9.4 Agrobacterium-mediated transformation

 

Kari Tompson, Kaori Miyawaki, Tetstuya Kurata,

and Mitsuyasu Hasebe

 

The PEG-mediated protoplast transformation is usually intended to target a specific locus via homologous recombination. This method is used to introduce a single kind of foreign DNA fragment containing homologous DNA sequences to the host DNA. When more than one kind of foreign DNA fragments, part of which contain the same nucleotide sequences such as a promoter or a terminator of a drug resistant marker gene, are mixed and introduced, different DNA fragments are often subjected to intermolecular recombination to form chimeric DNA fragments. If DNA fragments do not have the same nucleotide sequences each other, each DNA fragment is safely targeted to each homologous site, although the efficiency of double insertion is not high.

On the other hand, foreign DNA is inserted in one or more than one non-homologous sites with the agrobacterium-mediated transformation. The number of insertions varies from one to numerous, but on average in the following protocol, three to several insertions were observed. Insertion of a DNA fragment in non-homologous site often causes effects to the phenotype and we do not use the agrobacterium-mediated transformation to analyze gene functions, such as the chemical induction of mRNA or amiRNA. For the purpose, we established a relatively neutral site for targeting and always insert a foreign DNA fragment to the site using the PEG-mediated method. However, the disadvantage of multiple insertions by agro-transformation turns useful for efficient screening of genes with specific functions. A good point is that agrobacterium transformation does not appear to provide any chimeric insertions via intermolecular recombination. We have screened transcription factors necessary for the reprogramming of leaf cells to chloronema apical cells. A deletion mutant of a single transcription factor usually does not show any changes from the wild type likely because of its functional redundancy to other closely related genes. Therefore, we employed to introduce a cDNA fused with a nucleotide sequence encoding a transcriptional repression domain called SRDX. Even with this strategy, the induction of more than 90% of the fused cDNA does not show any change of the phenotype. For more efficient screening, we employed agrobacterium-mediated transformation to introduce several kinds of cDNA-SRDX fragments at the same time. Five different cDNA-SRDX DNA fragments were mixed and introduced to Physcomitrella. When we find any differences of phenotype from the wild type, we then introduced one kind of DNA fragment from the five kinds one by one to identify which cDNA-SRDX is responsible for the phenotype change.

 

Useful tips:

 

Vector

We found that longer vector shows higher transformation rates to Physcomitrella. The traditional binary vector pBI121-Hm with larger size is more effective than modern compact vectors including pGREEN.

 

Transformation efficiency of protonemata and protoplasts

Protonema transformation can yield up to 37 positive lines in the first selection and then 25 lines remained in the second selection in a co-culture experiment using the pBI121-Hm vector.

 

Protoplast transformation can yield up to 800 positive lines in the first selection and 200 lines remained in the second selection in a co-culture experiment when we used pBIFox-Dual-Gate/SRDX. However, the efficiency varies and we sometimes got only 45 positive lines in the second screening while 755 positive lines in the first screening. The larger colonies found in the first selection usually have the greatest stability in the second selection.

 

So, based on the above I would say that in best cases Protoplasts can yield about double the lines that protonema can.  However, we've used protoplasts for transformation more than the protonema so the procedure for protoplasts is optimized for the vector we are using.  It might be possible to adjust the conditions for protonema transformation to produce equally high efficiencies and potentially more stability of transformants.

 

Agrobacterium strain and a vector:

 

Agrobacterium Strain: C58 GV2260

Binary Vector: pBIFox-Dual-Gate/SRDX

 

Vector Construction:

 

*This vector contains a stop codon immediately following the SRDX sequence.

 

Clone the target gene ORF sequence into the pENTR/D-TOPO vector (Invitrogen).　 After a positive clone is confirmed, digest the pENTR plasmid with AsiSI enzyme to reduce the number of pENTR clones growing on the destination vector plate.　 TOPO clone the ORF into the destination vector using LR Clonase II (Invitrogen) and grow on LB containing 100 ug/mL Hygromycin B and 50 ug/mL Kanamycin Sulfate for 20 hours at 37°C.　

 

Agrobacterium transformation:

 

  1. Thaw the frozen the agrobacterium strain (C58[pGV 2260]) on ice.
  2. Add 1 mL of 0.5 mg/mL plasmid to 100 mL of competent cells.
  3. Mix gently by tapping and keep on ice for 1 minute.
  4. Transfer 100 mL of the mixture to a 2mm gap electroporation cuvette.
  5. Electroporate at 2500 volts.　
  6. Put the cuvette back on ice and add 700 mL of SOC.　 Mix gently by pipetting.
  7. Place the cuvettes at 30°C for 1 hour.　
  8. Spread 200 mL on a LB/Kanamycin plate.　
  9. Incubate the plates at 30°C for 2 days.

