Protoplast FAQ

We have compiled this list of frequently asked questions, and provided answers based on our knowledge and experience with Arabidopsis and maize protoplast experiments. Protocols mentioned here can be downloaded on our web site.

Q1. Is there a reason why you use protoplasts from leaves as opposed to from cell cultures? (We have found it easier to make protoplasts from cell cultures.)

A1. It takes time and patience to develop any protoplast assay and I believe that either leaf protoplasts or cell-culture protoplasts could work if people have sufficient knowledge about the physiology of plant cells and are willing to spend the time to figure things out. I have been working with mesophyll protoplasts for the past 15 years and I found them convenient to work with since they are abundant, easy to isolate, and can respond to numerous signals in a similar manner as intact leaves. I don't think there is a "corresponding cell type" for any of the plant cell culture lines. In reality, they are "tumor lines" (similar to many of the mammalian cell lines) because they need to continue to divide indefinitely to be useful. I also like the idea of keeping everything "stored" as seeds and just grow them whenever needed. It usually takes a skilled and dedicated person to keep the cell culture lines alive and to make sure the genetic stability in a lab. Seeds are low maintenance, genetically stable, and can provide all types of tissues/cells. There seems to be more labs spending the time to develop cell culture lines, especially for Arabidopsis. It may work out better when more people are doing it. The parsley, maize (Black Mexican Sweet and A188), soybean, carrot, tomato, and tobacco BY2 cell lines have been around for a long time and are all very robust systems. It's the matter of having someone committed to it. Choosing leaf mesophyll protoplasts is my personal preference. It's more "physiological" since one can use "differentiated cells" for specific responses and assays. For instance, etiolated or greening maize mesophyll protoplasts have the best response to light and photosynthesis gene promoters are most active in these protoplasts. For universal signaling pathways, it does not matter which type of cells are used. It should all work.

Q2. Does it matter whether you incubate your protoplasts in the light or dark after transformation?

A2. It depends on your purposes. You have to think about what kind of assays you are doing and whether it makes sense to do it in the dark or light and how much light and what kind of light are appropriate. If you are not sure about what condition to use, the only way to find out is to try different conditions. In general, strong light is not a good idea (less than 100 uE is better) for protoplasts. For most of our transient assays (we focus on early responses), the experiments are done between 2 to 12 hours. Just be aware that if the plant growth or cell culture condition changes, protoplast responses may change. The consistency of any assay is mostly relied on the plant materials and growth conditions. Our protoplast isolation and PEG transformation method has been streamlined and it usually is not the problem when an assay fails.

Q3. Do you find that PEG transformation works significantly better than electroporation on Arabidopsis protoplasts?

A3. I spent a lot of time trying to find the best electroporation condition for Arabidopsis protoplasts since I like it better than the PEG method (Machine is still more reliable than human hands and I was worried about the use of too much calcium needed for PEG transformation). With maize protoplasts, we can carry out 100 samples in 30 min with consistency. However, I don't think I can really recommend anything that works well for Arabidopsis at the moment. I did find a condition that is described in the Arabidopsis mesophyll protoplast protocol. However, I have pretty much given up using it as a routine protocol since it's not always easy to obtain reproducible results. For each cell type and each plant, you need to figure it out empirically and it takes a lot of time. I have not yet tried protoplasts isolated from culture lines. You may give it a try and it may turn out to be easier.

Q4. We have been using Arabidopsis protoplasts for resistance response studies. We generally do not get great gene expression changes (compared to using intact tissues) with protoplasts. After seeing your Nature paper, we are wondering whether our protoplast culture conditions are significantly different. We use a medium consisting of 4 mM MES (pH 5.6), 10 mM KCl (sometimes, using Mg salts), and 0.4-0.5 M mannitol and leave it at room temperature under light. What conditions do you use? Do you have any other suggestions?

A4. The details of the medium is written in our standard Arabidopsis mesophyll protoplast protocol that can be downloaded from our website (http://genetics.mgh.harvard.edu/sheenweb/). Our studies on innate immune responses have been focused on the use the flg22 peptide elicitor discovered by Thomas Boller's lab. This elicitor stimulates robust general defense responses and can activate both endogenous early defense response genes and corresponding reporter genes in Arabidopsis protoplasts. Similar gene activation is also found in intact leaves. I do not know which elicitor you used and which genes you are studying. Without any information, it is difficult for me to come up with a precise answer. However, we are planning to check other elicitors and pathogen-derived signals to test the protoplast system further.

Q5. Aren't protoplasts stressed already during isolation? Aren't protoplasts dead after isolation? What kind of physiological responses can you get in mesophyll protoplasts?

