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To facilate breeding process of Brassica napus, a microspore culture and molecular marker-assisted screening combined system were proposed in this research. At early flowering stage, F1 offspring of hybridized combination HY15A×HF06 was used as donor for microspore culture to analyze effects of colchicine concentration on embryogenic and diploid rates of microspore. Treatment with 50 mg/L colchicine resulted in embryogenic rate of 3.56 embryos/bud, which was substantially higher than control (0.78 embryos/bud). A total of 1387 embryos were obtained and 862 single plants were obtained after induction culture. Ploidy detection was performed for the regenerated plants by flow cytometry. Diploid rates of microspores treated with 50 mg/L and 70 mg/L colchicine were 17.2% and 21.0% respectively, which was significantly higher than control (10.5%). A total of 108 single plants that doubled successfully were randomly selected and screened using molecular marker BE10. Approximately 54 of 108 plants generated a 305 bp amplification product, whereas the other 54 plants showed a 398 bp band, thereby satisfying 1:1 separation ratio (x20.05=0.0093). These results coincided with field identification results. Findings of this study indicated that homozygous breeding material could be obtained by microspore culture in a short time, thereby remarkably accelerate the breeding.

Production of homozygous lines derived from transgenic plants is one of the important steps for phenotyping and genotyping transgenic progeny. The selection of homozygous plants is a tedious process that can be significantly shortened by androgenesis, cultivation of anthers, or isolated microspores. Doubled haploid (DH) production achieves complete homozygosity in one generation. We obtained transgenic homozygous DH lines from six different transgenic events by using anther culture. Anthers were isolated from T0 transgenic primary regenerants and cultivated in vitro. The ploidy level was determined in green regenerants. At least half of the 2n green plants were transgenic, and their progeny were shown to carry the transgene. The process of dihaploidization did not affect the expression of the transgene. Embryo cultures were used to reduce the time to seed of the next generation. The application of these methods enables rapid evaluation of transgenic lines for gene function studies and trait evaluation.

Production of homozygous lines derived from transgenic plants is one of the important steps for phenotyping and genotyping transgenic progeny. The selection of homozygous plants is a tedious process that can be significantly shortened by androgenesis, cultivation of anthers, or isolated microspores. Doubled haploid (DH) production achieves complete homozygosity in one generation. We obtained transgenic homozygous DH lines from six different transgenic events by using anther culture. Anthers were isolated from T0 transgenic primary regenerants and cultivated in vitro. The ploidy level was determined in green regenerants. At least half of the 2n green plants were transgenic, and their progeny were shown to carry the transgene. The process of dihaploidization did not affect the expression of the transgene. Embryo cultures were used to reduce the time to seed of the next generation. The application of these methods enables rapid evaluation of transgenic lines for gene function studies and trait evaluation.

This review provides a summary of haploid induction methods and factors affecting the efficacy of specific methodologies as applied to cucumber (Cucumis sativus L.), melon (Cucumis melo L.), watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai), winter squash (Cucurbita maxima Duch. ex Lam.), summer squash (Cucurbita pepo L.) and other cucurbits. This report is focused on studies that were carried out during the last 20 years. The main objective of the research on the production of haploid cucurbit plants is to accelerate breeding programs through the use of homozygous double haploid lines (DHL) and to facilitate the selection of desired (e.g. disease-resistant) genotypes for breeding. Unfortunately, currently used protocols result in a low number of double haploids (DH). The most common and best-known method of obtaining haploid cucurbit plants is via pollination with irradiated pollen, which induces parthenogenetic development of haploid embryos in planta. The embryos are extracted from immature seeds and cultured in vitro to facilitate the maturation and development of plants. The studies described below were primarily aimed at the determination of an appropriate dose of radiation and the evaluation of the impact of the genotype and the time of year on the number of haploid embryos and plants obtained. A less popular method of haploid production - ovule and ovary culture - is based on in vitro gynogenesis. The studies related to this method concentrated on optimising the composition of the medium and pre-treatment conditions (primarily temperature) to which the flower buds were subjected. Recently, increasing attention has been paid to anther and microspore culture. As in the case of in vitro ovule and ovary culture, the medium composition and flower bud pre-treatment conditions were optimised. The most recent studies suggest that anther culture is comparable in effectiveness to the irradiated pollen technique.

