The scientific roots of modern plant biotechnology plant. Bacterial and fungal contaminants create a major problem in plant tissue culture laboratories. These may be plants that we have genetically altered in some way or may be plants of which we need many copies all exactly alike. Is contamination in your plant tissue cultures still worrying you. Standard light microscopy will also reveal bacterial cells and fungal structures, so daily microscopic observation of cultures will. Plant research often involves growing new plants in a controlled environment. Microorganisms infecting plant tissue culture leads to depletion of nutrients in the medium and reduces chances of survival of the plant culture. Nineteen microbial contaminants consisting of eleven bacteria and eight fungi were found associated with the tissue culture plants and the laboratory. Contamination of tissue culture medium the microorganism is. The file will be downloaded and can be opened with adobe acrobat reader. I want to sterilize my banana shoot tips with naocl in the initial stage of plant tissue culture in largescale. How to control fungal infection in plant tissue culture.
Although contamination remains a serious challenge for in vitro plant cell, tissue or organ culture researchers, over the past 25 years a large body of evidence has. In eds a taji and r williams proceedings of the vth international association for plant tissue culture australian branch conference. Nov 12, 2020 plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant totipotency. Fungal contamination may also arises from explants itself or air or during culture 31, 32, also associated to the indoor air, tableswalls, and human skin 33. There was no evidence of similar contamination in the continuous cell lines carried by the cell production laboratory, or in the routine control checks of tissue culture medium, serum, and trypsin.
The tissue culture medium contains all the compounds needed for plant growth, including mineral nutrients, sucrose, vitamins, and plant growth regulators that result in shoot or root production. Pdf a novel surface sterilization method for reducing. A protocol for endophytefree callus tissue of the grape. Apr 01, 2021 microbes are hurdle of plant tissue culture. Microbial hazards in plant tissue and cell cultures. Miconazole at concentrations between 5 and 20 mgl 1 inhibited hyphal growth and sporulation in a wide range of fungi commonly associated with plants. Micropropagation, plant tissue culture, bacterial and fungal isolates. Contamination from tissue contamination from the air contamination from the operator. All cell culture laboratories and cell culture workers have experienced it. The phytotoxicity of 20 mgl1 miconazole was also tested against a wide range of in vitro plant cultures. They are exogenously found in soil, water and plant surfaces cassels, 1996 and also as endophytes in some plant species suryanarayanan, et al. A shaker system formulation of tissue culture medium 1. Pdf curbing the menace of contamination in plant tissue culture.
Jan 01, 1973 contamination became apparent 57 days after subculturing from primary cells. Plant tissue culture has revolutionized the field of plant biotechnology. The various common and emerging technologies used for manual and rapid decontamination and sterilization 9. Possible sources of contamination and means of eliminating these contaminants is discussed.
Several possible sources of in vitro tissue culture contamination are culture containers, media, explants, equipment, the environment of the culture room and the transfer area. Isothiazolone biocides and sodium dichloroisocyanurate have been used singly and in combination to reduce microbial contamination in bud explants derived from greenhouse and fieldgrown citrus trees. Several culturists are now regularly using it in their culturing processes to prevent contamination in the cultures and ensure good health and growth of the explants. Contaminants of planttissue and cell cultures springerlink. Nov 06, 2019 contamination control and removal are very important technical aspects of microbiological research.
Rifampicin effectively controls bacterial contaminants without affecting the growth of shortterm explant cultures of helianthus tuberosus. Antibiotics are currently used but are not always effective, can alter plant growth, and are costly, and resistant strains can result with extensive use. Bacterial and fungal pollutants generate a main problem in plant tissue culture research laboratory. Tissue culture is a series of activities undertaken to make plan. These fungi included 4 oomycetes, 11 deuteromycetes, 4 ascomycetes and 4 common airborne contaminants of plant tissue culture. Tissue culture contamination frequently originates with the introduction into culture of explants contaminated with endophytic microorganisms or surface sterilisationresistant microorganisms e. Microbial contamination is one of the major challenges in plant tissue culture.