A5. We are careful when cutting the leaves and not to "wound" them. When the cell walls are digested, there are signals released and there may be some stress responses occurring in protoplasts. However, don't forget that plant cells are pretty resilient and many stress responses are transient and reversible. Using vital staining and physiological assays, we found that isolated mesophyll protoplasts are alive and dynamic. The "stress levels" of protoplasts can be precisely monitored and quantitated by RT-PCR or by testing different stress responsive gene promoters fused to sensitive reporter genes, e.g., LUC encoding the firefly luciferase. We have shown in several published papers that mesophyll protoplasts respond sensitively to diverse stress signals, including heat, H₂O₂, salt, darkness, cold, elicitors, ETOH, and osmotic stress. They also respond to stress hormones, such as ABA and ethylene, and growth hormones, such as auxin and cytokinin, in similar manner as intact leaves.

Q6. Does the protoplasting procedure damage surface components such as plasma membrane receptors?

A6. Our studies of the flg22 peptide receptor FLS2, a plasma membrane LRR receptor kinase, demonstrate that the receptors on the surface of protoplasts are intact and can actively perceive specific extracellular signals to trigger intracellular signaling cascades. We further showed that the plasmid expressing WT FLS2 receptor can complement the fls2 protoplasts after 6 h transient expression and trigger similar downstream signaling responses.

Q7. Aren't the majority of Arabidopsis protoplasts in any given preparation so badly damaged that they are incompetent to undergo cell wall regeneration and further cell division? Aren't the protoplasts heterogeneous?

A7. Using proper plant materials and methods for protoplast isolation and culturing, several labs have demonstrated that Arabidopsis mesophyll protoplasts can undergo cell wall regeneration and further cell division (see references cited in the protocol). For our transient expression analyses that occur in 2-12 h after DNA transfection, we do not wish to stimulate cell wall regeneration and cell division, which can be carried out by proper plant hormone treatments and takes much longer to occur. Our purpose here is to maintain protoplasts as "differentiated" leaf cells similar to those in intact plants and not to alter the fate of mesophyll protoplasts. The cells we used for the experiments are pure (>95%) mesophyll protoplasts. Other types of cells (e.g., guard cells, epidermal cells, vascular tissue cells, etc.) cannot be released using the procedure. The homogeneity of the protoplast population can be easily examined under microscope.

Q8. I think my protoplast isolation is working OK because they appear healthy for at least 3-4 days (they are spherical with a clearly visible plasma membrane and green chloroplasts). I resuspend the protoplasts in 400 mM Mannitol, 15mM MgCl₂, 5mM MES (pH5.6) and then use 250 ul aliquots (5 x10⁵ cells) for each transformation but I did not get good results.

A8. For standard Arabidopsis transfection by PEG, we use 1-4 x 10⁴ cells for each transfection (10 times less than what you and other people used. See the protocol for details). So, one million protoplasts could be 100 samples for us but may only be used as one sample by other scientists. It's probably a good idea for you to test the best ratio for DNA/protoplasts. Depending on your DNA and protoplast quality, more cells are not always better, often worse.

Q9. Do you use specific PEG? Do you use carrier DNA? Is there any difference if I use CaCl₂ instead of Ca(NO₃)₂?

A9. We have used PEG 4000 from Fluka since 1996 (see the protocol). I think the quality of PEG is very important but you can try it out yourself. We don't use carrier DNA and CaCl₂ works fine.

Q10. I autoclaved the PEG/mannitol stock. Is it a good idea? Do I need to keep everything sterile?

A10. It is probably a bad idea to autoclave PEG as the PH can change dramatically. Please check the PH of your PEG solution, especially if all your cells die after PEG transfection. For 2-12 h of incubation time, sterilization is not necessary.

Q11. When I do the PEG transfection, I leave it for 30 min at RT then dilute with W5 buffer over another 30 min. Is it necessary?

A11. I have always wondered about the "old" procedure for PEG transfection. The best way to find out is to try it out yourself! When I was streamlining the protocol to make everything simple and fast, I found that 3 min transfection actually worked better than the "standard" 60 min transfection. Find it out yourself how much more time you can actually save!

Q12. What is the percentage of protoplasts that stay healthy after PEG transfection? What is your PEG transfection efficiency? I have stained for GUS activity and can only see a handful (<0.001%) that is clearly blue. I have also done MUG assays and these also show equally poor levels of GUS expression (barely above background!). I would be extremely grateful if you could provide me with some tips that might help me improve my abysmal transformation efficiency.

A12. For healthy protoplasts, over 50% protoplasts should stay intact after PEG transfection. If your protoplasts are all broken after PEG transfection, I recommend that you check out your plant materials and start again. If a protoplast experiment fails, the plant material is more likely the problem instead of the protocol (if you follow our protocol). We routinely obtain 40-80% transfection efficiency using Arabidopsis mesophyll protoplasts. It is a good idea to follow precisely the standard protocol that is now very simple. Many people claimed that they followed the protocol but very often changed some steps "unconsciously". For example, people would use ten times more cells than what is written in the protocol. More is not always equal to better!!

Q13. Is GUS a good reporter gene to assess protoplast transfection efficiency?

