Introduction
PPM ™ is a thermoset preservative/biocide that can be used to effectively prevent or reduce microbial contamination in plant tissue cultures. In optimal doses, PPM ™, which stands for Plant Preservative Mixture ™, is an extremely effective preservative/biocide that does not harm in vitro seed germination, callus proliferation and regeneration. Despite the strictest use of sterile techniques, contamination of plant cells and plant tissue cultures remains a persistent problem that can result in losses ranging from a small number of cultures to complete batches.
PPM ™ can and should be used as a standard ingredient in plant tissue culture media. It is substantially less expensive and potentially less dangerous than commonly used antibiotics. PPM ™ was designed primarily to inhibit air, water, and human contact-induced pollution. However, in many cases, it can also be used to reduce endogenous contamination.
Mechanism of action
PPM ™ is a broad spectrum preservative and biocide, which kills bacteria and fungi cells, prevent spore germination, and in higher concentrations, it can eliminate endogenous contamination of explants. It is heat stable and therefore can autoclave.
Previous research has shown that the active ingredients in PPM ™ penetrate the cells of fungi or bacteria and inhibit key enzymes in central metabolic cycles such as the citric acid cycle and the electron transport chain. Our data indicate that PPM ™ can also inhibit the transport of monosaccharides and amino acids from the medium in fungal or bacterial cells.
As with any biocide, a critical ratio of PPM ™ molecules per microbial cell is required to eliminate bacteria and fungi. Note that a given volume of PPM ™ doses has a constant number of PPM ™ molecules, while the number of spores introduced into tissue culture through endogenous contamination is highly varied. Therefore, explants must not be “squeezed” into a beaker. There must be enough volume to move freely of the solution around the explanted material.
Advantages of PPM over antibiotics
1. PPM ™ is broad-based and effective against fungi.
2. PPM ™ is less expensive than antibiotics, making it affordable for routine and extensive use.
3. Since PPM ™ targets and inhibits multiple enzymes, the formation of resistant mutants towards PPM ™ is highly unlikely.
4. PPM ™ is heat stable and can generally be autoclaved with media.
Procedures
1. Media containing PPM ™ can be dispensed outside of laminar flow hoods (LFH) under ambient conditions [however, the use of orchid hoods is recommended in laboratories]. Culture plates and containers should be covered shortly after the agar solidification. When dispensing media with pumps, PCT recommends passing Autoclave hot water through the hoses before and after dispensing the medium.
2. Heat sensitive or thermoset liquid media containing PPM ™ do not need to be sterilized by passing through Millipore filters or autoclaved provided that it should be stored in sterile containers and that the stock solutions are not contaminated. In rich media containing 200 mg l – 1 or more of amino acids or proteins, it is recommended to filter and sterilize the medium with PPM ™ [as few orchids the media is so rich that this should not be necessary].
3. When working on LFH, it is not necessary to flare tools (forceps or scalpels) [to use with orchids, flaming is recommended]. They should be periodically immersed in 70% alcohol. The LFH does not need to be certified [LFH used with orchids does not require certification anyway]. Work can be performed outside of LFH on a clean surface for a period not exceeding 1 h.
4. PPM ™ is an acidic liquid solution (pH 3.8) that should be stored at 4 ° C (see safety information below). At the recommended dose of 0.05 to 0.2% (v / v), PPM ™ was added to the medium before or after autoclaving to avoid air and endogenous contamination at low inoculum densities.
5. PPM ™ is less effective when exposed to high densities of bacteria or fungi spores that can be found in the seed coats. For in vitro germination, the seeds must surface conventionally sterilized with an EPA registered bleach [or other methods used for orchid seeds]. Therefore, in the presence of PPM ™ (in germination medium), the seeds can be rinsed under tap water in a non-sterile strainer and let dry, preferably in the LFH [see below for methods to use with orchid seeds]. Yes, the ends of the utensil have touched the culture of active bacteria or fungi or are suspected of being contaminated, should be sterilized in an autoclave or by using an electric heating element.
