Journal of Plant Biotechnology 2016; 43(1): 66-75
Published online March 31, 2016
https://doi.org/10.5010/JPB.2016.43.1.66
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: kkm@knu.ac.kr
Keywords
Rice (
The mature seeds of japonica rice (
Embryogenic callus ready for use as an explant transformant; the lines (a and b) show the cut side
Schematic of the plasmid vector
A primary culture of the
Different media were used at different stages of the experiment. Supplementary 1 presents a list of the media that were used. The pH of the medium was 5.8, unless otherwise stated in the table.
Supplementary 1 List of the media used in this study
Medium | Composition |
---|---|
Soaking | Luria-Bertani (LB) brotha, 50 ?g/mL kanamycinb, 10 ?g/mL rifampycinb, 50 ?g/mL gentamycinb |
Seedling MS | 4.41 g/L Murashige and Skoog (MS) vitamin powderc, 30 g/L sucrosec, 4 g/L gerlitec, 100 ?M acetosyringoneb (pH 5.2) |
Regeneration MS | 4.41 g/L MS vitamin powder, 30 g/L sucrose, 5 mg/L kinetin and 1 mg/L 1-naphthaleneacetic (NAA)d, 250 mg/L carbenecillinc, 50 mg/L geneticine, 4 g/L gerlite |
Growing MS | 4.41 g/L MS vitamin powder, 30 g/L sucrose, 250 mg/L carbenecillin, 50 mg/L geneticin, 4 g/L gerlite |
Callusing | 4.14 g/L MS vitamin powder, 2 mg/L 2.4-dichlorophenoxyacetic acid (2,4-D)b, 30 g/L sucrose, 2 g/L casein hydrolysatec, 4 g/L gerlite |
Co-cultivation | 4.41 g/L MS vitamin powder, 5 mg/L kinetin, 1 mg/L NAA, 2 g/L casein hydrosalate, 30 g/L sucrose; 5 g/L silver nitrateb, 10.5 mg/L L-cysteineb, 1 mM sodium thiosulfateb, 4 g/L gerlite, 100 ?M acetosyringone (pH 5.2) |
Infection liquid | MS + 100 ?M acetosyringone |
Callusing vacuum | 4 g/L N6 powderc, 30 g/L sucrose, 2.878 g/L L-prolineb, 0.3 g/L casein hydrolysate, 0.1 g/L myo-inositolb, 2 mg/L 2,4-D, 4 g/L gelrite |
Co-cultivation vacuum | 4 g/L N6 powder, 30 g/L sucrose, 10 g/L D-glucoseb, 0.3 g/L casein hydrolysate, 2 mg/L 2,4-D, 4 g/L gelrite, 100 ?M acetosyringone (pH 5.2) |
Selection and elimination | 4 g/L N6 powder, 30 g/L sucrose, 2.878 g/L L-proline, 0.3 g/L casein, 2 mg/L 2,4-D, 4 g/L gelrite, 250 mg/L carbenecillin, 150 mg/L kanamycin |
Regeneration | 4.41 g/L MS vitamin powder, 30 g/L sucrose, 30 g/L sorbitolb, 2 g/L casein hydrolysate, 2 mg/L kinetin, 1 mg/L NAA, 0.1 g/L myo-inositol, 4 g/L gelrite, 500 mg/L carbenecillin, 50 mg/L geneticin |
aBecton, Dickinson and Company, New Jersey, United States,
bSigma-Aldrich, St. Louis, Missouri, United States,
cDuchefa Biochemie, BH Haarlem, Netherlands,
dJunsei Chemical co., Ltd, Tokyo, Japan,
ePhytotechnology Laboratories, Overland Park, United States
fYakuri Pure Chemical, Kyoto, Japan
In this experiment, both cultivars were used in the transformations. The transformation procedure began after the sterilized seeds had been soaked in soaking medium for 1, 2, and 3 days at 28°C under dark conditions. The seeds were then dried and placed on seedling MS medium under continuous dark conditions for 3 days and under 16/8 h light/dark conditions for a further 2 days. Subsequently, the seeds were placed on regeneration medium under 16/8 h light/dark conditions for 2 weeks. Regenerated seedlings were transferred to growing MS medium for 1 week, and the leaves were cut for use as putative transformant samples.
In this experiment, Ilpum calli were used as transformation explants. The sterilized seeds were cultured on callusing medium at 28°C under continuous dark conditions for 30 days. The embryogenic calli were separated from the endosperm and shoot (Fig. 1), at which point they were considered ready for infection. The explants were shaken in infection liquid at 28°C for 30 min in a rotary incubator and dried on autoclaved filter paper for 45 min at room temperature. The dried calli were placed in co-cultivation medium under continuous dark conditions for different durations and at different temperatures as follows; A: 28°C for 3 days, B: 28°C for 1 day followed by 25°C for 2 days, and C: 25°C for 3 days. Each treatment was repeated three times. The calli were inoculated on selection and regeneration medium for 45 days, and were subcultured 3-4 times in the same selection and regeneration media at 2-week intervals. Subsequently, the calli were transferred to a test tube containing the selection and regeneration media and left for 45 days. After this time-period, the leaves were cut and used as samples for the putative transformant.
