Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression f...Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of (-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected with Agrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of (-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.展开更多
Genetic transformation is an important technique for functional genomics study and genetic improvement of plants. Until now, Agrobacterium-mediated transformation methods using cotyledon as explants has been the major...Genetic transformation is an important technique for functional genomics study and genetic improvement of plants. Until now, Agrobacterium-mediated transformation methods using cotyledon as explants has been the major approach for cucumber, and its frequency has been up to 23%. For example, significantly enhancement of the transformation efficiency of this plant species was achieved from the cotyledon explants of the cultivar Poinsett 76 infected by Agrobacterium strains EHA105 with efficient positive selection system in lots of experiments. This review is to summarize some key factors influencing cucumber regeneration and genetic transformation, including target genes, selection systems and the ways of transgene introduction, and then to put forward some strategies for the increasing of cucumber transformation efficiency. In the future, it is high possible for cucumber to be potential bioreactor to produce vaccine and biomaterials for human beings.展开更多
Switchgrass is native to the tallgrass prairie of North America. It is self-incompatible and has varied ploidy levels from diploid（2x） to dodecaploid（12x） with tetraploid and octoploid being the most common. The h...Switchgrass is native to the tallgrass prairie of North America. It is self-incompatible and has varied ploidy levels from diploid（2x） to dodecaploid（12x） with tetraploid and octoploid being the most common. The high yielding potential and the ability to grow well in marginal lands make switchgrass an ideal species as a dedicated biomass producer for lignocellulosic ethanol production. Genetic transformation is an important tool for studying gene function and for germplasm improvement in switchgrass, the genome of which has been sequenced recently. This paper intends to provide a comprehensive review on plant regeneration and genetic transformation in switchgrass. We first reviewed the effect of explants, basal medium and plant growth regulators on plant regeneration in switchgrass, which is a prerequisite for genetic transformation. We then reviewed the progresses on genetic transformation with either the biolistic or Agrobacterium-mediated method in switchgrass, and discussed various techniques employed to improve the transformation efficiency. Finally we reviewed the recent progresses on the use of genetic transformation in improving biomass quality such as the reduction of lignin, and in increasing biomass yield in switchgrass. We also provided a future perspective on the use of new genome editing technologies in switchgrass and its potential impact on regulatory processes.展开更多
Non-embryogenic calli （NEC） was inevitably and heavily produced when grape embryogenic calli （EC） was induced from explants or during the subculture of EC.A stable and highly efficient NEC transformation platform ...Non-embryogenic calli （NEC） was inevitably and heavily produced when grape embryogenic calli （EC） was induced from explants or during the subculture of EC.A stable and highly efficient NEC transformation platform is required to further sort out and verify key genes which determine/switch the identity of NEC and EC.In this research,a vector pA5 containing a chitinase signal sequence fused to gfp （green fluorescent protein） and an HDEL motive was used to target and immobilize into Agrobacterium strain EHA105 to establish a transformation platform for Vitis vinifera L.cv.Chardonnay NEC.It was determined that NEC 10 d after subculture was the best target tissue;30 min for inoculation followed by 3 d co-cultivation with the addition of 200 μmol L-1 acetosyringone （AS） was optimized as protocol.The use of bacterial densities as 1.0 at OD600 did not result in serious tissue hypersensitive reaction and it had higher efficiency.Kanamycin at 200 mg L-1 was picked for positive expression selection.The stable transformation of NEC was proved by reverse transcription-polymerase chain reaction techniques （RT-PCR） and fluorescent microscopy after three sub-cultures of the selected cell line.Highly efficient genetic transformation protocol of grape NEC was achieved and some of the optimized parameters were different from that reported for EC.This transformation platform could facilitate the verification of candidate somatic embryogenesis （SE） decisive genes,and the successfully transformed NEC with certain genes can also be used as bioreactors for the production of functional products,as NEC not only proliferates fast,but also keeps in a rather stable condition.展开更多
