Effects of Acetosyringone on Genetic Transformation of Waxy Maize Using Different Receptor Systems

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.

Rapid delivery of Cas9 gene into the tomato cv.‘Heinz 1706’through an optimized Agrobacterium-mediated transformation procedure

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.

Effects of Acetylacetone Solution Soaking on Agrobacterium - transformed Maize Seed Buds

[Objectives]The effect of acetosyringone seed soaking on the transformation of maize seed buds was analyzed,so as to improve the genetic transformation efficiency of maize and to provide technical support for transgenic breeding of maize.[Methods]The seeds of the"Zheng 58"maize inbred line were used as experimental materials.When the seeds were germinated,AS was added to the water at concentrations of 70,140,210,and 280μmol/L,respectively,and the seeds germinated without the addition of AS served as the CK.The Agrobacterium-mediated method was used to transform bud growth points of maize seeds,and green fluorescent protein detection was performed on the young shoots transformed with EGFP(enhanced green fluorescent protein)gene.The effect of soaking seeds with acetosyringone solution on the transformation of maize bud growth points by Agrobacterium was studied according to the detection results.[Results]Soaking seeds in acetosyringone solutions for germination had the effect of inhibiting the germination of maize seeds and inhibiting sprout elongation,and the higher the concentration of acetosyringone,the stronger the inhibition.When the concentration of acetosyringone solution was 280μmol/L,the germination rate of seeds was only 36.2%of the CK,while soaking seeds with 70-140μmol/L acetosyringone solution for germination could not only ensure a higher germination rate of maize seeds,but also significantly increased the transformation efficiency of maize bud growth points.When the seeds were soaked with 70μmol/L acetosyringone solution for germination,the positive rate of transformed maize buds was the highest,reaching 32.1%.[Conclusions]When maize bud growth points were used as the receptor of Agrobacterium transformation,soaking seeds with 70-140μmol/L acetosyringone for germination basically did not affect the germination of seeds,and was beneficial to the activation of Agrobacterium,thereby promoting the transformation.

二妙丸HPLC指纹图谱及UPLC-LTQ-Orbitrap成分分析

目的建立二妙丸的HPLC指纹图谱质量评价方法,并对二妙丸中的化学成分进行UPLC-LTQ-Orbitrap定性分析。方法指纹图谱采用HPLC法,色谱柱为Shiseido C18柱(250 mm×4.6 mm,5μm),乙腈-0.1%甲酸水溶液为流动相进行梯度洗脱,体积流量为1.0 m L/min,检测波长为330 nm,以黄柏、苍术混合对照药材生成的色谱图为标准对照图谱,采用"中国药典委员会相似度评价软件(2012A)"对样品进行相似度评价;采用UPLC-LTQ-Orbitrap技术对二妙丸中化学成分进行定性分析,色谱柱为Thermo Scientific Syncronis C18柱(100 mm×2.1 mm,1.9μm),乙腈-0.1%甲酸水溶液梯度洗脱,经DAD检测器后采用LTQ-Orbitrap Elite质谱正离子模式采集,各成分的一级全扫描离子在离子阱内进行CID多级质谱、FT检测高分辨质谱数据分析。结果所建立的指纹图谱有10个共有色谱峰,20批样品的相似度在0.869~0.992。通过对照品指认、文献比对以及高分辨质谱数据解析,鉴定了21个化学成分,分别为neo-chlorogenic acid、木兰箭毒碱、竹叶椒碱、木兰花碱、3-O-feruloylquinic acid、蝙蝠葛碱、demethyleneberberine、oxyberberine、非洲防己碱、药根碱、小檗红碱、巴马汀、小檗碱、丁香酸、咖啡酸、(E)-4-(3-hydroxyprop-1-en1yl)-2-methoxyphenol、白术内酯II、acetosyringone、白术内酯I、selina-4(14),7(11)-dien-8-one、苍术素。结论建立的二妙丸HPLC指纹图谱专属性强,重现性好,结合液质联用技术的定性分析,可全面评价二妙丸的内在质量。

Agrobacterium tumefaciens CAN TRANSFORM Triticum aestivum AND Hordeum vulgare OF GRAMINEAE

So far there is no suitable vector system for the genetic engineering of monocotyledonous plants, especially cereal crops. It is first reported in this paper that Agrobacterium tumefaciens strains T37, A208 and B6 can transform some varieties of Triticum aestivum and Hordeum vulgare to form swellings and tumors. Itis shown in the experiments that phenolic compound acetosyringone may promote the transformation process. In addition, inoculating agrobacteria on appropriate plant tissues is also proved to be the key step in achieving successful transformation. The host range of A. tumefaciens and the possibility of utilizing Ti plasmid as the vector for the gene transfer of cereal crops are discussed.

PHENOLIC COMPOUNDS CAN PROMOTE THE TRANSFORMATION OF MONOCOTYLEDONOUS Commelina communis BY Agrobacterium tumefaciens

Agrobacterium tumefaciens can infect wounded plant cells, transfer a part of its Ti plasmid (T-DNA) into plant genomes and cause crown gall tumors. At present the Ti plasmid of A. tumefaciens is the best vector available for the genetic engineering of higher plants.