 

1. Transformation of protoplasts

 

Day 1: Extract Protoplasts

 

Materials:

 

Autoclave　　　　　　　　　　　　　　　　　　　　　 Non-Autoclave

 

8% Mannitol　　　　　　　　　　　　　　　　　　　 3 mL sterile plastic pipette

Forceps (rounded end)　　　　　　　　　　　 Driselase/mannitol solution

108 mm mesh　　　　　　　　　　　　　　　　　　 Centrifuge, bucket-type rotor

Funnel　　　　　　　　　　　　　　　　　　　　　　　　 Hemacytometer slide

PRM-L　　　　　　　　　　　 　　　　　　　　　　　 Microscope

10- 200 mL Flasks (or other size)　　 Counter clicker

2- 50mL round bottom glass tubes　　 0.45 mm filter and syringe (for 30 mL), sterile

　　　　　　　　　　　　　　　　　　　　　　　　　　　　　 Parafilm, sterilized by EtOH

　　　　　　　　　　　

(Tip: Put a piece of the mesh in the funnel and wet it with Milli-Q water.　 Place the funnel with the mesh into one of the 50 mL round bottom glass tubes and autoclave together.　 This reduces the risk of contamination.

 

Protocol:

 

Start with 8-10 plates of 5-7 day protonema grown on BCDAT with cellophane.　 (The moss should be relatively well-grown and covering most of the plate.)　 It is important to use it before it produces gametophores or turns brown.　

 

  1. Dissolve 500 mg of driselase in 25 mL of 8% mannitol.　 Invert the tube gently using a rotator at a low setting to mix for 15 min.　 Centrifuge at 2500 rpm for 5 min and then filter sterilize into 50 mL round bottom glass tube with a 0.45 mm filter in the clean bench.
  2. Scrape the protonema tissue gently from the cellophane with forceps and suspend it in the 50 mL tube containing the sterilized Driselase/mannitol solution.
  3. Wrap the tube with foil, lay it on its side and keep it at room temperature (between 22-25°C).　 Every 5 minutes turn the tube 5x in the aluminum foil for a total of 30 min.　 Be very careful at this step because the protoplasts are very delicate.　
  4. To remove the protonema, gently pour the protoplast solution through the funnel containing 108 mm mesh into another 50 mL glass round bottom tube.　
  5. Centrifuge the 50 mL tube at 800 rpm for 4 min.　 Be careful that the centrifuge does not stop during the 4 minutes because it is important for proper packing of the protoplasts at the bottom of the tube.　
  6. Carefully pipette away the supernatant leaving a little in the bottom of the tube to gently swirl and re-suspend the pellet. Be careful not to leave clumps, but also be gentle because the protoplasts are very delicate.　 Add 40 mL of 8% mannitol.
  7. Centrifuge the protoplasts again at 800 rpm for 4 min.　
  8. Carefully pipette away the supernatant leaving a little in the bottom of the tube to gently re-suspend the pellet.　 Add 40 mL of 8% mannitol.
  9. Centrifuge again at 800 rpm for 4 min.
  10. Repeat steps 8 and 9 once more.　 Remove the supernatant leaving a little in the bottom to re-suspend the pellet.　 Add 10 mL PRM-L to the protoplast suspension.　
  11. Use a 3 mL sterile pipette to carefully transfer some protoplast solution to a Hemacytometer slide for determining the protoplast density.　
  12. Count the number of protoplasts inside the large squares made by the lines of the two boxes and average them.　 This is the number of cells per mL x 104.　 So if the average were 50 then there would be 50 x 104 cells/mL.　 Multiply that number of cells by the volume they are suspended in and get the total number of cells.　 So 50 x 104 suspended in 10 mL means that there are 5 x 106 cells in that sample total.
  13. Add more PRM-L to the solution until the protoplasts are at a density of 0.5x106/10mL.　
  14. Pipette 10 mL of protoplast solution to each of 10 sterile 200 mL Erlenmeyer flasks.　
  15. Gently parafilm the flasks and keep them in the dark for two days at 25°C.

 

Day 2: Incubating.

 

Day 3: Incubating.

 

Day 4: Begin co-culture.