A method of vehicle license plate recognition utilizing Karhunen-Loeve(K-L) transform is provided. The transform is used to extract features from a mass of image templates, to describe high-dimensional images with low...A method of vehicle license plate recognition utilizing Karhunen-Loeve(K-L) transform is provided. The transform is used to extract features from a mass of image templates, to describe high-dimensional images with low-dimensional ones, and moreover, to implement data compression and play down complexity of the neural network. With the character to reduce eigenspace dimensionality of K-L transform and the ability to map data of BP network, the method does effectively in recognizing license plates.展开更多
A transformation system was established for loblolly pine (Pimus taeda L ) mature zygotic embryos using Agrobacterium tumefaciens.The gene coding for the glucuronidase (GUS) gene was introduced into loblolly pine tiss...A transformation system was established for loblolly pine (Pimus taeda L ) mature zygotic embryos using Agrobacterium tumefaciens.The gene coding for the glucuronidase (GUS) gene was introduced into loblolly pine tissues andits transient expression was detected with histochemical staining. The influences of different genotypes. Agrobacterium concentrations. and cocultivation time on GUS expression and Kanamycin resistant callus and shoot regeheration were investigated. The results showed that the highest `GUS expression frequency (1 6.3%) and shoot regencration frequency(7.8%) wereobtained from genotype 9-1003 with, Agrobactemm concentration decreased 9 times and cocultivation time of 56 hours.respectively GUS expression was, obtained in all genotypes tested The successtul expression of the GUS gene in differentgenotypes suggested that it will be a useful transformation system for loblolly pine展开更多
Background: The carcinogenesis of hepatocellular carcinoma (HCC) is a multi-factorial, multistep and complex process. Its prognosis is poor and early detection is of the utmost importance. Transforming growth factor ...Background: The carcinogenesis of hepatocellular carcinoma (HCC) is a multi-factorial, multistep and complex process. Its prognosis is poor and early detection is of the utmost importance. Transforming growth factor β1 (TGF-β1) message RNA (mRNA) has been reported to be elevated in HCC patients using Northern blotting. However, little work has been done about the detection of TGF-β1 mRNA levels in peripheral blood of patients with HCC using the real-time polymerase chain reactions (PCR) method. Objective: To assess the prognostic value of quantitative levels of TGF-β1 mRNA in peripheral blood of patients with HCC, and to investigate the relationship between the expression of TGF-β1 mRNA in peripheral blood and many diagnostic and pathological factors. Methods: We developed an optimized Taqman real-time PCR to quantify TGF-β1 mRNA in peripheral blood of 53 patients with HCC and 44 healthy volunteers. In addition, blood was collected from patients with HCC for measuring levels of total bilirubin (TBil), prealbumin, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltranspeptidase (GGT), alpha-L-fucosidase (AFU), alpha fetoprotein (AFP), carcino-embryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), viral load and platelet counts. Statistical analysis was performed using the SPSS software system (SPSS 10.0). Results: In real-time PCR, fluorescence was detectable in all blood specimens from patients with HCC and healthy volunteers. The levels of TGF-β1 mRNA expression in patients with HCC were significantly higher compared to that in healthy volunteers (P<0.000 1), suggesting an association of the activated TGF-β1 gene transcription with hepato- carcinogenesis. Patients with HCC were divided into 2 groups according to their TGF-β1 mRNA above (group A, n=28)or below (group B, n=25) the mean level. Statistical results demonstrated that TGF-β1 mRNA expression level was correlated with patients age, serum levels of CEA, CA19-9 and viral copy number (P<0.05). Conclusion: Although this is a small sample size pilot study these findings imply that quantitative measurement of TGF-β1 mRNA level in peripheral blood may be a complementary serologic marker of HCC.展开更多
A transformation procedure of choline monooxygenase(CMO) gene, involved in stress tolerance, was established in white pine embryogenic tissue by using A. tumefaciens C58/pMP90. The CMO cDNA fragment(1.3 kb) was genera...A transformation procedure of choline monooxygenase(CMO) gene, involved in stress tolerance, was established in white pine embryogenic tissue by using A. tumefaciens C58/pMP90. The CMO cDNA fragment(1.3 kb) was generated by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) with primers based on the report sequence of CMO in gene bank. A chimerical gene composed of the cauliflower mosaic virus (CaMV) 35S promoter fused to CMO cDNA and β-glucuronidase (GUS-marker gene) was transferred into Ti-derived disarmed binary vector pBI121. The new vector, p35SCMOp, was transferred into Agrobacterium