The production of homozygous lines in citrus breeding programs by using conventional methods is difficult and time-consuming mainly because the problems related to the reproductive and biological characteristics of these species. The main characteristics are the polyembriony, longer juvenile period, high level of heterozygozity, sterility and some degree of auto-incompatibility. One faster possibility is to produce haploid plants, following to natural or artificial diploidization that will result in double-haploid plants, in one reproductive cycle only. The double-haploid plants can be use to increase the efficiency of citrus breeding programs, particularly for identification of QTL´s that are associated to quantitative characteristics, in genetic mapping researchs. The main techniques used to obtain haploid or double-haploid plants can be anther and microspore cultures and in situ parthenogenesis. These techniques were well developed to mandarins. Many factors that affect citrus anther culture were genotypes, anther pre-treatments, media and growth conditions of the donor plants. For in situ parthenogenesis experiments, it was also identified some factors that can affect the capacity to obtain haploid plants: genotype, irradiation doses used for pollen grains inactivation, stage of the embryos development at the moment of the rescue (mature and immature), media and in vitro growth conditions. The objectives of this project are to develop protocols for anther culture and in situ parthenogenesis of sweet orange varieties in order to obtain haploid and double-haploid plants. Several experiments of anther culture and in situ parthenogenesis will be installed to verify the effects of media, genotype and other factors, in callus, embryos and haploid plant regeneration. The regenerated plants will have its ploidy determined using flow citometry method. The diploidy level will be restored in haploid plants by the method of in vitro culture in medium containing colchicine. All plants obtained directly of experiments of anther culture and/or in situ parthenogenesis will be evaluated by molecular marker method to identify its origin, zygotic or nucellar.
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