 

Materials:

 

One overnight culture of agrobacteria grown at 30°C with 180 rpm shaking.　 The agrobacteria is grown in LB broth supplemented with appropriate antibiotics.　

200 mM Acetosyringone

PRM-L, autoclaved

Milli-wrap, autoclaved

 

Acetosyringone: Wako 320-29611

 

Protocol:

 

  1. Centrifuge the agrobacteria culture at 3,000 rpm for 7 min at room temperature and pour off the LB media.
  2. Re-suspend the agrobacteria in 5 mL of PRM-L.
  3. Centrifuge the agrobacterium culture at 3,000 rpm for 7 min at room temperature, pour off the PRM-L.
  4. Re-suspend in 5 mL of PRM-L.　
  5. Measure the OD600 of the culture re-suspended in PRM-L.　
  6. Add the agrobacteria as follows:

 

0.1 to 2 x 100/(OD600 x 1.8) mL for every 0.5 x 106 cells

 

The optimum amount to add greatly depends on the agro strain and plasmid. It is highly recommended that you optimize the amount of agrobacteria that you add in your own laboratory.

 

In our lab pBIFox-Dual-Gate/SRDX added at 0.1x produces the most transformants in the 1st selection.

 

  1. Add agrobacteria and 10mL of 200 mM Acetosyringone to each flask containing 10 mL of protoplast solution and gently swirl the flask.　 (Finally 200 mM Acetosyringone.)　
  2. Seal the flasks with Milliwrap.
  3. Keep the flasks in the dark at 25°C.
  4. Leave the flasks there for 2 days.　 If gently stir once or twice a day it may increase the efficiency.　

 

Days 5 and 6: Incubating.

 

Day 7: Washing and plating.

 

Materials:

 

10 mL Falcon Tubes (50 mL)

Centrifuge with a bucket rotor

8% Mannitol supplemented with 100 mg/mL Claforans

PRM-T with 250 ug/mL Claforans and 50 ug/mL Augmentin　

PRM-B Plates with cellophane

 

Protocol:

 

  1. Pour each culture into a 50 mL tube.　
  2. Centrifuge the protoplasts at 1000 rpm for 1 min. (It might be better and possible to use a lower speed because then less agrobacteria will go into the protoplast pellet.)
  3. Remove as much of the supernatant as possible and add 10mL of 8% mannitol supplemented with 100 ug/mL Claforans.　 Gently suspend and wash the protoplasts by pipetting repeatedly with a 10 mL pipette.
  4. Centrifuge the protoplasts at 1000 rpm for 1 min.　
  5. Remove as much of the supernatant as possible and add another 10 mL of 8% mannitol/Claforans solution.　 Gently suspend and wash the protoplasts by pipetting.
  6. Centrifuge the protoplasts at 1000 rpm for 1 min.
  7. Repeat washing steps up to 10x or more.
  8. Remove as much of the supernatant as possible and mix the protoplasts in 4 mL of 45°C PRM-T + 250 mg/mL Claforans and 50 ug/mL Augmentin.　 Spread 2 mL on each of 2 PRM-B plates with cellophane for each co-culture.　 (Usually each co-culture is mixed with 4 mL and spread on 2 plates, 2 mL each.　 If you have high transformation efficiency you may need to increase the amount of PRM-T and plate number so you spread the positives out more.)

 

 

4 Days after Plating the Co-culture

 

Transfer the cellophane with the transformants onto BCDAT selection plates supplemented with 100 ug/mL Claforans, 50 ug/mL Augmentin and proper antibiotic.

 

After another two weeks transfer positive lines to relaxed plates containing only 100 ug/mL Claforans and 50 ug/mL Augmentin.　

 

After about 2 weeks transfer a piece of colony from relaxed plate to second selection plate.　 Keep both sets of plates.　 Screen colonies grown on the relaxed plates that were positive on the 2nd selection plate.　

