J Plant Biotechnol 2017; 44(4): 388-393
Published online December 31, 2017
https://doi.org/10.5010/JPB.2017.44.4.388
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: cc31@knu.ac.kr
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
We report an
Keywords Genotype,
Grapevine (
Since the characterization of antifreeze protein (AFP) from Antarctic fishes (DeVries et al. 1970), many other
For isolation of carrot antifreeze protein gene (
Total RNA was extracted from carrot leaves using the method for pine trees (Chang et al. 1993) with some modification: use of extraction buffer consisting of 2% cetyltrimethylammonium bromide, 2% polyvinylpyrrolidone, 100 mM Tris-HCl (pH 8.0), 25 mM ethylenediaminetetraacetic acid, 2 M NaCl, 0.05% spermidine, and 0.24% DTT. Reverse transcription was performed from 3 μg of total RNA with oligo-dT by
Linear map of the T-DNA region of the recombinant pBI121-
To observe the effect of ethylene inhibitors, ethylene action inhibitor silver nitrate (AgNO3: 0.1, 0.5, 1.0, 3.0 mg/L) and ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG: 0.001, 0.01, 0.1, 0.5 mg/L) were additionally supplemented in a co-cultivation medium. Co-cultivation medium was consisted of MS inorganic salts, 0.1 g/L myo-inositol, 0.8 mg/L thiamine-HCl, 30 g/L sucrose, 0.1 mg/L IBA, 10 mg/L acetosyringone (Sigma-Aldrich), 1 mM proline (Sigma- Aldrich), and 8 g/L Plant Agar™, followed by adjusting pH to 5.9 before autoclaving at 121°C for 20 min. Culture flasks were sealed with rubber septum stoppers throughout co- cultivation. After co-cultivation, the explants were transferred to a selection medium. For the selection medium, 5 mg/L thidizuron (TDZ; Sigma-Aldrich) and 200 mg/L Clavamox™ (Pfizer, NY, USA) were supplemented in the co-cultivation medium, meanwhile acetosyringone, proline and ethylene inhibitors were removed from the co-cultivation medium. Regenerated shoots were observed five weeks after culturing on the selection medium.
Four kinds of antibiotics (cefotaxime, carbenicillin, Clavamox™ and kanamycin) at various concentrations were added to the selection media to decide efficient antibiotics types and their concentrations for suppressing the development of non- transgenic shoot development but minimizing the inhibition of transgenic shoot regeneration (
Table 1 Effects of ethylene inhibitors in co-cultivation medium on shoot regeneration from ‘Kyoho’ leaf explants sub-cultured in selection medium
Ethylene inhibitor (mg/L) in co-cultivation medium | Shoot regeneration (%) in selection medium | |
---|---|---|
AVG | 0.0 | 0.5 d |
0.001 | 2.9 c | |
0.01 | 3.3 c | |
0.1 | 5.0 a | |
0.5 | 4.1 b | |
AgNO3 | 0.1 | 4.3 b |
0.5 | 3.2 c | |
1.0 | 3.0 c | |
3.0 | 3.3 c |
For this experiment, 50 mg/L kanamycin and 200 mg/L Clavamox™ were also supplied in the selection medium. The data represent mean values of 9 replicates. Values followed by the same letter within the last column are not significantly different according to Duncan’s multiple range test at the 5% level
Table 2 Effects of antibiotics on shoot regeneration from leaf explants in grapevine ‘Kyoho’
Antibiotics (mg/L) | Shoot regeneration (%) | |
---|---|---|
0 | 30.9 a | |
Cefotaxime | 150 | 26.7 b |
250 | 16.4 c | |
Carbenicillin | 300 | 25.8 b |
500 | 18.5 c | |
Clavamox™ | 100 | 20.7 bc |
200 | 17.5 c | |
Cefotaxime/Clavamox™ | 100/100 | 18.9 c |
150/150 | 15.7 c | |
Kanamycin | 25 | 8.4 d |
50 | 2.0 e |
The data represent mean values of 6 replicates. Values followed by the same letter within the last column are not significantly different according to Duncan’s multiple range test at the 5% level
Genomic DNA was extracted from young leaves using a modification of CTAB method (Torres et al. 1993). Approximately 100 mg of fresh leaves was put into a pre-chilled mortar and grinded in liquid nitrogen. A 700 µL
CTAB buffer was added to the powder and the mixture was incubated at 65°C for 30 min. After incubation, equal volume of phenol:chloroform:isoamylalcohol (25:24:1) was added and centrifuged at 13,000 rpm for 10 min. The supernatant was transferred to a new tube, equal volume of chloroform: isoamylalcohol (24:1) was added and centrifuged at 13,000 rpm for 10 min. The supernatant was transferred to a new tube, equal volume of isopropanol was added and centrifuged at 13,000 rpm for 5 min. The precipitated DNA was washed with 1 mL of 70% ethanol and dried for 1 h. The DNA was dissolved in 100 µL of sterile H2O with 2 µL of RNase A and incubated at 37°C for 1 h. After determination of DNA concentration and purity, the extracted total DNA was stored at –20°C.