tumefaciens C58/pMP90 by freeze-thaw method. Somatic embryogenesis (SE) initiation of Pinus. Strobus L. and Pinus.Koraiensis Sieb. et Zucc. depended on the manipulation of plant growth regulator (PGR) concentrations in the GLH culture medium. Transgenic embryos and regenerated plants of two Pine species were produced after co-culture of embryogenic tissue with the disarmed strain of A. tumefaciens C58/pMP90/ p35SCMOp and selected on medium containing 25mg/L kanamycin. The transformed embryogenic tissue was initially confirmed by histochemical GUS assay followed by PCR. One copy of T-DNA was detected by transgenic lines analysis in Pinus. Strobus L. and transgenic plants were regenerated for two species using modified protocols for maturation and germination of somatic embryos.展开更多
In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was ...In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was constructed and the vector was introduced into tobacco with the agrobacterium-mediated method. PCR results showed that the RrGlu gene was integrated into the tobacco genome.展开更多
Using the hypocotyl and cotyledon explants of Brassica napus L. cuhivar Qingza No. 5 as receptors, hormone combinations in bud differentiation medi- um, bud growth medium and rooting medium were optimized to establish...Using the hypocotyl and cotyledon explants of Brassica napus L. cuhivar Qingza No. 5 as receptors, hormone combinations in bud differentiation medi- um, bud growth medium and rooting medium were optimized to establish an efficient plantlet regeneration system of B. napus cuhivar Qingza No. 5. The results showed that the highest differentiation efficiency of hypocotyls of B. napus cuhivar Qingza No. 5 reached about 90%, which was three times that of cotyledons. The appropriate differentiation medium was MSB ＋ 5 mg/L thidiazuron （TDZ） ＋7.5 mg/L AgNO3 ＋ 0.1 mg/L NAA ＋ 2 mg/L proline （L-pro） ＋ 250 mg/L casein acid hydrolysate （CH） ＋ 3% sucrose; the appropriate growth medium was 1/2 MSB ＋ 1 mg/L IBA ＋ 2 mg/L L-pro ＋ 250 mg/L CH ＋ 1.5% sucrose; the ap- propriate rooting medium was 1/2 MSB ＋ 0.2 mg/L IAA ＋ 1.5% sucrose. On this basis, a binary expression vector harboring insect resistance gene B12 was constructed and introduced into B. napus hypocotyls by Agrobacterium-mediated transformation. Positive plants were screened using hygromycin and carbenicillin. Transgenic plants were verified by PCR and GUS histochemical staining. The results showed that insect resistance gene B12 was successfully integrated into the nu- clear genome of B. napus plants and could be expressed normally. Leaves of transgenic plants with high expression levels were collected for indoor inoculation test with Plutella xylotella larvae to evaluate insect resistance of transgenic plants.展开更多
Shoot meristems used for the study were exercised from the in vitro regenerated shoots cultured on MS medium supplemented with 0.5 mg/L of BAP for multiplication. The sensitivity of the in vitro regenerated was studie...Shoot meristems used for the study were exercised from the in vitro regenerated shoots cultured on MS medium supplemented with 0.5 mg/L of BAP for multiplication. The sensitivity of the in vitro regenerated was studied using shoot meristems of 0.5 cm. Shoot meristems were cultured on medium containing 10-100 mg/l kanamycin to determine the concentration that was lethal for multiple shoot induction and root induction. The response of shoot multiplication decreased (66.2%-6.2%) as the concentration of kanamycin increased (10.0-70.0 mg/L) with complete inhibition of shoot proliferation at 100 mg/L kanamycin. The rooting phase was very sensitive to kanamycin compared to shoot multiplication. The percentage of shoots that rooted decreased (53.8%-4.8%) with increase in the concentration of kanamycin (10.0-70.0 mg/l) on IBA and 2,4-D supplemented medium. For transformation studies, the shoot tips that were infected with Agrobacterium strain were placed on selection medium containing MS medium with 0.5 mg/L BAP and 100 mg/L kanamycin and scored for the putative transformed shoots. An average of 62.2% of shoot tips developed shoot buds from the base and the shoots reached a length of 0.5-1.0 cm at the end of 30 days of culture on the selective medium in comparison to control which showed no response. An average of 66.7% of the regenerated plants showed GUS expression on selection medium where 43.2% and 65% of GUS expression was recorded in the leaves and callus. Leaves and callus induced from the controls did not show GUS activity. Stable integration of nptII gene with the genomic DNA from these transformed plants was confirmed through PCR analysis. Our result presents an efficient regeneration system using in vitro derived shoot meristems for Agrobacterium mediated gene transfer.展开更多
文摘Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of (-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected with Agrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of (-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.