Both cultivars were used for transformation. The sterilized seeds were cultured on callusing vacuum medium at 32 ± 1°C under continuous dark conditions for 10 days, and were then separated from the endosperm and shoot to prepare the explants for infection. Subsequently, the calli were transferred to a falcon tube containing the
Genomic DNA was isolated from the rice leaves of transgenic and non-transgenic plants for molecular analysis. DNA was isolated from rice leaves using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). For molecular confirmation, and to confirm the presence of the transgene in
Total RNA from the rice leaves was extracted and isolated from transgenic and non-transgenic plants (Qiagen, Hilden, Germany). The extracted total RNA samples were used for RT-PCR analysis. A SuperScript? III Reverse Transcriptase kit (Invitrogen, Carlsbad, United States) was used for the analysis. RT-PCR primer pairs used to amplify
Samples of total RNA were used to synthesize cDNA. Then, the samples were used for qRT-PCR. For the synthesis of cDNA from RNA, a qPCR SyGreen 1-Step kit from the PCR biosystem was used to analyze the expression of
About 100 ng of genomic DNA was digested with 4-5 units of
In this experiment, the soaking seeds method affected germination of infected seeds (Supplementary 2). The highest germination rate (114 ± 63%) was obtained by soaking in Ilpum for 1 day. Our data showed that Ilpum led to higher germination rates than Ilmi in all of the soaking treatments. For both cultivars, soaking treatment for 1 day was the optimal duration for germinating seeds. When using this method, multiple shoots were produced following 2 and 3 days of soaking.
Supplementary 2 Effect of the soaking method on seed germination
Cultivar | Duration of soaking (days) | ||||||||
---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | |||||||
No. of inoculated seeds | No. of germinated seeds | No. of transformant seeds | No. of inoculated seeds | No. of germinated seeds | No. of transformant seeds | No. of inoculated seeds | No. of germinated seeds | No. of transformant seeds | |
Ilmi | 328 | 54 ± 28.3a | 0 ± 0 | 312 | 33 ± 15.6 | 0 ± 0 | 312 | 28.5 ± 12.0 | 0 ± 0 |
Ilpum | 334 | 114 ± 63.6 | 0 ± 0 | 360 | 99 ± 43.8 | 0 ± 0 | 247 | 68.5 ± 20.5 | 0 ± 0 |
aMean ± standard deviation
Supplementary 3 shows that different types of co-cultivation had different effects on the initiation of the green spot and the number of plants that regenerated. The B-type co-cultivation method conditions (28°C for 1 day followed by 25°C for 2 days) were found to be optimal, but transformants were not produced by this method. Rice development under the different co-cultivation methods showed that 30-day-old calli were inducted in the callusing medium. The embryogenic callus was first cut (Fig. 1) for use as the explant. The initiation of a green spot was detected after 7 days in the regeneration medium, which initiated leaf growth 2 weeks later.
Supplementary 3 Effect of different types of co-cultivation methods on calli development
Type of co-cultivation | No. of inoculated calli | No. of green spots | No. of regenerated plants | No. of transformants |
---|---|---|---|---|
A | 204a | 9.3 ± 3.1b | 1.6 ± 0.9 | 0 ± 0 |
B | 251 | 30.3 ± 31.4 | 6.0 ± 4.2 | 0 ± 0 |
C | 229 | 11.7 ± 7.6 | 4.2 ± 1.3 | 0 ± 0 |
aNo. of inoculated calli,
bMean ± standard deviation, A: 28°C for 3 days, B: 28°C for 1 day and 25°C for 2 days, C: 25°C for 3 days
The duration of vacuum infiltration had a notable effect on the number of calli that regenerated throughout the whole experiment (Table 1) and in every subculture (Supplementary 4) of both cultivars. Vacuum infiltration for 5 min led to the production of 41.7 ± 33.2 plant regeneration (Table 1) for Ilpum, whereas 10 min of vacuum treatment produced 55.3 ± 21.6 plant regeneration for Ilmi. Calli were observed to regenerate at different subculture durations. Kim et al. (2012) suggested that the somatic embryos could be continuously proliferated for years through the repetitive subculture of embryogenic calli. In this study, plant regeneration greatly increased in the two cultivars at different subculture durations. Supplementary 4 shows that Ilpum was faster than Ilmi at initiating plant regeneration in the 0-min vacuum treatment after 28-days subculture.
