QUALITY ASSURANCE SYSTEMS FOR PLANT CELL AND _ TISSUE CULTURE; THE PROBLEM OF LATENT PERSISTENCE OF BACTERIAL PATHOGENS AND AGROBACTERIUM-BASED TRANSFORMATION VECTOR SYSTEMS C. Leifert Aberdeen University Centre for Organic Agriculture (AUCOA), MacRobert Building, King Street, Aberdeen AB24 SUA E-mail: c.leifert@abdn.ac.uk Abstract Contaminants may be introduced with the explant or during manipulations in the laboratory or by animal vectors (Leifert et al., 1994a; 1991a; Berger et al., 1992) Contaminants may express themselves immediately or can remain latent for long periods of time (Leifert et al., 1994a; Leifert and Waites, 1992; Cooke et al., 1992). This often makes it difficult to identify the source of contamination. Itis extremely difficult to eliminate bacterial and fungal pathogens from established plant tissue cultures using antibiotics, fungicides and other biocides. In many cases treatments only inhibit contaminants and low levels of contamination persist. In particular, the use of antibiotics against Gram negative bacteria (including Agrobacterium tumefaciens vector systems used in genetic engineering) was frequently shown to be extremely difficult or unsuccessful. Hazard Analysis Critical Control Point (HACCP) systems are therefore now used in many commercial plant tissue culture and micropropagation laboratories (Leifert and Woodward, 1998; Leifert et al., 1994a) which aim at preventing contamination at sources. Detection of latent contamination may involve the use of general and semi-selective microbial growth media or serological and PCR-based molecular techniques for specific pathogens. It is often impossible to detect low numbers of latent bacterial contaminants (c.g. levels present following antibiotic treatment or when acidified plant media are used) This poses a particular risk in the production of transgenic plants where neither the elimination or detection of Agrobacterium tumefaciens-based vector systems can be guaranteed with the currently available methodologies: 1.___Introduction Microbial contamination is one of the most serious problems of plant cells and tissue culture and the problem has been reviewed in 3 previous conferences in Cork, Ireland, in 1987, 1997 and 1999 (Cassels 1988; 1997; 1999). Our understanding of the identity of contaminants and the sources and epidemiology of contamination has increased considerably during this time (Leifert er al., 1994a). Also. microbiological quality assurance (HACCP) ‘systems which aim at preventing contamination have successfully been introduced into larger scale commercial micropropagation and cell culture systems (Leifert and Waites, 1994; Leifert et al., 1998). However two principle problems remain: (i) the detection of latent bacterial contaminants (including plant pathogens and Agrobacterium tumefaciens based plant transformation vectors) (ii) the elimination of fungal and bacterial contamination from established plant tissue cultures (including Agrobacterium tumefaciens based plant transformation vectors) This review will therefore briefly introduce the reader to recent reviews describing the identity, sources and epidemiology of contamination and HACCP-based microbial ros nt Symp. on Meth and Mis ‘for Qual, Assur. in Micropropagation 87 ELA Cau Bogle RE Comy ‘eta Hor 30 iis 200,quality assurance schemes designed to prevent contamination. However, it will in more detail describe the problem of assuring the absence of latent microbial contaminants and Agrobacterium vector systems which are used in genetic engineering Identity, sources and epidemiology of contamination Contaminants may be introduced with the explant or during manipulations in the laboratory or by animal vectors (Leifert ef al., 1994a; 1991a; Berger ef al., 1992). Gram negative bacteria such as Pseudomonas (in particular Ps. fluorescens), Erwinia and Agrobacterium spp. are usually found when the initial disinfection procedure is inefficient, Gram positive species on the other hand indicate inefficient sterilisation of media (Bacillus spp.) or inadequate training of operators in aseptic techniques (Staphylococcus spp.). Descriptions of the different species of contaminants found in plant tissue and cell cultures and information on the sources for specific contaminants can be found in Leifert er al. (1988a; 1990; 1991a; 1994) and Danby er al. (1994). Contaminants may express themselves immediately after introduction into plant tissue culture or can remain latent (not producing symptoms on plants and/or visible growth on plant growth media) for long periods of time. Fungal contaminants rarely remain latent in vitro because plant tissue culture media provide all essential nutrients required for growth of most fungi. Bacteria on the other hand often require growth factors/vitamins which are not present in plant tissue culture media (most bacterial species tested cannot grow or persist in plant tissue culture media in the absence of plant materials) (Leifert and Waites, 1992) Latent contamination reduces the reliability of plant cell/tissue culture systems, since even small changes in the environmental conditions (temperature, pH, medium composition, or transfer to the glasshouse) may allow rapid proliferation of the contaminant. This can result in reduced