Meiotic chromosome preparation techniques of pollen mother cells for laser micro-dissection in Populus spp.

In order to isolate meiotic chromosomes of Populus species meiotic chromosome preparation techniques of pollen mother cells for laser micro-dissection were studied. Pollen mother cells at diakinesis ofPopulus canadensis Moench were used as samples. Two methods were used to prepare meiotic chromosomes： in the first, cell suspensions were dropped on polyethylene-naphthalate or polyester membrane slides which had just been incubated at -20~C; in the second method, cell suspensions were also dropped on polyethylene-naphthalate or polyester membrane slides, but spread with the aid of high temperatures. The cells did not completely spread by the first method and chromosomes at diakinesis could not be individually distinguished. In contrast, well-spread diakinesis chromosomes were obtained by the second method, where chromosomes, connected with their nucleolus, were successfully isolated with the laser micro-dissection system. As well, we discuss the prospect of applications of laser micro-dissection in cytogeneties and molecular genetics in Populus species.

Genotoxic Effects of Superconducting Static Magnetic Fields(SMFs)on Wheat(Triticum aestivum)Pollen Mother Cells(PMCs)

The effects of superconducting static magnetic fields （SMFs） on the pollen mother cells （PMCs） of wheat were investigated in order to evaluate the possible genotoxic effect of such non-ionizing radiation. The seeds of wheat were exposed to static magnetic fields with either different magnetic flux densities （0, 1, 3, 5 and 7 Tesla） for 5 h or different durations （1, 3 and 5 h） at a magnetic flux density of 7 Tesla. The seeds were germinated at 23℃ after exposure and the seedlings were transplanted into the field. The PMCs from young wheat ears were taken and slides were made following the conventional method. The genotoxic effect was evaluated in terms of micronucleus （MN）, chromosomal bridge, lagging chromosome and fragments in PMCs. Although the exposed groups of a low field intensity （below 5 Tesla） showed no statistically significant difference in the aberration frequency compared with the unexposed control groups and sham exposed groups, a significant increase in the chromosomal bridge, lagging chromosome, triple-polar segregation or micronucleus was observed at a field strength of 5 Tesla or 7 Tesla, respectively. The analysis of dose-effect relationships indicated that the increased frequency of meiotic abnormal cells correlated with the flux density of the magnetic field and duration, but no linear relationship was observed. Such statistically significant differences indicated a potential genotoxic effect of high static magnetic fields above 5 T.

High temperature treatment induced production of unreduced 2n pollen in Camellia oleifera

Unreduced gametes through chromosome doubling play a major role in the process of plant polyploidization.Our previous work confirmed that Camellia oleifera can produce natural 2n pollen,and it is possible to induce the 2n pollen formation by high temperature treatment.This study focused on the optimization of the 2n pollen induction technique and the mechanisms of high temperature-induced2n pollen formation in C.oleifera.We found that the optimal protocol for inducing 2n pollen via high temperature was to perform 45℃with4 h at the prophaseⅠstage of the pollen mother cells(PMCs).Meanwhile,high temperature significantly decreased the yield and fertility of2n pollen.Through the observation of meiosis,abnormal chromosome and cytological behaviour was discovered under high-temperature treatment,and we confirmed that the formation of 2n pollen is caused by abnormal cell plate.Based on weighted gene co-expression network analysis,fifteen hub genes related to cell cycle control were identified.After male flower buds were exposed to heat shock,polygalacturonase gene(CoPGX3)was significantly upregulated.We inferred that high temperature causes the CoPGX3 gene to be overexpressed and that CoPGX3 is redistributed into the cytosol where it degrades cytoplasmic pectin,which leads to an abnormal cell plate.Furthermore,abnormal cytokinesis resulted in the formation of dyads and triads,and PMCs divided to produce 2n pollen.Our findings provide new insights into the mechanism of 2n pollen induced by high temperature in a woody plant and lay a foundation for further ploidy breeding of C.oleifera.

Microsporogenesis and meiotic behavior in nine species of the genus Pinus

The meiotic behavior of 10 taxa （nine species and one variety） of the genus Pinus was investigated using pollen mother cells （PMCs） to reveal the differentiation among karyotypes. Chromosome spreads were prepared by conventional squashing. The meiotic index and the average configuration were higher, whereas the frequency of aberrance （chromosomal bridges, fragments, or micronuclei） was lower, in all 10 taxa compared with other gynmosperms. The meiotic index, average configuration, and frequency of irregularity were found to be uniform among the species. It was shown that the genomes of the Pinus species investigated were highly stable, confirming results of previous mitotic analyses in this genus. However, slight differentiation of homologous chromosomes among genomes was revealed by analysis of meiotic configurations in Pinus nigra var. poiretiana. Quadrivalents were observed in 9.31% of PMCs in this species. This is the first time that quadrivalents have been observed in gymnosperms.