Occurrence and cytological mechanism of numerically unreduced pollen in diploid Populus euphratica

Unreduced gametes are the driving force for the polyploidizaiton of plants in nature, and are also an important tool for breeding of triploid individuals. The final heterozygosity of a 2n pollen grain depends on the cytological mechanism behind 2n pollen formation. In this study, meiotic abnormalities were analysed using fluorescent chromosome staining and indirect immunofluorescence during the microsporogenesis of 18 genotypes of diploid P. euphratica Oliv. (2n = 2x = 38). Among the 18 genotypes, 16 genotypes produce 2n pollen and two genotypes produce only normal n pollen. In all 2n pollen producers, we found that the first meiotic division was normal but that the second division was characterized by frequent abnormal spindle orientation (parallel, tripolar, and fused spindles) and premature cytokinesis. The parallel, fused spindles and premature cytokinesis were considered to be leading dyad formation, and tripolar spindles seemed to be causing triad formation at the tetrad stage. There was a higher frequency of parallel spindles than other spindle forms, but no significant correlations between parallel spindles and dyads were observed. However, a significant association (r = 0.68, P < 0.05) between the tripolar spindles and dyads was found. In some Microspore mother cells (MMCs), an indirect immunofluorescence examination of meiosis II revealed that the parallel spindles led to the gathering of one or two non-sister groups of chromosomes, causing an incorporation of RMSs from two daughter nuclei. Therefore, the incorporated RMSs established two nuclear cytoplasmic domains for the control of division plane, resulting in either triad or dyad formation.

Distribution of Microtubular Cytoskeletons and Organelle Nucleoids During Microsporogenesis in a 2n Pollen Producer of Hybrid Populus

The distribution of microtubular cytoskeletons and organelle nucleoids during microsporogenesis in Populus simonii Carr. x P. nigra L. ‘Tongliao’ was studied by indirect immunofluorescence and 4’,6-diamidino-2- phenylindole (DAPI) staining. Our purpose was to reveal the relationship between changes in microtubule and chromosome behavior and cytokinesis and to explain the mechanism of 2n pollen production. We observed stable frequencies of 2n pollen production, ranging from 1.56% to 2.29%, between 2006 and 2008. Moreover, conjoined pollen grains were also observed. Meiotic abnormalities, including univalents, lagging chromosomes and micronuclei, were observed during microsporogenesis. Triads and dyads were also detected in meiotic products. Parallel spindles in metaphase II were unable to fuse, owing to the existence of an organelle band. Regularly, in telophase II, primary phragmoplasts were organized between sister nuclei, and secondary phragmoplasts formed between non-sister nuclei. Cell plates were initiated by centrifugal expansion of phragmoplasts and cytoplasmic infurrowing started at the junctions between the microsporocyte wall and the expanded phragmoplasts. However, a secondary phragmoplast was absent in some microsporocytes. These observations suggest that the occurrence of 2n pollen may result from the partial failure of cytokinesis caused by the absence of secondary phragmoplasts in this hybrid.

Cytological basis of 2n pollen formation in a wide range of 2x, 4x, and 6x taxa from tuber-bearing Solanum species

Cytological mechanisms of 2n pollen formation were investigated in microsporogenesis and sporad development in 2x, 4x, and 6x taxa of tuber-bearing Solanum species that were originally collected from Mexico, and Central and South America. Parallel spindles and tripoles were seen at anaphase II in the majority of genotypes observed. The occurrence of parallel spindles and tripoles were well correlated with the occurrence of dyads and triads at the sporad stage. Further, the frequency of 2n pollen predicted by frequencies of parallel spindles and tripoles and by frequencies of dyads and triads were also well correlated with observed 2n pollen frequencies. Premature cytokinesis at telophase I to prophase II was seen only in 2x and 4x species from series Conicibaccata. The frequency of premature cytokinesis corresponded well with the frequency of dyads leading to 2n pollen formation. It was concluded that the major mechanisms of 2n pollen formation in tuber-bearing Solanum species is parallel spindles.Key words: cytological mechanisms, parallel spindles, ploidy series, tuber-bearing Solanum species, 2n pollen.

2n GAMETES IN DIPLOID SOLANUM: FREQUENCY AND TYPES OF SPINDLE ABNORMALITIES

Cytological disturbances resulting in 2n pollen were examined in four clones of diploid (2x = 24) Solanum phureja Juz. and Buk.; two (148-17 and 127-14) were variable and two (154-1 and B2-11) were consistently high in 2n pollen production. Three types of abnormal spindle orientation were observed at the second meiotic division: fused, tripolar, and parallel spindles (fs, tps, and ps, respectively). All four genotypes had fs. Three had tps, and ps was least frequent, occuring in two genotypes only. Cross-sections of buds revealed that all three abnormalities can occur in adjacent cells of a locule. In all genotypes, fs predominated at high levels of expression of 2n gametes. At lower levels of expression (i.e. less than 60%, which occurred only in 148-17 and 127-14), however, the frequencies of tps and ps increased. In general, there was little evidence for localization of dyads (resulting from fs or ps) or tetrads (resulting from normal divisions) within cross sections of locules, but 52.4% of all tetrads which occurred in a single locule of genotype 127-14 were found in 25% of the locular area. Cross-sections of buds revealed variation in frequency of 2n gametes among anthers (61.7 vs. 5.6% in 148-17) and between locules of an anther (73.1 vs. 90.0% in B2-11). Implications of the anatomical study were applied to current genetic theory regarding the development of 2n gametes in potato species.

THREE MECHANISMS OF 2n POLLEN FORMATION IN DIPLOID POTATOES

Three mechanisms of 2n pollen formation observed in diploid hybrids between Solatium tuberosum Group Phureja (2x) and haploids of S. tuberosum Group Tuberosum (4x) are termed parallel spindles (ps), premature cytokinesis 1 (pc 1) and premature cytokinesis 2 (pc 2). A II spindles are parallel to each other in many cells of clones producing 2n pollen by ps. A single cleavage furrow follows and forms a dyad which generates two 2n microspores. The other two mechanisms, pc 1 and pc 2, produce 2n pollen by omission of the second meiotic division. Premature cytokinesis 1 (pc 1) involves asynchronized movement of bivalents to and from the M I plate and falling apart of chromatids at T I. A cleavage furrow follows the first meiotic division, no second division occurs and products of meiosis are dyads. Premature cytokinesis 2 (pc 2) exhibits no abnormalities at the first meiotic division. However, cytokinesis occurs prematurely at P II, no second division occurs, and only dyads are formed. Genetic data suggested that these three mechanisms may be controlled by single loci, and are inherited independently as simple recessives.