• Mycobiology
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• v.49 no.5
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• pp.491-497
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• 2021

An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pretreatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.

• Journal of Korean Society of Forest Science
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• v.77 no.2
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• pp.199-207
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• 1988

Three clones of Populus alba ${\times}$ P. grandidentata have been tested for susceptibility to Agrobacterium tumefaciens strains A281 and A348. We determined the optimum concentration of kanamycin sulfate for effective selection of leaf disc-derived, transgenic tissues transformed using Agrobacterium binary vector pGA472 containing a neomycin phosphotransferase gene (NPT-II) which confers kanamycin resistance. Of the wild type Ti plasmids contained by the two Agrobacterium strains, pTiBo542 of strain A281 appears to be best suited to serve as a helper plasmid for binary vector systems. A relatively low concentration (10mg/l) of kanamycin sulfate inhibited adventitious shoot initiation from leaf discs on regeneration medium. Transformed kanamycin-resistant calli were obtained by culturing Agrobacterium inoculated leaf discs on selective regeneration medium. The transformed kanamycin-resistant calli continued to grow on regeneration media supplemented with kanamycin sulfate to levels of 50 and 200mg/l. The growth of non-co-cultivated control calli was severely inhibited on regeneration medium containing 50mg/l kanamycin sulfate.

• Research in Plant Disease
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• v.12 no.3
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• pp.189-196
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• 2006

Crown gall on lower stem of weeping fig(Ficus benjamina Roxb.) was first observed at Daejon in 2003. Tumors were about 15 cm in size and semi-round with rough surface texture of dark brown color. Two virulent isolates among ten bacteria isolated from the tumor tissues were characterized. Their colonies were convex, glistening, circular with an entire edge, and white or tannish cream in color on potato dextrose agar supplemented with 0.5% $CaCO_3$. They were rod shape with peritrichous flagellae, gram-negative, aerobic growth, oxidase-positive, and grew on D1M agar. The isolates were identified as Agrobacterium larrymoorei and A. tumefaciens based on biochemical and physiological characteristics, fatty acid profiles and substrate utilization patterns. Seedlings of some host plants excepting grapevine produced typical galls two to three weeks after inoculation with cell suspensions of the virulent strains. This is the first report on crown gall of weeping fig in Korea.

• Current Research on Agriculture and Life Sciences
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• v.11
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• pp.11-17
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• 1993

The transfer of genetic material into pea tissue was accomplished by using an avirulent strain of Agrobacterium tumefaciens containing the binary vector. The method used for transformation requires non-tissue culture steps as it involves the inoculation of the site of the shoot removed of germinating seeds. The identification of ${\beta}$-glucuronidase activity in the tissues of $T_0$ pea plants indicates that the plant expressible ${\beta}$-glucuronidase gene, contained the T-DNA region from pLPBO2, had been transferred at least into somatic tissues. Putative transformed $T_0$ pea plants were advanced to produce $T_1$ plants which were also assayed for the presence of the transferred ${\beta}$-glucuronidase gene. The presence of the ${\beta}$-glucuronidase gene in DNAs isolated from $T_1$ plant was demonstrated by DNA gel blot hybridization. This analysis revealed that the transformed plants contained ${\beta}$-glucuronidase gene.

• Korean Journal of Plant Tissue Culture
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• v.21 no.6
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• pp.319-326
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• 1994

For the gene tagging of Armoracia rusticana, maize controlling element Ac and Ds were introduced into A.rusticana via Agrobacterium-mediated transformation method. We established an efficient in via regeneration and transformation system for gene transfer in A. rusticana. The optimum in via regeneration condition has been obtained from leaf, petiole and root organs on modified MS medium supplemented with NAA 0.1 mg/L plus BA 1.0 mg/L for direct shooting and with free growth regulators for root induction for transformation, the leaf, petiole and root explants of A. rusticana were concultivated with Agrobacterium tumefaciens, LBA4404 which carries a binary vector pEND4K containing maize controlling element Ac or Ds, respectively: Selections were performed in the shoot induction medium supplemented with 100 mg/L kanamycin, and 500 mg/L carbenicillin transformation frequency showed about 8 to 10% in case of leaf disks. PCR md Southern blot analyses showed that the Ac and the Ds elements were integrated into the chromosome of donor plants.

• Journal of Microbiology and Biotechnology
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• v.9 no.5
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• pp.637-645
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• 1999

Two putative regulator genes, mocR and mocS, of the moc (mannityl opine catabolism) operons in pTi15955 of the octopine-/mannityl opine-type Agrobacterium tumefaciens strain 15955, were tested for their possible roles as repressors in the moc operons. The regions upstream of macC and mocD, the first structural genes in the two divergently oriented moc operons, were transcriptionally fused into the promoterless lacZ reporter gene. Each of the lacZ-fusions was introduced into Agrobacterium strain UIA5, a Ti plasmid-cured derivative, harboring either a mocR or a mocS clone. The resulting strains were grown in media containing various sugar sources, and the $\beta$-galactosidase activities were quantitatively measured. The results suggested that MocR repressed the expression of macC and macD. The expression of the fused $\beta$-galactosidase was not induced by mannopine (MOP) or possible catabolic intermediates of the opine, e.g. santhopine (SOP), glucose, mannose, or glutamine. However, the repression was significantly relieved by the supplementation of MOP and the concomitant introduction of the agcA gene encoding MOP cyclase that catalyzes the lactonization of MOP to agropine (AGR). These results suggested that AGR, rather than MOP or the other catabolic intermediates, is the inducer for the expression of the operon. On the contrary to previous report showing that the induction levels of macC and macD were lowered by the supplementation of inorganic nitrogen in media, the expression of these genes was not affected by the level of nitrogen in our reporter system. MocS did not strongly repress the expressions of macC and mocD. It is possible that MocS may be involved in the regulation of the operons present downstream of the moc operon, which are responsible for the utilization of mannopinic acid and agropinic acid.

• Journal of Plant Biology
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• v.38 no.4
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• pp.365-371
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• 1995

Binary vectors, pBI-ActR1, pBI-ActF1 and pBSH-ActR1, were constructed using pGA642, the replication origin of pTi12 and the rice actin promoter. The sizes of pBI-ActR1, pBI-ActF1 and pBSH-ActR1 were 12.9 kb, 13.2 kb and 11.95 kb, respectively. These vectors containing a rice actin promoter followed by a GUS structural gene could induce stronly the expression of GUS gene in transformed rice cells. Rice explants from 3-4 day old seedlings after germinatin were cocultured with A. tumefaceins harboring pBI-ActR1, pBI-ActF1 or pBSH-ActR1, and then GUS expression in the explants was assayed. Transformation of rice explants by these binary vectors was tissue-specific, such that the meristematic regions of shoot apex, root and hypocotyl were transformed by these binary vectors.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2003.04a
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• pp.101-101
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• 2003

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2005.04a
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• pp.98-98
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• 2005

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.324-325
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• 2005