Martinsencarney2175

Cork oak (Quercus suber L.) is a forest tree species of the family Fagaceae. It is characterized by long life cycles which hamper doubled haploid plant production to obtain homozygotes and pure lines. The time-consuming method of repeated backcrossings by conventional breeding techniques to produce pure lines is impractical in woody species. Nevertheless, biotechnology has offered new tools to make it possible. A doubled haploid plant or embryo is one that is developed by the doubling of a haploid set of chromosomes. A protocol to produce doubled haploids of cork oak has been developed through microspore embryogenesis. By a heat stress treatment, the microspores inside the anther leave the gametophytic pathway and react shifting their development to the sporophytic pathway by means of which haploid embryos are obtained. Chromosome duplication of haploids from cork oak anther cultures occurs either spontaneously or may be induced by the application of antimitotic agents (e.g., colchicine, oryzalin, amiprophos-methyl). Furthermore, a genetic test is designed through microsatellite markers to elucidate whether the diploid embryos obtained are originally haploids which spontaneously duplicated their genome, or alternatively those embryos are generated from the diploid tissue of the anther wall. Here we describe a detailed protocol to produce doubled haploid individuals from cork oak anther cultures by using temperature stress and antimitotic agents.This chapter deals with induction of haploidy via parthenogenesis in Persian walnut and via microspore embryogenesis in almond and hazelnut. Haploid induction through in situ parthenogenesis using pollination with irradiated pollen to stimulate the embryogenic development of the egg cell, followed by in vitro culture of the immature haploid embryos. Microspore embryogenesis allows the induction of immature pollen grains (microspores), to move away from the normal gametophytic developmental route in the direction of the sporophytic one, yielding homozygous organisms (embryos in this case). Unlike other fruit crops (such as Citrus), regeneration of entire plants has not yet been obtained in our studied nut crops; however, it gives the methodology should be used to continue the roadmap.Doubled haploids have a high impact on the improvement of heterozygous crops through hybridization. Anther culture is a doubled haploid technique for producing homozygous lines. In coconut, a tree species reported to be recalcitrant for tissue culture, a successful doubled haploid protocol was established through anther culture. All the factors affecting androgenesis induction have been optimized. In this chapter, a stepwise protocol, from doubled haploid induction including palm selection, anther isolation, pretreatment, and culture initiation, up to plant regeneration and thereafter acclimatization of the regenerated plants, is described. Furthermore, the protocol for testing the anther-derived plants for the ploidy level is also presented.This chapter deals with microspore embryogenesis in Citrus. Microspore embryogenesis allows to induce immature gametes (microspores) and to deviate them, in this case, the male one, from the normal gametophytic developmental route in the direction of the sporophytic one, yielding homozygous organisms (embryos and plants).Due to their many superior agronomic traits (high yield and fruit quality, resistance/tolerance to biotic and abiotic stress factors, etc.), hybrid vegetable cultivars are widely used in vegetable production all over the world. The first stage of hybrid vegetable breeding is to obtain homozygous pure parental lines. Unfortunately, producing pure lines takes a long time by classical breeding methods, especially in open-pollinated vegetable species, and this period can be up to 8-10 years. Recently, doubled haploid (DH) technology, as a biotechnological method, has emerged as an alternative to classical breeding methods and allows for the generation of pure (100% homozygous) DH lines in one or two years.However, the DH technique needs labor-intensive efforts and experiences as well as the use of appropriate production technologies. The main objective of this chapter is to provide explanatory information on the technique of induction of parthenogenesis by irradiated pollen applied to several species of the Cucurbita genus. For this purpose , key points and details of methods and protocols of this technique are described in summer squash (Cucurbita pepo L.), pumpkin (Cucurbita moschata Duch.), and winter squash (Cucurbita maxima Duch.).The development of F1 hybrid vegetable varieties emerges as a result of a great effort, long time, investment, knowledge, and advanced technology. The first stage of hybrid vegetable breeding is obtaining pure lines. It is possible to obtain homozygous parent lines used in the production of hybrid varieties with traditional breeding methods. This period takes 8-10 years, especially in some vegetables which are highly open-pollinated, such as Cucurbita spp. Androgenetic- and/or gynogenetic-based dihaploidization methods provide 100% homozygous pure haploid lines in 1-2 years and save time and effort.The DH frequency by irradiated pollen technique and anther culture strongly depends on the genotypic response, whereby their practical use in a breeding program is still limited. As a possible alternative technique, gynogenesis (unfertilized ovule/ovarium cultures) switches on to produce haploid plants in some Cucurbita species. In the Cucurbita genus, gynogenesis has been one of the most studied and popular DH techniques and presented remarkable results in recent years.Production of homozygous pure parental lines is the first stage of hybrid vegetable breeding. Unfortunately, producing pure lines takes a long time by classical breeding methods, especially in open-pollinated vegetable species, and this period can be up to 8-10 years. Recently, doubled haploid (DH) technology, as a set of biotechnological methods, has emerged as an alternative to classical breeding methods and allows for the generation of 100% homozygous pure double haploid lines in 1 or 2 years. Although haploid plants were successfully produced via irradiated pollen technique and gynogenesis in some Cucurbita species, haploid plants have not been obtained from some lines due to genotype dependency, and haploidy frequency is still not sufficient for use in a breeding program. Thus, anther culture technique has emerged as an alternative technique in the DH process. The main objective of this chapter is to provide explanatory information on anther culture technique applied in the Cucurbita genus. Adenosine 5′-diphosphate For this purpose , key points and details of methods and protocols of the anther culture technique are described in summer squash (Cucurbita pepo L.