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A Novel Phenolic Compound, Chloroxynil, Improves Agrobacterium-Mediated Transient Transformation in Lotus japonicus.

Kimura M, Cutler S, Isobe S - PLoS ONE (2015)

Bottom Line: We identified a phenolic compound, chloroxynil, which increased the genetic transformation of L. japonicus by Agrobacterium tumefaciens strain EHA105.In addition, transgenic L. japonicus lines were successfully generated by 5 μM chloroxynil treatment.Furthermore, we show that chloroxynil improves L. japonicus transformation by Agrobacterium strain GV3101 and rice transformation.Our results demonstrate that chloroxynil significantly improves Agrobacterium tumefaciens-mediated transformation efficiency of various agriculturally important crops.

View Article: PubMed Central - PubMed

Affiliation: Department of Frontier Research, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan.

ABSTRACT
Agrobacterium-mediated transformation is a commonly used method for plant genetic engineering. However, the limitations of Agrobacterium host-plant interactions and the complexity of plant tissue culture often make the production of transgenic plants difficult. Transformation efficiency in many legume species, including soybean and the common bean, has been reported to be quite low. To improve the transformation procedure in legumes, we screened for chemicals that increase the transformation efficiency of Lotus japonicus, a model legume species. A Chemical library was screened and chemicals that increase in transient transformation efficiency of L. japonicus accession, Miyakojima MG-20 were identified. The transient transformation efficiency was quantified by reporter activity in which an intron-containing reporter gene produces the GUS protein only when the T-DNA is expressed in the plant nuclei. We identified a phenolic compound, chloroxynil, which increased the genetic transformation of L. japonicus by Agrobacterium tumefaciens strain EHA105. Characterization of the mode of chloroxynil action indicated that it enhanced Agrobacterium-mediated transformation through the activation of the Agrobacterium vir gene expression, similar to acetosyringone, a phenolic compound known to improve Agrobacterium-mediated transformation efficiency. Transient transformation efficiency of L. japonicus with 5 μM chloroxynil was 60- and 6- fold higher than that of the control and acetosyringone treatment, respectively. In addition, transgenic L. japonicus lines were successfully generated by 5 μM chloroxynil treatment.Furthermore, we show that chloroxynil improves L. japonicus transformation by Agrobacterium strain GV3101 and rice transformation. Our results demonstrate that chloroxynil significantly improves Agrobacterium tumefaciens-mediated transformation efficiency of various agriculturally important crops.

No MeSH data available.


Related in: MedlinePlus

Growth inhibition of L. japonicus treated with CX.(A) Hypocotyl segments were incubated on co-cultivation medium containing different concentrations of CX for 5 days. (B) Fresh weight of hypocotyl segments treated with CX for 5 days. The segments were weighed in triplicate. The bars represent the mean ± SE of three replicates. Bar represents 1 cm. Asterisks indicate significant differences between the control and treated segments (**: P<0.01) by Student’s t-test.
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pone.0131626.g002: Growth inhibition of L. japonicus treated with CX.(A) Hypocotyl segments were incubated on co-cultivation medium containing different concentrations of CX for 5 days. (B) Fresh weight of hypocotyl segments treated with CX for 5 days. The segments were weighed in triplicate. The bars represent the mean ± SE of three replicates. Bar represents 1 cm. Asterisks indicate significant differences between the control and treated segments (**: P<0.01) by Student’s t-test.

Mentions: Benzonitrile herbicides, which include CX and bromoxynil (BX), have been known to induce rapid cell death in Arabidopsis thaliana[42]. Thus, the degree of CX-mediated growth inhibition was investigated in L. japonicus. Hypocotyl explants suffered serious damage with 50 μM CX treatment (Fig 2A), and the fresh weight per explant was significantly less than explants treated with 5 μM CX (Fig 2B). No significant differences were observed in the appearance of explants and fresh weight in 0.5 μM CX and 100 μM AS treatment (Fig 2). Based on these results, it was concluded that the growth inhibition in L. japonicus occurred at concentrations above 5 μM of CX treatment.


