PPM™ PLANT PRESERVATIVE MIXTURE
Ordering information
Plant Cell Technology, Inc.
1920 N Street, NW – Suite 601
Washington, DC 20036
202-778 8522
INTRODUCTION:
Plant Cell Technology has developed a new, heat stable preservative/biocide, which effectively prevents or reduces microbial contamination in plant tissue culture. At optimum doses, PPM™, which stands for Plant Preservative Mixture, is an extremely effective Preservative/Biocide, yet does not impair in vitro seed germination, callus proliferation and callus regeneration.
Despite the most stringent use of sterile techniques, the contamination of plant cell and plant tissue cultures remain a persistent problem that can result in losses ranging from small number of cultures to the loss of whole batches. PPM™ prevents the germination of both bacteria and fungi spores. It is heat stable and therefore can be autoclaved with the media. PPM™ can be, and should be used as a standard ingredient in plant tissue culture media, and is also substantially less expensive than commonly used antibiotics. While PPM™ was principally designed to inhibit airborne contamination, waterborne contamination and contamination introduced from human contact, it can also — in many cases — be used to reduce endogenous contamination.
The principal PCT scientist involved in the development of the PPM™ application is Dr. Assaf Guri. Dr. Assaf Guri holds degrees in genetics, applied genetics and plant breeding from the Hebrew University in Jerusalem and Michigan State University in the US. Before joining Plant Cell Technology, Inc., Dr. Guri worked with the Volcani Agricultural Research Center in Israel, Michigan State University in East Lansing, Michigan and DNAP in New Jersey.
MECHANISM OF ACTION:
PPM™ is a broad-spectrum preservative and biocide, which kills bacteria and fungi cells, prevents germination of spores, and in higher concentrations, can eliminate explants of endogenous contamination. Previous research has shown that the active ingredients of PPM™ penetrate the fungus or the bacterium cell wall and inhibit the activity of key enzymes in the citric acid cycle and in the Electron Transport Chain. Our data indicate that PPM ™ may also inhibit the transport of monosaccharides and amino acids from the medium into the fungus or bacterium cells.
As in any biocide, a critical ratio of PPM™ molecules per microbial cell is needed to eliminate bacteria and fungi. Keep in mind that a given volume of PPM™ dose has a constant number of PPM™ molecules while the number of spores introduced to tissue culture via endogenous contamination is highly varied. Therefore explants should not be “squeezed” into a beaker. There should be enough volume fro free movement of the solution around the explant material.
PROCEDURES AND ADVANTAGES:
PPM™ significantly simplifies the tissue culture working procedures as follows:
1. Media containing PPM™ may be dispensed outside the laminar flow hood (LFH) exposed to the ambient air. The plates should be covered soon after agar solidification. In the event that media dispensing is done by a pump, we recommend passing autoclaved hot water through the hoses prior to and after media dispensing.
2. Heat sensitive or heat stable liquid media containing PPM™ does not need to be sterilized by Millipore filters or autoclaved provided that it will be stored in sterile containers and that the stock solutions are not contaminated. In rich media containing 200 mg/liter or more of amino acids or proteins, it is recommended to filter the media with the PPM™.
3. If working in the LFH the utensils (forceps or scalpels) do not need to be flamed. They should be periodically dipped in 70% alcohol. The LFH does not need to be certified and the work can be done as well outside the LFH on a clean surface for a period not exceeding 1 hour.
4. PPM™ is an acidic liquid solution (pH 3.8). PPM™ should be stored at 4°C. (see Safety Information below). The recommended dose is 0.5 – 2.0 ml of PPM™ per liter of medium is added to the medium before or after autoclavation to prevent airborne and endogenous contamination at low inoculum densities. Higher doses are required to treat endogenous contamination or to obtain Agrobacteria free plant material.