Table 1 Number of calli regenerated under the vacuum treatment method
Cultivars | Vacuum treatment (min.) | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
0 | 5 | 10 | 15 | |||||||||
Na | PRb | Tc | N | PR | T | N | PR | T | N | PR | T | |
Ilmi | 249d | 8.5 ± 12.0e | 0 | 253 | 31.7 ± 16.0 | 0 | 266 | 55.3 ± 21.6 | 0 | 264 | 19.0 ± 17.3 | 0 |
Ilpum | 269 | 26.3 ± 45.6 | 0 | 276 | 41.7 ± 33.2 | 9 | 275 | 0 ± 0 | 0 | 287 | 0 ± 0 | 0 |
aNumber of inoculated calli,
bnumber of regenerated plants,
cnumber of transformants confirmed with PCR,
dsum of calli with each replication,
emean ± standard deviation
Supplementary 4 Number of calli regenerated after subculturing times
Sub-culture time (day) | cultivar | Vacuum treatment (min.) | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
0 | 5 | 10 | 15 | ||||||||||
Na | PRb | Tc | N | PR | T | N | PR | T | N | PR | T | ||
14 | Ilmi | 249d | - | - | 253 | 0.3 ± 0.6e | - | 266 | - | - | 264 | - | - |
Ilpum | 269 | - | - | 276 | 0.3 ± 0.6 | - | 275 | - | - | 287 | - | - | |
28 | Ilmi | 249 | - | - | 253 | 1.3 ± 2.3 | - | 266 | 1.0 ± 1.7 | - | 264 | 1.3 ± 2.3 | - |
Ilpum | 269 | 1.3 ± 2.3 | - | 276 | 1.7 ± 2.9 | - | 275 | - | - | 287 | - | - | |
42 | Ilmi | 249 | 1.0 ± 1.7 | - | 253 | 1.7 ± 2.9 | 266 | 2.7 ± 4.7 | - | 264 | 1.3 ± 2.3 | - | |
Ilpum | 269 | 2.0 ± 3.5 | - | 276 | 5.1 | 9 | 275 | - | - | 287 | - | - |
aNo. of inoculated calli,
bNo. of regenerated plants,
cNo. of transformants confirmed with PCR,
dSum of calli with each replication,
eMean ± standard deviation, -: no data
Plant regeneration was initiated after 14-days subculture for both cultures in the 5 min vacuum treatment group, but not the 10 and 15 min vacuum treatment groups. It was easy to regenerate Ilmi plants using this method. The notable plant regeneration observed in this study is shown in Figure 3. Vector backbone DNA sequences in transgene loci were detected at both left and right borders by flanking analysis. Nine transformed plants were tested and transformant numbers 74 and 90 were inserted into chromosome number 10 and 4 (Supplementary 5).
Noticeable plant regeneration. (a) Calli induction, (b) co-cultivation of calli, (c) regeneration of calli, (d) plant regeneration in a bottle, (e) individual plant in pot, and (f) transgenic plant after acclimatization in a green house
Supplementary 5 Results summary of flanking analysis
Query name | Read Length (bp) | Insert to | Adaptor Start | Insert Length (bp) | Chro-mosome | Mat-ching (%) | Mat-ching Length | Chr Start | Chr End | Type | Gene_ID | Description |
---|---|---|---|---|---|---|---|---|---|---|---|---|
74_A | 778 | 778 | -1 | 778 | 10 | 99.7 | 774 | 5777561 | 5776789 | Intergenic | Os10t0187366-01 downstream 8.416kb | Hypothetical protein. |
74_B | 507 | 495 | 496 | 495 | 4 | 99.2 | 495 | 24411663 | 24412156 | 5’Upstream- 1000 | Os04t0477900-01 upstream 0.394kb | Similar to Cysteine proteinase EP-B 1 precursor (EC 3.4.22.-). |
90_A | 766 | 766 | -1 | 766 | 10 | 99.6 | 759 | 5777560 | 5776802 | Intergenic | Os10t0187366-01 downstream 8.417kb | Hypothetical protein. |
90_B | 436 | 424 | 425 | 424 | 4 | 98.6 | 424 | 24411733 | 24412156 | 5’Upstream- 1000 | Os04t0477900-01 upstream 0.324kb | Similar to Cysteine proteinase EP-B 1 precursor (EC 3.4.22.-). |
The number of plants integrating the transgene based on PCR is shown in Supplementary 4 and Table 1. Approximately 125 plants, nine plants were positive for
PCR analysis of leaf tissue from a regenerated plant.
Successful rice transformation has been achieved via
The first method involved soaking seeds, which was inferred by phenomenal seed imbibition in nature. Imbibition is the phenomenon of water absorption, or any other liquid, by the solid particles of substances, without forming a solution. In plants, imbibition is one of the steps that triggers the activation of enzymes during the early germination stage. In the current study, we attempted to transfer
The second method involved different types of co-cultivation, whereby different temperatures were tested based on the optimal conditions for both
Vacuum infiltration is an
This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ011257012016). The authors thank Prof. Byung-Wook Yun for providing the
Journal of Plant Biotechnology 2016; 43(1): 66-75
Published online March 31, 2016 https://doi.org/10.5010/JPB.2016.43.1.66
Copyright © The Korean Society of Plant Biotechnology.
