| The species of Populus spp. are important trees planted worldwide, and play crucial roles in forest industry in our country. The application of transgenic technology in poplar genetic breeding has been greatly accelerating the process of selection and cultivation of new poplar varieties. To date, many transgenic poplar trees with improved traits such as insect resistance, salt tolerance and modified wood properties have been obtained, showing a broad application in the future. In our country, several new poplar varieties, such as insect resistance transgenic P. nigra and transgenic hybrid 741 poplar have been permitted to be commercialized. With the rapid progress of research in transgenic trees, potential ecological risks caused by foreign genes are getting to be concerned. Thus, ecological risk assessment of transgenic trees has significant meanings and application. However, most of the current works on risks assessment are foused on crops, few work are carried out in research field of forest trees. In this study, transgenic P. alba×P. glandulosa and P.×euramericana'Guariento'were used as materials to perform ecological risks assessment. Based on a 2-year-old field trail of transgenic poplar, preliminary evaluations were implementd on six aspects, including assay of the quantity of expressed protein of marker gene NPTII, stability of foreign genes, growth traits of transgenic trees, horizontal transfer of foreign genes, the effects of foreign genes on the community and density of target, non-target and parasite insects and on soil microorganisms ecosystems. The main results are described as following:1. Protein expression level of marker gene NPTII using ELISA method. A total of 15 transgenic lines and 1 non-transgenic line of P.×euramericana'Guariento', 18 transgenic lines and 1 non-transgenic line of P. alba×P. glandulosa growing in greenhouse were used for this assay. There were no significant differences were observed in protein contents among different transgenic lines both in the two poplar species. In P.×euramericana'Guariento', the quantities of NPTII protein in lines with different number of foreign genes of 1, 2, 3 and 5 are 27.47, 0.19.00, 19.69, and 16.72 ng per gram fresh leaf tissues, respectively. In P. alba×P. glandulosa, the quantities of NPTII protein in lines with different number of foreign genes of 1, 2, 3, 4 and 5 are 2.44, 3.51, 3.82, 7.54 and 5.60 ng per gram fresh leaf tissues, respectively. The results suggested that the quantity of protein would not increase with increased number of transgenes.2. Identification of genetic stability of foreign genes. Sets of PCR were performed on 3 lines of transgenic P.×euramericana'Guariento'and 3 lines of transgenic P. alba×P. glandulosa. Results showed that all transgens were identified in these lines and not observed in control plants, suggesting that target genes were stable present in the genome of transgenic plants, and these trees were suitable to be assessed further.3. Measurement of growth traits of transgenic poplar. Height, breast diameter and mortality were scored on 800 trees of 8 transgenic and non-transgenic lines of both 2-year-old poplar species. Higher and lower scores in different transgenic lines were observed compared with that of non-transgenic line. Among them, the average level of breast diameter in transgenic lines was higher than that of non-transgenic line in P. alba×P. glandulosa, but with no significant differeces. These results indicated that the expression of foreign genes conferred no advantage of growth ability to transgenic poplar compared with non-transgenic plants.4. Assessment of horizontal transfer of foreign genes. Using genomic DNA from weed grasses, insects and soil microorganisms, series of PCR were conducted to amplify 5 transgenes Vgb, SacB, BtCry3A, OC-I and JERF36 and marker gene NPTII. No any target PCR product was observed in all samples analyzed. This result suggested that no horizontal transfer of transgenes occurred between transgenic poplar and other plant species, insects or microorganisms.5. Assessment of the effects of transgenic poplar on target, non-target and parasite insects. In the stand of P.×euramericana'Guariento', the density of Lepidoptera in transgenic lines was lower than in non-transgenic line, and significant differences were observed among 7 transgenic lines. The developing trends of species and density of insects in transgenic and non-transgenic stands were the same with the change of time. The density of predator in transgenic lines was higher compared to that in control lines, which suggested that transgenic lines with resistance trait was favorable for the living of predator. In the stand of P. alba×P. glandulosa, the density of target insects of Coleoptera and Lepidoptera in C013-5 was lower compared with that of in control line S13-0, and also lower than that of in transgenic line C13-5 which has a single foreign gene, indicating that transgenic lines with two target genes had a higher resistance to insects. There was a similar character on the developing trend of the density of target insects in both transgenic and non-transgenic poplar stand. Furthermore, the density of parasite insects in transgenic poplar stand was slightly higher than that of non-transgenic plants.6. Assessment of the effects of transgenic poplar on soil microbial ecosystems. Dilution-plate method was used to determine the quantities of main soil microbes of bacteria, fungi and actinomycetes in 8 poplar lines in different months of 2 successive years. The results showed different developing trends of three groups of microorganisms with different month and different years. In the stand of P.×euramericana'Guariento', there was no significant difference in the quantities of these three kinds of microbes in all lines, whereas significant different was found in several lines of P. alba×P. glandulosa stand, but did not show any relation to the type and number of foreign genes. Significant differences were also found in some lines between transgenic and non-transgenic stands, which need to be studied further whether foreign genes have any effects. The results from the same month in different years showed that there were significant differences in the quantity of microorganisms among most lines with no clear pattern, which might be caused by changes of natural environmental conditions. Our preliminary results indicated that the transgenic poplar growing in different years had no significant negative effects on soil microorganisms.Our work is the first study on biosafety assessment of transgenic poplar with multiple transgenes with different functions. The results showed that the content of NPTII protein in poplar leave tissues did not increase with the increased number of foreign genes. Target genes were stable present in the poplar genome, and transgenic poplar did not showed advantage growth ability on non-transgenic plants. No horizontal gene transfer was found. No significant negative effects were found on non-target and predator insects, and on the soil microbial ecosystems. |
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