A13. Personally, I prefer to use the engineered GFP that is a vital marker, has no diffusion problem, and is100 fold brighter than the original jellyfish GFP (Chiu et al., 1996).

Q14. How early can I start to detect gene expression in transfected protoplasts?

A14. It depends on the amount of DNA you use and the type of genes you introduce. I can detect LUC reporter gene expression as early as 30 min after DNA transfection and as early as 1-2 h for GFP expression. However, each reporter gene and response may be different and you need to spend some time to figure out what the optimal incubation time is for you assays.

Q15. While I think about it, could you let me know what method of plasmid prep you routinely use? I have had some variable results using different preparation methods. For example, when I prepared the DNA using a Qiagen maxiprep my transformation efficiencies were really low.

A15. The columns are convenient but they are still too expensive for routine use since we make hundreds and thousands of maxipreps in the lab. I also heard from one postdoc in my lab that the Qiagen prep caused some problems. We always use the CsCl gradient for plasmid DNA purification but I encourage people to experiment themselves. We need a lot of DNA (at least 2 mg to carry our serious experiments). Make sure that you remove the salt though. Both mini- and maxi-prep protocols for plasmid DNA isolation from our lab can be downloaded from the new website.

9/5/02

Q16. Why the pH of the PEG solution is not specified? Should it be in the range of 8-9 "as usual"? With tobacco mesophyll protoplast (and PEG from Merck) we routinely adjust to 9 with KOH.

A16. As I indicated in the protocol, we have streamlined the protocol: which means to eliminate whatever steps that are not necessary and time consuming (which takes years of work). We don't just do experiments "as usual". We ask questions constantly whether any step is necessary and whether there is a better way to do it more efficient and obtain better results. We then test ideas and find answers. For instance, have you asked the simple question "why pH 9"? Without adjusting the pH, our PEG solution pH is around 6, which is very close to the pH of all solutions that we are using (pH 5.7). At least for our protocol, using PEG at pH 9 is unnecessary. You can do a comparison yourself and that's always the best way to find reliable answers.

Q17. I wonder about the enzymes cellulase and macerozyme R10, what does R10 mean?

A17. I think it's the name of the fungal strain. You can write to the Japanese company for a sure answer of your question.

Q18. For my barley protoplast prep I used enzymes that were purchased from Calbiochem but you have referred to a company in Japan. Are the enzymes from Calbiochem too strong?

A18. You can try the ones from Calbiochem or any other companies and see whether you like the results. We only use the enzymes from Japan routinely now.

Q19. For my fractionations (I use different size filters) do you think W5 or WI is the best buffer to use?

A19. Arabidopsis protoplasts look better in W5 (with lots of calcium). However, we avoid W5 when we do experiments that are calcium sensitive. We don't usually use W5 for monocot protoplasts (maize, rice, barley and wheat) but you can give it a try (again the best way of learning).

6/9/03

Q20. Do we need to visit your lab to learn how to carry out experiments using protoplasts?

A20. The truth is that there is no "magic" in my lab. After we test a new protocol extensively, we publish and share it with the plant community. We even got rejected when we wanted to share our experience of using GFP as a vital maker many years ago. The reason for rejection was that no one else could show that it worked in plants! Now, using GFP in plants is a common practice. I imagine that using protoplasts will be common for plant experiments in the future. For anybody who has the desire to establish the protoplast system in her/his lab, the detailed protocols published on our lab website should provide sufficient instruction. The protocol has been streamlined and is very simple now. The key for success is all the same basic stuff in conducting good plant research: grow healthy plants, make clean DNA, practice until you feel comfortable with the system, and repeat the experiments until you figure out the story. The standard recommendation is to grow plants every week, observe them every day, and practice as often as you could. My observation of the lab members is that it could take a few months to a year to master the skills (faster than learning to play a violin and it's quiet), depending on how observing, patient, and committed you are. Arabidopsis plants are very sensitive to changes in growth conditions, thus most of the time the problem is the starting plant material. The quality of your DNA is also extremely important for the success of the experiments. We always compare the protoplast responses to intact plant responses to make sure that we are seeing physiological activities. For gene expression, you can compare the reporter gene with endogenous gene activities in the same protoplasts. To start with, you can request a GFP or LUC construct from our lab or find it on your campus. We have sent out clones for numerous requests worldwide over the years and these controls are good for testing both dicot and monocot protoplasts. You will always get a permission to use them freely for basic research.

Q21. We have been using your protocol for monocot protoplast isolation and have had very good success. We are now trying to electroporate the protoplasts and are having problems with protoplast survival. Our electroporator is a Bio-Rad Gene Pulser II. We have tried a range of settings from 200-400 Volt and 100-200 uF. We cannot set the pulse time, but the result is a range of 56.9-25.2 msec. When we observed the cells after electroporation, we saw clumps of lysed cells. Any suggestions?