Fika Ayu Safitri, Mohammad Ubaidillah, and Kyung-Min Kim
Division of Plant Biosciences, School of Applied Biosciences, College of Agriculture & Life Science, Kyungpook National University, Daegu, 41566, Korea
Correspondence to:e-mail: kkm@knu.ac.kr
Keywords:
Rice (
The mature seeds of japonica rice (
Embryogenic callus ready for use as an explant transformant; the lines (a and b) show the cut side
Schematic of the plasmid vector
A primary culture of the
Different media were used at different stages of the experiment. Supplementary 1 presents a list of the media that were used. The pH of the medium was 5.8, unless otherwise stated in the table.
Supplementary 1. List of the media used in this study.
Medium | Composition |
---|---|
Soaking | Luria-Bertani (LB) brotha, 50 ?g/mL kanamycinb, 10 ?g/mL rifampycinb, 50 ?g/mL gentamycinb |
Seedling MS | 4.41 g/L Murashige and Skoog (MS) vitamin powderc, 30 g/L sucrosec, 4 g/L gerlitec, 100 ?M acetosyringoneb (pH 5.2) |
Regeneration MS | 4.41 g/L MS vitamin powder, 30 g/L sucrose, 5 mg/L kinetin and 1 mg/L 1-naphthaleneacetic (NAA)d, 250 mg/L carbenecillinc, 50 mg/L geneticine, 4 g/L gerlite |
Growing MS | 4.41 g/L MS vitamin powder, 30 g/L sucrose, 250 mg/L carbenecillin, 50 mg/L geneticin, 4 g/L gerlite |
Callusing | 4.14 g/L MS vitamin powder, 2 mg/L 2.4-dichlorophenoxyacetic acid (2,4-D)b, 30 g/L sucrose, 2 g/L casein hydrolysatec, 4 g/L gerlite |
Co-cultivation | 4.41 g/L MS vitamin powder, 5 mg/L kinetin, 1 mg/L NAA, 2 g/L casein hydrosalate, 30 g/L sucrose; 5 g/L silver nitrateb, 10.5 mg/L L-cysteineb, 1 mM sodium thiosulfateb, 4 g/L gerlite, 100 ?M acetosyringone (pH 5.2) |
Infection liquid | MS + 100 ?M acetosyringone |
Callusing vacuum | 4 g/L N6 powderc, 30 g/L sucrose, 2.878 g/L L-prolineb, 0.3 g/L casein hydrolysate, 0.1 g/L myo-inositolb, 2 mg/L 2,4-D, 4 g/L gelrite |
Co-cultivation vacuum | 4 g/L N6 powder, 30 g/L sucrose, 10 g/L D-glucoseb, 0.3 g/L casein hydrolysate, 2 mg/L 2,4-D, 4 g/L gelrite, 100 ?M acetosyringone (pH 5.2) |
Selection and elimination | 4 g/L N6 powder, 30 g/L sucrose, 2.878 g/L L-proline, 0.3 g/L casein, 2 mg/L 2,4-D, 4 g/L gelrite, 250 mg/L carbenecillin, 150 mg/L kanamycin |
Regeneration | 4.41 g/L MS vitamin powder, 30 g/L sucrose, 30 g/L sorbitolb, 2 g/L casein hydrolysate, 2 mg/L kinetin, 1 mg/L NAA, 0.1 g/L myo-inositol, 4 g/L gelrite, 500 mg/L carbenecillin, 50 mg/L geneticin |
aBecton, Dickinson and Company, New Jersey, United States,
bSigma-Aldrich, St. Louis, Missouri, United States,
cDuchefa Biochemie, BH Haarlem, Netherlands,
dJunsei Chemical co., Ltd, Tokyo, Japan,
ePhytotechnology Laboratories, Overland Park, United States
fYakuri Pure Chemical, Kyoto, Japan
In this experiment, both cultivars were used in the transformations. The transformation procedure began after the sterilized seeds had been soaked in soaking medium for 1, 2, and 3 days at 28°C under dark conditions. The seeds were then dried and placed on seedling MS medium under continuous dark conditions for 3 days and under 16/8 h light/dark conditions for a further 2 days. Subsequently, the seeds were placed on regeneration medium under 16/8 h light/dark conditions for 2 weeks. Regenerated seedlings were transferred to growing MS medium for 1 week, and the leaves were cut for use as putative transformant samples.
