Genetic transformation

Agrobacterium-mediated Tragopogon Transformation

Protocol

Agrobacterium Culture

  • Streak the Agrobacterium tumefaciens onto a LB + Kan + Rif plate. Grow at 28 °C for 2 days.
    • pCambia 1300 vector has Kan- and Hyg-resistance genes; Agrobacterium stain EHA105 has Rif-resistance gene
    • To knock out the PDS gene, bacteria stock 093 (1300-AtCas9-GmUbi-GFP-AtU6-1-gTraPDS2-AtU6-29-gTraPDS1) will be used
  • Agrobacterium colonies are isolated and inoculated into 20 ml liquid Ty medium with AS and appropriate antibiotics (in an autoclaved flask). Shake (dark condition) at 100 rpm, 28 °C for ~24 h.
    • Avoid large lumps of bacteria; brush the bacteria against the inside surface of the flask
    • The streaked Agrobacterium plate can be used for 1-2 weeks, but no longer than that (e.g., 1 month)

 

Infection Medium Preparation

  • Resuspend Agrobacterium pellet in liquid AAM infection medium (containing AS).
  • Add 1 ml Agrobacterium culture to a 1.5 ml microcentrifuge tube
  • Centrifuge at 2,000 rpm for 10 min
  • Remove the Ty medium, and re-suspend the Agrobacterium pellet with 1 ml AAM medium
  • Adjust the Agrobacterium cell density to OD600 = 0.1 (blank: AAM + AS without Agrobacterium)

Explant Preparation

  • Place wounded leaf segments (i.e., explants; Figure 1, step 1) on M5 medium in dark for 1-2 days at room temperature.
    • Wounded-side down; shallow wounds act as sites for Agrobacterium infection

 Explant Infection

  • Immerse explants in a Petri dish containing Agrobacterium suspension culture for 30 min at room temperature.
    • Transfer ~20 ml AAM infection medium (containing Agrobacterium) into 100 ml X 15 ml Petri dish
    • Swirl the Agrobacterium suspension culture (with the explants) every 10 min
    • Blot explants completely on a sterile filter paper; this step usually takes ~20 min
  • Transfer infected explants to Cocultivation medium covered with autoclaved filter paper (preventing rampant growth of Agrobacterium, and therefore, avoid tissue necrosis/browning) and put the plates in dark for 2-3 days at room temperature (Figure 1, step 2).

 Callus induction, shooting and rooting are carried out at 25 °C with 14 h photoperiod in an incubator

Callus induction

  • Transfer the explants to Selective M5 medium for callus induction; it usually takes 3 weeks (Figure, step 3).

Shooting

  • Transfer the callus to Selective COCO-1 medium for 2 weeks for shoot induction.
  • Transfer the callus to Selective WPM medium for shoot formation; shoots will start forming after 2 weeks (Figure 1, step 4).

Rooting

  • Excise regenerated shoots from the callus and transfer the shoots to COCO-R medium (Figure 1, step 5).
  • Transfer the rooted shoots to soil.
    • Transfer rooted shoots to soil inside a tray with a lid on. Put the tray in the growth chamber for 2 weeks. Repot the plants and put the pots inside the greenhouse (Figure 1, step 6)

Timeline

Day

Agrobacterium preparation

Explant preparation

1

Streak the stock on plate in the morning; take ~2 days to grow

 

2

 

Place leaf segments on M5 medium in dark for 1-2 days at RT

3

Pick colonies and shake in Ty medium for 24 hours

 

4

Prepare the AAM with Agrobacterium and transform the explants; cocultivation of explants and Agrobacterium in dark for 2-3 days at RT

5

 

6

Transfer explants to selective M5 medium

27

Transfer callus to selective COCO-1 medium

41

Transfer callus to selective WPM medium for shooting

62

Transfer shoots to COCO-R medium for rooting

83

Move regenerated seedlings to soil

 

Abbreviations

Abbreviation

Description

AS

Acetosyringone (乙酰丁香酮): a signal (phenolic compound) exuded by wounded eudicot plant cells and involved in plant-pathogen recognition; AS is recognized by a receptor encoded by the virA gene, which facilitates the processing and transfer of T-DNA from Agrobacterium to the nuclear genome of the plant host; AS, a vir gene inducer, allows higher transformation efficiency

Cefo

Cefotaxime (头孢噻肟): often used in transformation of plant species for the elimination of Agrobacterium tumefaciens

Hyg

Hygromycin B (潮霉素 B): an antibiotic produced by Streptomyces hygroscopicus; active against both prokaryotic and eukaryotic cells by inhibiting polypeptide synthesis

Kan

Kanamycin (卡纳霉素): works by interfering with protein synthesis – it binds to the 30S subunit of the bacterial ribosome

LB

Lysogeny broth

Rif

Rifampicin (利福平): an antibiotic works by decreasing the production of RNA by bacteria

Tim

Timentin (特美汀): used most commonly in the regeneration medium for elimination of the Agrobacterium post-transformation

Medium

 

Ty medium (liquid)

 

For 1 L

For 200 ml

Tryptone

5 g

1 g

Yeast extract

3 g

0.6 g

pH 5.5 (use 1 M HCl)

Before use add:

·      Rif (1,000 X; 50 mg/ml); final [c] = 50 mg/L

·      Kan (1,000 X; 50 mg/ml); final [c] = 50 mg/L

·      AS (1,000 X; 100 mM); final [c] = 0.1 mM

 

AAM infection medium (liquid)

 

For 1 L

For 500 ml

AA macro (10 X)

100 ml

50 ml

AA micro (1,000 X)

1 ml

0.5 ml

AA amino acid (100 X)

