Transient Expression Assay and Microscopic Observation in Kumquat Fruit

Materials and reagents

Biological materials

1. Kumquat (F. crassifolia Swingle) fruit (collected from Guangxi Institute of Citrus Research, located at Guilin city, Guangxi, China)

   
   

Reagents

1. Actin cytoskeleton marker GFP-Lifeact containing a 35S::GFP cassette as a visual reporter [13]

2. P19 Agrobacterium

3. Yeast extract (OXOID, catalog number: LP0021)

4. Tryptone (OXOID, catalog number: LP0042)

5. NaCl (HUSHI, catalog number: 10019318, CAS: 7647-14-5)

6. D-glucose (HUSHI, catalog number: 10010518, CAS: 14431-43-7)

7. MES (Sigma-Aldrich, catalog number: M8250)

8. Na₃PO₄ (HUSHI, catalog number: 20040928, CAS: 7601-54-9)

9. Acetosyringone (Sigma-Aldrich, catalog number: D134406)

10. GV3101 Chemically Competent Cell (Shanghai Weidi Biotechnology, catalog number: AC1001)

11. Antibiotics: Kanamycin (Kan) (Genview, catalog number: AK177-10G) and Rifampicin (Rif) (Genview, catalog number: AR280-1G)

12. Antibodies: Polyclonal mouse antibody against GFP (Biorbyt, catalog number: orb688437) as the primary antibody and goat anti-mouse HRP (Biorbyt, catalog number: orb670233) as the secondary antibody

13. Dimethyl sulfoxide (DMSO) (MACKLIN, catalog number: C13178602)

14. Mannitol (Solarbio, catalog number: M8141)

15. KCl (Rhawn, catalog number: R051965)

16. CaCl₂ (MACKLIN, catalog number: C832203)

17. BSA (MACKLIN, catalog number: B928042)

18. Cellulase R10 (Yakult, catalog number: 220908-02)

19. Sodium hypochlorite (MACKLIN, catalog number: S935360)

    
    

Solutions

1. Luria Broth (LB) medium (see Recipes)

2. Infiltration buffer (see Recipes)

3. Enzymatic solution (see Recipes)

4. Antibiotics (see Recipes)

   
   

Recipes

1. LB media

   10 g of tryptone, 10 g of NaCl, 5 g of yeast extract; make up to 1 L with dH ₂O, following autoclave sterilization.

   
   

2. Infiltration buffer

   
   

   Infiltration medium stock solutions:

   500 mM MES: 4.88 g of MES in 50 mL of dH₂O. Store at 4 °C.

   20 mM Na₃PO₄: 0.17 g of Na₃PO₄ in 50 mL of dH₂O. Store at 4 °C.

   1 M Acetosyringone (3’,5’-dimethoxy-4’-hydroxyacetophenone): 0.196 g of Acetosyringone in 1 mL of DMSO. Divide into single-use aliquots and store at -20 °C.

   
   

   50 mL infiltration buffer

   250 mg of D-glucose, 5 mL of MES stock solution, 5 mL of Na₃PO ₄ stock solution, 5 μL of Acetosyringone stock solution; make up to 50 mL with dH₂O.

   
   

3. Enzymatic solution

   Enzymatic solution stock solutions:

   0.8 M mannitol stock: 7.29 g of mannitol in 50 mL of dH₂O.

   2 M KCl stock: 7.455 g of KCl in 50 mL of dH₂O.

   0.2 M MES stock: 1.952 g of MES in 50 mL of dH₂O. Store at 4 °C.

   1 M CaCl₂ stock: 1.11 g of CaCl₂ in 10 mL of dH₂ O.

   10% BSA stock: 0.1 g of BSA in 1 mL of dH₂O.

   10 mL enzymatic solution: 0.15 g of cellulase R10, 0.04 g of macerozyme R1, 5 mL of 0.8 M mannitol stock, 100 μL of 2 M KCl stock, and 1 mL of 0.2 M MES (pH 5.7) stock. Heat solution in heat block or oven set to 55 °C (maximum) for 10 min to help enzymes dissolve (or place in 55 °C water) and then cool to room temperature (RT). Next, add 100 μL of 1 M CaCl ₂ stock, 100 μL of 10% BSA stock, and dH₂O up to 10 mL. Sterilize by filtration (0.45 μm).

   
   

4. Antibiotics

   50 mg/L Rif

   50 mg/L Kan

   
   

Laboratory supplies

1. Tips (Axygen, catalog number: AXYT1000B)

2. 1 mL syringe with needle (GEMTIER, catalog number: 0.45X16 RW LB)

3. Glass microscope slides, coverslips (Corning, catalog number: CLS294875X25, CLS285522), and Vectashield (Vectorlabs, catalog number: H-1000) for microscopy

4. Test tubes (Eppendorf, catalog number: EP0030122178) and flask (Corning, catalog number: CLS4444250) for culturing Agrobacterium

5. Fine forceps and razor blades (Artis Tweezer, catalog number: Z742671; smartSlicer, catalog number: Z740503)

6. Marker pen (STATMARK, catalog number: Z648191)

7. Tray (PureSolv, catalog number: Z681687) and Falcon tube cap (Corning, catalog number: 352070) for placing fruit

8. Petri dishes (60 mm, optional, for enzymatic digestion of protoplasts) (Nalgene, catalog number: TMO5921-0060)

9. Pipette (Eppendorf, catalog number: GN686271)

10. Rubber gloves (Microflex, catalog number: Z265179)

11. Bibulous paper (for uptaking the extravasated Agrobacterium)

12. 50 mL Falcon tubes (Corning, catalog number: 352070)

Equipment

1. Shaking incubator (Radobio, model: Stab M1T) used for the growth of Agrobacterium at 28 °C

2. Centrifuge (Eppendorf, model: 5424)

3. Clean workbench (AIRTECH, model: SW-CJ-2FD)

4. Fluorescence dissecting stereomicroscope (Olympus, model: SZX7)

5. Confocal microscope (Leica, model: SP8)

6. Nanodrop spectrophotometer (or equivalent) to determine optical density of bacterial culture

Procedure