基金financially supported by grants from the Biogreen 21 Program, RDA, Korea (PJ00810304)the Agricultural Science and Technology Innovation Program (ASTIP) of Chinese Academy of Agricultural Sciences (2014–2015)the Beijing Municipal Education Commission, China (KM200910011001)
文摘Genetic transformation is an important technique for functional genomics study and genetic improvement of plants. Until now, Agrobacterium-mediated transformation methods using cotyledon as explants has been the major approach for cucumber, and its frequency has been up to 23%. For example, significantly enhancement of the transformation efficiency of this plant species was achieved from the cotyledon explants of the cultivar Poinsett 76 infected by Agrobacterium strains EHA105 with efficient positive selection system in lots of experiments. This review is to summarize some key factors influencing cucumber regeneration and genetic transformation, including target genes, selection systems and the ways of transgene introduction, and then to put forward some strategies for the increasing of cucumber transformation efficiency. In the future, it is high possible for cucumber to be potential bioreactor to produce vaccine and biomaterials for human beings.
基金supported by a grant from the Bill Melinda Gates FoundationNational Institute of Food and Agriculture of the United States Department of Agriculture for support (Award number 2013-33522-21091)
文摘Switchgrass is native to the tallgrass prairie of North America. It is self-incompatible and has varied ploidy levels from diploid（2x） to dodecaploid（12x） with tetraploid and octoploid being the most common. The high yielding potential and the ability to grow well in marginal lands make switchgrass an ideal species as a dedicated biomass producer for lignocellulosic ethanol production. Genetic transformation is an important tool for studying gene function and for germplasm improvement in switchgrass, the genome of which has been sequenced recently. This paper intends to provide a comprehensive review on plant regeneration and genetic transformation in switchgrass. We first reviewed the effect of explants, basal medium and plant growth regulators on plant regeneration in switchgrass, which is a prerequisite for genetic transformation. We then reviewed the progresses on genetic transformation with either the biolistic or Agrobacterium-mediated method in switchgrass, and discussed various techniques employed to improve the transformation efficiency. Finally we reviewed the recent progresses on the use of genetic transformation in improving biomass quality such as the reduction of lignin, and in increasing biomass yield in switchgrass. We also provided a future perspective on the use of new genome editing technologies in switchgrass and its potential impact on regulatory processes.
基金supported by the National Natural Science Foundation of China (30471212,30500347)the Earmarked Fund for Modern Agro-Industry Technology Research System,Ministry of Agriculture,China (NYCYTX-3-CY-04)
文摘Non-embryogenic calli （NEC） was inevitably and heavily produced when grape embryogenic calli （EC） was induced from explants or during the subculture of EC.A stable and highly efficient NEC transformation platform is required to further sort out and verify key genes which determine/switch the identity of NEC and EC.In this research,a vector pA5 containing a chitinase signal sequence fused to gfp （green fluorescent protein） and an HDEL motive was used to target and immobilize into Agrobacterium strain EHA105 to establish a transformation platform for Vitis vinifera L.cv.Chardonnay NEC.It was determined that NEC 10 d after subculture was the best target tissue;30 min for inoculation followed by 3 d co-cultivation with the addition of 200 μmol L-1 acetosyringone （AS） was optimized as protocol.The use of bacterial densities as 1.0 at OD600 did not result in serious tissue hypersensitive reaction and it had higher efficiency.Kanamycin at 200 mg L-1 was picked for positive expression selection.The stable transformation of NEC was proved by reverse transcription-polymerase chain reaction techniques （RT-PCR） and fluorescent microscopy after three sub-cultures of the selected cell line.Highly efficient genetic transformation protocol of grape NEC was achieved and some of the optimized parameters were different from that reported for EC.This transformation platform could facilitate the verification of candidate somatic embryogenesis （SE） decisive genes,and the successfully transformed NEC with certain genes can also be used as bioreactors for the production of functional products,as NEC not only proliferates fast,but also keeps in a rather stable condition.