In this experiment, Ilpum calli were used as transformation explants. The sterilized seeds were cultured on callusing medium at 28°C under continuous dark conditions for 30 days. The embryogenic calli were separated from the endosperm and shoot (Fig. 1), at which point they were considered ready for infection. The explants were shaken in infection liquid at 28°C for 30 min in a rotary incubator and dried on autoclaved filter paper for 45 min at room temperature. The dried calli were placed in co-cultivation medium under continuous dark conditions for different durations and at different temperatures as follows; A: 28°C for 3 days, B: 28°C for 1 day followed by 25°C for 2 days, and C: 25°C for 3 days. Each treatment was repeated three times. The calli were inoculated on selection and regeneration medium for 45 days, and were subcultured 3-4 times in the same selection and regeneration media at 2-week intervals. Subsequently, the calli were transferred to a test tube containing the selection and regeneration media and left for 45 days. After this time-period, the leaves were cut and used as samples for the putative transformant.
Both cultivars were used for transformation. The sterilized seeds were cultured on callusing vacuum medium at 32 ± 1°C under continuous dark conditions for 10 days, and were then separated from the endosperm and shoot to prepare the explants for infection. Subsequently, the calli were transferred to a falcon tube containing the
Genomic DNA was isolated from the rice leaves of transgenic and non-transgenic plants for molecular analysis. DNA was isolated from rice leaves using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). For molecular confirmation, and to confirm the presence of the transgene in
Total RNA from the rice leaves was extracted and isolated from transgenic and non-transgenic plants (Qiagen, Hilden, Germany). The extracted total RNA samples were used for RT-PCR analysis. A SuperScript? III Reverse Transcriptase kit (Invitrogen, Carlsbad, United States) was used for the analysis. RT-PCR primer pairs used to amplify
Samples of total RNA were used to synthesize cDNA. Then, the samples were used for qRT-PCR. For the synthesis of cDNA from RNA, a qPCR SyGreen 1-Step kit from the PCR biosystem was used to analyze the expression of
About 100 ng of genomic DNA was digested with 4-5 units of
In this experiment, the soaking seeds method affected germination of infected seeds (Supplementary 2). The highest germination rate (114 ± 63%) was obtained by soaking in Ilpum for 1 day. Our data showed that Ilpum led to higher germination rates than Ilmi in all of the soaking treatments. For both cultivars, soaking treatment for 1 day was the optimal duration for germinating seeds. When using this method, multiple shoots were produced following 2 and 3 days of soaking.
Supplementary 2. Effect of the soaking method on seed germination.
Cultivar | Duration of soaking (days) | ||||||||
---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | |||||||
No. of inoculated seeds | No. of germinated seeds | No. of transformant seeds | No. of inoculated seeds | No. of germinated seeds | No. of transformant seeds | No. of inoculated seeds | No. of germinated seeds | No. of transformant seeds | |
Ilmi | 328 | 54 ± 28.3a | 0 ± 0 | 312 | 33 ± 15.6 | 0 ± 0 | 312 | 28.5 ± 12.0 | 0 ± 0 |
Ilpum | 334 | 114 ± 63.6 | 0 ± 0 | 360 | 99 ± 43.8 | 0 ± 0 | 247 | 68.5 ± 20.5 | 0 ± 0 |
aMean ± standard deviation
Supplementary 3 shows that different types of co-cultivation had different effects on the initiation of the green spot and the number of plants that regenerated. The B-type co-cultivation method conditions (28°C for 1 day followed by 25°C for 2 days) were found to be optimal, but transformants were not produced by this method. Rice development under the different co-cultivation methods showed that 30-day-old calli were inducted in the callusing medium. The embryogenic callus was first cut (Fig. 1) for use as the explant. The initiation of a green spot was detected after 7 days in the regeneration medium, which initiated leaf growth 2 weeks later.
Supplementary 3. Effect of different types of co-cultivation methods on calli development.
Type of co-cultivation | No. of inoculated calli | No. of green spots | No. of regenerated plants | No. of transformants |
---|---|---|---|---|
A | 204a | 9.3 ± 3.1b | 1.6 ± 0.9 | 0 ± 0 |
B | 251 | 30.3 ± 31.4 | 6.0 ± 4.2 | 0 ± 0 |
C | 229 | 11.7 ± 7.6 | 4.2 ± 1.3 | 0 ± 0 |
aNo. of inoculated calli,
bMean ± standard deviation, A: 28°C for 3 days, B: 28°C for 1 day and 25°C for 2 days, C: 25°C for 3 days
The duration of vacuum infiltration had a notable effect on the number of calli that regenerated throughout the whole experiment (Table 1) and in every subculture (Supplementary 4) of both cultivars. Vacuum infiltration for 5 min led to the production of 41.7 ± 33.2 plant regeneration (Table 1) for Ilpum, whereas 10 min of vacuum treatment produced 55.3 ± 21.6 plant regeneration for Ilmi. Calli were observed to regenerate at different subculture durations. Kim et al. (2012) suggested that the somatic embryos could be continuously proliferated for years through the repetitive subculture of embryogenic calli. In this study, plant regeneration greatly increased in the two cultivars at different subculture durations. Supplementary 4 shows that Ilpum was faster than Ilmi at initiating plant regeneration in the 0-min vacuum treatment after 28-days subculture.
Table 1. Number of calli regenerated under the vacuum treatment method.
