RESUMO
Mutation of the sperm-specific phospholipase A and treatment of pollen with reactive oxygen species (ROS) reagents lead to the induction of maize haploids. ZmPOD65, a gene associated with sperm-specific ROS metabolism, also exhibits a haploidization effect.
Assuntos
Pólen , Zea mays , Haploidia , Pólen/genética , Pólen/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Zea mays/genética , Zea mays/metabolismoRESUMO
In this chapter we describe protocols for immunolabeling and FISH of pollen grains undergoing postmeiotic mitosis using Aegilops speltoides, Secale cereale, and Hordeum vulgare as models. Tissue sectioning of pollen overcomes the problem of the pollen grain wall impermeability that interferes with immunolocalization and in situ hybridization. The crucial element of the protocol is the generation and immobilization of tissue sections. Our method facilitates the investigation of the microspore cell divisions and pollen grain maturation.
Assuntos
Imunofluorescência , Hibridização in Situ Fluorescente , Meiose , Mitose , Pólen/genética , Secale/genética , Cromossomos de Plantas , Flores/genéticaRESUMO
B chromosomes (Bs) are supernumerary chromosomes, which are often preferentially inherited. When transmission rates of chromosomes are higher than 0.5, not obeying the Mendelian law of equal segregation, the resulting transmission advantage is collectively referred to as 'chromosome drive'. Here we analysed the drive mechanism of Aegilops speltoides Bs. The repeat AesTR-183 of A. speltoides Bs, which also can be detected on the Bs of Aegilops mutica and rye, was used to track Bs during pollen development. Nondisjunction of CENH3-positive, tubulin interacting B sister chromatids and an asymmetric spindle during first pollen grain mitosis are key for the accumulation process. A quantitative flow cytometric approach revealed that, independent of the number of Bs present in the mother plant, Bs accumulate in the generative nuclei to > 93%. Nine out of 11 tested (peri)centromeric repeats were shared by A and B chromosomes. Our findings provide new insights into the process of chromosome drive. Quantitative flow cytometry is a useful and reliable method to study the drive frequency of Bs. Nondisjunction and unequal spindle organization accompany during first pollen mitosis the drive of A. speltoides Bs. The prerequisites for the drive process seems to be common in Poaceae.