No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Endophytic bacteria expressing ?-glucuronidase cause false positives in transformation of Dioscorea species
Abstract
False positive transformants obtained during plant transformation experiments on species of the monocotyledonous genus Dioscorea (yam) are described. The false positive results were found to be due to endophytic bacteria which exist within aseptically micropropagated shoot cultures and which express β-glucuronidase (GUS). The bacteria were isolated and identified as two species of Curtobacterium. The expression of GUS in these organisms was found to be induced by a variety of glucuronide substrates. The induction of GUS activity in the bacteria can be inhibited by chloramphenicol, tetracycline, ticarcillin and sodium azide. Implications of these results for use of the gus gene in plant transformation work are discussed.
... As results of these preliminary experiments showed that plasmid DNA could be introduced into yam protoplasts and still allow their survival, experiments were conducted to investigate whether or not genes carried on the plasmid could be expressed in cells. The GUS reporter gene system was used to measure the expression of the introduced GUS gene using both a MUG assay and the standard histochemical GUS assay following addition of appropriate substrates described and evaluated in our previous yam transformation studies (Tor et al., 1992(Tor et al., , 1993. Protoplasts were isolated from the EC3 and EC5 cell lines and the uptake of plasmid pBI221.2 ...
... Homogenates of protoplast batches of both EC3 and EC5 cell lines that were either untreated, treated with PEG solution only, carrier DNA only or with PEG plus carrier DNA only did not contain detectable GUS activities. This result confirmed the consistent lack of an endogenous GUS enzyme originating from the plant cells and, in view of the recent report by us (Tor et al., 1992) that some food yams contain endogenous bacteria that can produce false positives in genetic transformation experiments, that no GUS-positive endogenous bacteria were present in the cell lines used for the current Figure 4. Determination of DNA uptake into Dioscorea alata cv. Oriental Lisbon protoplasts by dot-blot hybridisation. ...
... Previous studies by Tor et al. (1992) showed that bacteria could frequently be associated with cultured tissues of yams and could create problems with generation of false positives during routine histochemical tests for GUS, since bacteria such as Curtobacterium flaccumfasciens present in D. cayenensis shoot cultures were found to contain a gene fragment with homology to the uidA gene (Tor et al., 1992). Therefore, as a precaution during the current transformation studies on D. alata, negative controls were included. ...
Leaf mesophyll protoplasts from immature leaves of in vitro shoot cultures of a range of cultivars of three species of food
yam (Dioscorea alata, D. bulbifera and D. cayenensis-rotundata) were isolated and their responses to culture in agarose-solidified
media compared. Leaves at early stages of development (< 1.0 cm in length) proved most suitable for production of active yam
protoplasts capable of cell division. Formation of cell colonies to the 50-cell stage was observed in protoplast cultures
in five of ten cultivars of D. alata and to the 30-cell stage in two cultivars of D. cayenensis-rotundata but not in cultures
of D. bulbifera. Embryogenic cell suspension protoplasts of D. alata cv. Oriental Lisbon were successfully transformed with
plasmids pBI 221.2, pBI 221.54, pBSGUS1 and pJT137 using a standard polyethylene glycol-mediated uptake method. Levels of
transient expression of the uidA gene varied according to the plasmid used and the cell lines from which yam protoplasts were
derived. This is the first report of yam protoplast culture leading to cell regeneration and direct gene transfer into protoplasts
of this monocotyledonous genus.
... Endophytic Curtobacterium species have been isolated from many host plants including citrus (Araújo et al. 2002;Araújo et al. 2001), red clover (Sturz and Christie 1998), rice (Elbeltagy et al. 2000), potato (Sturz and Matheson 1996), yam (Tor et al. 1992), and prairie plants (Zinniel et al. 2002). They have also been associated with the control of diseases in tobacco and cucumber (Raupach and Kloepper 2000) and potato plants (Sturz and Matheson 1996), which suggests that the diversity and/or plasticity of this genus is high, allowing it to colonize different host plants. ...
Endophytic bacteria were isolated from Citrus plants and based on the similarity of 16S rRNA gene sequence, 20 isolates were included in the genus Curtobacterium in the family Microbacteriaceae , class Actinobacteria. Amplified Fragment Length Polymorphism (AFLP) analysis indicated that these strains formed four clusters with low variability that were separated from Curtobacterium flaccumfaciens . The isolates were Gram-positive, non-motile, non-spore forming, pale-yellow to orange-pink colonies. Analysis of eleven isolates showed that the major fatty acids of these strains were anteiso-C 15:0 , anteiso-C 17:0 and iso-C 16:0 and MK-9 and MK-8 as the major isoprenoid quinone, supporting the affiliation into Curtobacterium genus. The similarity of 16S rRNA gene between these isolates ranged from 99.9 to 100%, suggesting that this endophytic population present a low variability and similarity to species with validity published names within this genus, forming a distinct group in the phylogenetic tree. The DNA–DNA relatedness values to closest species were less than 48% for ER1/6 T , suggesting that this strain does not belong to already described species. Analysis of the ER1/6 T genome detected genes predicted to be involved in secondary metabolites synthesis, such as siderophore Desferrioxamine-like, bacteriocin Lactococcin 972-like, and terpene C50 carotenoid-like. Based on genome sequencing, the G + C content of the strain ER1/6 T was 72.2%. Therefore, the polyphasic taxonomic characterization demonstrated that the strains ER1/6 T , ER1.4/2, SR4/1 and SR4/8 dominant in the citrus tissues, represent a new species of the genus Curtobacterium , for which the name Curtobacterium uspiensis sp. nov. is proposed, with strain ER1/6 T = CBMAI 2131 as the type strain.
