Wang et. al.·Silvae Genetica (2009) 58-5/6, 220-226 Distribution of Microtubular Cytoskeletons and Organelle Nucleoids During Microsporogenesis in a 2n Pollen Producer of Hybrid Populus By J. WANG1),2) and X. Y. KANG1),2),*) (Received 2nd August 2008) Abstract 1991; SHAMINA et al., 2007). The arrangement of four The distribution of microtubular cytoskeletons and daughter nuclei in telophase II is determined by the ori- organelle nucleoids during microsporogenesis in Popu- entation of metaphase II spindles (BROWN and LEMMON, lus simonii Carr. x P. nigra L. ‘Tongliao’ was studied by 1992). Abnormal orientation of spindles may cause mei- indirect immunofluorescence and 4’,6-diamidino-2- otic nuclear restitution, resulting in the production of phenylindole (DAPI) staining. Our purpose was to unreduced gametes (MOK and PELOQUIN, 1975; RAMANA, reveal the relationship between changes in microtubule 1974, 1979; KANG, 2002). and chromosome behavior and cytokinesis and to explain the mechanism of 2n pollen production. We Cytokinesis is mediated by the cytokinetic apparatus, observed stable frequencies of 2n pollen production, including preprophase band of microtubules (PPB) and ranging from 1.56% to 2.29%, between 2006 and 2008. phragmoplast microtubules (GUNNING, 1982). In meiosis Moreover, conjoined pollen grains were also observed. of higher plant, phragmoplast microtubules play a role Meiotic abnormalities, including univalents, lagging in successive cytokinesis and in some instances of simul- chromosomes and micronuclei, were observed during taneous cytokinesis (SHELDON and DICKINSON, 1986; microsporogenesis. Triads and dyads were also detected BROWN and LEMMON, 1991a, b; SCHOPFER and HEPLER, in meiotic products. Parallel spindles in metaphase II 1991; SHAMINA et al., 2007). However, in another form of were unable to fuse, owing to the existence of an simultaneous cytokinesis, the formation of cell plates organelle band. Regularly, in telophase II, primary does not depend on phragmoplasts and cytokinesis is phragmoplasts were organized between sister nuclei, and secondary phragmoplasts formed between non-sis- accomplished by the centrifugal expansion of cell plates ter nuclei. Cell plates were initiated by centrifugal (BROWN and LEMMON, 1988c, 1996). BROWN and LEMMON expansion of phragmoplasts and cytoplasmic infurrow- (1989, 1991a, b, 1996) consider that nuclear-based radi- ing started at the junctions between the microsporocyte al microtubule systems (RMSs) function in the organiza- wall and the expanded phragmoplasts. However, a sec- tion and apportionment of cytoplasm, in the location of ondary phragmoplast was absent in some microsporo- division planes, and in the definition of spore domains. cytes. These observations suggest that the occurrence of The genus Populus L. is widely distributed and culti- 2n pollen may result from the partial failure of cytokine- sis caused by the absence of secondary phragmoplasts in vated throughout the world (RAE et al., 2007). As one of this hybrid. the most intensively studied forest tree species, the genus has become a model for forest tree biotechnology Key words: microsporogenesis, microtubular cytoskeleton, and genetic studies (JANSSON and DOUGLAS, 2007). The organelle nucleoids, Populus, 2n pollen. Populus genome is characterized by a high level of het- erozygosity, which results in complicated chromosome Introduction behavior (SMITH, 1943; WANG et al., 2006). Although Meiosis is essential for sexual reproduction and genet- many cytological studies of microsporogenesis in Popu- ic recombination, and plays an important role in species lus have been carried out using squashed preparation, evolution (KLECKNER, 1996). The chromosomes of sporo- the distribution and structure of microtubular cytoskele- phytes are doubled and apportioned equally into four tons during meiosis continue to remain unclear. microspores during the process of microsporogenesis. P. simonii Carr. x P. nigra L. ‘Tongliao’ is a hybrid These fertile microspores serve to produce the next between two different sections (Tacamahaca and sporophyte generation via fertilization with eggs. Aigeros) within the genus Populus; it has the properties Microtubular cytoskeletons play an important role in of fast growth and high adaptability, and is easily propa- nuclear division, cytokinesis, and apportionment of gated by cuttage. In this hybrid, meiosis of pollen moth- organelles during meiosis (SHELDON and DICKINSON, er cells exhibits a lack of complete homology between 1986; BROWN and LEMMON, 1988c, 1991a, b; TANAKA, the genomes of the parental species. Some natural 2n pollen grains have been observed and the occurrence of this 2n pollen is assumed to be related to the existence 1) National Engineering Laboratory for Tree Breeding, Beijing of parallel spindles at metaphase II (WANG et al., 2006). Forestry University, 100083, Beijing, P. R. China. In this investigation, in order to reveal the relation- 2) Key Laboratory of Genetics and Breeding in Forest Trees and ship between the change in microtubule and chromo- Ornamental Plants, Ministry of Education, Beijing Forestry some behavior and cytokinesis and to explain the mech- University, 100083, Beijing, P. R. China. anism of 2n pollen production, we analyzed the distribu- *) Address correspondence to: P.O. Box 118, Beijing Forestry Uni- versity, 100083, Beijing, P. R. China; Tel.: +86-10-62336104; tion of microtubules and organelle nucleoids during E-Mail: [email protected] microsporogenesis in P. simonii x P. nigra ‘Tongliao’. 