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Hildegard Crous -- Specialist Horticulturist, Cape Institute of Micropropagation
"I hope this is not too elaborate. It is a rather long, sad story, but with a happy ending.
Two years ago I started noticing a contamination in many of my cultures. My first concern was that the media was not being adequately steralised, or that the culture containers were not sealing properly. Both these concerns were laid to rest, and on closer inspection I noticed that the contamination was entering the culture vessel via the plant material. (A week after replating, a white "cloud" would develop around the roots of the plants and increase steadily in size. Eventually it would surface and grow as layer on top of the media. On opening the culture vessel, a sickly, sour smell was evident.)
After replacing all the HEPA filters in the laminar flow bench, and ensuring new un-used sterilized working surface for each replate, I started a rigorous rinsing programme of all the affected cultures. Many of the orchids I grow represent a genepool from as far back as 1996. I was anxious to not lose any if I could. New initiations were clean and were not becoming contaminated.
On replating, all plant material was rinsed in a solution of household bleach, (3,5%m/v), mixing 25ml bleach with 75ml water and adding another 5ml Dettol. After rinsing, the material was trimmed down, removing all outer layers. The plant material was then rinsed in a new solution mixed to the same strength as above and then given a final rinse in sterile water before being placed into a fresh culture vessel on new medium. This was done on average every 28 days, needless to say a very time consuming and costly excercise. In this way I managed to keep the contamination at bay, but I was steadily losing the battle. It is extremely demoralising to carry on desperately "cleaning" material, when you know its not going to work and that you will again be faced with the situation of either throwing out the material or trying to think of another method to stave off the contamination in order to rescue the plants. Although some of the plants continued to make offshoots, once these were harvested, they soon succumbed to the toxic effects of the contamination.
On two seperate occasions I sent a sample of the contamination to a pathology lab for identification, but in both cases nothing was forthcoming.
Although I was pretty sure that I had to do with a bacterial contamination, I was hesitant to apply antibiotics, as this can lead to mutation. Not much is known concerning the effect of antibiotics on disas (the orchids that I work with), and so I was hesitant to experiment with the material I was trying to save.
Somewhere in the depths of desperation I remember recieving a sample of PPM when I still worked in the tissue culture laboratory at Kirstenbosch Botanic Garden. I didn't have a need for it then, and therefore not tried it our, but on finding it on the internet I was intrigued by the accounts given by various people who had used it. I managed to obtain a 200ml sample from within South Africa. By this time I was desperate, and time was of the essence. I started incorporating it in all my media and started rinsing all my most desperate cases in the recommended PPM and citric acid solution. And then I waited. This was Dec 2005.
I am happy to say that I can now walk into my growth room with confidence and admire the lovely strong, green growth on all the plants. Although the contamination is still slightly visible on the larger, older plants, all the progeny that we are harvesting are culturing pure and clean. For some rhyme or reason the smaller the plant volume, the more effective PPM is. In the cases where the contamination was serious, I am finding that the contamination remains encased around the affected plant material and plants around it can be safely removed and cultured on."
Julie Richards - Research Officer, Crop Biology Resource Centre, South African Sugarcane Research Institute
Use of PPM in the SASRI Tissue Culture Laboratory
"The tissue culture laboratory at the South African Sugarcane Research Institute (SASRI) at Mount Edgecombe, KwaZulu Natal, South Africa uses tissue culture for both the transformation and micropropagation of sugarcane. While microbial contamination levels are generally low enough to be acceptable, we have experienced instances where contamination levels rise and become problematic. Such instances are generally attributed to changing environmental conditions.
We initially started using PPM primarily as a precautionary measure during the initiation of sugarcane somatic embryo cultures. Explant material consists of immature leaf roll tissue from the apical region of the sugarcane plant. The outermost leaves are aseptically removed, and the young, innermost, tightly furled leaves are sliced into transverse sections approximately 1.5mm thick. The explants were initially sliced “dry” (i.e. on a sterile glass plate), however this method resulted in the relatively high production of phenolics, which often inhibited the explant growth. We therefore modified our approach to slicing the explants in liquid medium (the same MS-based formulation as the semi-solid embryo-initiation medium, minus the agar). A small volume of liquid medium is poured into a petri dish, the leaf roll is placed in the liquid medium and the explants sliced off. These are then transferred to the semi-solid embryo-initiation medium. This approach resulted in the significant reduction in phenolic production. However, a drawback to slicing the explants in liquid medium is that if one or two explants from a leaf roll are contaminated, the risk exists for that contamination to spread to the remaining explants. To overcome this, the liquid medium is supplemented with 0.2% PPM, and our microbial contamination levels are subsequently very low.
