PPM Frequently Asked Questions
1. Is PPM effective against algae?
a. Yes, use 0.1% v/v
2. When dealing with aquarium explants, what is the PPM dosage?
a. Aquarium plants have very thin cell walls and as a result are very sensitive to PPM exposure. We recommend starting with the 0.5 -1 ml range.
3. When going through peak rainy/fungi season, what is the preferred dosage?
a. For a mild issue use 0.1% = 1ml/L or 0.2%v/v if issue is mild.
4. Is PPM able to disinfect seeds from seed borne fungi and bacteria?
a. Absolutely yes. Rinse the seeds thoroughly with tap water then take the seeds and stir them in 4% PPM solution for 12 to 16 hours. Without rinsing place them on the proper medium + 0.1% PPM.
5. Does PPM cause plant malformations?
a. No malformations were detected at the dose of 1-2ml/l.
6. Does freezing PPM cause efficacy issues?
a. Freezing PPM for a short period of time (24hrs or less) is not change its efficacy. For best results wait until the PPM is back up to optimal temperature before using.
7. What is the optimal seed sterilization technique for PPM?
a. Stir seeds in 4% PPM solution (4ml/96ml water) for 12 hours, after, with out rinsing, place in germination medium +0.1%. If results are not satisfactory try increasing/decreasing exposure time to the 4% solution until desired results are received.
8. What are the optimal dosages for PPM for best pre-treatment results?
a. With the exception of endogenous contamination, the recommended dose range is 0.05%-0.2%. (For callus proliferation, organogenesis and embryogenesis, the recommended range is 0.05-0.075%.) To eliminate higher endogenous contamination densities, higher doses of PPM are needed (see paragraph 6 below).
9. What are the optimal dosage techniques for eliminating endogenous contamination for explants?
a. Gently and routinely shake / stir 1 cm. long explants (or shorter) for 4-12 hours in 4-5% v/v PPM™ solution supplemented as above with full MS strength basal salts without pH ing and without Tween 20. Without rinsing, insert into a medium supplemented with 0.05 - 0.1% PPM™ for herbaceous plants and 0.2% PPM™ for woody plants.
10. What are the optimal dosage techniques for eliminating endogenous contamination for seeds?
a. Stir water rinsed seeds for 12-16 hours in 4% v/v non- pHed PPM solution without Tween 20. Without rinsing place onto a medium supp. with 0.1% PPM.
11. Can you use PPM during surface sterilization?
a. Yes
12. What is the suggested concentration, technique and incubation time during surface sterilization?
a. You can surface sterilize with 5% v/v PPM solution + 3x MS basal ( inorganic ) salts. Then gently stir/shake the water rinsed explants for 4-12 hours and without rinsing place the explants onto the proper medium with 0.1% v/v PPM. The role of the basal MS salts is to protect the explants during the exposure to 5% PPM. In case of seeds, 4% v/v PPM solution is sufficient and the MS salts are not needed.
Specific Questions
1. When studying plant explants transformed with Agrobacterium, and using PPM in medium for these explants (Transformed by Agrobacterium), does PPM effect negatively, positively or have no effect on these explants(I thought PPM will suppress microbial growth of Agrobacterium in vitro culture, but have to be sure)
a. With Agrobaterium, the explants after the co- cultivation are too soft and too sensitive too high dose ( 5%) PPM. The only way is to add 0.1% PPM to the regular antibiotic cocktail to enhance its potency.
2. When should we add PPM? Before or after autoclave cycle?
a. PPM is only preventative to rid the explants from bacteria and fungi, before culture in media. Once your material is already infected in culture it is impossible to use high dose of PPM ( up to 100 ml/Liter without killing the plantlets too. Once plantlets are "clean" a dose of 1.0-1.5 ml/Liter added before autoclavation will not affect rooting. From experience with Agrobacterium, adding PPM at 1 ml/l to antibiotics might enforce there effect.
3. I'm working on a leaf symbiosis (Dioscorea sansibarensis/Orrella dioscorea) and my goal is to obtain aposymbiotic plants via node cuttings. During this process, I experience a lot of endophytic contamination. I saw that you suggest a protocol on the website to eliminate endophytic bacteria, where 5% PPM is used for 4-12 hours. I tried this protocol, 5% PPM overnight (18 hrs) but saw that my plants suffered too much and the plant tissue is turning brown. Prior to this step, I soak the node cuttings in a fungicide (carbendazim) for 3 hours and do a surface sterilization ( 5' 70% Ethanol and 15' 1.4% bleach). Do you have any suggestions on what would be a fitting protocol to get rid of the endophytic bacteria without putting the cuttings under too much pressure? Is the protocol as effective when done in a lesser percentage, but overnight (> 12hrs), or is it possible you think to skip the fungicide step, as it is redundant to the PPM step?
a. Avoid any pre-treatment just use PPM.
b. Add 3X MS basal salts ( preferably as powder ) to the non- pHed 5% PPM solution.
c. Once UN rinsed and on medium + 0.1% PPM checked the ex-plants after a week. If too damaged but clean lower the exposure time for example from 12 to 8 hours. You should adjust the exposure time by trial and error.
d. From my experience, 4% PPM is too low but you can test it too.
4. I am using Plant Cell Technology "Plant Preservative Mixture" in my tissue culture media; however, it doe not seem to be working. I am struggling with a lot of endophytic contamination (Penicillium, Bacillus, and Pseudomonas among other microbes) and I just thought that I should reach out and ask how to formally use this product. I had read and followed the instructions sent with the product but it has not worked.
My main questions are:
a.) Do I need to add it to my media then pH the media to 5.6-5.8 or should I pH the media first then add the PPM (before autoclaving)?
a. Add 0,1-0.2 % PPM v/v before or after pHing the medium (it makes no difference ) and before autoclavation unless the medium contains any source of proteins in which she should add it after.
b.) What concentration of PPM should I be using in 1L of media to effectively "clean out" a plant of endophytic microbes? According to the literature that was sent with the product, PPM has the potential to clean out endophytic contaminants and produce clean tissue culture plants.
a. It seems to me that the sterilization with bleach, ethanol, etc. are not efficient and in this case she should use 4% for (seeds) and 5% for (explants) non- pHed PPM solutions. She should gently stir/shake the seeds /explants in these solution from 4-8 hours then without rinsing place onto solid medium + 0.1% PPM. With explants only, she should add 3x MS basal ( inorganic ) salts from commercial source or from freshly made stock solutions, in order to protect the explants during the exposure to the 5% ( high dose ) of PPM.
5. We are producing 10 M young clonal plant material annually by TC and
we are interesting in knowing about solutions to the well known phytotoxicity of the PPM, could you end up with some alternatives at this regard?
a. To grow on medium , the dose is 0.1-0.2% v/v and for sterilization 4 and 5% for seeds and explants respectively shake or stirred between 4-12 hours. For explants , 3x MS inorganic salts preferably as powder should be added to the 5% PPM solution.
