PPM Product Information PPM TM - Plant Preservative Mixture
FOR RESEARCH USE ONLY
Not for use in diagnostic procedures
Product Name：PPM TM- Plant Preservative Mixture
Product Number：PPM
Storage Conditions：4º C
Physical Properties：Solution
Appearance: Clear, colorless to amber
pH: 3.8
Expiration Date: See label on the bottle
Product Description：
1. PPM is a heat stable preservative/biocide which can be used to effectively prevent or reduces microbial contamination in plant tissue culture.
2. At optimum doses, PPMT TM, 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.windowblinds 6
上海大学bbs3. 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.
4. PPMT TM prevents the germination of both bacteria and fungi spores. It is heat stable and
therefore can be autoclaved with the media.
5. PPMT TM 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.
6. While PPMT TM 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.
Mechanism of action:
1. PPMT TM 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.
2. The active ingredients of PPMT TM penetrate the fungus or the bacterium cell wall and inhibit the
activity of key enzymes within the central metabolic cycles such as the citric acid cycle and the electron transport chain.
3. PPMT TM may also inhibit the transport of monosaccharides and amino acids from the medium into the fungus or bacterium cells.
4. As in any biocide, a critical ratio of PPMT'" molecules per microbial cell is needed to eliminate bacteria and fungi. Keep in mind that a given volume of PPMT"' dose has a constant number of PPMT TM 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 for free movement of the solution around the explant material.
Advantages of PPM over antibiotics:
1. PPMT TM is broad ‐based and effective against fungi.
2. PPMT TM is less expensive than antibiotics, making it affordable for wide and routine use.
3. Since PPMT"' targets and inhibits multiple enzymes, the formation of resistant mutants towards PPMT TM is very unlikely.
4. PPMT TM is heat stable and in general can be autoclaved with media.
General Usage:
The instructions listed below are for general use. Optimization may be required. 0.05%.
Higher concentrations
may result in toxicity or
uptake. with other sterilization techniques. Do not adjust pH.
Treated tissues should be
Protocols:
The procedures described below are generic. Slight modifications might be needed for your specific plant species. For assistance, contact Dr. Assaf Guri at guri1@erols
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 filter sterilized 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 less effective when exposed to high density of bacteria or fungi spores found regularly on seed's coat. For in vitro germination, seeds should be conventionally surface sterilized with EPA registered 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 autoclave or by use of an electric heating element.
5. 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%.) To eliminate higher endogenous contamination densities, higher doses of PPM are needed (see paragraph 6 below).
6. Endogenous Contamination: (a) For explants: 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.
Note
Paragraphs 6(b) through 10 below are intended for ornamental plants only.
(b) For tubers, bulbs and scales: shake / stir the entire tuber / bulb / scale in bleach. Rinse with water (can be done under non-sterile conditions). Slice the tuber / bulb / scale to thin slices. Shake / stir for 12-24 hours in 4 - 5% PPM™ solution supplemented with full strength basal salts without pH ing and Tween 20. Without rinsing, insert into a medium supplemented with 0.1 - 0.2% PPM™.
7. In cases where the above protocols do no yield satisfying results (especially thick explants, highly infested explants, seeds), we recommend the following:
(a) Shake / stir the explants in water (1hr for soft tissues and 2 hr for hard tissues).
(b) Shake / stir the explants in (50%) PPM™ supplemented with full strength MS basal salts (without pH ing and without Tween 20) for 5 -10 minutes.
(c) Without rinsing, insert the explants into the medium. In fungal contamination, the addition of PPM™ to the medium is optional. However, with bacterial or mixed contamination, the addition of 0.05 - 0.2% PPM™ to the medium in the first month is essential. Do not discard highly oxidized explants as approximately 50% of the explants will recover within 4 - 6 weeks.
Note
Refer to notes 2 and 3 below.
8. To decontaminate "in culture" contaminated plant material (rescue treatment):
(Note: The culture should not be left visibly contaminated longer than one week.)
(a) Clean the material mechanically using a soft tooth-brush under a stream of tap water. Shake / stir in a 50% PPM™ solution (diluted with sterile water) for 5 - 15 minutes. For bacterial or mixed contamination we recommend to lower the solution pH to the range of 2.8 - 3.2 by mixing 1:1 full strength PPM™ (100%) with 0.6 gr./liter Citric acid solution (use sterile water).
(b) Without rinsing insert into a medium with 0.05 - 0.2% PPM™ for at least one month. 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 as approximately 50% should recover.
In some cases the fungal or the bacterial spores are located deep within the ex-plants beyond PPM's reach. In such cases, and after the water-soaking period, slice the ex-plants along and then stir/shake in 50% PPM for 5 -15 minutes.
THROUGH ALL THE ABOVE STERILIZATION PROCESSE(S), ENSURE THAT THE PPM PROFUSELY REACHES THE ENTIRE SURFACE OF THE EX-PLANT.