Chrysanthemum is one of important ornamental species in the world. Its highly heterozygous state complicates molecular analysis, so it is of interest to derive haploid forms. A total of 2,579 non-fertilized chrysanthemum ovules pollinated by Argyranthemum frutescens were cultured in vitro to isolate haploid progeny. One single regenerant emerged from each of three of the 105 calli produced. Chromosome counts and microsatellite fingerprinting showed that only one of the regenerants was a true haploid. Nine doubled haploid derivatives were subsequently generated by colchicine treatment of 80 in vitro cultured haploid nodal segments. Morphological screening showed that the haploid plant was shorter than the doubled haploids, and developed smaller leaves, flowers and stomata. An in vitro pollen germination test showed that few of the haploid's pollen were able to germinate and those which did so were abnormal. Both the haploid and the doubled haploids produced yellow flowers, whereas those of the maternal parental cultivar were mauve. Methylation-sensitive amplification polymorphism (MSAP) profiling was further used to detect alterations in cytosine methylation caused by the haploidization and/or the chromosome doubling processes. While 52.2% of the resulting amplified fragments were cytosine methylated in the maternal parent's genome, the corresponding proportions for the haploid's and doubled haploids' genomes were, respectively, 47.0% and 51.7%, demonstrating a reduction in global cytosine methylation caused by haploidization and a partial recovery following chromosome doubling.

The selection of desirable genotypes with recessive characteristics, such as self-incompatible plants, is often difficult or even impossible and represents a crucial barrier in accelerating the breeding process. Molecular approaches and selection based on molecular markers can allow breeders to overcome this limitation. The use of self-incompatibility is an alternative in hybrid breeding of oilseed rape. Unfortunately, stable self-incompatibility is recessive and phenotype-based selection is very difficult and time-consuming. The development of reliable molecular markers for detecting desirable plants with functional self-incompatible genes is of great importance for breeders and allows selection at early stages of plant growth. Because most of these reliable molecular markers are based on discrimination of class I S-locus genes that are present in self-compatible plants, there is a need to use an internal control in order to detect possible PCR inhibition that gives false results during genotyping. In this study, 269 double haploid F2 oilseed rape plants obtained by microspore embryogenesis were used to verify the applicability of an improved PCR assay based on the detection of the class I SLG gene along with an internal control. Comparative analysis of the PCR genotyping results vs. S phenotype analysis confirmed the applicability of this molecular approach in hybrid breeding programs. This approach allows accurate detection of self-incompatible plants via a different amplification profile.

10 Págs., 3 figs. The definitive version is available at: http://link.springer.com/journal/497, Microspore embryogenesis involves reprogramming of the pollen immature cell towards embryogenesis. We have identified and characterized a collection of 14 genes induced along different morphological phases of microspore-derived embryo development in wheat (Triticum aestivum L.) anther culture. SERKs and FLAs genes previously associated with somatic embryogenesis and reproductive tissues, respectively, were also included in this analysis. Genes involved in signalling mechanisms such as TaTPD1-like and TAA1b, and two glutathione S-transferase (GSTF2 and GSTA2) were induced when microspores had acquired a 'star-like' morphology or had undergone the first divisions. Genes associated with control of plant development and stress response (TaNF-YA, TaAGL14, TaFLA26, CHI3, XIP-R; Tad1 and WALI6) were activated before exine rupture. When the multicellular structures have been released from the exine, TaEXPB4, TaAGP31-like and an unknown embryo-specific gene TaME1 were induced. Comparison of gene expression, between two wheat cultivars with different response to anther culture, showed that the profile of genes activated before exine rupture was shifted to earlier stages in the low responding cultivar. This collection of genes constitutes a value resource for study mechanism of intra-embryo communication, early pattern formation, cell wall modification and embryo differentiation. © 2013 Springer-Verlag Berlin Heidelberg., RA Sánchez-Díaz was recipient of a predoctoral fellowship, from Junta Ampliación de Estudios, Consejo Superior de Investigaciones Científicas (JAE-CSIC) of Spain. This work was supported by Project AGL2010-17509, from ‘Plan Nacional de Recursos y Tecnologías Agroalimentarias’ of Spain and by COST Action FAO0903 ‘Harnessing of Reproduction for Plant Improvement’ (HAPRECI)