Fungal associations and improving micropropagation of native spp. Microbial contaminants of cultured cannabinus and telfaria. Fungal contaminationssummary table of invivogens antimicrobial agentsreferences 3 49 1011 12 14 15 15 a practical guide to understand cell culture contamination this comprehensive guide highlights the different categories of cell culture contaminants and how they can impact your research. Pdf control of contamination during micropropagation. Introduction no cell culture problem is as universal as that of culture loss due to contamination.
Bacteria, fungi, moulds and yeasts are common contaminating microorganisms in tissue culture. Replace the cover of the petri dish and wrap with parafilm. Micropropagation, plant tissue culture, bacterial and fungal isolates contamination. How to solve plant contamination tissue culture plant.
This study meant at exploring the sources of microbial contamination in tissue. Study of bacteria and fungi isolate from contaminated. Microbial contamination in tissue culture and micropropagation facilities, oftentimes, affect the tissue culture micropropagation media and the explantsplantlets. The plant tissue culture medium used was murashige and. Biotic contamination and possible ways of sterilization. The movement of plants also involves accidental risk of introducing plant disease. However, there are certain obstacles which overall restrain the output of. They were previously treated mainly as contaminants that needed to be eliminated to obtain a sterile tissue culture with healthy plant growth ewald et al.
Jan 05, 2015 bacterial contamination is a recurrent problem in moss tissue culture. Pl p 421 general mycology medium for growing cultures of agaricus, pleurotus, lentinus, stropharia, flammulina, and some of the psilocybe species. The fungal contamination was considered as most predominant factor in b. The contaminants bacteria and fungi will use nutrients from the medium and the plant, which quickly destroys the plant tissue. Common cell culture problems cell culture contamination.
Plant tissue culture and micropropagation, especially in commercial, largescale facilities, are affected by microbial e. A method for eliminating bacterial contamination from in. Any environment that can support life has its bacterial or fungal population. Microbial contaminants of cultured hibiscus cannabinus. Pdf sterile culture techniques arpita mahobia academia. Pdf common fungi contamination affecting tissuecultured. Biological contamination in plant cell cultures originates from two sources, namely, from the tissue used to initiate the culture, and from. Plant preservative mixture is a trademark product of plant cell technology pct that has been tested in the tissue culture of multiple plant species to avoid contamination. Avoidance or prevention of microbial contamination in plant tissue culture is critical. Some consequences of contamination loss of time, money, and effort adverse effects on the cultures inaccurate or erroneous experimental results loss of valuable products personal embarrassment the most obvious consequence of cell culture contamination is the loss of your time, money for cells, culture vessels.
A very useful list of antibiotics, the organisms they are effective against, and recommended working concentrations compiled by the sigmaaldrich company can be found as a pdf document 129kb, 3 pages by clicking on the following link. Controlling fungal and bacterial contamination of woody plant material can be extremely difficult. For plant tissue cultures, nonautoclavable vessels are suggested to be disinfected by the solution of 0. Fungal contaminants of plant tissue cultures have also been reported kane, 2003. Download adobe acrobat reader free software to read pdf f. Sterilization process of explants depends on the plant species and. Fungal contamination may also arises from explants itself or air or during culture.
Paper open access browning treatment in tissue culture of. A wide range of microorganisms filamentous fungi, yeasts, bacteria, viruses and viroids and microarthropods mites and thrips have been identified as contaminants in plant tissue cultures. Microbial contamination is a constant problem, which often compromise development of all in vitro techniques. Common types of tissue culture contamination plant cell. Here we describe an agarembedding method that is reliable, rapid, and relatively inexpensive, using materials that are readily available in most laboratories that conduct sterile tissue culture. Although plant tissue culture has been a successful method to propagate many species, v. Microbial contaminants in plant tissue culture propagation. Tissue culture is a long and laborious process and it feels vexing when fungus or bacteria attack our lovely cultures.