6. General dose levels: With the exception of endogenous contamination, the recommended dose range is 0.05 to 0.2%. For callus proliferation, organogenesis and embryogenesis, the recommended range is 0.05–0.075% [different concentrations may have to be used for orchids; look down]. Add PPM mid pre or post autoclave sterilization to avoid endogenous and airborne contamination with low inoculum densities or slow-growing bacteria. To remove higher endogenous contamination densities, higher doses of PPM are needed (see point 7 below).
7. Endogenous contamination:
(a) Stir the unsterilized seeds for 8 to 12 hours in a 2 to 3% (v / v) PPM ™ solution supplemented with undiluted basal salts of the usual medium. Not to do add Tween-20 or pH to this solution. Later, and without clarifying, transfer to germination medium supplemented with 0.05–0.1% PPM ™ to herbaceous plants and with 0.2% PPM ™ for woody plants. Hard coating seeds (eg, asparagus, lupins, ornamental palms, roses, etc.) should be soaked in water for 2 to 4 hours before sterilization with PPM ™ [different procedures may it has to be used for orchids; look down].
(b) Shake/stir gently and routinely 1 cm long (or shorter) explants for 4 to 12 h at 4–5% v / v PPM ™ solution supplemented as above with full concentration basal MS salts [or orchid media salts] without adjusting the pH and without Tween-20. Without rinsing, insert explants into medium supplemented with 0.05–0.1% PPM ™ for herbaceous plants and 0.2% PPM ™ for woody plants [different procedures may have to be used with orchids; look down].
(c) 2 For tubers, bulbs and scales: shake/stir the entire tuber/bulb/scale in bleach. Rinse with water (can be performed in non-sterile conditions). Cut the tuber/bulb/scale thinly sliced. Shake/stir for 12 to 24 h in a 4-5% PPM ™ solution supplemented with unadjusted total concentration basal salts and Tween-20. Without rinsing, insert into medium supplemented with 0.1–0.2% PPM ™ [Different procedures may need to be used with orchids; look down].
8. In cases where the above protocols do not produce satisfactory results (especially explants, highly infested explants, seeds), we recommend the following:
(a) Shake/swirl the explants in water (1 hr for soft tissues and 2 hr for hard tissues).
(b) Shake/swirl explants in (50%) PPM ™ supplemented with full strength MS basal salts (without pH-ing and without Tween-20) for 5 to 10 min.
(c) Without rinsing, insert the explants into the medium. In cases of fungal contamination, the addition of PPM ™ to the medium is optional. However, with bacterial or mixed contamination, adding 0.05–0.2% PPM ™ to the medium during the first month is essential. Do not dispose of highly oxidized explants since approximately 50% of the explants will recover in 4-6 weeks. [Different procedures may need to be used with orchids; look down].
9 To decontaminate contaminated crops use the following rescue treatment (crops must not be left visibly contaminated for more than 1 week):
(a) Clean the material mechanically with a soft toothbrush under running water. Shake/stir in a 50% PPM ™ solution (diluted with sterile water) for 5-15 min. For bacterial or mixed contamination, we recommend reducing the pH of the solution in a range of 2.8 to 3.2 mixing it 1: 1 (v / v) PPM ™ of full concentration (100%) with 0.6 g l – 1 of citric acid solution in sterile water.
(b) Without rinsing, insert tissues into 0.05–0.2% PPM ™ medium for at least 1 month. Keep the crop away from high light intensity for the first time in 10 days. As mentioned above, do not discard the rusted explants. Wait 4-6 weeks since about 50% should be recovered.
10. In some cases, fungal or bacterial spores are found deep within the explants out of reach of PPM. In such cases, and after immersion in water period, cut the explants and then stir/shake in 50% PPM for 5 to 15 min [different
orchid procedures may be necessary]. For this decontamination procedure to be effective, it is important to ensure that the tissues are completely bathed in the PPM solution.