5. PPM™ is less effective when exposed to high density of bacteria or fungi spores found on seed’s coat. For in vitro germination, seeds should be conventionally surface sterilized with bleach. Therefore, in the presence of PPM™ (in the germination medium), the seeds can be rinsed under tap water in a non-sterile strainer and left to dry preferably in the LFH. If the utensil ends have touched active bacteria, fungi culture or otherwise suspected of being contaminated, they should be sterilized by autoclaving or by use of an electric heating element.
6. General dosage levels. 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%. Add PPM™ to medium pre or post autoclavation to prevent airborne contamination and endogenous contamination at low inoculum densities or slow growing bacteria. To eliminate higher endogenous contamination densities, higher doses of PPM™ are needed (see paragraph 7).
7. Endogenous Contamination:
(a) For seeds: stir non-sterilized seeds for 8-12 hours in 2% PPM™ solution (v/v) supplemented with full strength basal salts of your routinely used medium and don’t add Tween 20 or pH this solution. Subsequently, without rinsing, transfer to germination medium supplemented with 0,05% – 0,1% PPM™ for herbaceous plants and 0,2% PPM™ for woody plants. Hard coated seeds such as Asparagus, Lupine, Ornamental Palm, Rose etc. should be soaked in water for 2-4 hours prior to the sterilization with PPM.
(b) For explants: gently shake / stir 1 cm long explants (or shorter) routinely in bleach solution to remove surface contamination. Rinse with water (can be done under non sterile conditions) and shake / stir for 12 hours in 4-5% v/v PPM™ solution supplemented as above with full MS (or 2X) 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% for woody plants
If possible, collect woody plant material after dormancy is broken. This will substantially decrease contamination.
(c) For tubers: shake /stir the entire tuber routinely in bleach solution. Rinse with water (can be done under non sterile conditions) and slice the tuber (bulb or scale) into thin slices. Shake / stir for 12 – 24 hours in 4-5% PPM™ solution supplemented with full strength (or 2X) basal salts without pH ing and add Tween 20. Without rinsing insert into a medium supplemented with 0,1-0,2% PPM™.
8. In cases that the above protocols don’t yield satisfied results (especially with thick explants or highly infested explants and seeds), we recommend the followings:
(a) Shake / stir the explants (or seeds) in water (does not have to be sterile), 1 hour for soft tissues and 2 hours for hard tissues
(b) Shake / stir the explants in 50% PPM™ (dilute with sterile water) supplemented with 2X full strength MS basal salts without pH ing and Tween 20 for 10 – 15 minutes.
(c) Without rinsing, insert the explants in to the medium. In fungal contamination, the addition of PPM™ to the medium is optional. However in bacterial contamination or mixed contamination, the addition of 0,05-0,2% PPM™ to the medium in the first month is essential. Don’t discard highly oxidized explants, approximately 50% will recover after 4-6 weeks.
9. To decontaminate “in culture” contaminated explants or plants (rescue treatment), the cultures shouldn’t be left visibly contaminated longer than one week.
Clean the material mechanically with the aid of a soft brush under stream of tap water. Shale / stir in a 50% PPM™ solution (diluted with sterile water) for 5 – 15 minutes. In bacterial contamination or mixed contamination, we recommend to lower the pH to the range of 2.8 – 3.2 by mixing 1:1 full strength 100%PPM™ with 0,6 g/l citric acid solution (use sterile water).
Without rinsing insert into a medium with 0,05-0,2% PPM™ for at least one month and keep the culture away from high light intensities for the first 10 days. As mentioned above, don’t discard oxidized explants. Wait 4 – 6 weeks.
In some cases the fungal or the bacterial spores are located deep within the explants beyond PPM™ reach. In those cases after the water soaking period, slice the explants along and then stir / shake in 50% PPM™ for 10 – 15 minutes.
THROUGH ALL THE ABOVE STERILIZATION PROCESSE(S), ASURED THAT PPM™ PROFUSLY REACHS THE ENTIRE EXPLANTS SURFACES.
Note:
For the first transfer following the sterilization with PPM™, we advise to insert the explants entirely into semi solid medium.