A21. When using electroporation, you need to test the condition for each plant species and cell types. My 1991 Plant Cell paper provided some general guidelines. The protocol is also available on our lab website. It is important to set the pulse time for less than 5 msec. Capacitance does not matter too much when using 200 uF or more, but the higher settings slow downs the re-charge. For smaller protoplasts (10-20 u), higher voltage (400-500 Volt) could be applied. The bigger the cells, the lower the voltage should be used. We use an old Promega machine that was no longer available in the market. However, there are many other companies that produce electroporators with a pulse time control. Home-made ones will work too and are cheaper. For electroporating monocot leaf protoplast, we found that the use of etiolated and greening tissues is easier. Adding more KCl could help with dicot leaf protoplasts (Arabidopsis and tobacco).

Q22. I am using the protoplast transfection protocol (Yoo et al., 2007) and encountering a problem. I hope you could help me troubleshoot this. The protoplast isolation works very well, however, after transfection most of the protoplasts are 'exploded'. I am trying to express a GFP-expressing construct and I need to incubate the transfected protoplast for 20 hours. After this incubation, most of the protoplasts are destroyed. I should mention that I am using PEG 4000 from Sigma to transfect Arabidopsis mesophyll protoplasts.

A22. The health of protoplasts is mainly determined by the plant growth conditions. When others have encountered problems, we have found that often what we have recommended has not been followed, e.g., not more than 12 h light photoperiod and less than 75 u mol/m2s light along with choice of correct leaves at the correct age, and water only without salt. You can download the movie we provided on the Sheen lab website, showing what the plants should look like. You can run a "no PEG control" to see whether the protoplast burst is due to PEG or DNA or just the protoplasts alone. If the protoplasts did not last, then it's a problem with plant growth. If protoplasts are fine without PEG or DNA, then the problem is with the quality of your PEG or DNA (both are very critical). Our protocol has specific recommendations for the quality of PEG and DNA, and caution should be taken before deviating from them. However, there is not a uniform way that will work for everyone's needs, so you can figure out what you can do in your own lab by trying some variations. Growing healthy plants and following the protocol precisely is the key. Sigma sells many types of PEG and most of them are not good. We also suggest using the protoplasts for 3-6 h for "transient" expression assays. Any experiment taking 20 hr to do means you have additional considerations to address, as we don't recommend this long a time period with our protocol. Many more issues need to be considered. It will be useful to read this FAQ webpage completely and download the protocol and movie. There are lots of details and troubleshooting tips from our 25 years of experience. http://molbio.mgh.harvard.edu/sheenweb/protocols_reg.html

Q23. I find that sometimes it is difficult to pass to the enzyme solution through a filter for sterilization, and sometimes it is easy. When it is easy, I am able to get many protoplasts, but when it is difficult the digestion does not work well - the enzyme solution is not very green, even after an overnight incubation. I believe I am following the protocol in the same way each time. Is this something that you have experienced, or do you know what may be the cause?

A23. Your observation was consistent with what we have experienced in the past, and the answer can be found in the "Enzyme solution" part of the protocols (both the web protocol and 2007 Nature Protocol) and also highlighted in the protoplast movie ( which can be downloaded from the Sheen Lab website. http://molbio.mgh.harvard.edu/sheenweb/protocols_reg.html ). Heat the enzyme solution at 55^oC for 10 min to enhance enzyme solubility and cool to room temperature before adding CaCl₂ and carrying out the filtering step. I'd recommend studying the protocols carefully, keeping the tips in mind, and watching the 22-min movie before starting your protoplast experiments. We also do not recommend carrying out protoplast digestion for overnight as indicated in your e-mail.

Q24. I'm about to start working on protoplast preparation and transfection. I'm now ordering essential chemicals and enzymes and I would like to know where you purchased your cellulase R-10 and macerozyme R-10 ? I would like to order from Yakult but there is no catalog # and price information on their web site, so I sent an email but I'm still waiting for an answer. I also tried to call but I was unable to speak to somebody who speaks English. So I would like to know if you are ordering these enzymes from Yakult directly or if another company sells these enzymes manufactured by Yakult. Also, if you buy these through Yakult directly maybe you already have the information I need to order them and maybe you can share this information with me.

A24. Please download our updated protocol, "Transient Expression in Arabidopsis Mesophyll Protoplasts". You can do this after a quick free registration. On page 6, section E. Enzymes and nylon filters, of the updated protocol you will find the following:

Here is what we ordered:(They call it "DESCRIPTION OF GOODS")

CELLULASE"ONOZUKA"R-10 (100g) (for Arabidopsis and dicot leaves)
CELLULASE"ONOZUKA"RS (100g) (for maize and monocot leaves)
MACEROZYME R-10 (100g) (for both dicot and monocot leaves)

The Cellulase and macerozyme are purchased from:

Yakult Pharmaceutical IND. CO., LTD.
Shinbashi MCV Building
5-13-5 Shinbashi Minato-Ku
Tokyo, Japan
Tel 03-5470-8911 (international call 81-3-5470-8911)
Fax 03-5470-8921 (international fax 81-3-5470-8921)

The purchasing process can take up to a few weeks.
You can pay for express mail delivery, which takes 3-7 days.