plant growth and/or rooting or kill of plant cells/tissues in vitro or the expression of disease pathogens after weaning of micropropagated plants, (Leifert & Waites, 1992; Cooke ef al., 1992). Additional growth factors for bacterial growth are thought to be provided by exudates from the plant cells or ues making the growth of bacterial contaminants closely dependent on the plant tissue itself (Leifert er al, 1994a; Leifert & Waites, 1992). Bacterial growth in plant tissue cultures is also closely dependent on the incubation temperature. Temperature regimes used for plant tissue culture are often lower (<25 °C) then those required for maximum growth of bacterial contaminants (235 °C). Latent bacterial contaminants can therefore become virulent (producing visible symptoms on plants and/or visible growth in the culture medium) when the culture environment is changed, For example, growing of plants at higher densities or transfer onto rooting media may increase the concentration of plant exudates, resulting in stimulation of bacterial growth. Similarly, a rise in growth room temperature may result in both increased exudation caused by high temperature stress and more ideal growth temperatures for bacteria (Leifert & Woodward, 1997a, Leifert ef al, 1994a), Initially, it was thought that only so called ‘vitropath’ (bacteria which may harm plants ii vitro, but do not cause disease in plants growing in natural or agricultural habitats in vivo) remain latent in vitro. More recently it was realised that most bacterial pathogens (bacterial which cause disease in plants growing in natural or agricultural habitats ‘in vivo") may also stay latent in vitro (Cooke ef al., 1992). Most significantly Agrobacterium tumefaciens, a bacterium which is the most commonly used vector system used in genetic engineering was shown to persist latent in vitro, but cause disease when plants are transferred to in vivo conditions on the nursery. This clearly demonstrated the potential risk, that Agrobacterium based vector systems may be released into the environment, if the contaminant is not eliminated or contaminated plants detected and discarded prior to the weaning of transformed tissue cultured plants. 883 Microbiological quality assurance (HACCP) system: Hazard Analysis Critical Control Point (HACCP) systems are used in many commercial plant tissue culture and micropropagation laboratories. These systems are based on: (i) an in depth analysis of all potential contamination sources, (ii) the establishment of monitoring system for all contamination sources (=critical control points or CCP’s), (iii) the improvement of methods for preventing contamination at the different CCP’s and (iv) the development of treatment methods (in case preventative strategies fail) The HACCP principles and their application to plant tissue and cell culture systems have been reviewed extensively in the past (Leifert & Woodward, 1998; Leifert and Waites, 1994; Leifert ef al., 1994a) and are therefore not described in detail here. 4. Removal of contamination with antimicrobials Preventative quality assurance (HACCP) systems were frequently shown to be the more efficient approach for contamination management, than strategies based on treatment of contamination in established tissue and cell culture with antibiotics and other biocides. Even broad-spectrum antibacterial and antifungal compounds fail to eliminate al] target organisms and often have negative side effects on the growth or rooting of plant tissue cultures (Leifert et al., 1991b and c; 1994b). Often bacterial growth is only suppressed (bacteriostatic effect) by antimicrobial treatment and when antimicrobials are removed the bacteria regrow. In particular, Gram negative bacteria, are extremely difficult to eradicate completely from in vitro plant cultures (Leifert et al., 1991b). The use of medium acidification was shown to prevent the establishment and/or proliferation of bacterial contaminants in plant tissue cultures and may provide a cheap alternative to antibiotics for certain in vitro systems (Leifert et al., 1994b). However, similar to antibiotic treatments, it may only suppress the growth of bacteria rather then eliminate them completely. There were many reports of failure to eliminate Agrobacterium tumefaciens based vector systems from tissue cultures of genetically modified plants (e.g. Martin et al., 1990, Barrett et al., 1997; Hammerschlag et al., 1999). It was shown to be extremely difficult to eliminate Gram-negative bacteria (such as A. tumefaciens), which are more resistant to many antibiotics, from plant tissue cultures (Leifert et al., 1994a, Leifert et al., 1991b). Also, although contaminants could not be detected by visual examination and indexing (placing of plant tissue samples into sterility test media), contaminants were found to be still present and could be detected after plants were transferred onto antibiotic-free media (Leifert et al., 1994a). Even when antibiotic treatment resulted in the successful removal of specific contaminants treatment was only successful for a certain proportion of treated plants. As a result individual plants had to be separated (to avoid cross-contamination by plants which remained infected during subculturing) and regularly tested