 anthers), so the percentage of spontaneous rediploidization was 34.4%. The spontaneous DH plants produced fertile spikes, while a few seeds were harvested from seven partially fertile plants.

Regenerasi dan Aklimatisasi Kultur Antera Enam Persilangan F1 Padi Sawah

ABSTRACT<br /><br />The breeding of rainfed rice tolerant to drought can be accomplished using anther culture. The objectives of this research were to determine regeneration abilities of six F1 anther culture and its acclimatization ability. The experiment was arranged in completely randomized design with 14 replications. The treatments consisted of six F1 derived from crossing: INPARI 18 x IR83140-B-11-B (G1), INPARI 18 x B12825E-TB-1-25 (G2), INPARI 18 x IR87705-14-11-B-SKI-12 (G3), INPARI 22 x IR83140-B-11-B (G4), Bio-R81 x O18b-1 (G5), Bio-R82-2 x O18b-1 (G6). Media for callus induction was based on N6 medium + 2.0 mg L-1 NAA + 0.5 mg L-1 kinetin + 1.0 mM putresin + 60 g L-1 sucrosa, media for regeneration was based on MS + 0.5 mg L-1 NAA + 2.0 mg L-1 kinetin + 1.0 mM putresin, and media for rooting was based on MS + 0.5 mg L-1 IBA + 30 g L-1 sucrosa. The result indicated that all six F1 had different ability in anther culture. Bio-R82-2 x O18-b1 (G6) and Bio-R81 x O18-b1 (G5) F1 genotype had good response both of callus induction and plant regeneration. These two F1 genotypes also gave the highest ratio of green planlet production to number of anther inoculated (GP:AI) were 5.50% and 4.65%, respectively. In this research, there were identified doubled haploid plants were developed from 4 F1 derived cross namely G2 (2 plants), G3 (4 plants), G5 (21 plants), and G6 (26 plants).<br /><br />Keywords: Callus induction, doubled haploid, rice<br /><br />

Fully-Automated High-Throughput NMR System for Screening of Haploid Kernels of Maize (Corn) by Measurement of Oil Content.

One of the modern crop breeding techniques uses doubled haploid plants that contain an identical pair of chromosomes in order to accelerate the breeding process. Rapid haploid identification method is critical for large-scale selections of double haploids. The conventional methods based on the color of the endosperm and embryo seeds are slow, manual and prone to error. On the other hand, there exists a significant difference between diploid and haploid seeds generated by high oil inducer, which makes it possible to use oil content to identify the haploid. This paper describes a fully-automated high-throughput NMR screening system for maize haploid kernel identification. The system is comprised of a sampler unit to select a single kernel to feed for measurement of NMR and weight, and a kernel sorter to distribute the kernel according to the measurement result. Tests of the system show a consistent accuracy of 94% with an average screening time of 4 seconds per kernel. Field test result is described and the directions for future improvement are discussed.

Identification of loci associated with embryo yield in microspore culture of Brassica rapa by segregation distortion analysis.

We identified three physical positions associated with embryo yield in microspore culture of Brassica rapa by segregation distortion analysis. We also confirmed their genetic effects on the embryo yield. Isolated microspore culture is well utilized for the production of haploid or doubled-haploid plants in Brassica crops. Brassica rapa cv. 'Ho Mei' is one of the most excellent cultivars in embryo yield of microspore culture. To identify the loci associated with microspore embryogenesis, segregation analysis of 154 DNA markers anchored to B. rapa chromosomes (A01-A10) was performed using a population of microspore-derived embryos obtained from an F1 hybrid between 'CR-Seiga', a low yield cultivar in microspore-derived embryos, and 'Ho Mei'. Three regions showing significant segregation distortion with increasing 'Ho Mei' alleles were detected on A05, A08 and A09, although these regions showed the expected Mendelian segregation ratio in an F2 population. The additive effect of alleles in these regions on embryo yield was confirmed in a BC3F1 population. One region on A08 containing Br071-5c had a higher effect than the other regions. Polymorphism of nucleotide sequences around the Br071-5c locus was investigated to find the gene possibly responsible for efficient embryogenesis from microspores.

Structural Equation Model as a Tool to Assess the Relationship Between Grain Yield Per Plant and Yield Components in Doubled Haploid Spring Barley Lines (Hordeum vulgare L.)

Abstract The aim of this study was to describe and characterize the relationships between yielding factors and grain yield per doubled haploid (DH) plant of spring barley as well as relation between yield components and duration of each stage of plant development. To describe these relations structure equation modeling was used. The study included plants of doubled haploid spring barley lines (Hordeum vulgare L.) derived from two-rowed form of Scarlett cultivar. The SAS® system was used to analyze the model of relationships between grain yield per plant and yield components. Our results indicate that the number of spikes per plant and grain yield per spike had a direct and decisive influence on the grain yield of the investigated DH plants of spring barley. Based on the path model analysis it was found that the most important factor determining grain yield per DH plants of spring barley was the number of spikes per plant and the duration of tillering and shooting stages.