Rapid Generation of Selectable Marker-Free Transgenic Rice with Three Target Genes by Co-Transformation and Anther Culture

The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.

Development of Marker-Free Transgenic Cry1Ab Rice with Lepidopteran Pest Resistance by Agrobacterium Mixture-Mediated Co-transformation

CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and transformation efficiency. Different mixtures of Agrobacterium strains （EHA105 and EHA101） contained Hpt and CrylAb genes resulted in different frequencies of resistant calli. There was no correlation between the frequency of transformants with the ratio of the Agrobacterium strain mixture contained Hpt and CrylAb genes. A total of 509 transgenic plants were obtained from the four rice varieties, and 272 T2 progenies were analyzed for CrylAb and Hpt genes. PCR analysis revealed that 412 regenerated plants were Hpt positive （80.94%）, 62 plants were also CrylAb co-transformants （15.05% in total frequency）, and 42 plants among the 272 T2 progenies were CrylAb positive but Hpt negative. This suggests that marker-free transgenic plants could be produced by co-transformation mediated by mixed Agrobacterium strains with the selectable marker gene and target gene Southern blot analysis of five independent marker-free T2 transgenic lines co-transformed from Zhejing 22 showed that CrylAb gene had been inserted into rice genome with a single copy. The transgenic plants showed significantly stronger resistance to lepidopteron than the non-transgenic plants under no application of insecticides against lepidopteron.

Development and drought tolerance assay of marker-free transgenic rice with OsAPX2 using biolistic particle-mediated co-transformation

Abiotic stresses such as drought, salinity, and low temperature cause–losses in rice production worldwide. The emergence of transgenic technology has enabled improvements in the drought resistance of rice plants and helped avert crop damage due to drought stress.Selectable marker genes conferring resistance to antibiotics or herbicides have been widely used to identify genetically modified plants. However, the use of such markers has limited the public acceptance of genetically modified organisms. Marker-free materials (i.e., those containing a single foreign gene) may be more easily accepted by the public and more likely to find common use. In the present study, we created marker-free drought-tolerant transgenic rice plants using particle bombardment. Overall, 842 T_0plants overexpressing the rice ascorbate peroxidase-coding gene OsAPX2 were generated. Eight independentmarker-free lines were identified from T_1 seedlings using the polymerase chain reaction.The molecular characteristics of these lines were examined, including the expression level,copy number, and flanking sequences of OsAPX2, in the T_2 progeny. A simulated drought test using polyethylene glycol and a drought-tolerance test of seedlings confirmed that the marker-free lines carrying OsAPX2 showed significantly improved drought tolerance in seedlings. In the field, the yield of the wild-type plant decreased by 60% under drought conditions compared with normal conditions. However, the transgenic line showed a yield loss of approximately 26%. The results demonstrated that marker-free transgenic lines significantly improved grain yield under drought-stressed conditions.

Co-transformation to tobacco of Cre/lox site-specific recombination system and its precise recombination

For the temporally and spatially regulated expression of the barnase gene in plant, two kinds of plasmids with ere gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure. The transgenic plants were conformed by PCR analysis. The blocking fragment between the two lox directly repeat sites was excised by Cre protein in the transgenic plant genome. Cloning and seguencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre//ox site-specif ic recombination .

Co-overexpression of genes for nitrogen transport, assimilation, and utilization boosts rice grain yield and nitrogen use efficiency

Nitrogen(N)fertilization is necessary for obtaining high rice yield.But excessive N fertilizer reduces rice plant N efficiency and causes negative effects such as environmental pollution.In this study,we assembled key genes involved in different nodes of N pathways to boost nitrate and ammonium uptake and assimilation,and to strengthen amino acid utilization to increase grain yield and nitrogen use efficiency(NUE)in rice.The combinations OsNPF8.9a×OsNR2,OsAMT1;2×OsGS1;2×OsAS1,and OsGS2×OsAS2×OsANT3 optimized nitrate assimilation,ammonium conversion,and N reutilization,respectively.In co-overexpressing rice lines obtained by co-transformation,the tiller number,biomass,and grain yield per plant of the OsAMT1;2×OsGS1;2×OsAS1-overexpressing line exceeded those of wild-type ZH11,the OsNPF8.9a×OsNR2×OsGS1;2×OsAS1-overexpressing line,and the OsGS2×OsAS2×OsANT3-overexpressing line.The glutamine synthase activity,free amino acids,and nitrogen utilization efficiency(NUt E)of the OsAMT1;2×OsGS1;2×OsAS1-overexpressing line exceeded those of ZH11 and other lines that combined key genes.N influx efficiency was increased in the OsAMT1;2×OsGS1;2×OsAS1-overexpressing line and OsNPF8.9a×OsNR2×OsGS1;2×OsAS1-overexpressing line under a low ammonium and a low nitrate treatment,respectively.We propose that combining overexpression of OsAMT1;2,OsGS1;2,and OsAS1 is a promising breeding strategy for systematically increasing rice grain yield and NUE by focusing on key nodes in the N pathway.

Efficiencies of Generating Selectable Marker-Free Transgenic Rice with Different Transformation Methods

To study the efficiency of generating selectable marker-free （SMF） transgenic rice, two transformation methods were employed for four rice varieties （Wuxiangjing 9, Longtefu, Xieqingzao and Zhenshan 97）. One method is by using a single twin T-DNA binary vector pYH592 in one Agrobacterium strain, which is composed of two separate T-DNA regions （one carrying an antisense Wx gene and the other carrying a HPTgene）. The other one, named as two-strain/two-vector system, is by using two separate binary vectors in two separate Agrobacterium cultures. The results indicated that the average co-transformation frequencies of the antisense Wx gene and the HPT gene were 10.1% and 45.0%, respectively, for the four rice varieties. And the SMF transgenic plants selected from the offsprings of co-transformants were 55.6% and 60.0% in the two-strain/two-vector and twin T-DNA vector binary systems, respectively.