Cultivars | Vacuum treatment (min.) | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
0 | 5 | 10 | 15 | |||||||||
Na | PRb | Tc | N | PR | T | N | PR | T | N | PR | T | |
Ilmi | 249d | 8.5 ± 12.0e | 0 | 253 | 31.7 ± 16.0 | 0 | 266 | 55.3 ± 21.6 | 0 | 264 | 19.0 ± 17.3 | 0 |
Ilpum | 269 | 26.3 ± 45.6 | 0 | 276 | 41.7 ± 33.2 | 9 | 275 | 0 ± 0 | 0 | 287 | 0 ± 0 | 0 |
aNumber of inoculated calli,
bnumber of regenerated plants,
cnumber of transformants confirmed with PCR,
dsum of calli with each replication,
emean ± standard deviation
Supplementary 4. Number of calli regenerated after subculturing times.
Sub-culture time (day) | cultivar | Vacuum treatment (min.) | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
0 | 5 | 10 | 15 | ||||||||||
Na | PRb | Tc | N | PR | T | N | PR | T | N | PR | T | ||
14 | Ilmi | 249d | - | - | 253 | 0.3 ± 0.6e | - | 266 | - | - | 264 | - | - |
Ilpum | 269 | - | - | 276 | 0.3 ± 0.6 | - | 275 | - | - | 287 | - | - | |
28 | Ilmi | 249 | - | - | 253 | 1.3 ± 2.3 | - | 266 | 1.0 ± 1.7 | - | 264 | 1.3 ± 2.3 | - |
Ilpum | 269 | 1.3 ± 2.3 | - | 276 | 1.7 ± 2.9 | - | 275 | - | - | 287 | - | - | |
42 | Ilmi | 249 | 1.0 ± 1.7 | - | 253 | 1.7 ± 2.9 | 266 | 2.7 ± 4.7 | - | 264 | 1.3 ± 2.3 | - | |
Ilpum | 269 | 2.0 ± 3.5 | - | 276 | 5.1 | 9 | 275 | - | - | 287 | - | - |
aNo. of inoculated calli,
bNo. of regenerated plants,
cNo. of transformants confirmed with PCR,
dSum of calli with each replication,
eMean ± standard deviation, -: no data
Plant regeneration was initiated after 14-days subculture for both cultures in the 5 min vacuum treatment group, but not the 10 and 15 min vacuum treatment groups. It was easy to regenerate Ilmi plants using this method. The notable plant regeneration observed in this study is shown in Figure 3. Vector backbone DNA sequences in transgene loci were detected at both left and right borders by flanking analysis. Nine transformed plants were tested and transformant numbers 74 and 90 were inserted into chromosome number 10 and 4 (Supplementary 5).
Noticeable plant regeneration. (a) Calli induction, (b) co-cultivation of calli, (c) regeneration of calli, (d) plant regeneration in a bottle, (e) individual plant in pot, and (f) transgenic plant after acclimatization in a green house
Supplementary 5. Results summary of flanking analysis.
Query name | Read Length (bp) | Insert to | Adaptor Start | Insert Length (bp) | Chro-mosome | Mat-ching (%) | Mat-ching Length | Chr Start | Chr End | Type | Gene_ID | Description |
---|---|---|---|---|---|---|---|---|---|---|---|---|
74_A | 778 | 778 | -1 | 778 | 10 | 99.7 | 774 | 5777561 | 5776789 | Intergenic | Os10t0187366-01 downstream 8.416kb | Hypothetical protein. |
74_B | 507 | 495 | 496 | 495 | 4 | 99.2 | 495 | 24411663 | 24412156 | 5’Upstream- 1000 | Os04t0477900-01 upstream 0.394kb | Similar to Cysteine proteinase EP-B 1 precursor (EC 3.4.22.-). |
90_A | 766 | 766 | -1 | 766 | 10 | 99.6 | 759 | 5777560 | 5776802 | Intergenic | Os10t0187366-01 downstream 8.417kb | Hypothetical protein. |
90_B | 436 | 424 | 425 | 424 | 4 | 98.6 | 424 | 24411733 | 24412156 | 5’Upstream- 1000 | Os04t0477900-01 upstream 0.324kb | Similar to Cysteine proteinase EP-B 1 precursor (EC 3.4.22.-). |
The number of plants integrating the transgene based on PCR is shown in Supplementary 4 and Table 1. Approximately 125 plants, nine plants were positive for
PCR analysis of leaf tissue from a regenerated plant.
