TITLE: Time-course live imaging of maize and sorghum protoplasts
AUTHORS: Rachel Baschieri, Thai Dao, Samuel Leiboff

[DESCRIPTION]
A protoplast is a living plant, fungal, or bacterial cell with the cell wall removed. Protoplasts offer a simplified system for studies of gene expression compared to more complex whole-plant systems and are promising for the interrogation of cellular physiology, metabolism and responses to stimuli. Performing live imaging of protoplast experiments in a time-course experiment provides more information about how these cellular processes progress over time. Protoplast time-course live imaging studies have previously explored intracellular auxin localization [1], auxin’s effect on gene expression [2], the cellular localization of a mobile transcription factor, and its dynamic response to chemical endomembrane system inhibitors [3], cell wall regeneration [4], and cell division and micro-callus formation [5]. 

Though time-course live imaging of protoplasts is a useful tool, it is also a skill that can be difficult to acquire. Practice and troubleshooting of various protoplast isolation, transfection, and immobilization protocols can be time-consuming and may deter many from performing experiments with protoplasts. This protocol combines protoplast isolation, transfection, and immobilization methods to form a cohesive protoplast time-course live imaging protocol with notes and modifications from our troubleshooting. 

This protocol was developed for time-course live imaging of maize and sorghum protoplasts. The isolation and transfection steps are based on methods published in Meng et al. 2020 with a few modifications. First, 50mM MES was added to the PEG-calcium transfection solution to buffer the transfection solution at pH 5.7 and increase protoplast transfection efficiency . Second, 1% bovine serum albumin(BSA) was added to the incubation solution based on Ohshima et al. 1989  to increase protoplast viability after overnight incubation. Third, 50ug/mL ampicillin is added to the incubation solution to decrease bacterial growth overnight [8]. The alginate immobilization steps were adapted from Middleton et al. 2018 and Dovzhenko et al. 1998. 


References
Middleton, A. M., Dal Bosco, C., Chlap, P., Bensch, R., Harz, H., Ren, F., ... & Dovzhenko, A. (2018). Data-driven modeling of intracellular auxin fluxes indicates a dominant role of the ER in controlling nuclear auxin uptake. Cell reports, 22(11), 3044-3057.
Maurel, C., Leblanc, N., Barbier-Brygoo, H., Perrot-Rechenmann, C., Bouvier-Durand, M., & Guern, J. (1994). Alterations of auxin perception in rolB-transformed tobacco protoplasts (time course of rolB mRNA expression and increase in auxin sensitivity reveal multiple control by auxin). Plant Physiology, 105(4), 1209-1215..
Wu, S., & Gallagher, K. L. (2014). The movement of the non-cell-autonomous transcription factor, SHORT-ROOT relies on the endomembrane system. The Plant Journal, 80(3), 396-409.
Kuki, H., Higaki, T., Yokoyama, R., Kuroha, T., Shinohara, N., Hasezawa, S., & Nishitani, K. (2017). Quantitative confocal imaging method for analyzing cellulose dynamics during cell wall regeneration in Arabidopsis mesophyll protoplasts. Plant Direct, 1(6), e00021.
Xu, M., Du, Q., Tian, C., Wang, Y., & Jiao, Y. (2021). Stochastic gene expression drives mesophyll protoplast regeneration. Science Advances, 7(33), eabg8466.
Meng, R., Wang, C., Wang, L., Liu, Y., Zhan, Q., Zheng, J., & Li, J. (2020). An efficient sorghum protoplast assay for transient gene expression and gene editing by CRISPR/Cas9. PeerJ, 8, e10077.
Ohshima, M., & Toyama, S. (1989). Studies on Culture of Cells and Tissues of Crop Plants: I. Survey on enzymatic isolation and culture of rice leaf sheath protoplasts. Japanese Journal of Crop Science, 58(1), 103-110.
Yoo, S. D., Cho, Y. H., & Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature protocols, 2(7), 1565-1572.
Dovzhenko, A., Bergen, U., & Koop, H. U. (1998). Thin-alginate-layer technique for protoplast culture of tobacco leaf protoplasts: shoot formation in less than two weeks. Protoplasma, 204(1), 114-118.



[STEPS]
SECTION: Day 1 - Protoplast Isolation and Transfection
3. Fill a glass petri dish with the filter sterilized enzyme solution, and set aside.
SECTION: Day 1 - Protoplast Isolation and Transfection
2. Harvest 10-14 day old seedlings and submerge in 5% bleach 1% Tween solution for 1min., remove and rinse seedlings thoroughly in autoclaved tap water 6x. Pat seedlings dry with autoclaved Kimwipes. 
SECTION: Day 1 - Protoplast Isolation and Transfection
4. Cut green leaf tissue from seedlings into ~1mm strips with a fresh razor blade and immediately transfer to glass petri dish pre-filled with filter sterilized enzyme solution.     