Detection of integrated transgenes was performed by PCR and Southern blot analyses (Southern 1975). Primer sets for PCR analysis were designed to amplify
A fragment of about 1,099 bp in length was amplified from the cDNA of carrot by RT-PCR, which was recombined into pBI121 binary vector, resulting in creation of pBI121-
Increase of shoot regeneration efficiency by using ethylene inhibitors in co-cultivation medium
Wounded plant tissues are able to produce significant amounts of ethylene during
For this experiment, 50 mg/L kanamycin and 200 mg/L Clavamox™ were also supplied in the selection medium. The data represent the mean values nine replicates. Values followed by the same letter within the last column are not significantly different according to Ducan’s multiple range test at the 5% level.
In almost
The data represent the mean values six replicates. Values followed by the same letter within the last column are not significantly different according to Ducan’s multiple range test at the 5% level.
With five batches of
Exhibition on generation of
Detection of transgenes and transcript of
Completely acclimatized plantlets were subjected to PCR, Southern blot, and RT-PCR analyses to detect the transgenes and the transcript of D
Together, we generated transgenic grapevine ‘Kyoho’ plants with carrot antifreeze protein gene (
J Plant Biotechnol 2017; 44(4): 388-393
Published online December 31, 2017 https://doi.org/10.5010/JPB.2017.44.4.388
Copyright © The Korean Society of Plant Biotechnology.
Hye Young Shin, Gi Hoon Kim, Sang Jae Kang, Jeung-Sul Han, and Cheol Choi
Department of Horticultural Science, College of Agriculture & Life Sciences, Kyungpook National University, Daegu 41566, Republic of Korea,
School of Applied Life Science, College of Agriculture & Life Sciences, Kyungpook National University, Daegu 41566, Republic of Korea,
Department of Ecological Environment, College of Ecology & Environmental Science, Kyungpook National University, Sangju 37224, Republic of Korea,
Institute of Agricultural Science & Technology, Kyungpook National University, Daegu 41566, Republic of Korea
Correspondence to: e-mail: cc31@knu.ac.kr
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
We report an
Keywords: Genotype,
Grapevine (
Since the characterization of antifreeze protein (AFP) from Antarctic fishes (DeVries et al. 1970), many other
For isolation of carrot antifreeze protein gene (
Total RNA was extracted from carrot leaves using the method for pine trees (Chang et al. 1993) with some modification: use of extraction buffer consisting of 2% cetyltrimethylammonium bromide, 2% polyvinylpyrrolidone, 100 mM Tris-HCl (pH 8.0), 25 mM ethylenediaminetetraacetic acid, 2 M NaCl, 0.05% spermidine, and 0.24% DTT. Reverse transcription was performed from 3 μg of total RNA with oligo-dT by
Linear map of the T-DNA region of the recombinant pBI121-
To observe the effect of ethylene inhibitors, ethylene action inhibitor silver nitrate (AgNO3: 0.1, 0.5, 1.0, 3.0 mg/L) and ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG: 0.001, 0.01, 0.1, 0.5 mg/L) were additionally supplemented in a co-cultivation medium. Co-cultivation medium was consisted of MS inorganic salts, 0.1 g/L myo-inositol, 0.8 mg/L thiamine-HCl, 30 g/L sucrose, 0.1 mg/L IBA, 10 mg/L acetosyringone (Sigma-Aldrich), 1 mM proline (Sigma- Aldrich), and 8 g/L Plant Agar™, followed by adjusting pH to 5.9 before autoclaving at 121°C for 20 min. Culture flasks were sealed with rubber septum stoppers throughout co- cultivation. After co-cultivation, the explants were transferred to a selection medium. For the selection medium, 5 mg/L thidizuron (TDZ; Sigma-Aldrich) and 200 mg/L Clavamox™ (Pfizer, NY, USA) were supplemented in the co-cultivation medium, meanwhile acetosyringone, proline and ethylene inhibitors were removed from the co-cultivation medium. Regenerated shoots were observed five weeks after culturing on the selection medium.
Four kinds of antibiotics (cefotaxime, carbenicillin, Clavamox™ and kanamycin) at various concentrations were added to the selection media to decide efficient antibiotics types and their concentrations for suppressing the development of non- transgenic shoot development but minimizing the inhibition of transgenic shoot regeneration (
Table 1 . Effects of ethylene inhibitors in co-cultivation medium on shoot regeneration from ‘Kyoho’ leaf explants sub-cultured in selection medium.
Ethylene inhibitor (mg/L) in co-cultivation medium | Shoot regeneration (%) in selection medium | |
---|---|---|
AVG | 0.0 | 0.5 d |
0.001 | 2.9 c | |
0.01 | 3.3 c | |
0.1 | 5.0 a | |
0.5 | 4.1 b | |
AgNO3 | 0.1 | 4.3 b |
0.5 | 3.2 c | |
1.0 | 3.0 c | |
3.0 | 3.3 c |
For this experiment, 50 mg/L kanamycin and 200 mg/L Clavamox™ were also supplied in the selection medium. The data represent mean values of 9 replicates. Values followed by the same letter within the last column are not significantly different according to Duncan’s multiple range test at the 5% level.