The objective of the study was to investigate efficiency of anther culture in the production of spontaneous double haploids from randomly selected heterozygous genotypes of wheat (Triticum aestivum L.). Anthers of 20 F1 wheat combinations were grown in vitro on a modified Potato-2 medium. All of the examined genotypes have shown the ability to produce pollen calluses as well as to regenerate green plants. On average for the whole experiment material, 47.2 calluses were produced per 100 cultured anthers. The green plant regeneration ranged from 0.8 to 13.4 green plants per spike, with an overall mean of 5.8. From the total of 582 regenerated green plants, 47.9% (279) were spontaneous double haploids. The final average yield from the study was 2.8 double haploids per spike. Cilj rada bio je da se ispita efikasnost kulture antera u proizvodnji spontanih dvostrukih haploida iz slučajno odabranih heterozigotnih genotipova pšenice (Triticum aestivum L.). Antere iz 20 F1 kombinacija pšenice gajene su in vitro na modifikovanoj Potato-2 podlozi. Svi ispitivani genotipovi pokazali su sposobnost da proizvedu kaluse, kao i da regenerišu zelene biljke. U proseku za ceo eksperimentalni materijal, proizvedena su 47.2 kalusa na 100 izolovanih antera. Regeneracija zelenih biljaka kretala se od 0.8 do 13.4 zelene biljke po klasu, sa ukupnim prosekom od 5.8. Od ukupno 582 regenerisane zelene biljke, 47.9% (279) bile su spontani dvostruki haploidi. U proseku, u ovom istraživanju, proizvedeno je 2.8 DH biljaka po klasu.

The objective of the study was to investigate the production of homozygous doubled haploid (DH) lines from heterozygous genotypes of wheat (Triticum aestivum L.) using an anther culture. Anthers were isolated from 20 randomly selected F1 wheat combinations and grown in vitro on a modified Potato-2 inductive medium. Plant regeneration was performed on the modified 190-2 medium. The results have shown that all genotypes had the ability to produce pollen calli, as well as, to regenerate green plants. The hybrid Mex.3 x MV 18 had the highest value for androgenous capacity (40.6%), while the combination Kutječanka x Slavija had the lowest androgenous capacity (6.3%). The average androgenous capacity of all genotypes was 19.4%. The regeneration frequency ranged from 1.3 to 21.6 green plants per 100 isolated anthers, with an overall mean of 9.6%. Out of a total of 582 regenerated green plants 279 (47.9%) were spontaneous double haploids. The average production of double haploids of wheat in this study amounted to 4.6 per 100 cultured anthers. Cilj rada bio je da se ispita mogućnost proizvodnje homozigotnih linija dvostrukih haploida (DH) iz heterozigotnih genotipova pšenice (Triticum aestivum L.) korišćenjem kulture antera. Antere su izolovane iz 20 slučajno odabranih F1 kombinacija pšenice i gajene u kulturi in vitro na modifikovanoj potato-2 indukcionoj podlozi. Regeneracija biljaka vršena je na modifikovanoj 190-2 podlozi. Rezultati su pokazali da su svi genotipovi posedovali sposobnost da proizvedu kaluse iz polena, kao i da regenerišu zelene biljke. Hibrid Mex.3 x MV 18 je imao najvišu vrednost za androgeni kapacitet (40,6%), dok je kombinacija Kutječanka x Slavija imala najniži androgeni kapacitet (6,3%). Prosečan androgeni kapacitet za sve genotipove bio je 19,4%. Frekvencija za regeneraciju zelenih biljaka kretala se od 1,3 do 21,6 zelenih biljaka na 100 izolovanih antera, sa prosekom od 9,6%. Od ukupno 582 regenerisane zelene biljke, 279 (47,9%) su bile spontani dvostruki haploidi. U ovom istraživanju prosečno je proizvedeno 4,6 dvostrukih haploida pšenice na 100 izolovanih antera.

WOS: 000483480000001, Advanced marker technologies are widely used for evaluation of genetic diversity in cultivated crops, wild ancestors, landraces or any special plant genotypes. Developing agricultural cultivars requires the following steps: (i) determining desired characteristics to be improved, (ii) screening genetic resources to help find a superior cultivar, (iii) intercrossing selected individuals, (iv) generating genetically hybrid populations and screening them for agro-morphological or molecular traits, (v) evaluating the superior cultivar candidates, (vi) testing field performance at different locations, and (vii) certifying. In the cultivar development process valuable genes can be identified by creating special biparental or multiparental populations and analysing their association using suitable markers in given populations. These special populations and advanced marker technologies give us a deeper knowledge about the inherited agronomic characteristics. Unaffected by the changing environmental conditions, these provide a higher understanding of genome dynamics in plants. The last decade witnessed new applications for advanced molecular techniques in the area of breeding, with low costs per sample. These, especially, include next-generation sequencing technologies like reduced representation genome sequencing (genotyping by sequencing, restriction site-associated DNA). These enabled researchers to develop new markers, such as simple sequence repeat and single- nucleotide polymorphism, for expanding the qualitative and quantitative information on population dynamics. Thus, the knowledge acquired from novel technologies is a valuable asset for the breeding process and to better understand the population dynamics, their properties, and analysis methods.