... All of them have their own advantages and limitations. For instance, the GUS gene is one of the most widely used reporter genes in many plant species, including Eucalyptus [19,20] . It can be simply detected by histochemical staining but material is consumable due to the destructive nature of the staining and de-staining procedure. ...
Eucalyptus is a genus of over 900 species and hybrids, and many of them are valuable fast-growing hardwoods. Due to its economic importance, Eucalyptus is one of the early tree species whose genomes were deciphered. However, the lack of efficient genetic transformation systems severely restricts the functional genomic research on the plant. The success of Eucalyptus regeneration and transformation depends greatly on the genotypes and explants. In this study, we systematically screened 26 genotypes from 12 Eucalyptus species in an attempt to obtain Eucalyptus genotypes with high regeneration potential. We developed two common regeneration media that can be applied to most tested Eucalyptus genotypes for both seeding hypocotyls and cloned internodes as explants. We then implemented DsRed2 as a visual marker for genetic transformation efficiency test. Our results suggest that E. camaldulen and E. robusta are amenable for genetic transformation. Finally, we successfully set up a stable Agrobacterium-mediated genetic transformation procedure for both E. camaldulen and E. robusta using seeding hypocotyls and cloned internodes respectively. Taken together, our study provides valuable means for vegetative propagation, gene transformation, CRISPR based gene mutagenesis, activation and suppression, as well as functional characterization of genes in Eucalyptus.
... Regulatory genes of the maize anthocyanin biosynthetic pathway, such as C1, B, and R, have also been successfully used in wheat transformation (Ludwig et al., 1990;Kloti et al., 1993;Dhir et al., 1994;McCormac et al., 1998;Chawla et al., 1999aChawla et al., , 1999b. Expression of anthocyanin genes in plant tissues can be useful where false positive results due to intrinsic GUS-like activity (Hu et al., 1990) and GUS production by endophytic micro-organisms have been reported (Tor and Ainsworth, 1992). A related transcription factor from Arabidopsis, AtMYB12, which is a flavonol-specific regulator of phenylpropanoid biosynthesis (Mehrtens et al., 2005), has been used successfully to activate anthocyanin expression in wheat and serve as a scorable marker (Gao et al., 2011). ...
Wheat is one of the most important staple food crops of the world, and continuous genetic improvement is vital to meet the demands of the rapidly growing world population. Conventional breeding has led to the development of current high yielding wheat varieties, and recent achievements in genetic engineering are expected to augment conventional breeding to further increase production. Advances in genome sequencing and molecular breeding have increased the rate of gene discovery, leading to a need for highly efficient and robust transformation systems. Targeted genome editing will require efficient delivery of sequence-specific nucleases, such as zinc fingers (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases such as CRISPR-Cas9. Since the first report of fertile transgenic wheat in 1992, optimization of plant tissue culture techniques, DNA delivery methods, gene expression cassettes, and marker genes have led to reliable transformation protocols for a range of wheat model cultivars. However, like other cereal crops, wheat transformation has also been hampered by genotype effects. The limited range of transformable tissues in wheat is considered another challenge. Several excellent review papers have described the progress made towards developing robust genetic transformation systems for wheat, so we have focused our attention on a detailed analysis of selectable markers and promoters that have been used. The choice of selectable marker and promoter can dramatically influence the outcome of a transformation project. Both Agrobacterium tumefaciens and microprojectile-mediated transformation systems have been employed successfully for genetic transformation of wheat using genes of agronomic importance. Since improvement in agronomic traits of wheat will affect a sizeable population, we have provided an analysis of the progress made towards developing genetically superior wheat containing gene(s) of agronomic importance. Recent efforts on targeted genome editing in wheat are also discussed.
... The bacterial enzyme GUS, encoded by the E. coli gusA gene, is one of the most widely used transgenic reporter genes. Despite its simplicity, false-positives are difficult to eliminate among the modified plants owing to the foreign GUS activity [8]. The high cost of the substrate (X-Gluc) for GUS staining is another disadvantage of GUS. ...