The 50% PPM™ solution can be reused approximately 10 times. The number of uses depends on the volume of the treated explants and the inoculum densities. Keeping the 50% PPM™ solution stored at 4º C will prolong its activity. If necessary we suggest to prepare two PPM™ solutions, one to disinfect endogenous contamination and second to disinfect “in culture” contamination. We recommend to filter the second solution through a 0,2 μm after each treatment. Filtration can be done in unsterile atmosphere and one filter can be reused for the entire duration of the solution.
In cases that the treatment with 50% PPM™ is still insufficient, full strength 100% PPM™ can be used. The treatment with 100% is similar to 50% PPM™ but the exposure time shouldn’t exceed 3 – 10 minutes.
10. To eliminate Agrobacterium:
After co-cultivation stage, rinse the leaf discs and deep (entirely)) the disks in a 100% PPM™ solution supplemented with full strength basal salts for approximately 2 minutes. Blot the discs between two sterile paper towels and place onto a medium supplemented with full strength of the commonly used antibiotics. After 3 weeks transfer to medium with solely PPM™ at 0,05 – 0,075%.
Advantages over Antibiotics:
1. PPM™ is broad-based and effective against fungi.
2. PPM™ is less expensive than antibiotics, making it affordable for wide and routine use.
3. Since PPM™ targets and inhibits multiple enzymes, the formation of resistant mutants towards PPM™ is very unlikely.
4. PPM™ is heat stable and in general can be autoclaved with media.
CONCLUSION:
PPM™ most definitely will facilitate the work in any plant tissue laboratory and should significantly increase technician and laboratory productivity. However, conditions in each lab may vary which could have a bearing on the effectiveness of PPM™.
It is advisable that staff follows the above guidelines and thus find out for themselves how much “freedom” they achieve by using PPM™ and whether or not PPM™ works for their particular application.
REFERENCES:
1. Randall P. Niedz HorTechnology 1998 8(4) 598-601
2. Sandra M. Reed J.Environ.Hort. 2000 18(1) 34-39
3. M. Babaoglu & M. Yorgancilar Plant Cell Tissue Organ Culture 2000 440: 31- 34
4. Compton and koch 2001 In vitro Cell Dev.Biol. – Plant 37: 259-261
Updated Instructions for PPM
For additional information /technical assistance, please contact:
Dr. Assaf Guri, Ph.D., Senior Vice President and Chief Scientist, Plant Cell Technology, Inc. Tel: 856 541 1141 Fax: 856-541-6967 E-mail: guri1@erols.com
SAFETY PROCEDURES:
Safety Issues: PPM™ is non-toxic, however, inhalation and contact with skin and eyes should be avoided since PPM™ is an acid.
PPM™ is non-toxic. It can be an irritant to skin, eyes, nose and throat. Precautions: We recommend that users wear gloves and splash goggles. Avoid contact with skin and eyes. Avoid inhalation. Use proper/adequate ventilation. The material should not be spray applied except with directed flow, positive pressure ventilation and protective equipment.
FIRST AID MEASURES:
Ingestion
If swallowed, give 2 glasses of water to drink. IMMEDIATELY see a physician. Never give anything by mouth to an unconscious person.
Eye and Skin Contact
IMMEDIATELY flush eyes with large amounts of water for at least 15 minutes. Wash affected skin areas thoroughly with soap and water. Remove and wash contaminated clothing thoroughly.
Inhalation
Move subject to fresh air.
DO NOT CONCENTRATE THE MATERIAL
DISPOSAL INFORMATION:
Dispose of media containing PPM™ in the same manner in which you dispose of media without PPM™.
In an emergency, contact one of the following numbers:
Dr. Assaf Guri 1 (856) 541-1141 or Martin Kalin: 1 (202) 463-0904 ext. 134 or ext. 0 (for operator). A toxicological assessment has been performed by a qualified toxicologist. This assessment is available upon request.
Patent n.5,750,402