10 ml

5 ml

MS vitamins (100 X)

10 ml

5 ml

Fe2EDTA (100 X)

10 ml

5 ml

Casamino acids

0.5 g

0.25 g

Sucrose

68.5 g

34.25 g

Glucose (0.5 g/ml)

72 ml

36 ml

pH:

·      5.33 before autoclaving (using KOH); add filter-sterilized glucose; the final pH 5.15 (should be around 5.2)

Before use add:

·      AS (1,000 X; 100 mM); final [c] = 0.1 mM

 

M5 medium

 

For 1 L

For 400 ml

MS salt

4.44 g

1.776 g

Sucrose

30 g

12 g

BAP (1 mg/ml)

1 mg

0.4 mg

NAA (1 mg/ml)

0.5 mg

0.2 mg

Gelrite

5 g

2 g

pH: set to 5.8

 

Cocultivation medium

 

For 1 L

For 400 ml

MS salt

4.44 g

1.776 g

Sucrose

30 g

12 g

BAP (1 mg/ml)

1 mg

0.4 mg

NAA (1 mg/ml)

0.5 mg

0.2 mg

Gelrite

5 g

2 g

pH: set to 5.35; will reach 5.2 (the desired pH) after autoclaving

Add AS (1,000 X; 100 mM) after autoclaving; final [c] = 0.1 mM

 

Selective M5 medium

 

For 1 L

For 400 ml

MS salt

4.44 g

1.776 g

Sucrose

30 g

12 g

BAP (1 mg/ml)

1 mg

0.4 mg

NAA (1 mg/ml)

0.5 mg

0.2 mg

Gelrite

5 g

2 g

pH: set to 5.8

After autoclaving, add:

·      Tim (500 X; 200 mg/ml); final [c] = 300 mg/L

·      Hyg (50 mg/ml stock)

o   15 mg/L Hyg for T. porrifolius; add 300 µl to 1 L medium

 

Selective COCO-1 medium

 

For 1 L

For 400 ml

Woody Plant Medium

2.41 g

0.964 g

Sucrose

30 g

12 g

BAP (1 mg/ml)

2.5 mg

1 mg

Agargellan

8.5 g

3.4 g

Volume

950 ml (+ 50 ml coco)

380 ml (+ 20 ml coco)

pH: set to 5.7

After autoclaving, add:

·      filter-sterilized coconut water (final 5%)

·      Tim (500 X; 200 mg/ml); final [c] = 300 mg/L

·      Hyg (50 mg/ml stock)

o   15 mg/L Hyg for T. porrifolius; add 300 µl to 1 L medium

 

Selective WPM medium

 

For 1 L

For 400 ml

Woody Plant Medium

2.41 g

0.964 g

Sucrose

30 g

12 g

Agargellan

5 g

2 g

pH: set to 5.7

After autoclaving, add:

·      Tim (500 X; 200 mg/ml); final [c] = 300 mg/L

·      Hyg (50 mg/ml stock)

o   15 mg/L Hyg for T. porrifolius; add 300 µl to 1 L medium

 

COCO-R medium

 

For 1 L

For 400 ml

Woody Plant Medium

2.41 g

0.964 g

Sucrose

30 g

12 g

IBA (1 mg/ml)

1.5 mg

0.6 mg

Agargellan

8.5 g

3.4 g

pH: set to 5.7

 

AA Macro 10X solution

 

For 1 L

For 500 ml

KCl

29.5 g

14.75 g

MgSO4·7H2O

2.5 g

1.25 g

CaCl2·2H2O (or CaCl2)

1.5 g (or 1.13 g)

0.75 g (or 0.565)

NaH2PO4·H2O

1.5 g

0.75 g

Store at 4 °C

 

AA Micro 1000X solution

 

For 1 L

For 500 ml

MnSO4·4H2O

10 g

5 g

H3BO3

3 g

1.5 g

ZnSO4·7H2O

2 g

1 g

KI

750 mg

375 mg

Na2MoO4·2H2O

250 mg

125 mg

CuSO4·5H2O (or CuSO4)

25 mg (or 15.98 mg)

12.5 mg (or 7.99 mg)

CoCl2·6H2O

25 mg

12.5 mg

Store at 4 °C

 

AA Amino Acids 100X solution

 

For 1 L

For 500 ml

L-glutamine

87.6 g

43.8 g

L-aspartic acid

26.6 g

13.3 g

L-arginine

17.4 g

8.7 g

Glycine

750 mg

375 mg

Store at 4 °C; heat to dissolve

 

MS Vitamins 100X solution

 

For 1 L

For 500 ml

Myo-inositol

10 g

5 g

Nicotinic acid

50 mg

25 mg

Pyridoxine-HCl

50 mg

25 mg

Thiamine-HCl (10 mg/ml)

1000 mL

500 mL

Glycine (100 mg/ml)

20 mL

10 mL

Store at 4 °C

 

Fe2EDTA (Iron Chelate) 100 X Solution

  • Dissolve 2.78 g of FeSO47H2O in 350 ml H2O
  • Dissolve 3.726 g of disodium EDTA (MW: 372.24) in 350 ml H2 Heating is required
  • Combine the solutions and bring up to the final volume of 1 L (clear yellow solution)
  • Storage: amber bottle (or covered by foil) to protect it from exposure to light; store at 4 °C

Additional Links

Acetosyringone’s function in plant transformation

https://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-018-0459-5

pCAMBIA 1300 Vector

https://www.snapgene.com/resources/plasmid-files/?set=plant_vectors&plasmid=pCAMBIA1300

T-DNA Binary Vectors

http://www.plantphysiol.org/content/146/2/325

 

Figure 1. Tragopogon transformation and regeneration.