Genes encoding reporter proteins are used as visual marker-assisted tools in genetic transformation as well as plant breeding. In this study, the red fluorescent protein identified in Discosoma sp. coral (DsRed2) was successfully used as a visual marker for cotton genetic
engineering. DsRed2 was successfully expressed in two cotton cultivars, JIN668 and YZ1, driven by the CaMV-35S promoter via the Agrobacterium-mediated transformation. Our results suggest that DsRed2 expression provides an early-stage selection tool for the
transgenic calli via visual observation. Red fluorescence can be detected not only in callus and somatic embryos
but also in most tissues and organs of mature plants. The transgenic line Yz-2-DsRed2 was crossed with four different cotton cultivars to assess the transgene heritability and stability in different genetic backgrounds. The heritability of the red color was highly stable when Yz-2-DsRed2 was used as a male parent. The DsRed2 gene expressed 100% in the F1 hybrids. To investigate the relationship between DsRed2 transcription and DNA methylation, a methylation-specific PCR approach was applied to the CaMV-35S promoter region. The results showed a negative association between DNA methylation level in the promoter region and the transgene transcription. Taken together, these findings suggest DsRed2 a visual reporter gene for cotton genetic transformation and molecular breeding program.
... Les réponses au photo-thermopériodisme semblent varier avec les espèces et avec la richesse du milieu en saccharose; de plus, elles se traduisent soit par une plus ou moins grande précocité et/ou fréquence de tubérisation, soit par un nombre et une masse de microtubercules plus ou moins élevés.La maîtrise des différents volets de la culture in vitro de l'igname, et surtout les phénomènes de régénération, peuvent faciliter le développement d'un programme de transformation génétique. Cette dernière orientation de la recherche a déjà permis la réalisation d'un nombre réduit de travaux sur l'igname(XINHUA et al., 1986;SCHAFER et al., 1987 ;TOR et al., 1992TOR et al., -1993. ...
Les ignames cultivées sont naturellement tributaires d'une conservation à court terme. Leur mode de propagation par multiplication végétative favorise l'accumulation des virus et la dispersion de pathogènes. Les risques d'érosion génétique sont importants et nécessitent de trouver des modes de conservation efficaces. Dans le cadre de la conservation des ressources génétiques des ignames, plusieurs aspects peuvent être envisagés. La conservation aux champs est la méthode la plus courante. Elle permet une application la plus large à toutes les espèces, mais est pénalisée par son coût d'entretien élevé et les risques d'érosion génétique d'ordre climatique ou d'ordre pathologique. La conservation sous la forme de banques de graines est envisagée, les graines d'igname étant considérées comme non récalcitrantes. Les techniques de culture in vitro, croissance ralentie et cryoconservation, permettent d'éviter les contraintes de la conservation aux champs. L'utilisation d'un procédé simple de cryoconservation (encapsulation-déshydratation), appliqué actuellement à un nombre réduit de génotypes d'igname, a permis d'obtenir de bons résultats. Il faut attendre son application à un nombre plus important de génotypes, plus représentatifs de la diversité, pour assurer une utilisation en routine. Les échanges de germoplasme ne pourront se faire qu'à partir de matériel végétal sain. Pour cela, il est nécessaire de détecter la présence de pathogènes, et de les éradiquer. Les problèmes majeurs proviennent de la présence de plusieurs virus dont la caractérisation a largement progressé ces dernières années. Une sanitation des plantes infectées est maintenant envisageable. (Résumé d'auteur)
... A grande aceitação do geneuidA ou GUS como gene marcador em plantas decorre da simplicidade e versatilidade do método para detecção de sua atividade enzimática e do fato das plantas não apresentarem atividade endógena significante 45 . O produto da enzima β-glucuronidase pode ser detectado por meio de ensaios histoquímicos e fluorimétricos 46 . ...
... The antibiotic timentin consists of ticarcillin and clavulanic acid (50:1) and has been used to kill systemic bacteria in in vitro studies (Tor et al. 1992;Buckley & Reed 1994) and to inhibit Agrobacterium in genetic transformation experiments (Cheng et al. 1998;Ling et al. 1998;Tang et al. 2004). Additionally, plant regeneration affected by these antibiotics was also assessed. ...
This research was conducted to develop genetic transformation of the recalcitrant chrysanthemum cv. Shinma by application of appropriate antibiotics and selective agents. Clavamox had the least inhibitory effect on shoot regeneration compared to timentin, carbenicillin, and cefotaxime. Clavamox, at a concentration of 125 mg L−1, was found to be the most suitable for shoot regeneration and production of quality shoots, suppressing the growth of Agrobacterium in explants infected with strains GV3101 or C58C1 for 3 and 4 weeks, respectively. The concentration of phosphinothricin (PPT) was found to be 1.0 mg L−1 for screening of putative transgenic shoots. Moreover, transgenic chrysanthemums were obtained by culturing explants co-cultivated with A. tumefaciens strain GV3101 harboring an anthocyanin regulatory gene RsMYB1 isolated from radish (Raphanus sativus), which was placed under the control of cauliflower mosaic virus promoter (CaMV) 35S and petal-specific promoter InMYB1 isolated from the morning glory (Ipomoea nil), on shoot regeneration medium supplemented with recommended concentration of antibiotic and selective agent. Flow cytometry analysis revealed that there was no variation in ploidy level between transgenic plants and donor plants (non-transformants). To our knowledge, this is the first report of the use of Clavamox and MYB transcription factor for genetic transformation of this chrysanthemum.