You can send an e-mail ( yakultph@nifty.com ) or call ( 81-3-5470-8911 ) to make an order request.
If you don�t speak Japanese (I do not), please be very patient and insist on asking for someone who can speak English with you to make the order. Speaking slowly and politely is the key!!

Hiroto Watanabe
Deputy General Manager
or
Haruhiko Kanai
Deputy Manager of Technical Department
(Note that over the years the managers may change.)

Yakult Pharmaceutical Ind.Co,LTD.
5-13-5,Shinbashi,Minato-Ku,Tokyo,Japan 105-0004
FAX:81-3-5470-8921
Email:yakultph@nifty.com
URL: hppt://www.yakult.co.jp/ypi/

You may order re-packaged Yakult enzymes from other companies (Google search), but our experience is that they are sold in a smaller package (10g) and are much more expensive.

11/12/2013

Q25. I have a simple question that at first sight was not important for me. How do you store and handle the enzymes (cellulase and macerase) in your lab? Do you prepare small aliquots? I'm using the enzymes from Yakult and the company suggested to keep them a room temp as they are reported to be stable for two years. Well, after one year their activity seems to be significantly reduced (leaf material requires longer and longer times to be digested and sometimes it is only partially digested, moreover transfection efficiency is getting worse).

A25. As a common practice, after opening each 100 g bottle, I store the enzyme powder in 5 g aliquots at -20^oC in tightly capped plastic tubes. I never keep any enzyme at room temperature for short-term or long-term (>1-2 years) storage. I've used these enzymes for over 30 years and have never observed a significant activity decrease when they have been stored over 1-2 years. Most of the digestion problems that people have reported were mainly due to incomplete dissolution of the enzyme powder, which can be improved by heating at 55^oC in solution for 10 min (see the protocol). For longer-term storage, I keep the enzyme powder at -20^oC, but I also keep a 5 g tube at 4^oC as the "working " stock. When aliquoting the 100 g enzyme powder bottle, I bring out the bottle from -20^oC and let it "thaw" to room temperature before aliquoting the powder into 5 g tubes. For regular use, a frozen 5 g tube is "thawed" and stored at 4^oC.

Q26. Does it matter whether the protoplast lysis buffer has 25 mM or 2.5 mM Tris-phosphate?

A26. For both LUC and GUS assays, the activities are much higher when 25 mM Tris-phosphate is used for protoplast lysis. In the Arabidopsis Protoplast Protocol that you can download from the Sheen Lab website, the lysis buffer was referred to the Promega LUC protocol (Promega Technical Bulletin TB281, p. 12) available on the website
( http://www.promega.com/resources/protocols/technical-bulletins/0/luciferase-assay-system-protocol/ ).

Luciferase Cell Culture Lysis Reagent, 1X

4/7/2020

Q27.Does etiolation enable easier digestion of the cell walls? Does it affect the quantity or arrangement of cellulose and other components? I have been researching factors that affect the yield and viability of protoplasts obtained from various crop species in our lab. We normally grow them in a GH with a 12 hour light/12 hour dark cycle (more light in summer), but I want to try growing them on media in a growth chamber. I have noted that some protocols grow their plants in the dark - rice and maize for example, and wonder if that is preferable to growing them in the light.

A27. Use of etiolated seedlings is limited to ‘monocots’, e.g., rice and maize, because most dicot leaves cannot develop without light. However, I usually treat imbibed (in water overnight) maize seeds covered under 0.5-1 cm of wet (water only) vermiculite/peat moss with low light for 3 days until the shoot tip is about 1 cm out of ‘soil’ to suppress excess elongation of the whole seedlings. If you grow maize seedlings in "complete darkness from the beginning" they will look very messy. I usually use the "newly and fully expanded" second maize leaf for protoplast isolation and only cut the middle part of the second leaf for a more uniform developmental stage (leaf tip cells are older and leaf base cells are younger).

Etiolated and greening (etiolated leaves were illuminated with 30 uE light for 6-24 h) protoplasts do not have ‘heavy’ and fully developed chloroplasts and perhaps the cell membrane is also different. These protoplasts are much more robust for handling or electroporation, i.e., they do not break as much as protoplasts from fully green leaves (grown under constant illumination or 12 h L/D). My 1991 paper in The Plant Cell (The Plant Cell, vol. 3, 225-245) PDF describes these different protoplasts.

If you want to try agar/medium for plant growth, you need to test them for protoplast isolation. I generally do not recommend high light (50-75 uE).