Successful rice transformation has been achieved via
The first method involved soaking seeds, which was inferred by phenomenal seed imbibition in nature. Imbibition is the phenomenon of water absorption, or any other liquid, by the solid particles of substances, without forming a solution. In plants, imbibition is one of the steps that triggers the activation of enzymes during the early germination stage. In the current study, we attempted to transfer
The second method involved different types of co-cultivation, whereby different temperatures were tested based on the optimal conditions for both
Vacuum infiltration is an
This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ011257012016). The authors thank Prof. Byung-Wook Yun for providing the
Embryogenic callus ready for use as an explant transformant; the lines (a and b) show the cut side
Schematic of the plasmid vector
Noticeable plant regeneration. (a) Calli induction, (b) co-cultivation of calli, (c) regeneration of calli, (d) plant regeneration in a bottle, (e) individual plant in pot, and (f) transgenic plant after acclimatization in a green house
PCR analysis of leaf tissue from a regenerated plant.
Supplementary 1. List of the media used in this study.
Medium | Composition |
---|---|
Soaking | Luria-Bertani (LB) brotha, 50 ?g/mL kanamycinb, 10 ?g/mL rifampycinb, 50 ?g/mL gentamycinb |
Seedling MS | 4.41 g/L Murashige and Skoog (MS) vitamin powderc, 30 g/L sucrosec, 4 g/L gerlitec, 100 ?M acetosyringoneb (pH 5.2) |
Regeneration MS | 4.41 g/L MS vitamin powder, 30 g/L sucrose, 5 mg/L kinetin and 1 mg/L 1-naphthaleneacetic (NAA)d, 250 mg/L carbenecillinc, 50 mg/L geneticine, 4 g/L gerlite |
Growing MS | 4.41 g/L MS vitamin powder, 30 g/L sucrose, 250 mg/L carbenecillin, 50 mg/L geneticin, 4 g/L gerlite |
Callusing | 4.14 g/L MS vitamin powder, 2 mg/L 2.4-dichlorophenoxyacetic acid (2,4-D)b, 30 g/L sucrose, 2 g/L casein hydrolysatec, 4 g/L gerlite |
Co-cultivation | 4.41 g/L MS vitamin powder, 5 mg/L kinetin, 1 mg/L NAA, 2 g/L casein hydrosalate, 30 g/L sucrose; 5 g/L silver nitrateb, 10.5 mg/L L-cysteineb, 1 mM sodium thiosulfateb, 4 g/L gerlite, 100 ?M acetosyringone (pH 5.2) |
Infection liquid | MS + 100 ?M acetosyringone |
Callusing vacuum | 4 g/L N6 powderc, 30 g/L sucrose, 2.878 g/L L-prolineb, 0.3 g/L casein hydrolysate, 0.1 g/L myo-inositolb, 2 mg/L 2,4-D, 4 g/L gelrite |
Co-cultivation vacuum | 4 g/L N6 powder, 30 g/L sucrose, 10 g/L D-glucoseb, 0.3 g/L casein hydrolysate, 2 mg/L 2,4-D, 4 g/L gelrite, 100 ?M acetosyringone (pH 5.2) |
Selection and elimination | 4 g/L N6 powder, 30 g/L sucrose, 2.878 g/L L-proline, 0.3 g/L casein, 2 mg/L 2,4-D, 4 g/L gelrite, 250 mg/L carbenecillin, 150 mg/L kanamycin |
Regeneration | 4.41 g/L MS vitamin powder, 30 g/L sucrose, 30 g/L sorbitolb, 2 g/L casein hydrolysate, 2 mg/L kinetin, 1 mg/L NAA, 0.1 g/L myo-inositol, 4 g/L gelrite, 500 mg/L carbenecillin, 50 mg/L geneticin |
aBecton, Dickinson and Company, New Jersey, United States,
bSigma-Aldrich, St. Louis, Missouri, United States,
cDuchefa Biochemie, BH Haarlem, Netherlands,
dJunsei Chemical co., Ltd, Tokyo, Japan,
ePhytotechnology Laboratories, Overland Park, United States
fYakuri Pure Chemical, Kyoto, Japan
Supplementary 2. Effect of the soaking method on seed germination.
Cultivar | Duration of soaking (days) | ||||||||
---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | |||||||
No. of inoculated seeds | No. of germinated seeds | No. of transformant seeds | No. of inoculated seeds | No. of germinated seeds | No. of transformant seeds | No. of inoculated seeds | No. of germinated seeds | No. of transformant seeds | |
Ilmi | 328 | 54 ± 28.3a | 0 ± 0 | 312 | 33 ± 15.6 | 0 ± 0 | 312 | 28.5 ± 12.0 | 0 ± 0 |
Ilpum | 334 | 114 ± 63.6 | 0 ± 0 | 360 | 99 ± 43.8 | 0 ± 0 | 247 | 68.5 ± 20.5 | 0 ± 0 |
aMean ± standard deviation
Supplementary 3. Effect of different types of co-cultivation methods on calli development.
Type of co-cultivation | No. of inoculated calli | No. of green spots | No. of regenerated plants | No. of transformants |
---|---|---|---|---|
A | 204a | 9.3 ± 3.1b | 1.6 ± 0.9 | 0 ± 0 |
B | 251 | 30.3 ± 31.4 | 6.0 ± 4.2 | 0 ± 0 |
C | 229 | 11.7 ± 7.6 | 4.2 ± 1.3 | 0 ± 0 |
aNo. of inoculated calli,
bMean ± standard deviation, A: 28°C for 3 days, B: 28°C for 1 day and 25°C for 2 days, C: 25°C for 3 days
Table 1. Number of calli regenerated under the vacuum treatment method.