 

Make a 3rd selection plate and transfer to 4°C for storage after about 2-3 weeks.　
Solutions

 

8% Mannitol

 

40 g D(-) Mannitol

Bring up to 500 mL w/H2O

 

Driselase/Mannitol

 

500 mg Driselase

25 mL of 8% Mannitol

 

PRM-L (From Dr. Yuji Hiwatashi’s PEG-Mediated Transformation Protocol)

Stock A

1mL

2mL

3mL

Stock B

1mL

2mL

3mL

Stock C

100mL

200mL

300mL

5g/L Ammonium tartrate

1mL

2mL

2mL

Mannitol

6.6 grams

13.2 grams

19.8 grams

Glucose

0.5 grams

1.0 gram

1.5 grams

H2O

Up to 100 mL

Up to 200 mL

Up to 300 mL

Autoclave

 

PRM-B (From Dr. Yuji Hiwatashi’s PEG-Mediated Transformation Protocol)

Stock B

1.5mL

3mL

6mL

Stock C

1.5mL

3mL

6mL

Stock D

1.5mL

3mL

6mL

Alternative TES

150mL

300mL

600mL

500 mM Ammonium tartrate

1.5mL

3mL

6mL

Mannitol

9 grams

18 grams

36 grams

CaCl2 2H2O

0.22 grams

0.441 grams

0.882 grams

Nacalai Tesque Agar: cat. no. 01028-85

1.2 grams

2.4 grams

4.8 grams

H2O

Up to 150 mL

Up to 300 mL

Up to 600 mL

Autoclave

 

PRM-T (From Dr. Yuji Hiwatashi’s PEG-Mediated Transformation Protocol)

Stock B

0.5mL

1mL

Stock C

0.5mL

1mL

Stock D

0.5mL

1mL

Alternative TES

50 mL

100 mL

500 mM Ammonium tartrate

0.5mL

1 mL

Mannitol

4 grams

8 grams

CaCl2 2H2O

0.0725 grams

0.145 grams

Sigma Agar (A6924: Low Melt Point)

0.4 grams

0.8 grams

H2O

Up to 50 mL

Up to 100 mL

Autoclave

Keep at 45°C until ready to use.

DO NOT FORGET TO ADD THE CLAFORANS AND AUGMENTIN!!!

 

 

Selection Plates

 

BCDAT supplemented with appropriate antibiotics.　

 

Stock B

3.0 mL

Stock C

3.0 mL

Stock D

3.0 mL

Alternative TES

300 mL

500 mM Ammonium Tartrate

3.0 mL

50 mM CaCl2 · 2H2O

6.0 mL

Agar (final 0.8%)

2.4 g

 

Bring up to 300 mL with Milli-Q

 

Autoclave

Antibiotic

Depends on your Antibiotic

Claforans 100 mg/mL (Sanofi-aventis)

300 mL (Finally 100 mg/mL.)

Augmentin 25 mg/mL (GlaxoSmithKline)

600 µL (Finally 50 ug/mL.)

Autoclave

 

For the antibiotics G418 (GIBCO) and Hygromycin (Invitrogen), they are used at a final concentration of 20 mg/mL.

 

 

 

2. Transformation of chloronemata

 

Culture and Co-Culture

 

1. Pick an Agrobacterium colony from the LB/Kanamycin plate

2. Grow the colony up in 5 mL LB/Kanamycin at 180 rpm.　 Grow for 24 hours (or longer depending on the growth rate).　

3. Centrifuge the culture at 3,000 rpm for 7 min at room temperature.

4. Go to the clean bench and pour off the supernatant.

5. Wash by adding 5 mL of BCDAT + 5% Glucose and re-suspend by vortexing.　

6. Centrifuge at 3,000 rpm for 7 min at room temperature.

7. 　 Prepare the co-culture media.　 Mix 50 mL of BCDAT + 5% Glucose with 50 mL 200 mM Acetosyringone.　 Final concentration of Acetosyringone is 200 mM.

8.　 Pour off the supernatant.　 Add 2 mL of BCDAT + 5% Glucose supplemented with Acetosyringone.　

9. Grow at 30°C for 2 hours with shaking at 180 rpm.

10. Check the OD600 of the bacteria cultures.

11. Calculate the amount of co-culture media to add to get 10 mL of OD600 = 0.1 agrobacteria in the final co-culture.　

12. Pipette the co-culture media into a 9 cm Petri dish.　 Swirl to cover the bottom.

13. Scrape protonema from 2 plates of about 3 or 4 days old moss protonema (homogenized moss OD600 = 0.1 when first made) and swirl it into the co-culture media in the Petri dish.

14. Pipette the appropriate amount of agrobacterium culture for a final OD600 = 0.1 into the Petri dish and mix by swirling, the total volume should now be 10 mL.　　