I use the same cellulase, R10 or RS, (for monocot) and macerozyme R10 (from Rhizopus sp, contains pectinase, hemicellulase and cellulase) for etiolated, greening and green leaf digestion. Three hours is sufficient for this digestion. For some tissues and plant species, you may need to add pectolyase, e.g., embryos/cotyledons with more pectin than cellulase in the cell walls.

I used to order these cell wall digestion enzymes from Japan (Yakult), but it's now available from the US. The quality and price for enzymes from Yakult are better than the products from many other companies. You may ask Libo Shan and Ping He at TAMU for ordering information if needed.

Information for ordering the enzymes in the US
Kanematsu USA
100 Randolph Road
Somerset, NJ 08873
Send inquires to: usv@kanematsuusa.com

Saya Kaneda
Life Science & Materials
Kanematsu USA Inc. Somerset Branch
100 Randolph Road Somerset,NJ 08873 USA

Phone: 732-271-7340

Email: skaneda@kanematsuusa.com
Web: www.kanematsuusa.com

Q28. I am isolating protoplasts from soybean and Arabidopsis leaves. I saw you use Macerozyme R10 (Yakult Pharmaceutical Ind. Co., Ltd., Japan) for Arabidopsis and maize protoplast isolation, but some other people use Pectolyase Y-23 for soybean leaves. I am not sure which one I have to choose for soybean. Which one do you think is better for soybean? Does the Macerozyme R10 also work well for soybean leaves?

A28. For many plant species and different tissues, cellulase and macerozyme (from Rhizopus sp, contains pectinase, hemicellulase and cellulase) work well, as long as you select the optimal plant tissue stage (newly expanded cells/tissues but not old tissues).

The original soybean protoplast protocols developed 30 years ago mostly used embryo/cotyledon tissues that contain more pectin, thus pectolyase was used. If you are using young leaves, macerozyme may be sufficient. It all depends on the developmental stages of tissues and organs with different cell wall compositions.

The best thing to do is to read thoroughly (see the three references below); think carefully about your experimental designs, and try it and find out the best protocol that works for your purposes. Also, remember to check for optimal mannitol concentrations.

References for soybean protoplast isolation
1. A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses.
Faqiang Wu, Yoshie Hanzawa
J.Vis.Exp., 2018, Jan 25;(131)
link to video DOI: doi:10.3791/57258

2. Stable transformation of soybean by electroporation and root formation from transformed callus (Glycine max/direct DNA transfer/protoplasts/organogenesis)
Paul Christou, Jean E. Murphy, and William F. Swain
Proc. Natl. Acad. Sci. USA., 1987, June, Vol. 84, pp. 3962-3966

3. Soybean Protoplast Culture and Direct Gene Uptake and Expression by Cultured Soybean Protoplasts
Willy Lin, Joan T. Odell and Ronald M. Schreiner
Plant Physiol., 1987, 84:856-861

Q29. I read and watched your teaching video, protocol manual. I would like to ask a piece of advice from you because I struggled to get a good quality protoplast from my Dendrobium. My previous colleague did get some good protoplasts from Phalaenopsis, another kind of orchid, but I cannot get the same. No one reported a good protocol for generating protoplasts from Dendrobium flowers.

First, some comments for the protoplast protocol sent by the scientist:
1. Use petal part then slide by razor blade into small pieces then put in the enzyme solution.
Comment 1: Plant growth conditions and tissue ages are the most important factors for protoplast isolation. For monocots, cut parallel to the vein to increase yield. One orchid protoplast paper showed the optimal mannitol concentration is 0.6 M. You may try and compare. Check out the details of the papers in the references in A29 below.

2. Lyse overnight around 16 hours in the dark condition room temperature.
Comment 2: Try using the minimal time needed for digestion; 2-6 hrs.

3. mid shaking around 25 rpm for 30 minutes and then agitate by cut tip pipetting after that mild shaking for 30 minutes more.
Comment 3: 5 min. is sufficient.

4. filter using a Millipore fiber (not really sure about mesh size)
Comment 4: Estimate the size of your protoplasts and use a 70-100 um nylon filter, but not a Millipore fiber (you can find the pore size under a microscope). Nylon mesh with the right pore size can be bought and reused for a long time. I cut the nylon to a convenient size for safe filtering and wash and reuse it for many years. It can be sterilized with 70-95% ethanol.

5. Centrifuge at 200 g for 2 minutes. Remove supernatant and resuspend with WI solution. Keep on ice for 30 minutes before visually checking the protoplasts.
Comment 5: Check for the optimal mannitol concentration and use ’minimal speed and time‘. We usually add the same volume of W5 (125 mM CaCl₂) to release the protoplasts as we do for centrifugation, as CaCl₂ can help settle protoplasts easily. If you do not want to add W5, which has high NaCl, then add 10-30 mM CaCl₂ in WI.

