Chapter 21 Agrobacterium-Mediated Transformation: Rice Transformation Inez H. Slamet-Loedin, Prabhjit Chadha-Mohanty, and Lina Torrizo Abstract Agrobacterium is a common soil bacterium with natural capacity for trans-kingdom transfer of genetic information by transferring its T-DNA into the eukaryotic genome. In agricultural plant biotechnology, combination of non-phytopathogenic strain of Agrobacterium tumefaciens with modified T-DNA and virgenes in a binary vector system is the most widely utilized system for genetic improvement in diverse plant species and for gene function validation. Here we have described a highly efficient A. tumefaciens-mediated transformation system for indica and japonica rice cultivars based on an immature embryo system. Key words Agrobacterium, Rice, Transformation, Indica, Immature embryo, Gene transfer
1
Introduction Functional genomic study and efficient production of transgenic crops require the establishment of an efficient transformation platform. The two most widely used methods for rice transformation are Agrobacterium-mediated transformation and particle gunmediated DNA delivery [1]. The Agrobacterium-mediated method generally results in higher transformation efficiency and higher frequency of single intact copy of the transgene [2, 3] with little rearrangement as compared to direct DNA delivery methods such as particle bombardment. Agrobacterium-mediated transformation has been widely reported in monocots including rice, but finding a reliable and efficient system for genetic transformation that can be applied across different major rice varieties remains a challenge. A mature seed transformation system, which is more convenient since it is not limited by the availability of immature embryo, is very efficient for japonica varieties (6.2–50 %) but generally results in a low transformation frequency for indica cultivars.
Robert J. Henry and Agnelo Furtado (eds.), Cereal Genomics: Methods and Protocols, Methods in Molecular Biology, vol. 1099, DOI 10.1007/978-1-62703-715-0_21, © Springer Science+Business Media New York 2014
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A major advance in rice transformation was reported by Hiei and Komari [4, 5] using Agrobacterium, describing an efficient transformation procedure for japonica and indica rice cultivars. The important factors for efficient rice transformation of immature embryos of both japonica and indica varieties [5] are use of fresh healthy embryos and optimization of media compositions. This chapter describes an improved and reproducible protocol, modified from the original method of Hiei and Komari for the production of transgenic rice plants for a range of indica rice genotypes, using immature embryos and giving a high transformation efficiency in indica rice. We have established a high-efficiency protocol for Agrobacterium-mediated transformation, routinely applied for functional genomic study [6], with efficiency for japonica varieties in the range of 90–100 % and elite indica variety IR64 in the range of 25–40 % and applicable to greenhouse-, screenhouse-, and fieldharvested immature embryos. To achieve higher efficiency we modified the bacterial cell culture density, altered the composition of the pre-regeneration and regeneration media, and used the open-source pCAMBIA-based binary vector system (http://www. cambia.org/daisy/cambialabs/home.htm) in combination with the LBA 4404 Agrobacterium strain.
2
Materials Prepare all solutions using ultrapure water (prepared by purifying RO water through the MQ system of Millipore™ attached with a 0.22 μm Millipore filter) or double-distillated water and analytical grade reagents. Prepare and store all solutions in a refrigerator (4–8 °C) unless indicated otherwise. Diligently follow all waste disposal and biosafety regulations when disposing of waste materials. Conduct bacterial work, transformation, and tissue culture process in a laminar airflow cabinet to maintain sterility; utilize the appropriate laminar airflow cabinet as requested by the regulation. Conduct all media autoclaving at 115 °C for 15 min.
2.1 Agrobacterium Culture Component
2.2 Other Components
The Agrobacterium plate culture medium components are listed in Table 1. Autoclave the three stock solutions separately. Add 25 mL each of stocks I and II to 450 mL of stock III. Add suitable antibiotics depending on the antibiotic-resistant gene present in the binary plasmids and Agrobacterium strain (e.g., spectinomycin 50 mg/L or kanamycin and hygromycin 50 mg/L). 1. Sterilizing solution component: 70 % ethanol, 1 % sodium hypochlorite (containing one to two drops of Tween 20) solution. 2. Platform shaker. 3. Falcon tubes, 50 mL. 4. Stereomicroscope.