文摘A method of vehicle license plate recognition utilizing Karhunen-Loeve(K-L) transform is provided. The transform is used to extract features from a mass of image templates, to describe high-dimensional images with low-dimensional ones, and moreover, to implement data compression and play down complexity of the neural network. With the character to reduce eigenspace dimensionality of K-L transform and the ability to map data of BP network, the method does effectively in recognizing license plates.
文摘A transformation system was established for loblolly pine (Pimus taeda L ) mature zygotic embryos using Agrobacterium tumefaciens.The gene coding for the glucuronidase (GUS) gene was introduced into loblolly pine tissues andits transient expression was detected with histochemical staining. The influences of different genotypes. Agrobacterium concentrations. and cocultivation time on GUS expression and Kanamycin resistant callus and shoot regeheration were investigated. The results showed that the highest `GUS expression frequency (1 6.3%) and shoot regencration frequency(7.8%) wereobtained from genotype 9-1003 with, Agrobactemm concentration decreased 9 times and cocultivation time of 56 hours.respectively GUS expression was, obtained in all genotypes tested The successtul expression of the GUS gene in differentgenotypes suggested that it will be a useful transformation system for loblolly pine
基金the National Natural Science Foundation of China (30770994)
文摘Background: The carcinogenesis of hepatocellular carcinoma (HCC) is a multi-factorial, multistep and complex process. Its prognosis is poor and early detection is of the utmost importance. Transforming growth factor β1 (TGF-β1) message RNA (mRNA) has been reported to be elevated in HCC patients using Northern blotting. However, little work has been done about the detection of TGF-β1 mRNA levels in peripheral blood of patients with HCC using the real-time polymerase chain reactions (PCR) method. Objective: To assess the prognostic value of quantitative levels of TGF-β1 mRNA in peripheral blood of patients with HCC, and to investigate the relationship between the expression of TGF-β1 mRNA in peripheral blood and many diagnostic and pathological factors. Methods: We developed an optimized Taqman real-time PCR to quantify TGF-β1 mRNA in peripheral blood of 53 patients with HCC and 44 healthy volunteers. In addition, blood was collected from patients with HCC for measuring levels of total bilirubin (TBil), prealbumin, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltranspeptidase (GGT), alpha-L-fucosidase (AFU), alpha fetoprotein (AFP), carcino-embryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), viral load and platelet counts. Statistical analysis was performed using the SPSS software system (SPSS 10.0). Results: In real-time PCR, fluorescence was detectable in all blood specimens from patients with HCC and healthy volunteers. The levels of TGF-β1 mRNA expression in patients with HCC were significantly higher compared to that in healthy volunteers (P<0.000 1), suggesting an association of the activated TGF-β1 gene transcription with hepato- carcinogenesis. Patients with HCC were divided into 2 groups according to their TGF-β1 mRNA above (group A, n=28)or below (group B, n=25) the mean level. Statistical results demonstrated that TGF-β1 mRNA expression level was correlated with patients age, serum levels of CEA, CA19-9 and viral copy number (P<0.05). Conclusion: Although this is a small sample size pilot study these findings imply that quantitative measurement of TGF-β1 mRNA level in peripheral blood may be a complementary serologic marker of HCC.
文摘A transformation procedure of choline monooxygenase(CMO) gene, involved in stress tolerance, was established in white pine embryogenic tissue by using A. tumefaciens C58/pMP90. The CMO cDNA fragment(1.3 kb) was generated by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) with primers based on the report sequence of CMO in gene bank. A chimerical gene composed of the cauliflower mosaic virus (CaMV) 35S promoter fused to CMO cDNA and β-glucuronidase (GUS-marker gene) was transferred into Ti-derived disarmed binary vector pBI121. The new vector, p35SCMOp, was transferred into Agrobacterium tumefaciens C58/pMP90 by freeze-thaw method. Somatic embryogenesis (SE) initiation of Pinus. Strobus L. and Pinus.Koraiensis Sieb. et Zucc. depended on the manipulation of plant growth regulator (PGR) concentrations in the GLH culture medium. Transgenic embryos and regenerated plants of two Pine species were produced after co-culture of embryogenic tissue with the disarmed strain of A. tumefaciens C58/pMP90/ p35SCMOp and selected on medium containing 25mg/L kanamycin. The transformed embryogenic tissue was initially confirmed by histochemical GUS assay followed by PCR. One copy of T-DNA was detected by transgenic lines analysis in Pinus. Strobus L. and transgenic plants were regenerated for two species using modified protocols for maturation and germination of somatic embryos.
