Stable transformation of suspension-cultured Glycyrrhiza inflata batalin cells with Agrobacterium tumefaciens. 2012

Yali Li, and Shutao Li, and Yanshan Dong, and Yu Zhang, and Chunhua Fu, and Longjiang Yu
Institute of Resource Biology and Biotechnology, Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, China.

A protocol for the efficient genetic transformation of licorice (Glycyrrhiza inflata Batalin) cells in suspension culture using Agrobacterium tumefaciens-mediated T-DNA delivery is described. G. inflata cells in suspension culture were infected with A. tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA1303, which contains the beta-glucuronidase (GUS) reporter gene and a hygromycin resistance gene (hpt II), respectively, under the transcriptional control of the CaMV35S promoter. Optimal transformation efficiency was achieved with an A. tumefaciens suspension having an OD600 of 0.4 and a period of 24 h of co-cultivation with 3-day-old cells in a medium supplemented with 200 microM acetosyringone. The transgenic cell lines have been maintained in suspension subculture for 5 months. PCR and Southern blot analyses confirmed the stable integration of transgenes into the G. inflata genome. The introduced genes had no discernable effect on cell growth or accumulation of total licorice flavonoids in the transgenic cell lines. This study provides the basis for the development of transgenic G. inflata cells.

UI MeSH Term Description Entries
D011401 Promoter Regions, Genetic DNA sequences which are recognized (directly or indirectly) and bound by a DNA-dependent RNA polymerase during the initiation of transcription. Highly conserved sequences within the promoter include the Pribnow box in bacteria and the TATA BOX in eukaryotes. rRNA Promoter,Early Promoters, Genetic,Late Promoters, Genetic,Middle Promoters, Genetic,Promoter Regions,Promoter, Genetic,Promotor Regions,Promotor, Genetic,Pseudopromoter, Genetic,Early Promoter, Genetic,Genetic Late Promoter,Genetic Middle Promoters,Genetic Promoter,Genetic Promoter Region,Genetic Promoter Regions,Genetic Promoters,Genetic Promotor,Genetic Promotors,Genetic Pseudopromoter,Genetic Pseudopromoters,Late Promoter, Genetic,Middle Promoter, Genetic,Promoter Region,Promoter Region, Genetic,Promoter, Genetic Early,Promoter, rRNA,Promoters, Genetic,Promoters, Genetic Middle,Promoters, rRNA,Promotor Region,Promotors, Genetic,Pseudopromoters, Genetic,Region, Genetic Promoter,Region, Promoter,Region, Promotor,Regions, Genetic Promoter,Regions, Promoter,Regions, Promotor,rRNA Promoters
D004269 DNA, Bacterial Deoxyribonucleic acid that makes up the genetic material of bacteria. Bacterial DNA
D006035 Glycyrrhiza A genus of leguminous herbs or shrubs whose roots yield GLYCYRRHETINIC ACID and its derivative, CARBENOXOLONE. Licorice,Glycyrrhiza glabra,Liquorice,Licorices,Liquorices
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D015139 Blotting, Southern A method (first developed by E.M. Southern) for detection of DNA that has been electrophoretically separated and immobilized by blotting on nitrocellulose or other type of paper or nylon membrane followed by hybridization with labeled NUCLEIC ACID PROBES. Southern Blotting,Blot, Southern,Southern Blot
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D016960 Agrobacterium tumefaciens A species of gram-negative, aerobic bacteria isolated from soil and the stems, leafs, and roots of plants. Some biotypes are pathogenic and cause the formation of PLANT TUMORS in a wide variety of higher plants. The species is a major research tool in biotechnology. Alcaligenes radiobacter,Bacillus radiobacter,Bacterium radiobacter,Bacterium tumefaciens,Phytomonas tumefaciens,Polymonas tumefaciens,Pseudomonas radiobacter,Pseudomonas tumefaciens,Rhizobium radiobacter,Agrobacterium radiobacter
D017931 DNA Primers Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques. DNA Primer,Oligodeoxyribonucleotide Primer,Oligodeoxyribonucleotide Primers,Oligonucleotide Primer,Oligonucleotide Primers,Primer, DNA,Primer, Oligodeoxyribonucleotide,Primer, Oligonucleotide,Primers, DNA,Primers, Oligodeoxyribonucleotide,Primers, Oligonucleotide

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