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Table 1 AB [7] medium for culture of Agrobacterium Components
Amount (g/500 mL)
Molarity
KH2PO4
30.0
0.22
NaH2PO4
10.0
0.083
10.0
0.187
MgSO4.7H2O
3.0
0.012
KCl
1.5
0.020
CaCl2
0.1
9.0 × 10−4
FeSO4.7H2O
0.025
9.0 × 10−5
Stock I
Stock II NH4Cl
Stock III
(g per 900 mL)
Glucose Agar
5.0
0.028
15.0
5. Sterile filer papers. 6. Sterile petri plates. 2.3 Transformation and Tissue Culture Medium, Rooting Media Component
The components of stock solutions used in the different media for rice transformation are shown in Table 2. Table 3 shows the preparation of culture medium coded as A200 for infection medium, A201 for cocultivation medium, A202 for resting medium, A203 for selection medium, A204 for the pre-regeneration medium, and A205 for regeneration medium. Rooting medium is the Murashige and Skoog medium as presented in Tables 2 and 3. Put about 700 mL of ultrapure water for every liter medium in a beaker with stirrer. Add the specified amount of stock solutions. Weigh amino acids, sucrose, and other components separately and add to the medium. Adjust medium to 1 L. Adjust the pH of the medium as specified.
2.4 Other Components
Refer to Table 4 for details.
3
Methods Prepare the culture media and store them in a refrigerator (4 °C) except for antibiotic stocks which should be stored at −20 °C. Carry out the Agrobacterium streak culture in a laminar air-downflow cabinet and tissue culture in a laminar airflow cabinet to maintain sterile conditions.
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Table 2 Preparation of stock solutions and media for immature embryo transformation Stock solution (volume)
Component
Weight
Molarity
N6 [8] major 1 (1 L)
KNO3
141.5 g
1.40
N6 major 2 (1 L)
MgSO4.7H2O (NH4)2SO4
18.5 g 46.3 g
0.075 0.35
N6 major 3 (1 L)
KH2PO4
40 g
0.294
N6 major 4 (1 L)
CaCl2.2H2O
16.6 g
0.11
B5 [9] minor 1 (1 L)
FeSO4.7H2O Na2EDTA.2H2O
2.785 g 3.725 g
0.01 0.01
B5 minor 2 (1 L)
MnSO4.4H2O ZnSO4.7H2O H3BO3
1g 0.2 g 0.3 g
4.4 × 10−3 7.0 × 10−4 4.85 × 10−3
B5 minor 3 (1 L)
KI
0.075 g
4.5 × 10−4
B5 minor 4 (1 L)
CuSO4.5H2O
0.0025 g
1.0 × 10−5
Na2MoO4.2H2O
0.025 g
1.0 × 10−4
CoCl2.6H2O
0.0025 g
1.05 × 10−5
B5 vitamins (100 mL)
Thiamine HCl Pyridoxine HCl Nicotinic acid Myoinositol
200 mg 20 mg 20 mg 2,000 mg
6.0 × 10−4 1.0 x10−4 1.6 × 10−5 0.011
AA [10] macro salts (1 L)
CaCl2.2H2O MgSO4.7H2O NaH2PO4.2H2O KCl
1.5 g 2.49 g 1.7 g 29.5 g
0.01 0.01 0.01 0.4
AA micro salts (1 L)
CoCl2.6H2O CuSO4.5H2O H3BO3 KI MnSO4 Na2MoO4.2H2O ZnSO4.7H2O
25 mg 25 mg 3,000 mg 750 mg 8.9 g 250 mg 2,000 mg
1 × 10−4 1.0 × 10−4 0.0485 4.5 × 10−3 0.059 1.0 × 10−3 7.0 × 10−3
AA iron stock (1 L) Same as B5 minor 1
FeSO.7H2O
2.78 g
0.01
Na2EDTA.2H2O
3.73 g
0.01 (continued)
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Table 2 (continued) Stock solution (volume)
Component
Glycine (1 L) (filter sterilize and store at −20 °C)
Weight
Molarity
7.5 g
0.10
MS [11] 1 (1 L)
KNO3 NH4NO3
95 g 82.5 g
0.94 1.03
MS2 (1 L)
MgSO4.H2O MnSO4.4H2O ZnSO4.7H2O CuSO4.5H2O
37 g 2.23 g 0.86 g 0.0025 g
0.267 0.01 0.003 1.0 × 10−5
MS3 (1 L)
CaCl2.2H2O KI CoCl2.6H2O
44 g 0.083 g 0.0025 g
0.3 5 × 10−4 1.0 × 10−5
MS4 (1 L)
KH2PO4 H3BO3 Na2MoO4.2H2O
17 g 0.62 g 0.025 g
0.125 0.010 1.0 × 10−4
MS vitamins (100 mL)
Nicotinic acid Pyridoxine HCl Thiamine HCl Glycine Myoinositol
10 mg 10 mg 2 mg 40 mg 2,000 mg
8.1 × 10−5 4.9 × 10−5 6 × 10−6 5.33 × 10−4 0.011
A1. Stock solutions: Components are dissolved in MQ water and stored at 4 °C
3.1 Agrobacterium Culture
1. Three days before immature embryo (IE) transformation, streak Agrobacterium from glycerol stock onto AB plate (see Table 1 for preparation) with suitable antibiotics depending on the plasmid. 2. Incubate Agrobacterium culture in the dark at 28 °C for 2–3 days.