文摘In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was constructed and the vector was introduced into tobacco with the agrobacterium-mediated method. PCR results showed that the RrGlu gene was integrated into the tobacco genome.
基金Supported by National Natural Science Foundation of China(31301703)Agricultural Science and Technology Independent Innovation Fund of Jiangsu Province[CX(14)5068]
文摘Using the hypocotyl and cotyledon explants of Brassica napus L. cuhivar Qingza No. 5 as receptors, hormone combinations in bud differentiation medi- um, bud growth medium and rooting medium were optimized to establish an efficient plantlet regeneration system of B. napus cuhivar Qingza No. 5. The results showed that the highest differentiation efficiency of hypocotyls of B. napus cuhivar Qingza No. 5 reached about 90%, which was three times that of cotyledons. The appropriate differentiation medium was MSB ＋ 5 mg/L thidiazuron （TDZ） ＋7.5 mg/L AgNO3 ＋ 0.1 mg/L NAA ＋ 2 mg/L proline （L-pro） ＋ 250 mg/L casein acid hydrolysate （CH） ＋ 3% sucrose; the appropriate growth medium was 1/2 MSB ＋ 1 mg/L IBA ＋ 2 mg/L L-pro ＋ 250 mg/L CH ＋ 1.5% sucrose; the ap- propriate rooting medium was 1/2 MSB ＋ 0.2 mg/L IAA ＋ 1.5% sucrose. On this basis, a binary expression vector harboring insect resistance gene B12 was constructed and introduced into B. napus hypocotyls by Agrobacterium-mediated transformation. Positive plants were screened using hygromycin and carbenicillin. Transgenic plants were verified by PCR and GUS histochemical staining. The results showed that insect resistance gene B12 was successfully integrated into the nu- clear genome of B. napus plants and could be expressed normally. Leaves of transgenic plants with high expression levels were collected for indoor inoculation test with Plutella xylotella larvae to evaluate insect resistance of transgenic plants.
文摘Shoot meristems used for the study were exercised from the in vitro regenerated shoots cultured on MS medium supplemented with 0.5 mg/L of BAP for multiplication. The sensitivity of the in vitro regenerated was studied using shoot meristems of 0.5 cm. Shoot meristems were cultured on medium containing 10-100 mg/l kanamycin to determine the concentration that was lethal for multiple shoot induction and root induction. The response of shoot multiplication decreased (66.2%-6.2%) as the concentration of kanamycin increased (10.0-70.0 mg/L) with complete inhibition of shoot proliferation at 100 mg/L kanamycin. The rooting phase was very sensitive to kanamycin compared to shoot multiplication. The percentage of shoots that rooted decreased (53.8%-4.8%) with increase in the concentration of kanamycin (10.0-70.0 mg/l) on IBA and 2,4-D supplemented medium. For transformation studies, the shoot tips that were infected with Agrobacterium strain were placed on selection medium containing MS medium with 0.5 mg/L BAP and 100 mg/L kanamycin and scored for the putative transformed shoots. An average of 62.2% of shoot tips developed shoot buds from the base and the shoots reached a length of 0.5-1.0 cm at the end of 30 days of culture on the selective medium in comparison to control which showed no response. An average of 66.7% of the regenerated plants showed GUS expression on selection medium where 43.2% and 65% of GUS expression was recorded in the leaves and callus. Leaves and callus induced from the controls did not show GUS activity. Stable integration of nptII gene with the genomic DNA from these transformed plants was confirmed through PCR analysis. Our result presents an efficient regeneration system using in vitro derived shoot meristems for Agrobacterium mediated gene transfer.