6. I use the same enzyme mix and WI as in the Arabidopsis protoplast protocol.
Comment 6: You may need to check the macerozyme requirement or add pectinase or pectolyase for some orchid tissues, e.g., embryos or very young tissues with more pectin than cellulase. You may add CaCl₂ (10-30 mM) for efficient centrifugation and to enhance protoplast stability.

7. My flower age is around 1 month after bloom, and not a new bloom flower. These flowers normally can bloom from 1.5-2 months, which, I think, will affect my results.
Comment 7: I would use flowers at the time of ‘opening’, and not wait for 1 month after blooming. However, it also depends on the purpose of your experiments. The reasons for isolating protoplasts and what you want to do with your protoplasts will affect the design the isolation accordingly. In general, old tissues do not work well for protoplast experiments.

A29. I'm listing three orchid protoplast papers, including one for Dendrobium leaves and ‘tepals’. The key seems to be using younger tissues (I think 1 month is too old) and fast digestion (less than 2 h), so 16 h would be too long. You may cut the tissue parallel to the vein, which will be more efficient for releasing protoplasts from ‘monocot’ tissues, unlike what I showed for dicot leaves that have a different vein pattern. ( Note: ‘tepal’ is a word for either a sepal or petal of an orchid flower, as sepals and petals often look similar. )

You do need to check the enzyme concentrations and combinations, pH and optimal mannitol concentrations, and I think the papers listed below will be helpful. The key is still to read papers reporting good results, pay attention to all the details, and try and figure out optimal plant growth conditions, plant tissue age (the best time is when the tissues are just expanded, not 1 month old and you need to try the flowers just starting to open), which was what I did when I started in 1987 by myself. I read everything I could find first and there was no google then. The petal of phalaenopsis is thicker and could be different from that in Dendrobium.

I did not have any experience with orchid protoplast isolation. I did a search for ‘Dendrobium protoplasts’ and you may check it yourself and find more information.
Google search for Dendrobium protoplasts

References
1. Dendrobium protoplast co-culture promotes phytochemical assemblage in vitro
Abitha Thomas, Ipsita Pujari, Vasudeep Shetty, Manjunath B. Joshi, Padmalatha S. Rai, Kapaettu Satyamoorthy and Vidhu Sankar Babu
Protoplasma, 2017, 254:1517-1528; DOI 10.1007/s00709-016-1043-2

2. A Protoplast Transient Expression System to Enable Molecular, Cellular, and Functional Studies in Phalaenopsis orchids
Hsiang-Yin Lin, Jhun-Chen Chen and Su-Chiung Fang
FrontPlantSci., 2018, Jun.22:9:843, doi: 10.3389/fpls.2018.00843

3. Embryo and Protoplast Isolation from Barlia robertiana Seeds (Orchidaceae).
American Journal of Plant Sciences, 2013, 4:1-8 http://dx.doi.org/10.4236/ajps.2013.46A001 Published Online June 2013 http://www.scirp.org/journal/ajps
Email: mehmetaybeke@yahoo.com Mehmet Aybeke

Q30. I am running some transient assays in maize protoplasts using inbred line B73.   The experiments are going well but it is very hard to get a hold of large quantities of B73 seeds.  Ultimately, I would like to obtain large quantities of viable seeds of a genotype that is amenable to protoplast experiments so I can continue work without interruption. I came across your maize protoplast isolation protocol online where you mentioned using Illinois Foundation Seeds as a supplier and recommended two hybrid lines for use.  I am wondering if there are any other hybrids you would recommend in the event the two you mentioned are unavailable, or if you have any alternative suggestions for suppliers.

A30. You may want to contact Illinois Foundation Seeds, Inc. (IFSI) to see what they have now to offer in the commercial production of their hybrid seeds. IFSI has been very helpful and supportive of our needs for maize protoplast experiments. Please request or order a small sample of seeds to screen with my maize protoplast protocol, e.g., grow etiolated seedlings for 10-11 days. Please check. the updated maize protoplast protocol online for details or two original papers, Sheen, Plant Cell. 3: 225-245, 1991; Plant Cell 2: 1027- 1038, 1990. You can use the PEG-Ca²⁺-MMg transfection method described in the Arabidopsis protoplast protocol (Yoo et al. Nature Protocols2:1565-1572) to reach high transfection efficiency. No need to do electroporation.

I hope you will find some good new commercial hybrid seed lines more convenient and much cheaper for your future maize protoplast assays. In the past, I had used two different hybrid lines from IFSI, but both were no longer available for routine commercial production.  It's very expensive and will take time to get the specific hybrid corn seeds I used in the past, but the protoplast protocol should work for most corn hybrid lines.

Contact Information at Illinois Foundation Seeds, Inc.

