Arabidopsis Leaf Explant Culture

Materials and Reagents

1. Plastic Petri dish
   
2. Parafilm
   
3. Millex-GP Filter Unit 0.22 μm (Merck Millipore Corporation, model: R4AA41572 )
   
4. 2-3 weeks old sterile Arabidopsis plants (Murashige & Skoog solid medium grown)
   
5. Murashige & Skoog medium (Duchefa Biochemie, catalog number: P11293.01 )
   
6. Phytagel (Sigma-Aldrich, catalog number: MFCD00131909 )
   
7. NaOH
   
8. Sucrose
   
9. 2, 4-dichlorophenoxyacetic acid (2, 4-D) (Sigma-Aldrich, catalog number: D7299 ) (see Recipes)
   
10. 6-benzylaminopurine (6-BA) (Sigma-Aldrich, catalog number: 83488 ) (see Recipes)
    
11. Murashige & Skoog solid medium (see Recipes)
    
12. Callus induction medium (see Recipes)
    

Equipment

1. Arabidopsis growth chamber
   
2. Magnetic stirrer and stirring bar
   
3. pH meter
   
4. Scissors
   
5. Tweezers
   
6. Flow cabinet
   

Procedure

1. Preparation of sterile Arabidopsis plants
   
   1. Prepare the Murashige & Skoog (MS) solid medium.
      
   2. Autoclave the Murashige & Skoog (MS) solid medium and pour it into Petri dishes when the temperature of the bottle reaches 50 °C.
      
   3. Put the Arabidopsis seeds in the 30% 84 bleach for 10 min for sterilization. Discard the bleach and wash the seeds with sterilized water for three times.
      
   4. Place 10~15 sterilized Arabidopsis seeds onto each Petri dish with the MS solid medium, and seal the Petri dishes with parafilm to keep sterile and moist.
      
   5. Store the Petri dishes in darkness at 4 °C for 2 to 4 d for vernalization.
      
   6. Transfer the Petri dishes to Arabidopsis growth chamber at 22 °C under a long-day condition (16 h light/8 h dark).
      
   7. Culture the Arabidopsis plants for about 2 to 3 weeks until the width of leaves reaches 3-5 mm (Figure 1).
      
      
      Figure 1. Arabidopsis plants culturing on the MS solid medium for 3 weeks. Bar = 1 cm.
   
   
2. Preparation of callus induction medium
   
   8. Prepare and autoclave the MS solid medium.
      
   9. Add 100 µl of 1 mg/L 2,4-D and 1 mg/L 6-BA into the MS solid medium, named callus induction medium, when the temperature of the bottle reaches 50 °C. Ensure that the performance is done in a flow cabinet.
      
   10. Pour the callus induction medium into Petri dishes. Ensure that the performance is done in a flow cabinet.
   
   
3. Leaf explant culture
   
   11. Ensure that all the performances from steps C11-13 are done in the flow cabinet.
       
   12. Cut across the leaf and midvein with sterilized scissors to yield small strips, which are about 5 mm in length and 2 mm in width, including the midvein going across the width. Ensure that similar mature rosette leaves are selected for the experiments.
       
   13. Select leaf strips in similar size and transfer them to the same callus induction medium with sterilized tweezers (Figure 2).
       
       
       Figure 2. Arabidopsis leaf strips. Bar = 3 mm.
       
       
   14. Seal the Petri dishes with parafilm to keep sterile and moist.
       
   15. Store the Petri dishes at 22 °C in darkness for 1 to 2 weeks.
       
   16. Observe the calli and take photographs (Figure 3).
       
       
       Figure 3. Callus growth in leaf explants (Wang and Guo, 2015). Callus growth in leaf explants of Col-0, 35S-miR₁₅₉847 transgenic (lines 8), iaa28-ko mutant, and 35S-IAA28-1 and PCsVMV-IAA28m transgenic plants. The explants were photographed on day 7 without changing the medium. Bar = 2 mm.

Recipes

1. 1 mg/ml 2,4-D
   Dissolve 20 mg 2,4-D power in 5 ml ethanol, and while stirring, gently add water to 20 ml
   Subsequently, sterilize it by filtration through a Millex-GP Filter Unit (0.22 µm), in the flow cabinet
   Stored at -20 °C
   
2. 1 mg/ml 6-BA
   Dissolve 20 mg 6-BA power in 5 ml 0.1 M NaOH, and while stirring, gently add water to 20 ml
   Subsequently, sterilize it by filtration through a Millex-GP Filter Unit (0.22 µm) in the flow cabinet
   Stored at -20 °C
   
3. Murashige & Skoog solid medium (1 L)
   4.4 g Murashige & Skoog medium
   30 g sucrose
   3.5 g phytagel
   Adjust pH to 5.9, using 1 M NaOH
   
4. Callus induction medium (1 L)
   Murashige & Skoog solid medium
   100 µl 1 mg/L 2,4-D
   100 µl 1 mg/L 6-BA
   

Acknowledgments

This protocol was developed from the following published paper: Hu et al. (2000). This research was supported by the Ministry of Science and Technology (Grant2014CB138402).

References

1. Hu, Y., Bao, F. and Li, J. (2000). Promotive effect of brassinosteroids on cell division involves a distinct CycD3-induction pathway in Arabidopsis. Plant J 24(5): 693-701.
   
2. Rashmi, R. and Trivedi, M. P. (2014). Effect of various growth hormone concentration and combination on callus induction, nature of callus and callogenic response of Nerium odorum. Appl Biochem Biotechnol 172(5): 2562-2570.
   
3. Wang, J. J. and Guo, H. S. (2015). Cleavage of INDOLE-3-ACETIC ACID INDUCIBLE28 mRNA by microRNA847 upregulates auxin signaling to modulate cell proliferation and lateral organ growth in Arabidopsis. Plant Cell 27(3): 574-590.