15. Seal the plates with two strips of parafilm to prevent contamination and leakage.

16. Place the plates in the 25°C growth chamber under continuous light for 2 days.

 

Solutions Needed

 

Co-culture Solution

 

BCDAT + 5% Glucose

 

Stock B

3.0 mL

Stock C

3.0 mL

Stock D

3.0 mL

Alternative TES

300 mL

500 mM Ammonium Tartrate

3.0 mL

50 mM CaCl2 · 2H2O

6.0 mL

Glucose

15 g

 

Bring up to 300 mL with Milli-Q

 

Selection Media 9 cm plates

 

BCDAT media supplemented with Claforans, Augmentin, and appropriate antibiotic.

 

Stock B

3.0 mL

Stock C

3.0 mL

Stock D

3.0 mL

Alternative TES

300 mL

500 mM Ammonium Tartrate

3.0 mL

50 mM CaCl2 · 2H2O

6.0 mL

Agar (final 0.8%) (Nacalai Tesque: cat. no. 01028-85)

2.4 g

 

Bring up to 300 mL with Milli-Q

 

Autoclave

Antibiotic for specific selection

Appropriate amount

Claforans 100 mg/mL

300 mL (Final 100 mg/mL)

Augmentin 25 mg/mL

600 µL (Final 50 ug/mL)

 

Acetosyringone (Wako 320-29611)

 

Kept in a 1M stock in DMSO at -20 to -30°C.　　 Working stocks are further diluted with DMSO.

 

Claforans (Sanofi-aventis [Paris, France], 1 g x 10 vials of powder [in NIBB, provided by Rikaken])

 

Kept in a working stock with concentration 100 mg/mL stock in water (filter sterilized) at -20 to -30°C.

 

Augmentin (GlaxoSmithKline, 100 pills [10 pills/1 sheet, includes 10 sheets], The package says Augmentin Tablets 125,  more specifically the tablets contain 62.5 mg of Clavulanic Acid Potassium and 125 mg of Amoxicillin)

 

Must be made fresh as it is unstable for long periods in water.　 Kept in a 25 mg/mL stock in water (filter sterilized).　 Maybe OK to keep at -20 to -30°C for 1-2 days, but we make it fresh each time.　
Washing and Plating

 

The washing step is very important because it washes away the excess agrobacteria.　 If agrobacteria are left with the moss then it will overgrow and kill the moss transformants.

 

  1. Add Claforans to the BCDAT washing media to a final concentration of 100 ug/mL.　
  2. Carefully place autoclaved funnels containing f 48 nylon mesh into 50 mL tubes.　
  3. Pour the protonema from the 9 cm Petri dish onto the mesh to filter out the excess media.　 If there is remaining protonema in the dish it can be rinsed with wash buffer or scraped off with forceps and put onto the mesh.　
  4. Use forceps to transfer the mesh to the top of a stack of 5-6 pieces of autoclaved blotting paper to soak away excess co-culture media.
  5. Carefully scrape the protonema from the surface of the mesh and suspend it in 10 mL of washing media (BCDAT) in a 50 mL tube.
  6. Mix the protonema gently in the media by pipetting with a 10 mL pipette.　 (This is important to remove agrobacteria from the surfaces of the moss.)　
  7. Centrifuge for about 1 min at no more than 1,000 rpm.　
  8. Pipette off as much as the supernatant as possible and add another 10 mL of washing media.
  9. Gently mix by pipetting and centrifuge again.　 (Repeat steps 8 – 10 ten times.)
  10. Finally suspend the moss protonema in 10 mL of washing media and plate on 3 selection plates.
  11. Pipette off the extra washing media as much as possible and try to spread evenly on the surface of the plate.　
  12. Seal the plates 1/4 surgical tape and 3/4 parafilm.　 Keep for about 2 weeks to identify positive transformant lines.　
  13. To eliminate transient transformant lines, transfer the resistant colonies to relaxed plates containing only Claforans and Augmentin (no specific antibiotic that was used for transformant selection) and keep for about 1-2 weeks.　
  14. Transfer a small piece of each line from the relaxed plates to selection plates.　 Moss lines growing well on these 2nd selection plates are positive lines!

 

Note:　 If the tissue is very small and it is difficult to pipette away the excess washing media then just spread on more plates to even it out and reduce the extra moisture of the washing media.　

 

Washing Media

 

BCDAT

 

Stock B

5.0 mL

Stock C

5.0 mL

Stock D

5.0 mL

Alternative TES

500 mL

500 mM Ammonium Tartrate

5.0 mL

50 mM CaCl2 · 2H2O

10.0 mL

 

Bring up to 500 mL with Milli-Q

 

Don’t forget to add 500 mL of 100 mg/mL Claforans just before using.