Hamptonmclean4783

This approach also enabled us to discriminate between homozygous and heterozygous or chimeric mutants. The heritability of induced mutations and their homozygous state were experimentally confirmed by progeny analyses. The major advantage of the method lies in the preferential production of genetically fixed primary mutants, which facilitates immediate phenotypic analyses and, relying on that, a particularly efficient preselection of valuable lines for detailed investigations using their progenies.The production of doubled haploids (DHs) has proved to be a highly valuable tool to obtain new cultivars. Among the cereals, barley (Hordeum vulgare L.) is the most successful species in large-scale haploid production. Techniques employed for this purpose are based on either the gynogenetic or the androgenetic pathway. Interspecific cross with Hordeum bulbosum L., haploid gene inducer (the hap gene), ovary culture, anther culture (AC), and isolated microspore culture (IMC) are the most used methods. Among all of them, IMC is regarded as a particularly effective system owing to the great increase in green plant numbers per spike and also the higher induction of chromosome doubling when compared with other methods. Thus, IMC provides the best way to mass scale production of new varieties.Leek (A. ampeloprasum L.) is an economically important vegetable crop from Alliaceae family. It is a non-bulb forming biennial species grown for its pseudostem and leaves. Mitoquinone molecular weight Leek is a tetraploid with one of the largest genomes known among cultivated plant species. It has enormous economic importance all around the world for many purposes such as vegetable, medicinal herb, and food seasoning. Production and consumption of leek is in rise all around the world and breeders are trying to develop new F1 hybrid varieties with desired agronomical traits. Although self-compatible, leek shows high tendency toward outcrossing and display severe inbreeding depression when selfed with its own pollen. Therefore, inbred development through classical breeding techniques is very difficult in this crop. Traditional leek genotypes are highly heterozygous, open pollinated varieties. There is a high demand for F1 hybrid varieties with resistance to biotic and abiotic stresses and high-quality plants. Our group is trying to incorporate gynogenesis-based doubled haploid technology to leek improvement programs. Over the years, many experiments were carried out to determine the gynogenic potential of donor leek genotypes of different genetic backgrounds in different induction media. Here, we report a protocol allowing production of green gynogenic leek plants via single step culture of unopened flower buds. Ploidy levels of gynogenic regenerants are determined by flow cytometry analysis. A majority of the gynogenic leek regenerants produced survived well in vivo.Onion (A. cepa L.) is an outcrossing biennial species with a very large genome. Development of genetically uniform (inbred) lines highly desired by onion breeders is a difficult process due to high level of heterozygosity. Inbred onion development may take up to five generations (~10 years) by classical selfing technique. Onion shows severe inbreeding depression, which further complicates production of lines with stabilized important agronomic traits. When applied successfully, haploidization technology can be useful in the development of fully homozygous onion lines in 2 years. Although production of haploid and doubled haploid (DH) onions via gynogenesis was reported more than three decades ago, successful production and utilization of DHs in onion breeding is still far behind of expectations of breeders. The main obstacles in front of the success include high variation in the response of donor materials to gynogenesis induction and difficulties faced in the process of obtaining DHs from haploid plants. We use a DH production procedure enabling us to develop DH plants from a wide range of onion donor materials. This procedure is based on production of haploid plants via single step culture of unopened flower buds, detection of haploid plants among gynogenic regenerants, and converting these plants to fecund DHs using a combination of ploidy manipulation techniques. The bulbs of DHs are produced in about 1 year after the initiation of induction cultures and selfed seeds are produced from fecund DH plants when they flower in the second year.The completely homozygous genetic background of doubled haploids (DHs) has many applications in breeding programs and research studies. Haploid induction and chromosome doubling of induced haploids are the two main steps of doubled haploid creation. Both steps have their own complexities. Chromosome doubling of induced haploids may happen spontaneously, although usually at a low rate. Therefore, artificial/induced chromosome doubling of haploid cells/plantlets is necessary to produce DHs at an acceptable level. The most common method is using some mitotic spindle poisons that target the organization of the microtubule system. Colchicine is a well-known and widely used antimitotic. However, there are substances alternative to colchicine in terms of efficiency, toxicity, safety, and genetic stability, which can be applied in in vitro and in vivo pathways. Both pathways have their own advantages and disadvantages. However, in vitro-induced chromosome doubling has been much preferred in recent years, maybe because of the dual effect of antimitotic agents (haploid induction and chromosome doubling) in just one step, and the reduced generation of chimeras. Plant genotype, the developmental stage of initial haploids, and type-concentration-duration of application of antimitotic agents, are top influential parameters on chromosome doubling efficiency. In this review, we highlight different aspects related to antimitotic agents and to plant parameters for successful chromosome doubling and high DH yield.Determination of the ploidy level is an essential step when trying to produce doubled haploids (DHs) in any species. Each species and method used to produce DHs has its own frequency of DH production, which means that the rest of plants produced stay haploid. Since haploids are of little use for breeding purposes, it is necessary to distinguish them from true DHs. For this, several methodologies are available, including flow cytometry, chromosome counting, chloroplast counting in stomatal guard cells, measurement of stomatal size and length, counting of nucleoli, evaluation of pollen formation and viability, analysis of cell size, and analysis of morphological markers. However, not all of them are equally easy to use, affordable, reliable, reproducible, and resolutive and therefore useful for a particular case. In this chapter, we revise these methods available to assess the ploidy level of plants, discussing their respective advantages and limitations, and provide some troubleshooting tips and hints to help decide which to choose in each case.