3.2 Panicle Harvesting and Immature Embryo Sterilization and Isolation
1. Harvest panicles at 8–12 days after anthesis (see Notes 6 and 7). Dehull developing seeds and put in 50 mL Falcon tubes. 2. Add sterilizing solution, and incubate the 50 mL tubes with constant shaking for 5 min. Rinse at least five times with plenty of sterile distilled water. 3. Isolate immature embryos (under a stereomicroscope, if needed).
5
B5 vitamins
10 10 10
MS3
MS4
10
A204
MS2
5
10
10
10
10
10
10
10
20
A203
20
1
5
10
10
10
10
10
10
10
20
A202
MS1
Glycine
10
10
B5 minor 4
AA iron stock
10
B5 minor 3
1
10
B5 minor 2
AA micro salts stock:
10
B5 [8] minor 1
100
10
N6 major 4
AA macro salts stock
10
N6 major 3
1
10
N6 major 2
A201
20
A200
Amount of stock to take per liter of medium (mL)
N6 major 1
Stock solution
Table 3 Media composition and amount of stock requirement for rice transformation media preparation
10
10
10
20
10
A205
10
10
10
20
10
MS0
266 Inez H. Slamet-Loedin et al.
−3
5.2
Acetosyringone
See Note 2
19.62 mg dissolved in 1 mL DMSO
100 μM (add before use)
5,500
See Note 1
Microwave medium to melt Gelrite
Gelrite
Agarose type 1
pH
Adjust volume to 1 liter
5.2
5,000
5.8
20,000 (0.058)
Maltose
Sorbitol
36,000 (0.197)
Mannitol
10,000 (0.056)
10,000 (0.056)
500
D-glucose
−3
20,000 (0.058)
500
200,000 (0.058)
500
Casamino acid
1.0 × 10−3
300
Sucrose
174 (0.001)
Arginine
2.0 × 10
−3
500 (4.3 × 10 ) 500 (4.3 × 10 )
260 (0.002)
Aspartic acid
6 × 10−3
L-proline
876 (0.006)
5,000
5.8
20,000 (0.058)
36,000 (0.197)
500 (4.3 × 10−3)
500
300
Amount of chemical in mg (mol) to add per liter of medium
L-glutamine
Chemical
MS vitamins
10,000
5.8
20,000 (0.11)
30,000 (0.088)
5
3,000
5.8
30,000 (0.088)
5
(continued)
See Note 3
Dispense in 20 mL/tube
2,000
5.8
3,000 (0.088)
5
Agrobacterium Transformation of Indica Rice 267
1
BAP
Dispense 25 mL in 100 × 15 petri dish to solidify
1
Carbenicillin
Hygromycin
1
Cefotaxime
Antibiotic
0.2
1
1
Amount to add per liter of medium (mL)—see Note 5
1
NAA
Kinetin
2
Amount to add per liter of medium (mL)—see Note 4
2,4-D
Growth regulator
(continued)
Table 3
1
1
2
5
1
1
2
1
Dispense 35 mL in 100 × 20 petri dish to solidify
0.6
1
1
0.2
1
1
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Agrobacterium Transformation of Indica Rice
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Table 4 Other media composition and amount of stock requirement for rice transformation media preparation Weight (molar Stock solution (vol) concentration)
Preparation
Storage temperature
Growth regulators BAP (10 mL)
10 mg (4.44 × 10−5)
Dissolve in 1N NaOH (0.2 mL per 10 mg BAP) and complete to volume with MQ water; filter sterilize and aliquot in Eppendorf tubes
4 °C
2,4-D (50 mL)
50 mg (2.26 × 10−4)
Dissolve in 1 M KOH (2 mL per 50 mL stock) and complete volume with MQ water; filter sterilize and aliquot in Eppendorf tubes
4 °C
Kinetin (25 mL)
25 mg (1.16 × 10−4)
Dissolve in 1N NaOH and complete volume with MQ water; filter sterilize and aliquot in Eppendorf tubes
−20 °C
NAA (20 mL)
20 mg (1.074 × 10−4)
Dissolve in 1N NaOH and complete volume with MQ water; filter sterilize and aliquot in Eppendorf tubes
4 °C
Carbenicillin (10 mL)1,000 mg
Dissolve in MQ water, filter sterilize and aliquot in sterile Eppendorf tubes
−20 °C
Cefotaxime sodium (50 mL)
5,000 mg
Dissolve in MQ water, filter sterilize and aliquot in sterile Eppendorf tubes
−20 °C
Hygromycin B
Ordered and used directly as 50 mg/L stock
Aliquot in sterile Eppendorf tubes
4 °C
Antibiotics
3.3 Immature Embryo Transformation: Cocultivation
1. 1 h prior to infection of IEs, take about one full loop (3 mm loop size) of Agrobacterium culture from AB plate and suspend in A200 medium contained in a Falcon tube. 2. Pipet or invert the tube gently several times for even mixing. Adjust bacterial density to 3 × 109 cfu/mL (OD 0.3). Incubate the suspension in the dark at 25 °C for 1 h (incubator) prior to infecting the IEs. 3. Place the IEs with the scutellum side up onto A201 medium. Drop 5 μL of Agrobacterium suspension to each IE. Incubate cultures in the dark at 25 °C for 7 days. Place 50 IEs per plate.
