| The transgenic herbicide-resistant oilseed rape(Brassica napus,AACC,2n=4x=38)is the leading genetically modified oil crop in the world.Although it has not released commercially in China,a large amount of transgenic oilseed rape seeds are imported for edible oil each year in China.In addition,there is a great possibility that transgenic oilseed rape become volunteers.Therefore,it should be paid attention to the risk of gene flow from transgenic oilseed rape to its weedy relatives.Whether resistance genes can introgress into related species depends on chromosome behaviur of hybridized and backcrossed offspring(Che`vre et al.,1998;Che`vre et al.,2007).Therefore,it is very important to analyze the number,composition,meiotic chromosome pairing behavior and genetic behavior of hybrid and backcross offspring when studying the gene flow from transgenic oilseed rape to its weedy relatives.Among wild relatives,wild Brassica junceas(AABB,2n=36)is the closest relative weed(AACC,2n=38)in China.Wild B.juncea is a common weed in the wasteland and farmland of the Northwest China and the Yangtze River basin.In order to explore whether the transgene of transgenic herbicide-resistant oilseed rape escape to wild B.juncea successfully,we have conducted experiments to evaluate potential gene flow from two transgenic oilseed rapes:glyphosate-resistant oilseed rape(DS-Roughrider,Roundup Ready,event RT73,resistance gene in A-chromosome),glufosinate-resistant oilseed rape(Swallow,Liberty Link,event HCN28,resistance gene in C-chromosome)to wild B.juncea.In this paper,based on the previous studies,the number and composition of the chromosomes from the first to the third progenies of the third backcross generation were observed,and the change of the chromosomes number with the generation increase were analyzed.The meiotic behavior of the third progenies of third backcross generations was observed.The chromosome numbers of the two kinds plants(glusosinate-resistance plant and plants carried glufosinate-resistant gene but was sensitive to glufosinate)of the fourth progeny of the third generation were also observed,and the chromosome karyotype of 2n=36 cells in these two kinds of plants was analyzed;Finally,the fitness of the two kinds plants was measured.The main results are as follow:1.The glyphosate-resistant progenies(BC3FR)were mixopliod,and could be set into 2n=36(AABB),similar to wild Brassica juncea.The glyphosate-resistant progenies of BC3F2R-BC3F4R between glyphosate-resistant oilseed rape and wild B.juncea were mixopliod.With the increase of generations,the chromosome numbers of cells with 2n=36 chromosomes increased.The plants displayed variable chromosome numbers in somatic tissues with a large proportion of cell in all tested plants with 2n=36 chromosomes.BAC-FISH observations showed that genomic constitution of the cell with 36 chromosomes was 20A+16B in three individual plants of BC3Fm4R and BC3Fp4R,respectively,which was the same as wild B.juncea.2.The glufosinate-resistant progenies(BC2FL)were mixopliod,and could be set into 2n=37(AABB+1C).The glufosinate-resistant progenies of BC2F2L-BC2F5L between glufosinate-resistant oilseed rape and wild B.juncea were also mixopliod.The plants displayed variable chromosome numbers in somatic tissues with a large proportion of cell in majority plants with 2n=37 chromosomes.BAC-FISH observations showed that the cell with 37 chromosomes was 20A+16B+1C in each five individual plants of BC2mF5L and BC2pF5L.In individual plants,2n=34,35 and 36 cells accounted for the highest proportion of observed cells.With the increase of generations,the proportion of 2n=37 cells did not increase significantly.3.Observation on chromosome abnormal behavior during meiosisThe abnormal meiosis,including asynchronous meiosisin,lagging chromosomes,chromatid bridge and emicronucleus,were observed in pollen mother cell(PMC)of both the third glyphosate-and glufosinate resistant progenies.Meiotic indexes of BC3mpF4R were among 98.11 to 99.29,displayed considerably genetic stability.Meiotic indexes of BC3mF4L were among 91.07 to 94.44,displayed considerably genetic stability.However,meiotic indexes of BC3pF4L were among 78.98 to 96.58,indicating considerably genetic instability.4.The chromosome numbers of the two kinds plants of the fourth progeny of the third generation(glusosinate-resistant plants and plants which carried glufosinateresistant gene but was sensitive to glusinate).Plants of resistant-no-expression BC3F5L has significantly less 2n>36 cells than plants of resistant-expression BC3F5L.Each two individual plants which had not 2n37 chromosomes cells were found in resistant-no-expression BC3mF5L and BC3pF5L,respectively.Preliminary studies showed that glufosinate-resistant gene located on the C chromosome.Under the glufosinate selective pressure,the C-chromosome carrying the resistance gene inherited in the progeny as an additional chromosome.These each two offspring without 2n≧37 chromosomes cells carried glufosinate resistance gene,the possible reason may be due to exchange of resistance genes on the C-chromosome to A or B genomes.5.Karyotype analysis of resistant-expression and resistant-no-expression progenies of BC3FL5.The chromosome karyotypes of 2n=36 between resistant-expression and resistant-noexpression plants in BC3FL5 were different.The karyotype formula of resistant-expression BC3mFL5 and BC3pFL5 is 2n=36=32m+4sm and 2n=36=28m+8sm.The karyotype formula of resistant-no-expression BC3FmL5 and BC3pFL5 is 2n=3 6=28m+8 sm and 2n=36=22m+14sm.The heterologous chromosome pairing and heterologous recombination,which occurred in resistant-no-expression plants of BC3FmL5 and BC3pFL5,may result in glufosinate-resistant gene silencing.6.Fitness analysis of resistant-expression and resistant-no-expression progenies of BC3FL5 in greenhouse.Fitness of resistant-expression and resistant-no-expression progenies of BC3FL5 were conducted by measuring vegetative variables(including plant height,stem diameter,the first branch number/plant,above-ground dry biomass/plant),and reproductive variables(including pollen viability,silique number,silique length and seed number/silique).The results showed that all measured fitness components of resistant-expression BC3mpF5L had no significant difference with these of wild B.juncea.While pollen viability and silique length of resistant-no-expression BC3mF5L were significantly lower than that of wild B.juncea.Pollen viability,silique length and silique length of resistant-no-expression BC3pF5L were also significantly lower than those of wild B.juncea.Two each individual plants of resistant-no-expression BC3mF5L and BC3pF5L without 2n≧37 chromosomes cells had similar fitness to wild B.juncea. |
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