... We used plants originally regenerated from tissue culture because they lacked endogenous GUS activity. GUS activity was obtained from stems of di¡erent sugarcane varieties coming from agricultural ¢elds, and it is most probably of bacterial origin, as described in Dioscorea [22]. Successful colonization was observed in all the sugarcane varieties used, but the inoculated strain and the GUS activity were only detected in plants grown under low levels of nitrogen fertilization. ...
... Die quantitative Analyse erfolgt meist durch einen fluorometrischen Nachweis des 4-Methylumbelliferons, das aus dem Substrat 4-Methylumbelliferyl-β-D-Glucuronid freigesetzt wird. Die Nachteile des GUS-Systems sind die destruktive Wirkung des Nachweisverfahrens auf die Zellen, die Tatsache, dass mehrere Pflanzen nennenswerte endogene Glucuronidaseaktivität besitzen, sowie mikrobielle Verunreinigungen, die eine Reportergenaktivität "vortäuschen" können (Tör et al., 1992). ...
... Bacteria of the genus Curtobacterium have been isolated as endophytes from many crops, including red clover (Sturz et al., 1998), rice (Elbeltagy et al., 2000), potato (Sturz and Matheson, 1996), yam (Tor et al., 1992), prairie plants (Zinnier et al., 2002), and citrus (Arau´jo et al., 2001). Several reports have indicated that C. flaccumfaciens can function as a biological control agent against many pathogens, and may function either by the triggering of induced systemic resistance (Raupach and Kloepper, 1998) or by antibiosis (Sturz and Matheson, 1996). ...
In vitro analyzing the antagonistic activity of seventy native bacterial isolates towards plant tumorigenic Agrobacterium tumefaciens resulted in a selection of eight potential biocontrol agents. These isolates were screened for their antagonistic effect in vitro as well as their efficacy in reducing gall formation in planta. They were identified using Biolog microplates system as Bacillus megaterium, Paenibacillus polymyxa, Pseudomonas fragi (two isolates), Pseudomonas viridilivd, Pseudomonas asplenii, Curtobacterium flaccumfaciens and Curtobacterum sp.All antagonists tested exhibited considerable inhibitory activity in vitro and significantly reduced incidence and size of galls in rose shoots, kalanchoe leaves and squash fruits with variable degrees on the tested hosts. C. flaccumfaciens reduced the incidence of crown gall up to 100% in the case of rose shoots and kalanchoe leaves whereas the same antagonist reduced galling of squash fruits to 75%. Likewise, P. asplenii, P. viridilivd and P. polymyxa reduced the incidence of crown gall up to 100% in the case of kalanchoe leaves and squash fruits, whereas they reduced galling of rose shoots to 66.7%, 55.6% and 44.5% respectively. In the same manner, the two isolates of P. fragi reduced galling up to 100% in squash fruits, while it was 88.9% in rose shoots and kalanchoe leaves. Interestingly, B. megaterium isolate completely suppressed the gall development in rose shoots, whereas the gall incidence was 100% in kalanchoe leaves and 25% in squash fruits. Bacterial isolates characterized in this study may be considered as potential sources of novel bioactive metabolites as well as promising candidates to develop new biocontrol agents for controlling crown gall disease.
... The isolates were small, yellow coccobacilli (0.15 to 0.25,um by 0.25 to 1.8,um) and strict aerobes, similar to authentic strains of Curtobacterium sp., such as C. luteum JCM 1480 T , C. albidum lAM 163F, C. citreum lAM 1614 T , and C. pusillum lAM 1479 T . Cultures of Curtobacterium sp. from Dioscorea (yam) displayed a f3-glucuronidase activity (Tor et al. 1992). Indeed, the five Curtobacterium-like isolates from rice (B8, B9, Bl4, B18, and B26) showed the f3-glucuronidase activity on NA medium supplemented with 5-bromo-4-chloro-3-indolyl-f3-o-glucuronide (data not shown). ...
Endophytic bacteria were isolated from surface-sterilized stems, seeds, and leaf sheaths of wild and traditionally cultivated rice varieties. Phylogenetic analyses based on 16S rDNA revealed a wide divergence among the isolates. However, the most frequently isolated groups were Methylobacterium sp. in the α-subdivision of Proteobacteria, and Curtobacterium sp. in the high G+ C Gram-positive group. Various phenotypic traits that are expected to be involved in the persistence and functions of the bacteria were analyzed: Most of the isolates from rice excreted pectinase, were motile, and showed an osmotic resistance to 0.6 M sucrose. These traits may be involved in endophytic characteristics in rice. About 50% of the isolates showed a cellulase activity. A few isolates fixed nitrogen, produced indole-3-acetic acid, and formed capsules. These activities were partially correlated with the phylogenetic group.
... The expression of PR-la by vira1 infection and SA treatment has been studied for more than 10 years, yet an understanding Severa1 studies have documented GUS artifacts associated with the histochemical GUS activity assay (Plegt and Bino, 1989;Mascarenhas and Hamilton, 1992;Tor et al., 1992). In RNA was isolated from the -903 T 3 lines indicated. ...
