Preparation and Transfection of Populus tomentosa Mesophyll Protoplasts

Materials and Reagents

1. 10 ml centrifugal tubes (HOUDIOR)

2. 1.5 ml microfuge tubes (Axygen)

3. Blades (Gillette blue, Super Gillette Blue Blades, 4.3 cm × 2.2 cm)

4. Culture dish (90 mm diameter)

5. Glass-bottom dish (In Vitro Scientific, catalog number: D35-10-1.5-N)

6. Strainers, 400 mesh (BIODEE, catalog number: DE2009)

7. Cellulase “ONOZUKA” R-10 (Yakult, catalog number: L0012-10g)

8. Macerozyme R-10 (Yakult, catalog number: L0021-5g)

9. Pectolase Y-23 (Yakult, product agent (BIODEE) catalog number: AOV0094-1g)

10. MES (Sigma-Aldrich, catalog number: M3671-50G)

11. PEG4000 (Sigma-Aldrich, catalog number: 81240-1KG)

12. D-glucose (Sigma-Aldrich, catalog number: G7021-100G)

13. β-thioglycol (Amresco, catalog number: 0482-100 ml)

14. BSA (Amresco, catalog number: 0332-100g)

15. D-Mannitol (BIODEE, catalog number: BN20023-250g)

16. KCl (BIODEE, catalog number: DE-0395A-250g)

17. NaCl (BIODEE, catalog number: DE0008-500g)

18. MgCl₂·6H₂O (BIODEE, catalog number: DE-0288A-500g)

19. CaCl₂ (BIODEE, catalog number: DE-0556A-500g)

20. Plasmid (dissolve in double distilled water, 2 µg/µl)

21. Enzymatic solution (see Recipes) (Table 1)

22. W5 solution (see Recipes) (Table 2)

23. MMg solution (see Recipes) (Table 3)

24. PEG/Ca²⁺ solution (see Recipes) (Table 4)

25. D-mannitol solution (see Recipes)

Equipment

1. 1,000 µl micropipette (Eppendorf)

2. 200 µl micropipette (Eppendorf)

3. 10 ul micropipette (Eppendorf)

4. Cell counting plate (MARIENFELD, model: MARIENFELD-0650030)

5. Erlenmeyer flasks (Bomex, 50 ml, 500 ml)

6. Plant incubator (Percival, model: AR-36L3)

7. Bacterial shaker incubator (Crystal Technology & Industries, model: IS-AX-190L)

8. Centrifuge (Eppendorf, model: 5804 R)

9. Confocal microscope (Carl Zeiss, model: Zeiss LSM780)

Procedure

1. Preparation of P. tomentosa materials and solutions

   1. One-month-old P. tomentosa young plants are grown in tissue culture media. Stem fragments are cultured on shoots propagation medium [Murashige and Skoog (MS) medium containing 0.1 mg/L α-naphthalene acetic acid (NAA), 0.02 mg/l thidiazuron (TDZ)] for 30 days to get plantlets, then the plantlets are transferred to rooting medium (1/2-strength MS containing 0.05 mg/L NAA) with a 16/8 h (light/dark) photoperiod. After 30 days, the plantlets are approximately 10 cm in height (Figure 1).

      
      
      Figure 1. Experimental scheme of the preparation and transfection of P. tomentosa mesophyll protoplasts. Cut the leaves of one-month-old P. tomentosa plants grown on tissue culture media into 1 mm strips pre-treated in 0.8 M D-mannitol for 20 min. Digest the leaf strips in enzyme solution for 3-5 h, then filter with 400-mesh cell strainer, centrifuge and wash the protoplasts with W5 solution twice, followed by wash with MMg buffer twice. The transfection reaction containing 10 μl plasmid, MMg resuspended protoplasts (100 μl), and 40% PEG/Ca²⁺ solution (110 μl) occurs for 50 min. Wash with W5 solution and incubate for 12-16 h. Centrifuge and observe the transfected protoplasts using a glass-bottom dish.

      
      

   2. Prepare enzymatic, W5, MMg, and PEG/Ca²⁺ solutions. Place the W5 and MMg solutions at 4°C for at least 1 h before use.

      
      

2. Isolation of P. tomentosa protoplasts

   1. Prepare D-mannitol solution (see Recipes).

   2. Use a sharp blade to cut 5 P. tomentosa young leaves into 1 mm wide strips, completely immerse them in the D-mannitol solution, and place the culture dish on the bench (room temperature, 23°C) for 20 min.

   3. Transfer the leaf strips into a 10 ml centrifuge tube with the prepared enzymatic solution, place the tube horizontally in the bacterial shaker incubator in the dark, and shake at 10 rpm for 3-5 h at 23°C until the color of solution changes to green.

