Solanum lycopersicum‘Heinz 1706’is a pioneer model cultivar for tomato research,whose whole genome sequence valuable for genomics studies is available.Nevertheless,a genetic transformation procedure for this cultiva...Solanum lycopersicum‘Heinz 1706’is a pioneer model cultivar for tomato research,whose whole genome sequence valuable for genomics studies is available.Nevertheless,a genetic transformation procedure for this cultivar has not yet been reported.Meanwhile,various genome editing technologies such as transfection of clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)ribonucleoprotein complexes into cells are in the limelight.Utilizing the Cas9-expressing genotype possessing a reference genome can simplify the verification of an off-target effect,resolve the economic cost of Cas9 endonuclease preparation,and avoid the complex assembly process together with single-guide RNA(sgRNA)in the transfection approach.Thus,this study was designed to generate Cas9-expressing‘Heinz 1706’lines by establishing an Agrobacterium tumefaciens-mediated transformation(ATMT)procedure.Here,we report a rapid and reproducible transformation procedure for‘Heinz 1706’by finetuning various factors:A.tumefaciens strain,pre-culture and co-culture durations,a proper combination of phytohormones at each step,supplementation of acetosyringone,and shooting/rooting method.Particularly,through eluding subculture and simultaneously inducing shoot elongation and rooting from leaf cluster,we achieved a short duration of three months for recovering the transgenic plants expressing Cas9.The presence of the Cas9 gene and its stable expression were confirmed by PCR and qRT-PCR analyses,and the Cas9 gene integrated into the T_(0) plant genome was stably transmitted to T_(1) progeny.Therefore,we anticipate that our procedure appears to ease the conventional ATMT in‘Heinz 1706’,and the created Cas9-expressing‘Heinz 1706’lines are ultimately useful in gene editing via unilateral transfection of sgRNA into the protoplasts.展开更多
Acetosyringone （AS） has great influences on Agrobacterium-mediated genetic transformation. In order to improve the transformation frequency of waxy maize, in this study, shoot tips and immature embryo-derived callus...Acetosyringone （AS） has great influences on Agrobacterium-mediated genetic transformation. In order to improve the transformation frequency of waxy maize, in this study, shoot tips and immature embryo-derived callus of Lainongnuo 38 were collected as receptor materials, to compare the effects of pH in suspension medium, addition modes and concentrations of acetosyringone on genetic transformation of waxy maize. Results showed that adding 2 μl of 150 μmol/L AS in the wound with suspension medimn at pH 5.2 can significantly improve the transformation frequency of shoot tips of waxy maize; in genetic transformation of maize callus, adding 5 mg/L AS in infection solution can significantly improve the percentage of resistant callus.展开更多
Several wood rotting fungi decolourized Remazol brilliant blue R (RBBR) and carminic acid (CA). Parallel activity of laccase in these fungi was studied. The addition of acetovanillone (AV) or acetosyringone (AS) inten...Several wood rotting fungi decolourized Remazol brilliant blue R (RBBR) and carminic acid (CA). Parallel activity of laccase in these fungi was studied. The addition of acetovanillone (AV) or acetosyringone (AS) intensified these processes: decolourization was more extensive than in the experiment omitting these compounds. At the presence of AS the decourization was more extensive than AV. However the level of decolorizing was relatively low in comparison to laccase activity on syringaldazine. The highly purified constitutive form of Cerrena unicolor and inducible form of Trametes versicolor laccases also destained both dyes. Anyway the addition of AV and AS improved the efficiency of dyes decolourization by wood rotting fungi and fungal laccase. Nitrogen starvation induced the laccase and decoloration activity in both organisms, irrespective of nitrogen availability. This fact indicates laccase not solely responsible for discoloration, and probably discoloration of dyes involves more than one mechanism.展开更多
文摘Solanum lycopersicum‘Heinz 1706’is a pioneer model cultivar for tomato research,whose whole genome sequence valuable for genomics studies is available.Nevertheless,a genetic transformation procedure for this cultivar has not yet been reported.Meanwhile,various genome editing technologies such as transfection of clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)ribonucleoprotein complexes into cells are in the limelight.Utilizing the Cas9-expressing genotype possessing a reference genome can simplify the verification of an off-target effect,resolve the economic cost of Cas9 endonuclease preparation,and avoid the complex assembly process together with single-guide RNA(sgRNA)in the transfection approach.Thus,this study was designed to generate Cas9-expressing‘Heinz 1706’lines by establishing an Agrobacterium tumefaciens-mediated transformation(ATMT)procedure.Here,we report a rapid and reproducible transformation procedure for‘Heinz 1706’by finetuning various factors:A.tumefaciens strain,pre-culture and co-culture durations,a proper combination of phytohormones at each step,supplementation of acetosyringone,and shooting/rooting method.Particularly,through eluding subculture and simultaneously inducing shoot elongation and rooting from leaf cluster,we achieved a short duration of three months for recovering the transgenic plants expressing Cas9.The presence of the Cas9 gene and its stable expression were confirmed by PCR and qRT-PCR analyses,and the Cas9 gene integrated into the T_(0) plant genome was stably transmitted to T_(1) progeny.Therefore,we anticipate that our procedure appears to ease the conventional ATMT in‘Heinz 1706’,and the created Cas9-expressing‘Heinz 1706’lines are ultimately useful in gene editing via unilateral transfection of sgRNA into the protoplasts.
基金Supported by National Natural Science Foundation of China(31201218)National Science and Technology Project of "Twelfth Five Year" Plan of China(2011BAD35B01)+3 种基金Taishan Scholars Program of Shandong Province(Crop Cultivation and Breeding)Basic Research Program of Science and Technology Project of Qingdao City[12-1-4-5-(11)-jch]Starting Fund for High-level Personnel of Qingdao Agricultural University(No.630740)Shandong Key Laboratory of Maize Breeding and Cultivation Techniques
文摘Acetosyringone （AS） has great influences on Agrobacterium-mediated genetic transformation. In order to improve the transformation frequency of waxy maize, in this study, shoot tips and immature embryo-derived callus of Lainongnuo 38 were collected as receptor materials, to compare the effects of pH in suspension medium, addition modes and concentrations of acetosyringone on genetic transformation of waxy maize. Results showed that adding 2 μl of 150 μmol/L AS in the wound with suspension medimn at pH 5.2 can significantly improve the transformation frequency of shoot tips of waxy maize; in genetic transformation of maize callus, adding 5 mg/L AS in infection solution can significantly improve the percentage of resistant callus.
文摘Several wood rotting fungi decolourized Remazol brilliant blue R (RBBR) and carminic acid (CA). Parallel activity of laccase in these fungi was studied. The addition of acetovanillone (AV) or acetosyringone (AS) intensified these processes: decolourization was more extensive than in the experiment omitting these compounds. At the presence of AS the decourization was more extensive than AV. However the level of decolorizing was relatively low in comparison to laccase activity on syringaldazine. The highly purified constitutive form of Cerrena unicolor and inducible form of Trametes versicolor laccases also destained both dyes. Anyway the addition of AV and AS improved the efficiency of dyes decolourization by wood rotting fungi and fungal laccase. Nitrogen starvation induced the laccase and decoloration activity in both organisms, irrespective of nitrogen availability. This fact indicates laccase not solely responsible for discoloration, and probably discoloration of dyes involves more than one mechanism.