3.4 Resting Culture and Selection
1. After cocultivation period, place IEs on a sterile filter paper and remove elongated shoots with a scalpel.
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2. Quick-dry the IEs several times by gently pressing these between two layers of sterile filter papers (see Note 8). 3. Transfer IEs with the scutellum side up onto A202 resting medium. Place 16 IEs per petri dish. Incubate under continuous light at 30 °C for 5 days. 4. Transfer IEs onto A203 selection medium and incubate under continuous light at 30 °C for 10 days; alternatively, each embryo may be cut into four pieces or more (first selection). Place eight to ten IEs per plate. 5. Transfer IEs onto fresh A203 medium and incubate under continuous light at 30 °C for 10 days (second selection). 6. Separate the embryogenic callus from the black callus tissue, and transfer the embryogenic calli to fresh A203 medium. Incubate under continuous light at 30 °C for 10 days (third selection). 3.5 Plant Regeneration
1. Transfer resistant calli to A204 pre-regeneration medium at 6–8 callus lines per petri dish. Incubate under continuous light at 30 °C for 10 days. 2. Select proliferating calli with green spots and transfer to A205 regeneration medium. Place four callus lines per petri dish. Label each regenerable callus line. 3. Carefully select one plantlet from each callus line and inoculate into a tube containing MS0 rooting medium. Barcode-label the plant ID on each test tube. Keep the plantlets under continuous light at 25 °C for 14 days. Keep original petri dishes with plantlets as backup. 4. If a callus line has not regenerated plants but has small shoots, transfer these to a fresh A205 medium to allow the shoots to grow into plantlets. Incubate under continuous light at 30 °C for 14 days, and once plantlets have developed, transfer to MS0 for a better rooting for 1–2 weeks.
4
Notes 1. A200 is filter sterilized and stored at 4 °C; add acetosyringone just before use for transformation. 2. For the rest of the media in solid form (A201–A205), add the gelling agent prior to autoclaving at 115 °C for 15 min. 3. For A201, add acetosyringone to the autoclaved medium and maintain at 55 °C prior to dispensing. 4. Likewise, add growth regulators and antibiotics to media and cool to 55 °C.
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5. Dispense solid media in appropriate size of petri dishes and store at 4 °C. 6. Embryo size at about 1.3–1.8 mm is crucial; a very-small-size embryo will not survive. 7. Screenhouse-grown material will be a better option for less occurrence of microbial contamination; however, a healthy field-grown material has been shown to also result in a good transformation frequency and manageable contamination. 8. Properly dry the embryos to avoid bacterial overgrowth, but do not damage the embryo and ensure a quick process to avoid drying.
Acknowledgement Establishment of the protocol was supported by USAID grant number DPPC2004-17. Technical support of F. Montecillo and facilitation of P. Herve and G. Barry are acknowledged. References 1. Tyagi AK, Mohanty A (2000) Rice transformation for crop improvement and functional genomics. Plant Sci 158:1–18 2. Iyer LM, Kumplata SP, Chandrasekharan MB et al (2000) Transgene silencing in monocots. Plant Mol Biol 43:323–346 3. Hiei Y, Ohta S, Komari T et al (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–2824 4. Hiei Y, Komari T (2006) Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 85:271–283 5. Hiei Y, Komari T (2008) Agrobacteriummediated transformation of rice using immature embryos or calli induced from mature seed. Nat Protoc 3:824–834 6. Gamuyao R, Joong Hyoun Chin JH, PariascaTanaka J et al (2012) The protein kinase Pstol1
7.
8.
9.