Induced transformation of antimony trioxide by Mn(II) oxidation and their co-transformed mechanism

Antimony(Sb)is a toxic and carcinogenic element that often enters soil in the form of antimony trioxide(Sb_(2)O_(3))and coexists with manganese(Mn)in weakly alkaline conditions.Mn oxides such as birnessite have been found to promote the oxidative dissolution of Sb_(2)O_(3),but few researches concerned the co-transformations of Sb_(2)O_(3) and Mn(II)in environment.This study investigated themutual effect of abiotic oxidation of Mn(II)and the coupled oxidative dissolution of Sb_(2)O_(3).The influencing factors,such as Mn(II)concentrations,pH and oxygen were also discussed.Furthermore,their co-transformed mechanism was also explored based on the analysis of Mn(II)oxidation products with or without Sb_(2)O_(3) using XRD,SEM and XPS.The results showed that the oxidative dissolution of Sb_(2)O_(3) was enhanced under higher pH and higher Mn(II)loadings.With a lower Mn(II)concentration such as 0.01 mmol/L Mn(II)at pH 9.0,the improved dissolution of Sb_(2)O_(3) was attributed to the generation of dissolved intermediate Mn(III)species with strong oxidation capacity.However,under higher Mn(II)concentrations,both amorphous Mn(III)oxides and intermediate Mn(III)species were responsible for promoting the oxidative dissolution of Sb_(2)O_(3).Most released Sb(∼72%)was immobilized by Mn oxides and Sb(V)was dominant in the adsorbed and dissolved total Sb.Meanwhile,the presence of Sb_(2)O_(3) not only inhibited the removal of Mn(II)by reducing Mn(III)to Mn(II)but also affected the final products of Mn oxides.For example,amorphous Mn oxides were formed instead of crystalline Mn(III)oxides,such as MnOOH.Furthermore,rhodochrosite(MnCO_(3))was formed with the high Mn(II)/Sb_(2)O_(3) ratio,but without being observed in the low Mn(II)/Sb_(2)O_(3) ratio.The results of study could help provide more understanding about the fate of Sb in the environment and the redox transformation of Mn.

FISH analysis of the integration patterns in transgenic rice co-transformed by micro-projectile bombardment

Using multi-color fluorescence in situ hybridization (FISH), we localized transferred barnase-psl and pHctinG DNA sequences onto chromosomes of two trans-genie rice plants, named Q12 and Q13, both of which were produced by micro-projectile bombardment. In both Q12 and Q13, each detected cell showed 2-3 signal spots on their chromosomes respectively. The signals of both barnase-psl and pHctinG were mostly detected in the adjacent chromosomal sites in which their signals were overlapped and could be recognized by the signal color on the metaphase chromosomes. Fiber FISH further demonstrated that the multiple copies in each of the two DNA sequences distributed adjacently on the DNA fiber in Q13. Combined with the results of Southern hybridization, the possible integration patterns in transgenic rice co-transformed by micro-projectile bombardment have been discussed.

Regeneration of foreign genes co-transformed plants of Medicago sativa L by Agrobacterium rhizogenes

Gene encoding sulphur amino acid-rich protein (HNP) and rol genes were transferred into Medicago sativa L (alfalfa) mediated by Agrobacterium tumafeciens. Regeneration of trans-genie plants was induced successfully from hairy root tissue of cotyledon in alfalfa. Cotyledon tissues were an ideally transformed recipient. There was a negative correlation between age of hairy roots and embryogenesis frequency in alfalfa. Production of co-transformed plants with greater yield and super quality was important for development of new alfalfa varieties.

Complementation and Expression Analysis of SoRablA and SoRab2A in Sugarcane Demonstrates Their Functional Diversification

Mammalian and plant Rabl and Rab2 are small GTPases that regulate vesicle trafficking in the endoplasmic reticulum （ER） to Golgi compartments. Little is known about their functional diversification or potential interaction. We cloned sugarcane （Saccharum officinarum L.） Rab1A and Rab2A genes and studied their functional differences by expression and complementation experiments. We found differential expression of the two genes during sugarcane leaf development： SoRab2A expression declined from the dividing base to the maturing tip of the growing leaves, whereas SoRab1A was constitutively expressed, suggesting that SoRab2A is required for cell division and expansion and SoRablA is required for cells at all developmental stages. We used a yeast temperature sensitive ypt1-A 136D mutant strain to further investigate these shared and unique functions. Yptl is a small GTPase that regulates vesicle transport in the same cellular location as Rabl and Rab2. Neither SoRab1A nor SoRab2A alone could restore the growth of the mutant at restrictive temperatures when SoRab1A and SoRab2A were transformed separately. However, SoRab1A transformants maintained normal morphology and viability at non-permissive temperature, and resumed growth when returned to permissive temperature, whereas SoRab2A transformants died at non-permissive temperature, suggesting that SoRablA function is required for a cell＇s viability. Mutant growth was fully restored when SoRab1A and SoRab2A were co-transformed, indicating that SoRablA and SoRab2A complement each other and they both are needed to restore the function of ypt1-A136D. These results demonstrate that SoRab1A and SoRab2A serve distinct but overlapping functions, mostly by regulating the transportation of different sets of proteins.