   4. Filter with a 400-mesh cell strainer, transfer the filtrate into a new 10 ml centrifuge tube, and centrifuge at 100 × g for 2 min at 23°C. Remove the supernatant and keep 2 ml of the precipitate.

      
      

3. Plasmid transfection

   1. Add 2 ml of pre-cooled W5 solution gently along the tube wall to wash the protoplasts, gently invert and mix, centrifuge at 100 × g for 1 min at 4°C, and remove the supernatant.

   2. Repeat C1 once.

   3. Resuspend the protoplasts with 2 ml of pre-cooled W5 solution and place on ice for 30 min.

   4. Centrifuge at 100 × g for 1 min at 4°C and remove the supernatant.

   5. Add 2 ml pre-cooled MMg solution to resuspend the protoplasts, centrifuge at 100 × g for 1 min at 4°C, and remove the supernatant.

   6. Resuspend the protoplasts to 1 × 10⁵-5 × 10⁵ per ml (determine their density with the cell counting plate) with pre-cooled MMg solution, and use a cut micropipette tip (0.5-1 cm) to add 100 µl of protoplast suspension for each plasmid of gene transfection.

   7. Handle the following steps at 23°C. Add 10 µl of plasmid (2 µg/µl) for a target gene to a 1.5 ml microfuge tubes, and add 100 µl of protoplast suspension liquid and gently mix with micropipette. Add 110 µl (equal volume) of the PEG/Ca²⁺ solution and gently mix with a cut micropipette tip (0.5-1 cm).

   8. Incubate in the dark at 23°C for 50 min (transfection step).

   9. Add 440 µl (twice volume) of W5 solution, gently mix with a cut micropipette tip (0.5 cm), centrifuge at 100 × g for 1 min at 23°C, and remove the supernatant.

   10. Add 100 µl of W5 solution, mix with cut micropipette tip (0.5-1 cm), centrifuge at 100 × g for 1 min at 23°C, and remove the supernatant.

   11. Repeat C10 once.

   12. Add 1 ml of W5 solution, mix with the 1,000 µl micropipette, and incubate in the dark at 23°C for 12-16 h.

   13. Centrifuge at 100 × g for 1 min at 23°C and remove the supernatant. Transfer the precipitate onto a glass-bottom dish for confocal microscopy observation (50-80% transfection efficiency, Figure 1). The results of transfection for PtHTB9 and EgHTB9 are displayed in Figures 2 and 3, respectively.

       
       
       Figure 2. Transfection of GFP-PtHTB9 in P. tomentosa mesophyll protoplasts. The coding sequence (CDS) of the P. tomentosa HTB9 gene (PtHTB9, Potri.008G030600) was inserted into a modified pUC19-GFP vector, and 10 μl of GFP-HTB9 plasmid (2 μg/μl) was used for transfection. Images were taken with a Zeiss 780 confocal microscopy. Scale bar = 50 μm.

       
       

       
       Figure 3. Transfection of GFP-EgHTB9 in Eucalyptus urophylla × E. grandis mesophyll protoplasts. The coding sequence (CDS) of the E. grandis HTB9 gene (EgHTB9, Eucgr.B03925) was inserted into a modified pUC19-GFP vector. Scale bar = 50 μm.

Notes

1. All the chemicals used in this study are Analytical Reagent (A.R.) standard.

2. For high transfection efficiency, we strongly recommend using PEG4000 (Sigma-Aldrich, catalog number: 81240-1KG) at a concentration of 40%.

3. Use scissors to cut off the tip (0.5-1 cm length) of all 100 µl micropipette tips for C6, C7, C10, and C11 steps, and cut off the tip (0.5 cm length) of 1,000 µl micropipette tips for C9 step.

4. Addition of β-mercaptoethanol to the enzymatic solution (Table 1) is optional.

5. Sterilized and separately stored W5 or MMg solutions can be placed at 4°C for 1 year, while enzymatic and PEG/Ca²⁺ solutions should be freshly prepared before use.

6. Set all the acceleration and deacceleration of centrifugation to 5 (max 10).

7. Leaves digested horizontally in 10 ml centrifuge tubes with 10 ml enzymatic solution provides better isolation. Too much air in the tubes (10 ml enzymatic solution in 15 ml centrifuge tubes) or higher speeds (>60 rpm) in step B3 lead to more broken protoplasts.

8. Smaller protoplast concentrations in the C6 step give higher transfection efficiency, while higher suspension concentration provides more yield (transfected cells).