9. To eliminate Agrobacterium:
After co-cultivation, rinse the leaf discs with water. Dip (entirely) the transfected discs 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
白细胞介素1
full-strength of the commonly used antibiotics. After 3 weeks, transfer to the medium with
solely PPM at 0.05 0.075%
General Notes:
1. For the first transfer following the sterilization with PPM™, we recommend to insert the explants entirely into a semi-solid medium.
2. The 50% PPM™ solution can be reused but is not recommended. The number of uses depends on the volume of the explants treated and the inoculum density. Keeping the 50% PPM™ solution stored at 4ºC prolongs its activity. If necessary, prepare two PPM™ solutions: one to disinfect endogenous contamination and the second, to disinfect "in-culture" contamination. The second solution should be filtered after each treatment, using 0.2 micrometer Millipore. The filtration process can be done in non-sterile atmosphere. A single filter can be used for the entire "lifespan" of the solution.
3. In cases where the treatment with 50% PPM™ is still insufficient, full strength PPM™ (100%) can be used. The treatment with 100% PPM™ is similar to the one described above for 50% PPM™, however, the exposure time should not exceed 10 minutes.
References:
1. Miyazaki J, Tan BH, Errington SG. Eradication of endophytic bacteria via treatment for axillary buds of Petunia hybrida using Plant Preservative Mixture (PPM TM). PCTOC. 2010;102(3):365-37
2.
2. Miyazaki J, Tan BH, Errington SG, Kuo JS. Bacterial endophyte in Macropidia fuliginosa: its localisation and eradication from in vitro cultured basal-stem callus. Aust J Bot. 2011;59(4):363-368.
3. Lata H, Chandra S, Khan IA, ElSohly MA. Propagation through alginate encapsulation of axillary buds of Cannabis sativa L. — an important medicinal plant. Phys and Mol Biol of Plants. 2009;15(1):79-86.
4. Greer SP, Rinehart TA. Dormancy and Germination In Vitro Response of Hydrangea macrophylla and Hydrangea paniculata Seed to Light, Cold-Treatment and Gibberellic Acid. J. Environ. Hort.
2010;28(1):41–47.
5. Moghaddam S, et al. Optimization of an Efficient Semi-Solid Culture Protocol for Sterilization and Plant Regeneration of Centella asiatica (L.) as a Medicinal Herb. Molecules. 2011;16(11): 8981-8991
6. Çolgecen H, Koca U, Toker G. Infl uence of diff erent sterilization methods on callus initiation and production of pigmented callus in Arnebia densifl ora Ledeb. Turk J Biol. 2011; 35:513-520
7. Pouvreau J, et al. A high-throughput seed germination assay for root parasitic plants. Plant Methods 2013;9:32
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边缘系统8. Marecik R, Bialas W, Cyplik P, Lawniczak L, Chrzanowski L. Phytoremediation Potential of Three Wetland Plant Species Toward Atrazine in Environmentally Relevant Concentrations. Pol. J. Environ. Stud. 2012;21(3):697-702
9. Pérez Flores J, Aguilar Vega ME, Roca Tripepi R. Assays for the in vitro establishment of Swietenia macrophylla and Cedrela odorata. Rev. Colomb. Biotecnol. 2012;14(1)
10. Nesterenko-Malkovskaya A, Kirzhner F, Zimmels Y, Armon R. Eichhornia crassipes capability to remove naphthalene from wastewater in the absence of bacteria. Chemosphere. 2012;87(10):1186-1191.
11. Kieffer M, Fuller MP. In Vitro Propagation of Cauliflower Using Curd Microexplants. Meth Mol Biol. 2013;994:329-339.
12. Kodym A, Temsch E, Bunn E, Delpratt J. Ploidy stability of somatic embryo-derived plants in two ecological keystone sedge species (Lepidosperma laterale and L. concavum, Cyperaceae). Aust J B
ot. 2012;60(5):396-404.
13. Peña-Ramírez YJ, et al. Induction of somatic embryogenesis and plant regeneration in the tropical timber tree Spanish red cedar [Cedrela odorata L. (Meliaceae)]. PCTOC. 2011;105(2):203-209.
14. Haddadi F, Adb Aziz M, Saleh G. Abd Rashid A, Kamaladini H. Micropropagation of Strawberry cv. Camarosa: Prolific Shoot Regeneration from In Vitro Shoot Tips Using Thidiazuron with
N6-benzylamino-purine. HortScience. 2010;45(3):453-456.
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15. Jimenez VM, Castillo J, Tavares E, Guevara E, Montiel M. In vitro propagation of the neotropical giant bamboo, Guadua angustifolia Kunth, through axillary shoot proliferation. PCTOC. 2006;86:389–395.
16. Compton ME, Koch JM. Influence of Plant Preservative Mixture (PPM) on adventitous organogenesis in Melon, Petunia, and Tobacco. In Vitro Cell. Dev. Biol.- Plant. 2001;37:259-261
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