Tina Swank
Customer Service Representative
Illinois Foundation Seeds, Inc.
Field Crops Business Unit
Phone: 765-448-9696
Email: TLSwank@ifsi.com

Time Johnson, President
Email: tljohnson@ifsi.com

Q31. I would like to ask you about the reason why NaCl is added to the W5 solution. According to Yoo et al. (2007), W5 contains 154 mM NaCl, 125 mM CaCl₂, and 5 mM KCl in 2 mM MES (pH 5.7). W5 is used in the purification steps of isolated protoplasts. The NaCl concentration seems toxic to many plants. I wonder if sugar alcohols such as mannitol or sorbitol which are metabolically less active would be convenient to obtain healthy (less stressed) protoplasts.

A31. Thanks for your question about W5. I'd recommend two method papers from the Sheen Lab with the protoplast Washing and Incubation solutions (WI or W5) that we typically use for protoplast culture in transient expression assays (1-24 h). This is not for long-term protoplast culture. Download pdf papers from https://molbio.mgh.harvard.edu/sheenweb/publications.html

1. Yoo et al., Nature Protocols 2:1565-1572 (the updated version corrected one error for the concentration of Protoplast lysis buffer from 2.5 mM to the original and correct 25 mM Tris–phosphate (pH 7.8). This turned out to be critical for LUC assays.

WI solution: Prepare 4 mM MES (pH 5.7) containing 0.5 M mannitol and 20 mM KCl.
W5 solution: Prepare 2 mM MES (pH 5.7) containing 154 mM NaCl, 125 mM CaCl₂, 5 mM KCl. (Note: we removed 5 mM Glucose because our lab study glucose signaling)

2. Niu & Sheen, Methods Mol. Biol. 876:195-206

1. W5 solution: 154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 2 mM MES, pH 5.7
2. WI solution: 0.5 M mannitol, 4 mM MES, pH 5.7, 20 mM KCl
3. MMg solution: 0.4 M mannitol, 15 mM MgCl₂, 4 mM MES, pH 5.7
4. Polyethylene glycol (PEG) solution: Add 4 g of PEG 4000 (Sigma-Aldrich-Fluka), 2.5 ml of 0.8 M mannitol, and 1 ml of 1 M CaCl₂ to 3 ml of ddH2O for a total volume of 10 ml.

When I first started developing the maize mesophyll protoplast transient expression system in 1987 (Sheen, Plant Cell. 3: 225-245, 1991; Plant Cell 2: 1027- 1038, 1990), I developed a very simple WI solution for protoplast incubation for 1-16 h at room temperature after conducting and comparing experiments for several years with various conditions and culture media.

WI (Washing & Incubation)

Although I started with much more complicated protoplast incubation media and tried many different kinds based on the literature since the 1970s, I realized later after several years of experiments that the simple WI worked well for maize, barley, rice, wheat, tobacco, and Arabidopsis mesophyll protoplasts for transient expression assays. As you may realize, I worked first with monocot mesophyll protoplasts and developed electroporation methods but not tobacco, thus did not use W5 first.

I only used mannitol and did not use W5 with high NaCl and high CaCl₂ until I started to develop the Arabidopsis mesophyll protoplast transient assay around 1997 with the goals of using the available Arabidopsis genome sequence to select, predict, screen, and evaluate universal regulatory genes in diverse plant signal transduction pathways. When I compared my methods with simple WI and the tobacco W5, I found that both worked well for protoplast incubation despite the high NaCl and high CaCl₂ in W5 using the PEG-Ca²⁺-MMg transfection method to introduce DNA, RNA, and protein.

The Sheen lab members usually try both and each decides whether WI or W5 is preferred for their assays and research projects. I even asked several new lab members around 2015 to directly compare WI and W5 incubation results from the same population of transfected mesophyll protoplasts. No significant differences were found. Most of our transient assays are done in 1-8 h. I recommended that people adjust their experimental clock timing as well as incubation conditions and duration as needed for each assay and project. Together, we have developed over 30 different biological/physiological assays over the decades. You may consider adding CaCl₂ and MgCl₂ (5-15 mM) in WI to increase protoplast stability if it helps with your biological and physiological assays.

If you treat intact plants with 154 mM NaCl, together with the endogenous osmoticum, this 154 mM salt would cause osmotic stress. However, with protoplasts that need 0.3-0.6 M osmoticum to stay active, the NaCl in W5 mostly acts as an osmoticum and may not be taken up extensively to affect protoplast physiology and signal responses too much. The 125 mM CaCl₂ may help protect protoplasts together with 154 mM NaCl.

Now the Yoo et al. NP2007 protocol we published (20 years after I started the protoplast transient expression system in 1987) has been used and cited by 4,467 using all kinds of fresh tissues from a broad range of plant species. Both WI and W5 work. It's up to the users to find their own preferences. Nutrients and hormones will be needed for longer cultures and different experimental purposes.

In general, there is no limit to what we can improve in new protoplast experiments for different purposes with innovation, creativity, basic logic, and common sense. There is so much more information available now.

Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. 4430, 2007 SD Yoo, YH Cho, J Sheen, Nature Protocols 2 (7), 1565-1572 PDF