Qin Guang-yong

The Effects of Low-Energy Nitrogen Ion Implantation on Pollen Exine Substructure and Pollen Germination of Cedrus deodara

The aim of this study is to investigate the biological effects of ion beams on pollen. Pollen grains of Cedrus deodara were implanted with 30 keV nitrogen ion beams at doses ranging from 1 × 1015 ions/cm2 to 15 × 1015 ions/cm2. The effects of N+ implantation on the pollen exine substructure were examined using an atomic force microscope (AFM), and the structure and morphology of pollen and pollen tubes were observed using a laser scanning confocal microscope (LSCM). AFM observations distinctly revealed the erosion of the pollen exine caused by N+ implantation in the micrometer to nanometer range. Typical results showed that the erosion degree was linearly proportional to the ion dose. Pollen germination experiments in vitro indicated that N+ implantation within a certain dose range increased the rate of pollen germination. The main abnormal phenomena in pollen tubes were also analyzed. Our results suggest that low energy ion implantation with suitable energy and dosage can be used to break the pollen wall to induce a transfer of exogenous DNA into the pollen without any damage to the cytoplasm and nuclei of the pollen. The present study suggests that a combination of the method of ion-beam-induced gene transfer and the pollen-tube pathway method (PTPW) would be a new plant transformation method.

Impact of Low-Energy Ion Beam Implantation on the Expression of Ty1-copia-like Retrotransposons in Wheat (Triticum aestivum)

Retrotransposon-like elements are major constituents of most eukaryotic genomes. For example, they account for roughly 90% of the wheat (Triticum aestivum) genome. Previous study on a wheat strain treated by low-energy N+ ions indicated the variations in AFLP (Amplified Fragment Length Polymorphism ) markers. One such variation was caused by the re-activation of Ty1-copia-like retrotransposons, implying that the mutagenic effects of low-energy ions might work through elevated activation of retrotransposons. In this paper an expression profile of Ty1-copia-like retrotransposons in wheat treated by low-energy N+ ions is reported. The reverse transcriptase (RT) domains of these retrotransposons were amplified by reverse-transcriptional polymerase chain reaction (RT-PCR) and sequentially cloned. 42 and 65 clones were obtained from the treated (CL) and control materials (CK), respectively. Sequence analysis of each clone was performed by software. Phylogeny and classification were calculated responding to the sequences of the RT domains. All the results show that there is much difference in the RT domain between the control sample and the treated sample. Especially, the RT domains from the treated group encode significantly more functional ORF (open reading frames) than those from the control sample. This observation suggests that the treated sample has higher activation of retrotransposons, possibly as a consequence of low-energy ion beam irradiation. It also suggests that retrotransposons in the two groups impact the host gene expression in two different ways and carry out different functions in wheat cells.

Study of Genetics and Embryology of Polyembryonic Mutant of Autotetraploid Rice Induced by N+Beam Implantation

In the present study autotetraploid rice IR36-4X was treated by an ion implantation technique with nitrogen ion beams. A polyembryonic mutant (named IR36-Shuang) was identified in the M2 generation. The mutant line and its offspring were systematically investigated in regard to their major agronomic properties and the rate of polyembryonic seedling in the M3-M6 generation. The abnormal phenomena in the embryo sac development and the cytological mechanism of the initiation of additional embryo in IR36-Shuang were observed by Laser Scanning Confocal Microscopy. The results were as follows. 1) The plant height, the panicle length and 1000 grain weight of IR36-Shuang were lower than that of its control by 35.41%, 5.08% and 15.72% respectively, Moreover, the setting percentage decreased 12.39% compared with that in normal IR36-4X plants. 2) The polyembryonic trait of IR36-Shuang was genetically stable and the frequency of the polyembryonic seedlings in the IR36-Shuang line was also relatively stable. 3) The rate of abnormal embryo sacs in IR36-Shuang was significantly higher than that in the control IR36-4X. 4) The additional embryo in IR36-Shuang might arise from the double set of embryo sacs in a single ovary, antipodal cells or endosperm cells. These results suggest that IR36-Shuang is a polyembryonic mutant and a new apomixis rice line induced by low energy ion implantation. The prospects for the application in production of the IR36-Shuang line are also discussed. The present study may provide a basis for future investigations of apomixis rice breeding via the ion implantation biotechnology.

Effects of Ion Implantation on in Vitro Pollen Germination and Cellular Organization of Pollen Tube in Pinus thunbergii Parl. (Japanese Black Pine)

Low-energy ion implantation, as a new technology to produce mutation in plant breeding, has been widely applied in agriculture in China. But so far there is a little understanding of the underlying mechanisms responsible for its biological effects at the cellular level. Here we report the biological effects of a nitrogen ion beams of 30 keV on the pollen grains of Pinus thunbergii Parl. In general, ion implantation inhibited pollen germination. The dose-response curve presented a particular saddle-like pattern. Ion implantation also changed the dimension of the elongated tubes and significantly induced tip swelling. Confocal microscopy indicated that the pollen tube tips in P. thunbergii contained an enriched network of microtubules. Ion implantation led to the disruption of microtubules especially in swollen tips. Treatment with colchicine demonstrated that tip swelling was caused by the disruption of microtubules in the tip, indicating a unique role for microtubules in maintaining the tip integrality of the pollen tube in conifer. Our results suggest that ion implantation induce the disruption of microtubule organization in pollen and pollen tubes and subsequently cause morphological abnormalities in the pollen tubes. This study may provide a clue for further investigation on the interaction between low-energy ion beams and pollen tube growth.