SECTION: Day 1 - Protoplast Isolation and Transfection
5. Incubate 4h in dark at RT w/ 40 rpm shaking.
SECTION: Day 1 - Protoplast Isolation and Transfection
7. Add 10mL W5 solution (or volume of W5 equal to the amount of enzyme solution). Gently swirl to mix, and shake additional 1h at 80 rpm.
SECTION: Day 1 - Protoplast Isolation and Transfection
8. Filter the W5 and enzyme solution mixture through 100 or 70um nylon mesh into 50 mL tube.
SECTION: Day 1 - Protoplast Isolation and Transfection
9. Centrifuge @ 1000-1200 rpm 5 min to collect protoplasts to the bottom of the 50mL tube.  

SECTION: Day 1 - Protoplast Isolation and Transfection
10. Use a pipette to remove the supernatant and resuspend the protoplast pellet in 600 uL of suspension solution if working on sorghum, 1ml of suspension solution if working on maize.
SECTION: Day 1 - Protoplast Isolation and Transfection
11. Use 9ul of resuspended protoplasts to count with a hemocytometer to determine the concentration of resuspended protoplasts.    

SECTION: Day 1 - Protoplast Isolation and Transfection
12. Add more suspension solution to reduce protoplast concentration to ~2-4 x 105 cells/mL for transfection.
SECTION: Day 1 - Protoplast Isolation and Transfection
13. In a 1.5mL tube add 20-40ug plasmid DNA and 200uL of protoplast suspension, let sit RT 5 min. 
SECTION: Day 1 - Protoplast Isolation and Transfection
14. Add 220uL PEG solution and mix immediately by gentle tube inversion, incubate 15 min. @ 28C.  
SECTION: Day 1 - Protoplast Isolation and Transfection
15. Quickly add 800 uL W5 solution to dilute the PEG, gently invert the tube 2x.
SECTION: Day 1 - Protoplast Isolation and Transfection
16. Collect the protoplasts by centrifuge 3min. @ 1000rpm, remove PEG and W5 supernatant. 
SECTION: Day 1 - Protoplast Isolation and Transfection
17. Suspend collected protoplasts in 1mL incubation buffer and incubate in the dark @ 28C overnight with gentle ~20rpm shaking. 1.5mL tubes should be laid on their side for overnight incubation.
SECTION: Day 2- Protoplast Immobilization in well-plate
20. Centrifuge transformed protoplasts after overnight incubation @ 1000rpm for 3minutes.
SECTION: Day 2- Protoplast Immobilization in well-plate
21. Remove the incubation buffer supernatant, resuspend the protoplast pellet in 100uL MMM and 100uL Alg-A solution. Mix by gently flicking the 1.5mL tube.
SECTION: Day 2- Protoplast Immobilization in well-plate
22. Pipette a large droplet of the protoplast solution onto Poly-L-lysine coated slide well (we use 8 well chamber, 50uL protoplast droplet in each well)
SECTION: Day 2- Protoplast Immobilization in well-plate
23. Allow the alginate droplet to sit for 10-15 minutes at RT for the protoplasts to settle to the surface of the glass slide.
SECTION: Day 2- Protoplast Immobilization in well-plate
24. Gently pipette 0.5-1uL droplets of W5 solution to the surface of the protoplast-alginate droplet so that the entire surface of the droplet gets coated in W5, but the protoplasts settled against the glass remain undisturbed as possible.   

SECTION: Day 2- Protoplast Immobilization in well-plate
25. Allow the W5 coated protoplast-alginate droplet to sit 15 minutes RT.
SECTION: Day 2- Protoplast Immobilization in well-plate
26. Pipette the equivalent volume of W5 as the volume of the protoplast-alginate droplet into the slide well containing the droplet (50uL W5: 50uL protoplast-alginate) and allow it to incubate for 45 minutes. Note: while pipetting, depress the W5 into the well very slowly and gently.  
SECTION: Day 2- Protoplast Immobilization in well-plate
27. Remove W5 and incubate the droplet in FPCN (350uL FPCN if protoplast droplet is 50uL) for 15 minutes. This is wash 1.
SECTION: Day 2- Protoplast Immobilization in well-plate
28. Remove FPCN and replace with another addition of fresh FPCN, incubate 15 minutes. This is wash 2.
SECTION: Day 2- Protoplast Immobilization in well-plate
29. Remove FPCN and incubate with the final FPCN or treatment solution. The alginate-embedded protoplasts are ready to be imaged.    

SECTION: Day 2- Protoplast Immobilization in well-plate
19. In a sterile hood, coat the bottom surface of the slide-well the protoplasts will be sitting in with 50ug/mL Poly-L-lysine, allow to sit for 30min-1hour, remove the poly-L-lysine, and allow the well-plate to air-dry. 
SECTION: Day 1 - Protoplast Isolation and Transfection
1. Prepare enzyme solution.
SECTION: Day 1 - Protoplast Isolation and Transfection
6. Prepare W5 solution, suspension solution, PEG solution, and incubation solution. 
SECTION: Day 1 - Protoplast Isolation and Transfection
18. Ensure MMM, Alg-A, and FPCN solutions are prepared for Day 2. The MMM and Alg-A solutions may be stored at 4C for 6 months or longer, FPCN may be stored at 20C for 6 months or longer.