for the presence of contaminants for at least 3 months after antibiotic treatment (to ensure the absence of latent contaminants) (Leifert ef al., 1994a). This data clearly showed that the sterility test media used were unable to detect very low numbers of bacterial contaminants and/or bacteria which were inhibited by antibiotic treatments. Barrett et al. (1997) reported the failure to eliminate Agrobacterium strain LBA4404 the binary vector PBI121 from potato, Brassica and Rubus tissue cultures, in fact they reported that microbiological testing revealed that contamination levels in explants increased from 12 to 16 weeks in potato cultures. Barrett ef al. (1997) reported that very few laboratories “actually test (using microbiological sterility test media) to ensure that the antibiotics succeed” in the elimination of Agrobacterium vector systems. In similar experiments Aster plants contaminated with a pathogenic strain of 89Agrobacterium tumefaciens were transferred onto acidified (pH_ 3.5) or antibiotic containing media (200 yg ml"! carbenicillin, cefotaxime or a combination of 50 jig mI" of gentamicin 200 ig ml! carbenicillin) contaminants could not be detected by indexing while plants grew on acidified or antibiotic containing media. In the gentamycin and carbenicillin treated plants all plants remained index negative (A. tumefaciens could not be detected by the indexing method used). In carbenicillin and cefotaxime treated plants between 86 and 100 % of plants remained index negative during growth on antibiotic containing media. Following transfer onto high pH (pH 5.3) and antibiotic free media, Aster cultures became index positive (A. tumefaciens could be detected by the indexing methods used) within 2 (plants grown on low pH media and carbenicillin or cefotaxime amended media) or 3 subcultures (Cooke, D.L., Camotta, H.M.S. and Leifert, C unpublished) This clearly indicates that antibiotic treatment itself may result in the latent persistence of Agrobacterium tumefaciens and failure to detect bacteria using the sterility tests commonly used in plant tissue culture. The risk of not detecting low levels of A twinefaciens was also highlighted by a study carried out at the Scottish Crop Research Institute (SCRD. The study also demonstrated that even 6 months after transformation and that approximately 50 % of contaminated plant material still harboured bacterial cells with the binary vector at levels of approx. 10’ colony forming units per gram (Barrett ef al., 1997). Iv is thought that even serological and/or molecular detection methods have a detection limit of between 10° and 10° cfu g" plant material, If bacterial cell numbers are below this level (e.g. because bacterial growth is suppressed by medium acidification or antibiotic use) contamination may not be detected by the currently available detection methods, resulting to the risk of plant contaminated with Agrobacterium-based vector systems being released into the environment Conclusions Contamination losses during the in vitro stages of plant tissue culture may be substantially reduced using microbiological quality assurance schemes based on HACCP principles. However, the use of antimicrobial, medium acidification (which can also be caused by the tissue cultures themselves), and the expression of resistance mechanisms by the plant cells/tissues cultured may suppress the growth of contaminants, resulting in Jatent persistence of contaminants in plant tissue cultures. This may cause problems when contaminants become virulent during in vitro culture, but more importantly may result in the transfer of plants contaminated with bacterial pathogens or Agrobacterium-based genetic transformation vectors into the environment, when plant are weaned. Currently available antibiotic treatments can not guarantee complete removal of Agrobacterium from plants. Furthermore, detection method for bacterial pathogens and Agrobacterium- based transformation vectors are not able to detect low levels of contamination. This makes it extremely difficult or impossible to guarantee complete elimination of Agrobacterium transformation vectors from tissue cultured plants and poses the risk of transfer of vectors into the environment and horizontal gene transfer (Lilley et al., 1994). It is unknown whether such low levels of contamination with Agrobacterium vector systems may result in transfer of genes (o non-target plants or other microorganisms in the environment (horizontal transfer of genes), but this potential risk will have to be taken extremely seriously. Due to the potentially catastrophic impact of widespread horizontal transmission, biotechnology companies will have to guarantee that transgenic plants are free of latent populations of the vector system, which requires a substantial improvement in the methods for both (a) eradication of A. tumefaciens and (b) the detection of latent bacteria down to the single cell level. 90References Barrett, C., Cobb, E., McNicol, R., & Lyon, G., 1997. A risk assessment study of plant genetic transformation using Agrobacterium and implications for analysis of transgenic plants. Plant Cell Tiss. Org. Cult. 47: 135-144. Berger, F., Keetley, J.. and Leifert, C., 1994. 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