10.
11.
from traditional rice confers tolerance of phosphorus deficiency. Nature 488:535–539 Clark DJ, Maaløe O (1967) DNA replication and the division cycle in Escherichia coli. J Mol Biol 1:99–112 Chu C-C (1978) The N6 medium and its application to anther culture of cereal crops. In: Proc. Symp. plant tissue culture. Science Press, Peking, pp 43–50 Gamborg OL, Miller RA, Ojima K (1968) Plant cell cultures 1, nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158 Toriyama K, Hinata K (1985) Cell suspension and protoplast culture in rice. Plant Sci 41:179–183 Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:473–497
1
Introduction Functional genomic study and efficient production of transgenic crops require the establishment of an efficient transformation platform. The two most widely used methods for rice transformation are Agrobacterium-mediated transformation and particle gunmediated DNA delivery [1]. The Agrobacterium-mediated method generally results in higher transformation efficiency and higher frequency of single intact copy of the transgene [2, 3] with little rearrangement as compared to direct DNA delivery methods such as particle bombardment. Agrobacterium-mediated transformation has been widely reported in monocots including rice, but finding a reliable and efficient system for genetic transformation that can be applied across different major rice varieties remains a challenge. A mature seed transformation system, which is more convenient since it is not limited by the availability of immature embryo, is very efficient for japonica varieties (6.2–50 %) but generally results in a low transformation frequency for indica cultivars.
Robert J. Henry and Agnelo Furtado (eds.), Cereal Genomics: Methods and Protocols, Methods in Molecular Biology, vol. 1099, DOI 10.1007/978-1-62703-715-0_21, © Springer Science+Business Media New York 2014
261
262
Inez H. Slamet-Loedin et al.
A major advance in rice transformation was reported by Hiei and Komari [4, 5] using Agrobacterium, describing an efficient transformation procedure for japonica and indica rice cultivars. The important factors for efficient rice transformation of immature embryos of both japonica and indica varieties [5] are use of fresh healthy embryos and optimization of media compositions. This chapter describes an improved and reproducible protocol, modified from the original method of Hiei and Komari for the production of transgenic rice plants for a range of indica rice genotypes, using immature embryos and giving a high transformation efficiency in indica rice. We have established a high-efficiency protocol for Agrobacterium-mediated transformation, routinely applied for functional genomic study [6], with efficiency for japonica varieties in the range of 90–100 % and elite indica variety IR64 in the range of 25–40 % and applicable to greenhouse-, screenhouse-, and fieldharvested immature embryos. To achieve higher efficiency we modified the bacterial cell culture density, altered the composition of the pre-regeneration and regeneration media, and used the open-source pCAMBIA-based binary vector system (http://www. cambia.org/daisy/cambialabs/home.htm) in combination with the LBA 4404 Agrobacterium strain.
2
Materials Prepare all solutions using ultrapure water (prepared by purifying RO water through the MQ system of Millipore™ attached with a 0.22 μm Millipore filter) or double-distillated water and analytical grade reagents. Prepare and store all solutions in a refrigerator (4–8 °C) unless indicated otherwise. Diligently follow all waste disposal and biosafety regulations when disposing of waste materials. Conduct bacterial work, transformation, and tissue culture process in a laminar airflow cabinet to maintain sterility; utilize the appropriate laminar airflow cabinet as requested by the regulation. Conduct all media autoclaving at 115 °C for 15 min.
2.1 Agrobacterium Culture Component
2.2 Other Components
The Agrobacterium plate culture medium components are listed in Table 1. Autoclave the three stock solutions separately. Add 25 mL each of stocks I and II to 450 mL of stock III. Add suitable antibiotics depending on the antibiotic-resistant gene present in the binary plasmids and Agrobacterium strain (e.g., spectinomycin 50 mg/L or kanamycin and hygromycin 50 mg/L). 1. Sterilizing solution component: 70 % ethanol, 1 % sodium hypochlorite (containing one to two drops of Tween 20) solution. 2. Platform shaker. 3. Falcon tubes, 50 mL. 4. Stereomicroscope.