Plant tissue culture is an expensive method, so contamination frees cultures are need to be developed to get desirable profit. Introduction plant tissue culture micropropagation is a tool, which allows the rapid production of many genetically identical. Some cause human diseases such as typhoid fever, syphilis, athletes foot, tuberculosis, and leprosy, while. There several antibiotics were already reported by several authors to eliminate the endophytic contaminants during tissue culture of many plants. It was not until the early 1980s that research articles were predicting the use of the ti plasmid as a vehicle for transfer of genes of choice into plants. This study aimed at investigating the sources of microbial contamination in tissue culture laboratories in southwestern nigeria. For surface disinfection typically 70% alcohol, sodium hypochlorite, calcium hypochlorite, and mercuric chloride is used. Antibiotic agar aause for isolating fungi from substrates not readily surface sterilized, or to clean up a culture contaminated with. Bacterial contamination is very common in fungal cultures. Summary of media and common use water agar wause for isolating fungi from surfacesterilized substrates. Prevention of fungal contamination in plant tissue culture using. But microbial contamination of plant tissue culture is a common problem that leads to gap between demand and production.
Microbial contamination is one other challenge facing plant tissue culture practices. Contamination in tissue culture covers the sources, prevention, detection, and elimination of contamination in tissue culture. Composed of 12 chapters, the book describes the frequency of occurrence of contamination and the many different effects of contamination on cultured cells. Pdf philippines is the worlds largest producer of abaca fiber, accounting for about 85% share of the global production in 20. Miconazole at concentrations between 5 and 20 mgl1 inhibited hyphal growth and sporulation in a wide range of fungi commonly associated with plants. Contamination problems can be divided into 3 classes. Minor annoyanceswhen up to several plates or flasks are occasionally to contamination. Fungal contaminants of the oil palm tissue culture in nigerian. Contamination detection and elimination in plant cell culture. Among fungal contamination, presence of systematic fungal contamination is the most problematic issue of micropropagation of mature woody species 31. Pdf plant tissue culture contaminants identification and its. Molecular and microscopic identification of fungi in. Microbial contamination is one of the most important problems in plant tissue culture and various methods are employed to reduce it.
Isolation, characterization and identification of contaminant. A handy method to remove bacterial contamination from fungal. Sources of microbial contamination in tissue culture. A photo of fungus on cells used to illustrate contamination in cell culture. A frequently used method is the addition of antibiotics to the culture medium to suppress bacterial growth kneifel and leonhardt 1992, asif et al. Is contamination in your plant tissue cultures still. Controlling bacterial and fungal contamination in plant tissue cultures is a serious problem.
Serious problemswhen contamination frequency increases or entire experiments or cell culture are. Using isothiazolone biocides to control microbial and fungal. In plant tissue culture, small pieces of plant tissue are placed on or in a medium rich in nutrients and sugar. Agrobacterium, biotization, in vitro culture, meristem culture, pathogen elimination. Identification and management of microbial contaminants. These things can be accomplished through tissue culture of small tissue pieces from the plant of interest. Micropropagation, plant tissue culture, bacterial and fungal isolates, contamination. In the cases that the purpose of tissue culture is commercially amplification of plant, bacterial and fungal contamination can cause irreparable damages. Historical events in plant tissue culture basic requirements for tissue culture laboratory 1. Currently, the commonly used approach for inhibiting bacteria is antibiotic treatment. Dec 15, 2009 biological contamination in plant cell cultures originates from two sources, namely, from the tissue used to initiate the culture, and from the laboratory environment.
In fungal contamination, the addition of ppm to the medium is optional. Pdf plant cell and tissue culturea tool in biotechnology. Adding the biocide, plant preservativemixtureppm,0. Fungal contamination may also arises from explants itself or air or during culture 31, 32. Contaminants transferred in or on the plant material include plant pathogens and environmental microorganisms. The culture materials used were leaf explants, callusembroid and plantlet. Effect of nanoparticles on biological contamination of in. A fumigationbased surface sterilization approach for. Microorganisms and their reproductive structures e. These observations included but were not limited to. Google scholar cassells ac 1991 problems in tissue culture. Although some latest technology and information on modern plant biotechnology has been included in this volume. Effects of antimicrobial activity of silver nanoparticles. Understanding and managing cell culture contamination.