Study on Screening of TaGA2ox1 Mutants in Wheat by Ion Beam Irradiation

As a kind of mutagen, ion beam irradiation can create abundant biological mutations. A population of about 2000 lines was generated by irradiating dry wheat seeds of Xiao Yan 81 with low-energy nitrogen ion beams. The traits of the plant, such as height, spike type, fertility, stem color and awn length, were investigated. The mutation rate in terms of the plant height in M2 was 2.9%. Eighteen deletion mutants of TaGA2ox1 were obtained. Associate analysis showed that TaGA2ox1 was closely related to the plant height. Most of the TaGA2ox1-deleted mutants were higher than the control, suggesting that the biological function of TaGA2ox1 is similar to its homologues in other plants. These results demonstrate that ion beam irradiation is an efficient tool in the construction of a mutant library for wheat.

Ion Implantation Hampers Pollen Tube Growth and Disrupts Actin Cytoskeleton Organization in Pollen Tubes of Pinus thunbergii

Pollen grains of Pinus thunbergii Parl. (Japanese black pine) were implanted with 30 keV nitrogen ion beams and the effects of nitrogen ion implantation on pollen tube growth in vitro and the organization of actin cytoskeleton in the pollen tube cell were investigated using a confocal laser scanning microscope after fluorescence labeling. Treatment with ion implantation significantly blocked pollen tube growth. Confocal microscopy showed that ion implantation disrupted actin filament cytoskeleton organization in the pollen tube. It was found that there was a distinct correlation between the inhibition of pollen tube growth and the disruption of actin cytoskeleton organization, indicating that an intact actin cytoskeleton is essential for continuous pollen tube elongation in Pinus thunbergii. Although the detailed mechanism for the ion-implantation-induced bioeffect still remains to be elucidated, the present study assumes that the cytoskeleton system in pollen grains may provide a key target in response to ion beam implantation and is involved in mediating certain subsequent cytological changes.

Screening of Bioflocculant-Producing Strain by Ion Implantation and Flocculating Characteristics of Bioflocculants

A bioflocculant-producing mutator strain, NIM-192, was screened out through nitrogen ion implanting into FJ-7 strain. The results showed that NIM-192 had good genetic stability and high flocculating activity, and the flocculating rate increased by 34.26% than that of the original. Sucrose, complex nitrogen source contained yeast extract, urea and pH 7.0 ~ 9.0 were chosen as the best carbon source, nitrogen source and initial solution pH for bioflocculant production, respectively. The bioflocculant kept high and stable flocculating activity at alkalinous reaction mixture with a pH beyond 7.0, while the flocculating activity was remarkably reduced when the reaction pH was lower than 7.0. Addition of many cations could obviously increase the flocculating rate, among which Ca2+ demonstrated the best effect. The bioflocculant had very strong acid-base stability and thermo-stability. The flocculating rate kept over 86% when pH of the bioflocculant was in a range of 3.0 ~ 12.0, and the change of flocculating activity was not great when heated at 100°C for 60 min.

MOSSBAUER STUDY OF THE WARRIOR FIGURES OF POTTERY IN THE GRAVE SITE OF THE FIRST CHINESE EMPEROR (221 B.C.)

Mossbauer spectroscopy measurements at both room and liquid nitrogen temperature of potsherd samples of terracotta figures of warriors and horses excavated from within the pit at the grave site of the First Emperor (221 B.C.), lead us to conclude that the No. 4 potsherd sample was fired at a temperature of 980±50°C in a reducing atmosphere.

Differential Expression of Retrotransposon WIS 2–1A Response to Vacuum, Low-Energy N+ Implantation and 60Coγ-ray Irradiation in Wheat

Mutagenesis and retrotransposons have a close relationship, but little attention has been paid yet to the activity of retrotransposons produced by physical mutagens. The variation of retrotransposon WIS 2–1A activity in wheat (Triticum aestivum L.) embryos at three different growth times (30 h, 45 h and 60 h) was investigated after they had been treated with N+ implantation in a vacuum of 5 × 10−2 Pa and irradiation by 60Coγ-ray respectively. For each of the three growth times the expression of WIS 2–1A showed almost entirely a same trend of downregulation, upregulation, then downregulation, and upregulation again with the increase in dose of N+ implantation, but the expression appeared irregular with the increase in irradiation of 60Coγ-ray. In conclusion, the acutely activating effect of WIS 2–1A stimulated by vacuum and high dose N+ implantation within a shorter incubation time may provide a convenient tool to advance the research on mutagenic breeding and function genes.

Proteome Changes in Maize Embryo (Zea mays L) Induced by Ion Beam Implantation Treatment

Low energy ion beam implantation was applied to the maize (Zea mays L) embryo proteome using two-dimensional gel electrophoresis. Protein profile analysis detected more than 1100 protein spots, 72 of which were determined to be expressed differently in the treated and control (not exposed to ion beam implantation) embryos. Of the 72 protein spots, 53 were up-regulated in the control and 19 were more abundantly expressed in the ion beam-treated embryos. The spots of up- or down-regulated proteins were identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Among the identified proteins, 11 were up-regulated in the treated embryos. Four of these up-regulated proteins were antioxidant molecules, three were related to stress response, two to sugar metabolism and two were associated with heat shock response. Of the five proteins up-regulated in the control embryos, three were functionally related to carbohydrate metabolism; the functions of the remaining two proteins were unknown. The data collected during this study indicate that treatment of maize embryos with low energy ion beam implantation induces changes in stress tolerance enzymes/proteins, possibly as a result of alterations in metabolism.