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Table 1 AB [7] medium for culture of Agrobacterium Components
Amount (g/500 mL)
Molarity
KH2PO4
30.0
0.22
NaH2PO4
10.0
0.083
10.0
0.187
MgSO4.7H2O
3.0
0.012
KCl
1.5
0.020
CaCl2
0.1
9.0 × 10−4
FeSO4.7H2O
0.025
9.0 × 10−5
Stock I
Stock II NH4Cl
Stock III
(g per 900 mL)
Glucose Agar
5.0
0.028
15.0
5. Sterile filer papers. 6. Sterile petri plates. 2.3 Transformation and Tissue Culture Medium, Rooting Media Component
The components of stock solutions used in the different media for rice transformation are shown in Table 2. Table 3 shows the preparation of culture medium coded as A200 for infection medium, A201 for cocultivation medium, A202 for resting medium, A203 for selection medium, A204 for the pre-regeneration medium, and A205 for regeneration medium. Rooting medium is the Murashige and Skoog medium as presented in Tables 2 and 3. Put about 700 mL of ultrapure water for every liter medium in a beaker with stirrer. Add the specified amount of stock solutions. Weigh amino acids, sucrose, and other components separately and add to the medium. Adjust medium to 1 L. Adjust the pH of the medium as specified.
2.4 Other Components
Refer to Table 4 for details.
3
Methods Prepare the culture media and store them in a refrigerator (4 °C) except for antibiotic stocks which should be stored at −20 °C. Carry out the Agrobacterium streak culture in a laminar air-downflow cabinet and tissue culture in a laminar airflow cabinet to maintain sterile conditions.
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Table 2 Preparation of stock solutions and media for immature embryo transformation Stock solution (volume)
Component
Weight
Molarity
N6 [8] major 1 (1 L)
KNO3
141.5 g
1.40
N6 major 2 (1 L)
MgSO4.7H2O (NH4)2SO4
18.5 g 46.3 g
0.075 0.35
N6 major 3 (1 L)
KH2PO4
40 g
0.294
N6 major 4 (1 L)
CaCl2.2H2O
16.6 g
0.11
B5 [9] minor 1 (1 L)
FeSO4.7H2O Na2EDTA.2H2O
2.785 g 3.725 g
0.01 0.01
B5 minor 2 (1 L)
MnSO4.4H2O ZnSO4.7H2O H3BO3
1g 0.2 g 0.3 g
4.4 × 10−3 7.0 × 10−4 4.85 × 10−3
B5 minor 3 (1 L)
KI
0.075 g
4.5 × 10−4
B5 minor 4 (1 L)
CuSO4.5H2O
0.0025 g
1.0 × 10−5
Na2MoO4.2H2O
0.025 g
1.0 × 10−4
CoCl2.6H2O
0.0025 g
1.05 × 10−5
B5 vitamins (100 mL)
Thiamine HCl Pyridoxine HCl Nicotinic acid Myoinositol
200 mg 20 mg 20 mg 2,000 mg
6.0 × 10−4 1.0 x10−4 1.6 × 10−5 0.011
AA [10] macro salts (1 L)
CaCl2.2H2O MgSO4.7H2O NaH2PO4.2H2O KCl
1.5 g 2.49 g 1.7 g 29.5 g
0.01 0.01 0.01 0.4
AA micro salts (1 L)
CoCl2.6H2O CuSO4.5H2O H3BO3 KI MnSO4 Na2MoO4.2H2O ZnSO4.7H2O
25 mg 25 mg 3,000 mg 750 mg 8.9 g 250 mg 2,000 mg
1 × 10−4 1.0 × 10−4 0.0485 4.5 × 10−3 0.059 1.0 × 10−3 7.0 × 10−3
AA iron stock (1 L) Same as B5 minor 1
FeSO.7H2O
2.78 g
0.01
Na2EDTA.2H2O
3.73 g
0.01 (continued)
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Table 2 (continued) Stock solution (volume)
Component
Glycine (1 L) (filter sterilize and store at −20 °C)
Weight
Molarity
7.5 g
0.10
MS [11] 1 (1 L)
KNO3 NH4NO3
95 g 82.5 g
0.94 1.03
MS2 (1 L)
MgSO4.H2O MnSO4.4H2O ZnSO4.7H2O CuSO4.5H2O
37 g 2.23 g 0.86 g 0.0025 g
0.267 0.01 0.003 1.0 × 10−5
MS3 (1 L)
CaCl2.2H2O KI CoCl2.6H2O
44 g 0.083 g 0.0025 g
0.3 5 × 10−4 1.0 × 10−5
MS4 (1 L)
KH2PO4 H3BO3 Na2MoO4.2H2O
17 g 0.62 g 0.025 g
0.125 0.010 1.0 × 10−4
MS vitamins (100 mL)
Nicotinic acid Pyridoxine HCl Thiamine HCl Glycine Myoinositol
10 mg 10 mg 2 mg 40 mg 2,000 mg
8.1 × 10−5 4.9 × 10−5 6 × 10−6 5.33 × 10−4 0.011
A1. Stock solutions: Components are dissolved in MQ water and stored at 4 °C
3.1 Agrobacterium Culture
1. Three days before immature embryo (IE) transformation, streak Agrobacterium from glycerol stock onto AB plate (see Table 1 for preparation) with suitable antibiotics depending on the plasmid. 2. Incubate Agrobacterium culture in the dark at 28 °C for 2–3 days.
