Callose depositions underlie the incompatible reaction in intergeneric crosses of rice

Distant hybridization of cereals is often impaired by fertilization barriers. Haploid induction through intergeneric crossing is well developed in wheat but has not been successful in rice due to incompatibility issues. The present study was thus undertaken to identify fertilization barriers that hinder the compatibility of the rice cultivar Punjab Rice 121 with maize and pearl millet lines as pollinators. A total of 37,357 spikelets were pollinated, yielding 494 caryopses upon supplementation with auxins. The resultant caryopses, arising from true intergeneric crosses, lacked embryos. Imaging of the pollinated pistils at different intervals indicated that intense callose depositions block the release of generative nuclei to the ovule in these wide crosses. Rice spikelets pollinated with rice pollen (cis-generic crosses) exhibited positive indicators of fertilization reaction at the micropyle. While the cis-generic crosses initiated true caryopsis formation after 24 h, no comparative reaction was observed in the intergeneric crosses. The current survey underlines that the rice female gametophyte presents a strong pre-fertilization barrier to foreign pollen. This barrier may be modulated in the future by altering genotype and auxin combinations.

Nondisjunction and unequal spindle organization accompany the drive of Aegilops speltoides B chromosomes

Supernumerary B chromosomes (Bs), which are often preferentially inherited, deviating from usual Mendelian segregation. This chromosome drive is one of the most important features of Bs. Here we analyzed the drive mechanism of Aegilops speltoides Bs and provide direct insight into its cellular mechanism. Comparative genomics resulted in the identification of the tandem repeat AesTR-183 of Ae. speltoides Bs, which also can be found on the Bs of Ae. mutica and rye, was used to track Bs during microgametogenesis. Nondisjunction of CENH3-positive, tubulin interacting B sister chromatids and an asymmetric spindle during first pollen grain mitosis are likely components of the accumulation process. A quantitative flow cytometric approach revealed, that independent on the number of Bs present in the mother plant Bs accumulate in the generative nuclei with more than 93%. Nine of eleven tested (peri)centromeric repeats were shared by A and B chromosomes. A common origin of the drive process in Poaceae is likely.

Morphological characterization and in vitro germination of heat-treated pollen in Eucommia ulmoides

Polyploid breeding has the potential to increase the economic secondary metabolites of Eucommia ulmoides. However, pollination with induced ploidy-mixed pollen has failed to produce polyploids (GAO, 2006). In this investigation, the morphological characterization and in vitro germination of heat-induced ploidy-mixed pollen of E. ulmoides were analysed to determine why there is no polyploid production. Heat-treated pollen grains were easily distinguished as large and small according to their length. The large pollen grains were significantly longer than both untreated and heat-treated small samples, suggesting that they were probably 2n pollen. Rather than the three germinal pores in small pollen, the large grains typically had four pores and, in some cases, shallow furrows, which might affect their germination. Although the maximal germination rates of the treated small and large pollen were not significantly different, the large pollen germinated tardily during the early stages of incubation. The small pollen maintained its growth during the incubation, but the tube growth of large pollen almost stopped after 24 h incubation. Both vegetative and generative nuclei in the large pollen moved into tubes later than in small pollen and the frequency of mitosis in generative nuclei of large pollen was low. Therefore, the tardy germination, poor tube growth, and weak activity of both vegetative and generative nuclei probably caused the poor competition of large pollen in certation. Finally, techniques to increase the competition of highploidy pollen and the prospect of 2n female gamete induction in the polyploid breeding program of E. ulmoides are discussed.

Ultrastructural transformations of nuclei in differentiating Hyacirlthus orientalis L. pollen grain cells

The ultrastructure of the pollen nuclei was studied during pollen maturation in <em>Hyacinthus orientalis</em> L., in which both pollen cells go through full interphase (G₁ S, G₂). The particular stages of interphase, however, do not proceed simultaneously in the vegetative and generative nuclei. The ultrastructural transformations of both pollen nuclei were analysed with reference to the dynamics of variations in the level of RNA and protein synthesis, investigated earlier. A distinction is drawn between the structural changes common for both nuclei, linked immanently with the interphase and the transformations connected with the differentiation of pollen cells.