Recipes

1. Enzymatic solution (Table 1)

   
   Table 1. Recipe for the enzymatic solution

   Stock Solutions	Weight or volume	Final Concentration
   Cellulase R-10	0.15 g	1.5%
   Macerozyme R-10	0.04 g	0.4%
   Pectolase Y-23	0.05 g	0.5%
   D-mannitol	1.092 g	0.6 M
   200 mM KCl	1 ml	20 mM
   200 mM MES	1 ml	20 mM
   5 ml ddH2O, vortex, 55°C 10 min, leave to cool at room temperature
   1 M CaCl2	100 μl	10 mM
   7.5 mM β-thioglycol	66.67 μl	0.05 mM
   1% BSA	1 ml	0.1%
   Fill the volume to 10 ml with ddH2O

   
   

2. W5 solution (Table 2)

   
   Table 2. Recipe for the W5 solution
   

   Stock Solutions	Weight or volume	Final Concentration
   2 M NaCl	38 ml	154 mM
   1 M CaCl2	62.5 ml	125 mM
   200 mM KCl	12.5 ml	5 mM
   200 mM MES	5 ml	2 mM
   D-glucose	0.45 g	5 mM
   Fill the volume to 500 ml with ddH2O

   
   

3. MMg solution (Table 3)

   
   Table 3. Recipe for the MMg solution
   

   Stock Solutions	Weight or volume	Final Concentration
   D-mannitol	7.29 g	0.6 M
   150 mM MgCl2	10 ml	15 mM
   200 mM MES	2 ml	4 mM
   Fill the volume to 100 ml with ddH2O

   
   

4. PEG/Ca²⁺ solution (Table 4)

   
   Table 4. Recipe for the PEG/Ca²⁺ solution

   Stock Solutions	Weight or volume
   PEG4000	3 g
   ddH2O	2.25 ml
   0.8 M D-mannitol	1.785 ml
   1 M CaCl2	0.75 ml

   
   

5. D-mannitol solution

   Dissolve 2.912 g mannitol in 20 ml ddH₂O in 90 mm diameter culture dish.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31900173 to H.-L.W.; 31970196 to Z.L.; 31770649 to X.X.). This protocol was modified from previous work (Yoo et al., 2007; Guo et al., 2012; Tan et al., 2013; Lin et al., 2014), and we would like to thank all who contributed to these previously published procedures.

Competing interests

No financial, personal, or professional interests have influenced the work.

References

1. Chen, H., Wang, J. P., Liu, H., Li, H., Lin, Y. J., Shi, R., Yang, C., Gao, J., Zhou, C. and Li, Q. (2019). Hierarchical transcription factor and chromatin binding network for wood formation in black cottonwood (Populus trichocarpa). Plant Cell 31(3): 602-626.
2. Guo, J., Morrell-Falvey, J. L., Labbe, J. L., Muchero, W., Kalluri, U. C., Tuskan, G. A. and Chen, J. G. (2012). Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays. PLoS One 7(9): e44908.
3. Jansson, S. and Douglas, C. J. (2007). Populus: a model system for plant biology. Annu Rev Plant Biol 58435-458.
4. Lin, Y. C., Li, W., Chen, H., Li, Q., Sun, Y. H., Shi, R., Lin, C. Y., Wang, J. P., Chen, H. C., Chuang, L., Qu, G. Z., Sederoff, R. R. and Chiang, V. L. (2014). A simple improved-throughput xylem protoplast system for studying wood formation. Nat Protoc 9(9): 2194-2205.
5. Muller, N. A., Kersten, B., Leite Montalvao, A. P., Mahler, N., Bernhardsson, C., Brautigam, K., Carracedo Lorenzo, Z., Hoenicka, H., Kumar, V. and Mader, M. (2020). A single gene underlies the dynamic evolution of poplar sex determination. Nat Plants 6(6): 630-637.
6. Munné-Bosch, S., (2008). Do perennials really senesce. Trends Plant Sci 13(5): 216-220.
7. Tan, B., Xu, M., Chen, Y. and Huang, M. (2013). Transient expression for functional gene analysis using Populus protoplasts. Plant Cell, Tissue Organ Cult 114(1): 11-18.
8. Tylewicz, S., Petterle, A., Marttila, S. and Miskolczi, P. (2018). Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication. Science 360(6385): 212-215.
9. Wang, H. L., Zhang, Y., Wang, T., Yang, Q., Yang, Y., Li, Z., Li, B., Wen, X., Li, W., Yin, W., Xia, X., Guo, H. and Li, Z. (2021). An alternative splicing variant of PtRD26 delays leaf senescence by regulating multiple NAC transcription factors in Populus. Plant Cell 33(5): 1594-1614.
10. Xue, L., Wu, H., Chen, Y., Li, X., Hou, J., Lu, J., Wei, S., Dai, X., Olson, M. S., Liu, J., Wang, M., Charlesworth, D. and Yin, T. (2020). Evidences for a role of two Y-specific genes in sex determination in Populus deltoides. Nat Commun 11(1): 5893.
11. Yoo, S. D., Cho, Y. H. and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2(7): 1565-1572.