3.2 Panicle Harvesting and Immature Embryo Sterilization and Isolation
1. Harvest panicles at 8–12 days after anthesis (see Notes 6 and 7). Dehull developing seeds and put in 50 mL Falcon tubes. 2. Add sterilizing solution, and incubate the 50 mL tubes with constant shaking for 5 min. Rinse at least five times with plenty of sterile distilled water. 3. Isolate immature embryos (under a stereomicroscope, if needed).
5
B5 vitamins
10 10 10
MS3
MS4
10
A204
MS2
5
10
10
10
10
10
10
10
20
A203
20
1
5
10
10
10
10
10
10
10
20
A202
MS1
Glycine
10
10
B5 minor 4
AA iron stock
10
B5 minor 3
1
10
B5 minor 2
AA micro salts stock:
10
B5 [8] minor 1
100
10
N6 major 4
AA macro salts stock
10
N6 major 3
1
10
N6 major 2
A201
20
A200
Amount of stock to take per liter of medium (mL)
N6 major 1
Stock solution
Table 3 Media composition and amount of stock requirement for rice transformation media preparation
10
10
10
20
10
A205
10
10
10
20
10
MS0
266 Inez H. Slamet-Loedin et al.
−3
5.2
Acetosyringone
See Note 2
19.62 mg dissolved in 1 mL DMSO
100 μM (add before use)
5,500
See Note 1
Microwave medium to melt Gelrite
Gelrite
Agarose type 1
pH
Adjust volume to 1 liter
5.2
5,000
5.8
20,000 (0.058)
Maltose
Sorbitol
36,000 (0.197)
Mannitol
10,000 (0.056)
10,000 (0.056)
500
D-glucose
−3
20,000 (0.058)
500
200,000 (0.058)
500
Casamino acid
1.0 × 10−3
300
Sucrose
174 (0.001)
Arginine
2.0 × 10
−3
500 (4.3 × 10 ) 500 (4.3 × 10 )
260 (0.002)
Aspartic acid
6 × 10−3
L-proline
876 (0.006)
5,000
5.8
20,000 (0.058)
36,000 (0.197)
500 (4.3 × 10−3)
500
300
Amount of chemical in mg (mol) to add per liter of medium
L-glutamine
Chemical
MS vitamins
10,000
5.8
20,000 (0.11)
30,000 (0.088)
5
3,000
5.8
30,000 (0.088)
5
(continued)
See Note 3
Dispense in 20 mL/tube
2,000
5.8
3,000 (0.088)
5
Agrobacterium Transformation of Indica Rice 267
1
BAP
Dispense 25 mL in 100 × 15 petri dish to solidify
1
Carbenicillin
Hygromycin
1
Cefotaxime
Antibiotic
0.2
1
1
Amount to add per liter of medium (mL)—see Note 5
1
NAA
Kinetin
2
Amount to add per liter of medium (mL)—see Note 4
2,4-D
Growth regulator
(continued)
Table 3
1
1
2
5
1
1
2
1
Dispense 35 mL in 100 × 20 petri dish to solidify
0.6
1
1
0.2
1
1
268 Inez H. Slamet-Loedin et al.
Agrobacterium Transformation of Indica Rice
269
Table 4 Other media composition and amount of stock requirement for rice transformation media preparation Weight (molar Stock solution (vol) concentration)
Preparation
Storage temperature
Growth regulators BAP (10 mL)
10 mg (4.44 × 10−5)
Dissolve in 1N NaOH (0.2 mL per 10 mg BAP) and complete to volume with MQ water; filter sterilize and aliquot in Eppendorf tubes
4 °C
2,4-D (50 mL)
50 mg (2.26 × 10−4)
Dissolve in 1 M KOH (2 mL per 50 mL stock) and complete volume with MQ water; filter sterilize and aliquot in Eppendorf tubes
4 °C
Kinetin (25 mL)
25 mg (1.16 × 10−4)
Dissolve in 1N NaOH and complete volume with MQ water; filter sterilize and aliquot in Eppendorf tubes
−20 °C
NAA (20 mL)
20 mg (1.074 × 10−4)
Dissolve in 1N NaOH and complete volume with MQ water; filter sterilize and aliquot in Eppendorf tubes
4 °C
Carbenicillin (10 mL)1,000 mg
Dissolve in MQ water, filter sterilize and aliquot in sterile Eppendorf tubes
−20 °C
Cefotaxime sodium (50 mL)
5,000 mg
Dissolve in MQ water, filter sterilize and aliquot in sterile Eppendorf tubes
−20 °C
Hygromycin B
Ordered and used directly as 50 mg/L stock
Aliquot in sterile Eppendorf tubes
4 °C
Antibiotics
3.3 Immature Embryo Transformation: Cocultivation
1. 1 h prior to infection of IEs, take about one full loop (3 mm loop size) of Agrobacterium culture from AB plate and suspend in A200 medium contained in a Falcon tube. 2. Pipet or invert the tube gently several times for even mixing. Adjust bacterial density to 3 × 109 cfu/mL (OD 0.3). Incubate the suspension in the dark at 25 °C for 1 h (incubator) prior to infecting the IEs. 3. Place the IEs with the scutellum side up onto A201 medium. Drop 5 μL of Agrobacterium suspension to each IE. Incubate cultures in the dark at 25 °C for 7 days. Place 50 IEs per plate.