Pathogenesis-related protein-la (PR-la) is a protein of unknown function that is strongly induced during the onset of systemic acquired resistance (SAR) in tobacco. The expression of PR-la is under complex regulation that is controlled at least partially by the rate of transcription. In this study, we demonstrated that 661 bp of 5'flanking DNA was sufficient to impart tobacco mosaic virus and salicylic acid inducibility to a reporter gene. The PR-la promoter did not respond significantly to treatments with either auxin or cytokinin. Experiments with the protein synthesis inhibitor cycloheximide indicated that protein synthesis is required for salicylate-dependent mRNA accumulation. At flowering, the PR-la gene was expressed primarily in the mesophyll and epidermal tissues of the leaf blade and the sepals of the flower. Several artifacts, most importantly ectopic expression in pollen, were associated with the use of the P-glucuronidase reporter gene.
... This danger is avoided by the use of constructs with an intron inserted in the uidA gene (28,29). Tör (30) has reported false positive results caused by endogenous Corynebacterium, possessing GUS activity. In the indigogenic reaction, false positive results could also appear due to the formation of colored product from ferri/ferrocyanide. ...
... We used plants originally regenerated from tissue culture because they lacked endogenous GUS activity. GUS activity was obtained from stems of di¡erent sugarcane varieties coming from agricultural ¢elds, and it is most probably of bacterial origin, as described in Dioscorea [22]. Successful colonization was observed in all the sugarcane varieties used, but the inoculated strain and the GUS activity were only detected in plants grown under low levels of nitrogen fertilization. ...
Acetobacter diazotrophicus is a nitrogen-fixing endophytic bacterium, originally isolated from sugarcane. Its colonizing ability was evaluated in high and low N-fertilized sugarcane plants by inoculating stem-cuts with a β-glucuronidase marked A. diazotrophicus strain. Bacterial quantification by the most probable number technique showed a severe decrease of A. diazotrophicus cells in plants fertilized with high levels of nitrogen. The inoculated strain was detected inside low N-fertilized sugarcane plants by histochemical staining of β-glucuronidase and scanning electron microscopy. A. diazotrophicus was found mainly inside cortical cells of stems and inside xylem vessels. No β-glucuronidase activity was observed in non-inoculated plants. High nitrogen fertilization of fields might be a threat to maintaining naturally occurring endophytic associations.
... GFP fluorescence in cells was a good indicator of transformation with further indication provided by the increased level of green fluorescence in nuclei of green-fluorescent cells, which is consistent with the use of a non-targeted gfp version. Tör et al. (1992) reported that endophytic bacteria could express β-glucuronidase and Growth and proliferation of transgenic cells after transformation can be followed using GFP whether exogenous DNA is delivered using Agrobacterium or microprojectile bombardment techniques. With microprojectile bombardment (where genes are frequently introduced via separate plasmids), application of GFP as a secondary marker requires a high frequency of cotransformation and coexpression of both genes. ...
Early detection of plant transformation events is necessary for the rapid establishment and optimization of plant transformation
protocols. We have assessed modified versions of the green fluorescent protein (GFP) from Aequorea victoria as early reporters of plant transformation using a dissecting fluorescence microscope with appropriate filters. Gfp-expressing cells from four different plant species (sugarcane, maize, lettuce, and tobacco) were readily distinguished, following
either Agrobacterium-mediated or particle bombardment-mediated transformation. The identification of gfp-expressing sugarcane cells allowed for the elimination of a high proportion of non-expressing explants and also enabled visual
selection of dividing transgenic cells, an early step in the generation of transgenic organisms. The recovery of transgenic
cell clusters was streamlined by the ability to visualize gfp-expressing tissues in vitro.
... While cells that have been damaged by micro projectile bombardment can still express GUS, thereby producing false positives, similarly damaged cells would not accumulate anthocyanin, since this system requires an intact vacuole and the coordinated expression of many genes. Expression of anthocyanin genes in plant tissues would thus be useful where false positive results have been reported due to intrinsic GUS-like activity (Hu et al., 1990) and GUS production by endophytic micro-organisms (Tor and Ainsworth, 1992). The gfp gene encodes a protein, green fluorescent protein (GFP), which emits green fluorescence (509 nm) when exposed to blue light (488 nm) and hence GFP activity can only be detected by using specialized fluorescence equipment (Furtado and Henry, 2002). ...
Three barley tissue-specific promoters (Dhn12, Itr1, and Ltp1) were transcriptionally fused to the C1 and/or the B-peru genes involved in anthocyanin biosynthesis. Transient expression of these constructs was examined in different wheat tissues.
Constructs with tissue-specific promoters were only active in embryos. A green fluorescent protein (GFP) driven by the actin
promoter was used as an internal standard to monitor the effectiveness of each bombardment. Further, normalization of the
transient expression assay using the GFP reference significantly reduced the variability between separate bombardments, and
the intensity of anthocyanin pigmentation quantified by image analysis allowed for a rapid and accurate evaluation of different
promoters. Compared to CaMV35S promoter, tissue-specific promoters were more effective in directing anthocyanin production, specifically in wheat embryos,
and the C1 gene was more effective than B-peru. Among the tissue-specific promoters, the Ltp1 was superior to ltr1 and Dhn12 in combination with C1 and/or B-peru gene(s). Analysis of nucleotide sequences of all three tissue-specific promoters revealed the presence of G-box, E-box, and
RY elements, which might trigger embryo-specific expression in wheat.