220 Silvae Genetica 58, 5–6 (2009) DOI:10.1515/sg-2009-0028 edited by Thünen Institute of Forest Genetics Wang et. al.·Silvae Genetica (2009) 58-5/6, 220-226 Material and Methods gel at 4°C. Pollen grains were removed from the vial Plant material using a needle, and mounted in a drop of 2% aceto- carmine on a microscopic slide. The frequency of 2n Male floral branches of P. simonii x P. nigra ‘Tongliao’ pollen was estimated based on pollen diameter. were collected in spring from a plantation, approximate- ly 40 years old, located to the west of Tongliao City air- port (Inner Mongolia Autonomous Region, P. R. China). Indirect immunofluorescence of microtubules and DNA The branches were cultured in a greenhouse (20–30°C) staining in order to induce meiosis. The male flower buds were For indirect immunofluorescence of microtubules and then collected every 3 h. The meiotic stage was deter- DNA staining, our experimental material was fixed in mined by a 2% aceto-orcein squash of an anther. 4% paraformaldehyde in a PEM buffer (50 mM PIPES, Anthers around the one used for the squash preparation 5 mM EGTA, 2 mM MgSO4, pH 6.9) for 45 min. After were used for indirect immunofluorescence microscopy three 5-min rinses in the PEM buffer, the material was of microtubules and DNA staining. dipped in 10% dimethylsulfoxide (DMSO) for 15 min and then extracted with 1% Triton X-100 for 30 min. Evaluation of 2n pollen The material was then rinsed in the PEM buffer, fol- Pollen samples were collected from male catkins in lowed by three 5-min rinses in a PBS buffer (137 mM the greenhouse and stored in a vial with allochroic silica NaCl, 2.7 mM KCl, 7 mM Na2HPO4, 1.5 mM KH2PO4, Table 1. – Frequency of 2n pollen and conjoined one of P. simonii x P. nigra ‘Tongliao’ from 2006 to 2008. Table 2. – Meiotic abnormalities of P. simonii x P. nigra ‘Tongliao’ in 2007 and 2008. 221 DOI:10.1515/sg-2009-0028 edited by Thünen Institute of Forest Genetics Wang et. al.·Silvae Genetica (2009) 58-5/6, 220-226 pH 7.3). Thereafter, microsporocytes were squeezed recorded in each year from 2006 to 2008, and conjoined out from the material and transferred to a slide coated pollen (Fig. 1b) was also observed. Compared with the with 0.1% poly-L-lysine (Sigma, P1274). The cells were years 2006 and 2008, there were relatively high fre- incubated in a monoclonal anti-α-tubulin antibody quencies of 2n and conjoined pollen grains in 2007 (Sigma, T9026), diluted 1:100 with the PBS buffer, (Table 1). for 2 h at 37°C in a moist chamber. Following further washing with the PBS buffer, the microsporocytes Abnormality of meiosis were incubated in a FITC-conjugated anti-mouse IgG It was found that meiosis in P. simonii x P. nigra (Sigma, F0257), diluted 1:50 with the PBS buffer, for 2 h ‘Tongliao’ proceeded to completion with a typically at 37°C in a dark chamber. After a final wash in the simultaneous cytokinesis. Abnormalities were, however, PBS buffer, the microsporocytes were mounted with a observed during microsporogenesis. There were steady mounting medium containing 4’,6-diamidino-2-phenylin- frequencies of abnormalities between 2007 and 2008 dole (DAPI) (Vectashield, H-1200). The preparations (Table 2). In 2007, although more than 18% cells were were observed and photographed using a Leica TCS-SP2 observed with lagging chromosomes in anaphase I, only confocal laser scanning microscope. approximately half of these developed into cells with micronuclei in telophase I. After the second meiosis, the Results frequency of telophase II cells with micronuclei declined to less than 5%. Parallel spindles in anaphase II were Occurrence of 2n and conjoined pollen observed at a stable frequency of approximately 15%. In In P. simonii x P. nigra ‘Tongliao’, a low frequency of the meiotic products, apart from tetrads with tetragonal 2n pollen production (1.56% to 2.29%; Fig. 1a) was and tetrahedral arrangements (Figs. 1c and d), triads were observed in both 2007 and 2008, and a few dyads were detected in 2008. Figure 1e shows an undivided spore of a triad, in which two nuclei are observed in the process of fusion. Figure 1f shows that in one of the undivided spores of a dyad, two nuclei are about to fuse, and that in the other spore, one fused nucleus has already formed. Changes of microtubule and organelle nucleoids During the microsporogenesis of P. simonii x P. nigra ‘Tongliao’, the distribution of microtubular cytoskeletons and organelle nucleoids (plastid and mitochondrial) changed regularly.