3.4 Resting Culture and Selection
1. After cocultivation period, place IEs on a sterile filter paper and remove elongated shoots with a scalpel.
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Inez H. Slamet-Loedin et al.
2. Quick-dry the IEs several times by gently pressing these between two layers of sterile filter papers (see Note 8). 3. Transfer IEs with the scutellum side up onto A202 resting medium. Place 16 IEs per petri dish. Incubate under continuous light at 30 °C for 5 days. 4. Transfer IEs onto A203 selection medium and incubate under continuous light at 30 °C for 10 days; alternatively, each embryo may be cut into four pieces or more (first selection). Place eight to ten IEs per plate. 5. Transfer IEs onto fresh A203 medium and incubate under continuous light at 30 °C for 10 days (second selection). 6. Separate the embryogenic callus from the black callus tissue, and transfer the embryogenic calli to fresh A203 medium. Incubate under continuous light at 30 °C for 10 days (third selection). 3.5 Plant Regeneration
1. Transfer resistant calli to A204 pre-regeneration medium at 6–8 callus lines per petri dish. Incubate under continuous light at 30 °C for 10 days. 2. Select proliferating calli with green spots and transfer to A205 regeneration medium. Place four callus lines per petri dish. Label each regenerable callus line. 3. Carefully select one plantlet from each callus line and inoculate into a tube containing MS0 rooting medium. Barcode-label the plant ID on each test tube. Keep the plantlets under continuous light at 25 °C for 14 days. Keep original petri dishes with plantlets as backup. 4. If a callus line has not regenerated plants but has small shoots, transfer these to a fresh A205 medium to allow the shoots to grow into plantlets. Incubate under continuous light at 30 °C for 14 days, and once plantlets have developed, transfer to MS0 for a better rooting for 1–2 weeks.
4
Notes 1. A200 is filter sterilized and stored at 4 °C; add acetosyringone just before use for transformation. 2. For the rest of the media in solid form (A201–A205), add the gelling agent prior to autoclaving at 115 °C for 15 min. 3. For A201, add acetosyringone to the autoclaved medium and maintain at 55 °C prior to dispensing. 4. Likewise, add growth regulators and antibiotics to media and cool to 55 °C.
Agrobacterium Transformation of Indica Rice
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5. Dispense solid media in appropriate size of petri dishes and store at 4 °C. 6. Embryo size at about 1.3–1.8 mm is crucial; a very-small-size embryo will not survive. 7. Screenhouse-grown material will be a better option for less occurrence of microbial contamination; however, a healthy field-grown material has been shown to also result in a good transformation frequency and manageable contamination. 8. Properly dry the embryos to avoid bacterial overgrowth, but do not damage the embryo and ensure a quick process to avoid drying.
Acknowledgement Establishment of the protocol was supported by USAID grant number DPPC2004-17. Technical support of F. Montecillo and facilitation of P. Herve and G. Barry are acknowledged. References 1. Tyagi AK, Mohanty A (2000) Rice transformation for crop improvement and functional genomics. Plant Sci 158:1–18 2. Iyer LM, Kumplata SP, Chandrasekharan MB et al (2000) Transgene silencing in monocots. Plant Mol Biol 43:323–346 3. Hiei Y, Ohta S, Komari T et al (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–2824 4. Hiei Y, Komari T (2006) Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 85:271–283 5. Hiei Y, Komari T (2008) Agrobacteriummediated transformation of rice using immature embryos or calli induced from mature seed. Nat Protoc 3:824–834 6. Gamuyao R, Joong Hyoun Chin JH, PariascaTanaka J et al (2012) The protein kinase Pstol1
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