A Novel Phenolic Compound, Chloroxynil, Improves Agrobacterium-Mediated Transient Transformation in Lotus japonicus.

Kimura M, Cutler S, Isobe S - PLoS ONE (2015)

Growth inhibition of L. japonicus treated with CX.(A) Hypocotyl segments were incubated on co-cultivation medium containing different concentrations of CX for 5 days. (B) Fresh weight of hypocotyl segments treated with CX for 5 days. The segments were weighed in triplicate. The bars represent the mean ± SE of three replicates. Bar represents 1 cm. Asterisks indicate significant differences between the control and treated segments (**: P<0.01) by Student’s t-test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4503419&req=5

pone.0131626.g002: Growth inhibition of L. japonicus treated with CX.(A) Hypocotyl segments were incubated on co-cultivation medium containing different concentrations of CX for 5 days. (B) Fresh weight of hypocotyl segments treated with CX for 5 days. The segments were weighed in triplicate. The bars represent the mean ± SE of three replicates. Bar represents 1 cm. Asterisks indicate significant differences between the control and treated segments (**: P<0.01) by Student’s t-test.
Mentions: Benzonitrile herbicides, which include CX and bromoxynil (BX), have been known to induce rapid cell death in Arabidopsis thaliana[42]. Thus, the degree of CX-mediated growth inhibition was investigated in L. japonicus. Hypocotyl explants suffered serious damage with 50 μM CX treatment (Fig 2A), and the fresh weight per explant was significantly less than explants treated with 5 μM CX (Fig 2B). No significant differences were observed in the appearance of explants and fresh weight in 0.5 μM CX and 100 μM AS treatment (Fig 2). Based on these results, it was concluded that the growth inhibition in L. japonicus occurred at concentrations above 5 μM of CX treatment.

Bottom Line: We identified a phenolic compound, chloroxynil, which increased the genetic transformation of L. japonicus by Agrobacterium tumefaciens strain EHA105.In addition, transgenic L. japonicus lines were successfully generated by 5 μM chloroxynil treatment.Furthermore, we show that chloroxynil improves L. japonicus transformation by Agrobacterium strain GV3101 and rice transformation.Our results demonstrate that chloroxynil significantly improves Agrobacterium tumefaciens-mediated transformation efficiency of various agriculturally important crops.

View Article: PubMed Central - PubMed

Affiliation: Department of Frontier Research, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan.

ABSTRACT
Agrobacterium-mediated transformation is a commonly used method for plant genetic engineering. However, the limitations of Agrobacterium host-plant interactions and the complexity of plant tissue culture often make the production of transgenic plants difficult. Transformation efficiency in many legume species, including soybean and the common bean, has been reported to be quite low. To improve the transformation procedure in legumes, we screened for chemicals that increase the transformation efficiency of Lotus japonicus, a model legume species. A Chemical library was screened and chemicals that increase in transient transformation efficiency of L. japonicus accession, Miyakojima MG-20 were identified. The transient transformation efficiency was quantified by reporter activity in which an intron-containing reporter gene produces the GUS protein only when the T-DNA is expressed in the plant nuclei. We identified a phenolic compound, chloroxynil, which increased the genetic transformation of L. japonicus by Agrobacterium tumefaciens strain EHA105. Characterization of the mode of chloroxynil action indicated that it enhanced Agrobacterium-mediated transformation through the activation of the Agrobacterium vir gene expression, similar to acetosyringone, a phenolic compound known to improve Agrobacterium-mediated transformation efficiency. Transient transformation efficiency of L. japonicus with 5 μM chloroxynil was 60- and 6- fold higher than that of the control and acetosyringone treatment, respectively. In addition, transgenic L. japonicus lines were successfully generated by 5 μM chloroxynil treatment.Furthermore, we show that chloroxynil improves L. japonicus transformation by Agrobacterium strain GV3101 and rice transformation. Our results demonstrate that chloroxynil significantly improves Agrobacterium tumefaciens-mediated transformation efficiency of various agriculturally important crops.

No MeSH data available.


Related in: MedlinePlus