... It is a mixture of ticarcillin and clavulanic acid and is commonly used at a ratio of 50 (ticarcillin):1 (clavulanic acid) (w/w) (Sweeney et al., 1988). In plants, some researchers have used ticarcillin or Timentin to inhibit systemic bacteria in tissue cultures (Tor et al., 1992;Buckley and Reed, 1994) and to suppress Agrobacterium in genetic transformation (Zimmerman, 1995). The objective of the research reported here was to determine the efficiency of Timentin as an alternative antibiotic to carbenicillin and Cef for the inhibition of A. tumefaciens in the Agrobacterium-mediated genetic transformation of P. acerifolia. ...
The effects of Timentin and cefotaxime (Cef) on shoot regeneration of the London plane tree (Platanus acerifolia Willd.) and their use for the suppression of Agrobacterium tumefaciens in Agrobacterium-mediated genetic transformation were compared. Shoot regeneration was significantly reduced on the media with Cef at concentrations
from 100 to 500 mg·L−1. Timentin showed negative effect on plant regeneration at concentrations of 100 and 500 mg·L−1; however, 300 mg·L−1 Timentin was shown to facilitate shoot regeneration significantly and the regeneration frequency increased from 64% (control)
to 88%. Effective suppression of A. tumefaciens could be obtained with 500 mg·L−1 Cef, but plant regeneration was completely inhibited at this level. The A. tumefaciens on infected P. acerifolia leaf tissues was visually undetectable after three subcultures on a medium with 300 mg·L−1 Timentin. Considering the effect of Cef and Timentin on plant regeneration and suppression of Agrobacteria, Timentin at 300
mg·L−1 is the preferred application in A. tumefaciens-mediated transformation of P. acerifolia.
Cur.to.bac.te'ri.um L. adj. curtus shortened; L. neut. n. bacterium a small rod; N.L. neut. n. Curtobacterium a short rodlet
Actinobacteria / Actinobacteria / Micrococcales / Microbacteriaceae / Curtobacterium
Short irregular rods (0.3–0.6 × 0.5–3.0 µm); pleomorphism is not distinctive. Cells become shorter to coccoid in older cultures. Branching is not found, bending type cell division . Nonsporeforming. Not all species are motile; generally motile species show peritrichous flagellation. Gram‐stain‐positive, but staining properties can be lost with age. Non‐acid‐fast. Colonies are generally ivory, yellow, or orange. Strictly aerobic chemoorganotrophs. Acid is produced weakly from glucose, fructose, and some other carbohydrates. Acetate, pyruvate, and lactate are assimilated in addition to other organic acids. Catalase and DNase are positive; gelatin and esculin are hydrolyzed. Nitrate is not reduced; oxidase and urease are not produced. The cell‐wall peptidoglycan, based on the presence of d ‐ornithine, is type B2 β. Acetyl is the peptidoglycan acyl type . Mycolic acids are not found. Nonhydroxylated fatty acids, specifically anteiso‐methyl branched chains fatty acids predominate ; ω‐cyclohexyl undecanoic acid is found in some species. Phosphatidylglycerol, diphosphatidylglycerol, and some glycosyldiacylglycerols are the major polar lipids. Spermidine and spermine are the major polyamines; putrescine and cadaverine are absent. Isoprenoid quinones, comprising menaquinones with 9 isoprene units ( MK‐9 ), predominate .
DNA G + C content ( mol% ): 68.8–75.2 ( T m , HPLC).
Type species : Curtobacterium citreum (Komagata and Iizuka, 1964) Yamada and Komagata 1972b, 425 AL ( Brevibacterium citreum Komagata and Iizuka 1964, 498).
This study was an attempt to isolate endophytic microorganism with antimicorbial properties from sweetpotato that are grown in the Philippines. Endophytic microorganims were isolated from surface-sterilized stem cuttings of selected Philipine sweetpotato varieties such as BSP-SP-17, BSP-SP-22, and NSIC-SP-25. The isolates were purified and tested for antimicrobial activities using spot and streak inoculation methods against Lasiodiplodia theobromae (sweetpotato rot-causing mold), Colletotrichum gloesporoides (yam anthracnose-causing mold)
Although dozens of stress inducible promoters have been identified in rice, detailed and comparative investigations under uniform condition are still limited. In this study, we selected eight previously reported drought-inducible genes [Oshox24, hypothetical protein (hp), OsLEA3-1, OsNAC5, OsNCED3, Rab21, RRJ1 and Wsi18] and analyzed their transcriptional responses to drought and osmotic stresses in rice. Based on their transcription patterns and the required standards for inducible promoters, Oshox24, Rab21 and OsLEA3-1 were chosen for cloning of their promoters for detailed analyses at both the RNA and protein/activity levels, with rice oxalate oxidase gene (OsOxO3) as a reporter gene. By generating transgenic plants and determining oxalate oxidase activity that is activated under different stresses, we defined more quantitatively that the promoters of Oshox24, OsLEA3-1 and Rab21 are ideally applicable to transgene expression for inducibly controlling drought resistance genes or other functional genes in rice. The results provided a quantitative assessment of the strength, sensitivity, stress specificity and time course of the three promoters. Such detailed information is essential for the selection of promoters for use in improving stress resistance.
The bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC) and has been associated with important losses in commercial orchards of all sweet orange [Citrus sinensis (L.)] cultivars. The development of this disease depends on the environmental conditions, including the endophytic microbial community associated with the host plant. Previous studies have shown that X. fastidiosa interacts with the endophytic community in xylem vessels as well as in the insect vector, resulting in a lower bacterial population and reduced CVC symptoms. The citrus endophytic bacterium Methylobacterium mesophilicum can trigger X. fastidiosa response in vitro, which results in reduced growth and induction of genes associated with energy production, stress, transport, and motility, indicating that X. fastidiosa has an adaptive response to M. mesophilicum. Although this response may result in reduced CVC symptoms, the colonization rate of the endophytic bacteria should be considered in studies that intend to use this endophyte to suppress CVC disease. Symbiotic control is a new strategy that uses symbiotic endophytes as biological control agents to antagonize or displace pathogens. Candidate endophytes for symbiotic control of CVC must occupy the xylem of host plants and attach to the precibarium of sharpshooter insects to access the pathogen. In the present review, we focus on interactions between endophytic bacteria from sweet orange plants and X. fastidiosa, especially those that may be candidates for control of CVC.
Verticordia grandis is a native Australian plant of the Myrtaceae family. The genus Verticordia (feather flower) consists of 79 species, most of which are endemic to the southwest of Western Australia. Members of this genus are perennial woody shrubs adapted to sandy soils and low rainfall areas; they occur mainly in heathland and shrubland communities. Their floral display is impressive with loose to tight clusters of showy flowers and feathery calices. Flower color is diverse, ranging from red, orange, yellow, mauve, or white. All have great horticultural value, but some species, including V. grandis, are difficult to propagate.
Both gram positive and gram negative bacteria were isolated from yam (Dioscorea spp) tubers obtained from Jamaica and Nigeria. The total bacterial count and species indicated that different types of bacteria exist to varying extents in different yam tubers. An intrinsic antibiotic resistance profile showed that up to 83% of the strains were resistant to ampicillin (50 μg mL-1), but most were sensitive to streptomycin (15 μg mL-1) and kanamycin (50 μg mL-1). Fourteen of the endophytes contained plasmid deoxyribonucleic acid (DNA), seven of which possessed a single plasmid of 22 kb while the other seven had two plasmids of 22 kb and 38 kb. Bacterial isolates from D. rotundata cv. Ikale containing single and double plasmids were identified by fatty acid profiling as Pantoea dispersa belonging to the Erwinia-Pantoea complex. Endonuclease restriction showed the 22 kb plasmid from endogenous P. dispersa to possess multiple restriction sites for EcoRI, PstI, BamHI, SalI, HincII, MluI, and SmaI. Their apparent non-pathogenic existence in the yam host and their genetic elements could be further explored for possible exploitation as vectors in the genetic engineering of yam.
Regulatory genes of the maize anthocyanin biosynthetic pathway have proven useful as scorable markers for transformation because of their high sensitivity, ease of visualization, cell-autonomous expression and lack of requirement for exogenous substrates. This is particularly advantageous when using particle bombardment as a DNA delivery system, since cells expressing genes can be counted easily and unambiguously (Bowen 1992). Furthermore, the cells that have been damaged by microprojectile bombardment can still express GUS (giving false positive), but they will not accumulate anthocyanin since the latter requires an intact vacuole and coordinate expression of many genes. Expression of anthocyanin genes in plant tissues will be useful where false positive results have been reported due to intrinsic GUS-like activity in several plant parts (Hu et al. 1990) and GUS production by endophytic microorganisms (Tor et al. 1992). The anthocyanin markers permit visualization of transgenic tissue from the beginning and throughout development without sacrificing the tissues. In addition, expression of anthocyanin genes in transformed tissues can provide a system for investigating the regulation of gene expression in plants, which is difficult with GUS (Ben-fey et al. 1990). Transient and stable expression of anthocyanin genes in various corn intact tissues (Klein et al. 1988; Ludwig et al. 1990; Wong et al. 1991; Bowen 1992; Dunder et al. 1993) and transient expression in wheat seedling tissues (Wong et al. 1991) has been reported previously. We have used anthocyanin as a visual marker for optimization of bombardment conditions using different target tissues (transiently) and evaluation of different selectable markers (stably) for wheat transformation by selecting the transformants visually in the early stage of development.