Improved Nuclear Staining of Asparagus Microspores for Cytological Analysis

Clear visualization of asparagus (Asparagus officinalis L.) microspore nuclei with common stains such as acetocarmine or DAPI is difficult, hindering cytological analyses. The addition of saturated aqueous ferric chloride solution to Carnoy's I fixative (30 μL·mL-1) resulted in clear visualization of nuclei. A distinct nucleus was observed in uninucleate cells and the vegetative and generative nuclei were clearly visible in binucleate microspores. This method can be used reliably for determination of asparagus microspore developmental stage. Chemical name used: 4′,6-diamidino-2-phenylindole-2HCL (DAPI).

Analysis of Nondisjunction Induced by the R-X1 Deficiency during Microsporogenesis in Zea Mays L.

The r-X1 deficiency in maize induces nondisjunction at the second mitotic division during embryo sac formation. However, it was not known if this deficiency also induces nondisjunction during the microspore divisions. Microsporogenesis in plants lacking or containing this deficiency was compared using two approaches. First, chromosome numbers were determined in generative nuclei. Many (8.3%) of the generative nuclei in r-X1-containing plants were aneuploid; however, those from control plants were all haploid. Thus, this deficiency induces nondisjunction during the first microspore division. Second, nucleoli were analyzed in microspores. The only nucleolar organizing region in maize is on chromosome 6. If chromosome 6 underwent nondisjunction during the first microspore division, one nucleus in binucleate microspores would contain no nucleolus and the other would contain two nucleoli (or one nucleolus if the nucleoli fused). Only one (0.03%) microspore of this type was observed in control plants while 1.12% were found in r-X1-containing plants. Thus, the r-X1 deficiency induces nondisjunction of chromosome 6 during the first microspore division. However, both of the sperm nuclei in trinucleate microspores contained one nucleolus in r-X1-containing and control plants; thus, this deficiency does not induce nondisjunction of chromosome 6 (and presumably other chromosomes) during the second microspore division.

Specific end-to-end attachment of chromosomes in Ornithogalum virens

C-banding of nonhomologous chromosomes in haploid generative nuclei of Ornithogalum virens (n = 3) reveals a high degree of specificity with respect to end-to-end connexions. The centromeric end of chromosome 2 preferentially associates with the centromeric end of chromosome 3 and the telomeric end of chromosome 3 associates preferentially with the telomeric end of chromosome 1. This same association of nonhomologous chromosomes persists in prophase nuclei of diploid root tips. In addition, the telomeric ends of the 2 chromosome 2s are connected to one another as are the centromeric ends of the chromosome 1s. This results in a ring of chromosomes in which homologues lie opposite one another. Centromeric ends lie on one side of the nucleus and telomeric ends on the other. It is proposed that this specific association of chromosome ends reflects an order which was probably established at the preceding anaphase or telophase and which persists throughout interphase. The suggestion is made that the proximity of homologous ends and consequently homologous alignment may facilitate initiation of pairing at meiosis.

Cytochemical and ultrastructural peculiarities of embryogenic pollen grains and of young androgenic embryos in Datura innoxia

Cytochemical and ultrastructural studies of androgenic embryogenesis in Datura innoxia Mill. have been performed on (a) uncultured pollen grains collected at the stage favourable to embryo formation, i.e., at the time just before and just after the first haploid mitosis of the microspore nucleus (flower bud length of 4–5 cm); and (b) pollen grains and young androgenic embryos picked from the medium after 5, 9, and 11 days in culture.Only features presumed to play a role in the induction of androgenic embryogenesis are described.The phase of pollen development favourable to embryogenesis is very short but it involves important changes in the cytochemical features and in the ultrastructure of pollen grains. The changes are particularly clear in RNA and nucleohistone stainabilities and in the number and activity of the cytoplasmic organelles.It is suggested that it is not one particular metabolic or ultrastructural state of the cell which is responsible for the embryogenesis competence but that it is the fact that the changes occur which is important. Pollen which is in an unstable ultrastructural and metabolic state will be more sensitive to external stimuli.The study of very young androgenic embryos shows several features which are presumed to be responsible for the induction of embryogenesis: (a) Changes in cytoplasmic distribution and in nuclear organization consecutive to the first haploid mitosis in the microspore. These modifications sometimes lead to the formation of two equal pollen nuclei instead of differing vegetative and generative nuclei. The two identical nuclei are located in two equal-sized cells or in a single pollen cell. (b) Variations in pollen RNA content and in the organization of the endoplasmic reticulum which are related to changes in the pollen metabolism. (c) Modifications of the plasmalemma within pollen grains and of the cell wall surrounding the pollen grains. Such modifications are presumed to cause variations in the intercellular exchanges as well as in the exchanges between pollen and medium.