Cultivars | Vacuum treatment (min.) | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
0 | 5 | 10 | 15 | |||||||||
Na | PRb | Tc | N | PR | T | N | PR | T | N | PR | T | |
Ilmi | 249d | 8.5 ± 12.0e | 0 | 253 | 31.7 ± 16.0 | 0 | 266 | 55.3 ± 21.6 | 0 | 264 | 19.0 ± 17.3 | 0 |
Ilpum | 269 | 26.3 ± 45.6 | 0 | 276 | 41.7 ± 33.2 | 9 | 275 | 0 ± 0 | 0 | 287 | 0 ± 0 | 0 |
aNumber of inoculated calli,
bnumber of regenerated plants,
cnumber of transformants confirmed with PCR,
dsum of calli with each replication,
emean ± standard deviation
Supplementary 4. Number of calli regenerated after subculturing times.
Sub-culture time (day) | cultivar | Vacuum treatment (min.) | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
0 | 5 | 10 | 15 | ||||||||||
Na | PRb | Tc | N | PR | T | N | PR | T | N | PR | T | ||
14 | Ilmi | 249d | - | - | 253 | 0.3 ± 0.6e | - | 266 | - | - | 264 | - | - |
Ilpum | 269 | - | - | 276 | 0.3 ± 0.6 | - | 275 | - | - | 287 | - | - | |
28 | Ilmi | 249 | - | - | 253 | 1.3 ± 2.3 | - | 266 | 1.0 ± 1.7 | - | 264 | 1.3 ± 2.3 | - |
Ilpum | 269 | 1.3 ± 2.3 | - | 276 | 1.7 ± 2.9 | - | 275 | - | - | 287 | - | - | |
42 | Ilmi | 249 | 1.0 ± 1.7 | - | 253 | 1.7 ± 2.9 | 266 | 2.7 ± 4.7 | - | 264 | 1.3 ± 2.3 | - | |
Ilpum | 269 | 2.0 ± 3.5 | - | 276 | 5.1 | 9 | 275 | - | - | 287 | - | - |
aNo. of inoculated calli,
bNo. of regenerated plants,
cNo. of transformants confirmed with PCR,
dSum of calli with each replication,
eMean ± standard deviation, -: no data
Supplementary 5. Results summary of flanking analysis.
Query name | Read Length (bp) | Insert to | Adaptor Start | Insert Length (bp) | Chro-mosome | Mat-ching (%) | Mat-ching Length | Chr Start | Chr End | Type | Gene_ID | Description |
---|---|---|---|---|---|---|---|---|---|---|---|---|
74_A | 778 | 778 | -1 | 778 | 10 | 99.7 | 774 | 5777561 | 5776789 | Intergenic | Os10t0187366-01 downstream 8.416kb | Hypothetical protein. |
74_B | 507 | 495 | 496 | 495 | 4 | 99.2 | 495 | 24411663 | 24412156 | 5’Upstream- 1000 | Os04t0477900-01 upstream 0.394kb | Similar to Cysteine proteinase EP-B 1 precursor (EC 3.4.22.-). |
90_A | 766 | 766 | -1 | 766 | 10 | 99.6 | 759 | 5777560 | 5776802 | Intergenic | Os10t0187366-01 downstream 8.417kb | Hypothetical protein. |
90_B | 436 | 424 | 425 | 424 | 4 | 98.6 | 424 | 24411733 | 24412156 | 5’Upstream- 1000 | Os04t0477900-01 upstream 0.324kb | Similar to Cysteine proteinase EP-B 1 precursor (EC 3.4.22.-). |
A-Ra Cho ・Dong-Kil Lee ・Kyung-Min Kim
J Plant Biotechnol 2015; 42(2): 83-87Do-Sin Lee · Dong-Young Kim · Kwang-Hyun Jo · Jeong-Ho Baek · Sung-Hwan Jo
J Plant Biotechnol 2024; 51(1): 344-353Do-Sin Lee , Dong-Young Kim , Kwang-Hyun Jo , Jeong-Ho Baek , Sung-Hwan Jo
J Plant Biotechnol -0001; ():
Journal of
Plant BiotechnologyEmbryogenic callus ready for use as an explant transformant; the lines (a and b) show the cut side
|@|~(^,^)~|@|Schematic of the plasmid vector
Noticeable plant regeneration. (a) Calli induction, (b) co-cultivation of calli, (c) regeneration of calli, (d) plant regeneration in a bottle, (e) individual plant in pot, and (f) transgenic plant after acclimatization in a green house
|@|~(^,^)~|@|PCR analysis of leaf tissue from a regenerated plant.