Composition of plant tissue culture media formulated by. Pdf plant tissue culture contaminants identification and. Mitigating risks of commercial plant tissue culture the utilization of plant tissue culture for commercial production is limited by two major risks viz. Is contamination in your plant tissue cultures still worrying.
For the control of bacterial contamination, the use of substances antagonistic to these. Although contamination remains a serious challenge for in vitro plant cell, tissue or organ culture researchers, over the past 25 years a large body of evidence has accumulated that the presence of microorganisms in plant in vitro cultures, while often harmful, may also, depending on the specific organism, have no effect on cultures or even be beneficial, presenting new opportunities for micropropagation or secondary metabolite production. This study reports a viable protocol for obtaining fungus free tissue culture callus. The phytotoxicity of 20 mgl 1 miconazole was also tested against a wide range of in vitro plant cultures. Cleanroom contamination including mouldfungal contamination. Contaminant may be introduced with the explant, during manipulations in the laboratory or by microarthropod vectors. Control of in vitro contamination of explants from greenhouse. There were also black spots, cottonlike substance as contaminated fungi. Introduction the practice of plant tissue culture has contributed towards the propagation of large number of plant from small pieces of stock plant in relatively short period of time daniel, 1998.
Plant culture tested pct naocl 4% in himedia is too costly 500 ml costs rs. B a c t e r i ol gy p f ra o s l a it n o r u issn. Fungal associations and improving micropropagation of. Pdf contaminants of planttissue and cell cultures carlo.
However, with bacterial or mixed contamination, the addition of 0. Bacterial, fungal including molds and yeast contamination are usually visible to the unaided eye as rapidonset turbidity and color change of the culture medium provided that the medium is supplemented with phenol red, the most common nontoxic ph indicator. Disinfecting effects of nano silver fluids in gerbera. Therefore, it is considered that the effective control of browning is the key to the success of plant tissue culture. Single cells, plant cells without cell walls protoplasts, pieces of leaves, stems or roots can often be used to generate a new plant on culture media given the required nutrients and plant hormones. Culture contaminants may be biological or chemical, seen or unseen, destructive or seemingly benign, but in all cases they adversely affect both the use of your cell cultures and the quality of your research. Standard light microscopy will also reveal bacterial cells and fungal structures. The isolated fungal contaminants found in this study have been frequently reported in plant tissue cultures msogoya et al. Microbial contaminants were isolated from banana cultures at sua plant tissue culture laboratory.
Most of these bacteria contaminants have been reported to increase culture mortality. Determination of the sources of microbial contaminants of. Bacterial contamination remains a continuing thrt to plant tissue culture, but techniques for reducing con,taminatton are available. Microbial contamination is one of the most serious problems of plant tissue culture. Bacterial and fungal contamination during node culture is a major cause for cultural failure. Tissue culture contamination and 7 easy steps of prevention. These things can be accomplished through tissue culture of small tissue pieces from the plant. Obviously not in 1902, 1907, or 1939, when the three founding articles in crown gall disease causation, single plant cell culture, and plant tissue culture, respectively, were published. If bacteria or fungi come in contact with the plant tissue or the medium, the culture becomes contaminated. Microbial contamination is an important barrier in the development of gerbera tissue culture and micropropagation procedure even after disinfecting with normal methods. In recent years, plant tissue culture technology has developed rapidly, and research on browning has become more and more. Our results indicate that ppm can be routinely added to tissue culture medium to control air and waterborne bacterial and fungal contaminants effectively. No cell culture problem is as universal as that of culture loss due to contamination.
Detection and control of bacterial contaminants of plant. Using isothiazolone biocides to control microbial and. Plant tissue culture can be initiated from almost any part of a plant however. Explants from greenhousegrown trees were effectively disinfested contamination using a standard disinfestation sd procedure followed by culture on medium containing 5 ml l. Plant tissue culture historical events in plant tissue culture basic requirements for tissue culture laboratory 1.
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