Table 2 . Effects of antibiotics on shoot regeneration from leaf explants in grapevine ‘Kyoho’.
Antibiotics (mg/L) | Shoot regeneration (%) | |
---|---|---|
0 | 30.9 a | |
Cefotaxime | 150 | 26.7 b |
250 | 16.4 c | |
Carbenicillin | 300 | 25.8 b |
500 | 18.5 c | |
Clavamox™ | 100 | 20.7 bc |
200 | 17.5 c | |
Cefotaxime/Clavamox™ | 100/100 | 18.9 c |
150/150 | 15.7 c | |
Kanamycin | 25 | 8.4 d |
50 | 2.0 e |
The data represent mean values of 6 replicates. Values followed by the same letter within the last column are not significantly different according to Duncan’s multiple range test at the 5% level.
Genomic DNA was extracted from young leaves using a modification of CTAB method (Torres et al. 1993). Approximately 100 mg of fresh leaves was put into a pre-chilled mortar and grinded in liquid nitrogen. A 700 µL
CTAB buffer was added to the powder and the mixture was incubated at 65°C for 30 min. After incubation, equal volume of phenol:chloroform:isoamylalcohol (25:24:1) was added and centrifuged at 13,000 rpm for 10 min. The supernatant was transferred to a new tube, equal volume of chloroform: isoamylalcohol (24:1) was added and centrifuged at 13,000 rpm for 10 min. The supernatant was transferred to a new tube, equal volume of isopropanol was added and centrifuged at 13,000 rpm for 5 min. The precipitated DNA was washed with 1 mL of 70% ethanol and dried for 1 h. The DNA was dissolved in 100 µL of sterile H2O with 2 µL of RNase A and incubated at 37°C for 1 h. After determination of DNA concentration and purity, the extracted total DNA was stored at –20°C.
Detection of integrated transgenes was performed by PCR and Southern blot analyses (Southern 1975). Primer sets for PCR analysis were designed to amplify
A fragment of about 1,099 bp in length was amplified from the cDNA of carrot by RT-PCR, which was recombined into pBI121 binary vector, resulting in creation of pBI121-
Increase of shoot regeneration efficiency by using ethylene inhibitors in co-cultivation medium
Wounded plant tissues are able to produce significant amounts of ethylene during
For this experiment, 50 mg/L kanamycin and 200 mg/L Clavamox™ were also supplied in the selection medium. The data represent the mean values nine replicates. Values followed by the same letter within the last column are not significantly different according to Ducan’s multiple range test at the 5% level.
In almost
The data represent the mean values six replicates. Values followed by the same letter within the last column are not significantly different according to Ducan’s multiple range test at the 5% level.
With five batches of
Exhibition on generation of
Detection of transgenes and transcript of
Completely acclimatized plantlets were subjected to PCR, Southern blot, and RT-PCR analyses to detect the transgenes and the transcript of D
Together, we generated transgenic grapevine ‘Kyoho’ plants with carrot antifreeze protein gene (
Linear map of the T-DNA region of the recombinant pBI121-
Exhibition on generation of
Detection of transgenes and transcript of
Table 1 . Effects of ethylene inhibitors in co-cultivation medium on shoot regeneration from ‘Kyoho’ leaf explants sub-cultured in selection medium.
Ethylene inhibitor (mg/L) in co-cultivation medium | Shoot regeneration (%) in selection medium | |
---|---|---|
AVG | 0.0 | 0.5 d |
0.001 | 2.9 c | |
0.01 | 3.3 c | |
0.1 | 5.0 a | |
0.5 | 4.1 b | |
AgNO3 | 0.1 | 4.3 b |
0.5 | 3.2 c | |
1.0 | 3.0 c | |
3.0 | 3.3 c |
For this experiment, 50 mg/L kanamycin and 200 mg/L Clavamox™ were also supplied in the selection medium. The data represent mean values of 9 replicates. Values followed by the same letter within the last column are not significantly different according to Duncan’s multiple range test at the 5% level.
Table 2 . Effects of antibiotics on shoot regeneration from leaf explants in grapevine ‘Kyoho’.
Antibiotics (mg/L) | Shoot regeneration (%) | |
---|---|---|
0 | 30.9 a | |
Cefotaxime | 150 | 26.7 b |
250 | 16.4 c | |
Carbenicillin | 300 | 25.8 b |
500 | 18.5 c | |
Clavamox™ | 100 | 20.7 bc |
200 | 17.5 c | |
Cefotaxime/Clavamox™ | 100/100 | 18.9 c |
150/150 | 15.7 c | |
Kanamycin | 25 | 8.4 d |
50 | 2.0 e |
The data represent mean values of 6 replicates. Values followed by the same letter within the last column are not significantly different according to Duncan’s multiple range test at the 5% level.
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Journal of
Plant BiotechnologyLinear map of the T-DNA region of the recombinant pBI121-
Exhibition on generation of
Detection of transgenes and transcript of