A recent problem we have experienced involves the transformation of sugarcane tissue, more specifically, the pre-emergent floral initials. Since flowering of sugarcane in the field under South African environmental conditions can be unpredictable, sugarcane plants in this particular instance were induced to flower by growing them in specific conditions where longer day-lengths and elevated temperatures were controlled in a growth room. During the week of culture prior to transformation, no contamination of the pre-emergent inflorescent tissue, which was placed on semi-solid embryo-initiation medium, was observed. During the week following the transformation, however, a specific bacterial contaminant frequently emerged from within the tissue. This contaminant was not observed when pre-emergent inflorescent material from field-grown sugarcane was transformed. It was hypothesized that the elevated temperature and humidity inside the growth room encouraged the growth of the observed contaminant in the region of the inflorescence tissue. This contaminant remained latent during the initial culture period. However, the stressful conditions of transformation (including biolistic gene delivery and a short period of exposing the explants to culture medium with high osmotic pressure) caused the manifestation of the contaminant. A high proportion of potentially transformed explants were lost due to this specific contaminant. This problem was effectively brought under control with the use of PPM. According to our transformation protocol, four hours prior to transformation explants are placed on a high osmotic pressure medium. PPM (at a concentration of 25%) was either pipetted directly onto the explants on the osmotic medium, or the PPM was incorporated into the osmotic medium at a concentration of 1% (the latter method is easier when large numbers of transformations are to be carried out). Following biolistic transformation and a further four hours on the osmotic medium the explants are transferred to selection medium, which was supplemented with 1% PPM. Following this regime, the bacterial contamination of the transformed tissue was eliminated.
The third case where we use PPM in our laboratory is with the micropropagation of sugarcane plants in liquid medium. The system used for this involves a temporary immersion routine using commercially available RITA® vessels. With this system, 30 explants obtained from one sugarcane leaf roll are pooled into one vessel. A major disadvantage of this technique is that if one or two of those explants are contaminated, the liquid medium in the vessel rapidly becomes contaminated and all 30 explants are lost. A preliminary culture period of one week on semi-solid medium can assist with the detection of contamination, but often the contamination is latent or slow growing and only manifests later in the culturing regime. To reduce contamination levels in the liquid culture vessels, between 0.75 and 1% PPM is added to the liquid growth medium. Preliminary studies showed that concentrations of PPM greater than 25% are phytotoxic to sugarcane tissue, however concentrations of about 1% are quite adequate to satisfactorily reduce microbial contamination in the RITA® liquid culture system.
The use of PPM in the above instances has proven very successful in controlling the microbial contamination levels in our tissue culture programme. Furthermore, with the development of new techniques and protocols, it is reasonable to assume that additional problems with contamination are likely to be encountered. PPM will therefore continue to be stocked as a standard consumable item in our laboratory."
Michael Tanabe - Professor of Plant Science, U. of Hawaii at Hilo, College of Agriculture, Forestry and Natural Resource Management
"My responsibilities at UHH-CAFNRM include directing a Certificate of Achievement in Plant Tissue Culture program, teaching 4 courses in the area of plant tissue culture and plant propagation and conducting research pertaining to micropropagation of especially tropical and subtropical plants.
Contamination is frequently the greatest concern for many labs in the State of Hawaii. Our year-round comfortable weather is great for attracting tourists but also for growth of microbes. As such, the plant TC lab is frequently challenged to develop disinfection protocols that maintain asepsis from initial explanting to growth in the culture growth room. This problem is compounded in the teaching environment due to the broad range of techniques and attitudes that the students bring to the lab.
I decided to try [Plant Preservative Mixture] as a medium-incorporated biocide several years ago. Initially, I conducted a simple air test using FS M&S and nutrient agar with and without the recommended level of PPM. The media combinations were sterilized and exposed to the open air environment for 3 hours. Absolutely no growth of microbes was evident after the cultures were incubated at 29° C for 4 days. I was convinced that PPM had biocide activity so I incorporated it into culture media for various tropical plants to determine possible phytotoxic effects. No obvious phytotoxic effects such as chlorosis or necrosis were observed. Additionally, contamination from airborne contaminants in the culture storage room was significantly reduced."