Peanut (Arachis hypogaea L.) transformation via particle bombardment of somatic embryos is now well established in several laboratories. However, transgenic peanut cultivars do not exist in part due to lengthy transformation protocols, and low transformation efficiencies. In order to develop more rapid peanut transformation methods, a reliable and easily detectable reporter gene is necessary. In this report two reporter genes, uidA + intron encoding β-glucuronidase (GUS) and sGFPS65T encoding the green fluorescent protein (GFP), were used in either Agrobacterium-mediated or biolistic transformation experiments to evaluate the efficacy of their expression in peanut meristematic tissue isolated from mature zygotic embryos. Reporter gene expression was analyzed transiently by a histochemical assay (GUS) or by fluorescence microscopy (GFP). Negative control explants in the Agrobacterium-mediatcd transformation experiment often exhibited false positive GUS expression. Staphylococcus, an endophytic bacterium, was identified as a contaminant within the explants and most likely resulted in the false GUS activity observed. Expression of GFP could not be detected following Agrobacterium co-cultivation. However, GFP expression was easily detectable following particle bombardment. Several peanut cultivars were used for particle bombardment experiments and examined for GFP expression at various time intervals post-bombardment. As expected, the number of cells transiently expressing GFP decreased as the time following bombardment increased. In light of these results, a reporter system based on GFP is being developed for biolistic transformation of peanut.
Contributions of biotechnology to selected tropical and subtropical crops are reviewed.
The presence of foreign genes that were transferred into Citrus nucellar cells by the particle bombardment system has been expressed with the GUS assay. Tungsten microcarriers coated with plasmid DNA containing the gus A gene (pBI221.2) were accelerated at high velocity using a biolistic device into lemon (Citrus limon var. Kütdiken) target cells. Histochemical examination of the bombarded nucellar cells revealed that only several cell aggregates had cells expressing the gus A gene. Control cell aggregates that were not bombarded did not exhibit any cells expressing the gus A gene.
A biolistic particle gun was used to deliver genetic material into intact yam cells. Cultured suspension cells of D. alata were bombarded with microprojectiles coated with pBI221.2 DNA and histochemical assays were carried out to show transient GUS expression in bombarded cells. Stably transformed D. alata cells were recovered from cultured cells after bombardment with microprojectiles coated with pRT99gus harbouring both the nptII and uidA genes. Bombarded cells were selected on a medium containing geneticin (G418). Two months after bombardment, calli resistant to G418 were assayed for GUS expression. There was a 100% correlation between resistance to G418 and GUS expression. From these calli, four cell lines were established and GUS activity in each line was determined fluorometrically. The use of a specific GUS inhibitor showed that the GUS activity was due to the introduced uidA gene rather than to any intrinsic GUS-like activity originating from the plant. Incorporation of the introduced DNA into the plant genomic DNA was confirmed by Southern analysis.
Microprojectile bombardment was used to introduce the GUS reporter gene into sugarcane axillary meristems. Chimeric expression of this gene was observed in 20-40% of shoots regenerated from sugarcane meristems one month after particle bombardment. The linear pattern of GUS expression observed is consistent with periclinal division from single transformed meristematic cells. Meristems have advantages over callus cells as targets for microprojectile transformation, and have potential for introducing agronomically important genes into current commercial sugarcane varieties.
Some conditions related to the transient expression of β-glucuronidase in biolistically-treated Coffea spp. tissues were investigated, and subsequently used in a promoter study. Bombardments were performed on different types of tissue (leaves, somatic embryos and suspension cultures) of genotypes of C. arabica, C. canephora and Arabusta, using 4 different promoter sequences. Tobacco leaves were used as a comparison. In general, similar large variation and mean values of transient expression were observed between coffee and tobacco leaves. With regard to the coffee tissues effect, transient expression was best detectable and most frequently observed with bombarded leaves of microcuttings. Disturbing endogenous light blue staining was found with control treatments of somatic embryos. For the three coffee species tested, the most effective promoter was the EF1α-A1 promoter of Arabidopsis thaliana.
This chapter discusses the reporter genes. Differential gene expression is essential for the development of higher organisms. Transcriptional regulation appears to play a key role in this process. However, the relevant gene products may be difficult to detect due to low abundance or chemical properties. Therefore, the analysis of developmental processes is greatly simplified if promoters or other regulatory sequences are linked to reporter genes. Likewise, reporter enzymes that remain active when fused to other peptides allow the study of protein trafficking in the cell. To compare enzyme activities, standardization of extracts is necessary. The most convenient method is determining the protein content of extracts—for example, using the assay described by Bradford. Enzyme kinetics are characterized by a lag, linear, and stationary phase. During the linear range, substrate accumulation is proportional to enzyme concentration. Enzyme assays are quantitative only within the linear phase, which may have to be determined initially. The frequent use of GUS is mainly justified because it allows the histochemical localization of enzyme activity in tissues or complete plantlets. Also, after the initial investment in a fluorimeter, a fluorogenic assay provides a relatively inexpensive and highly sensitive method to quantify GUS activity.
A significantly damaging problem encountered by the plant tissue culture industry is the presence of covert microbial contaminations [1]. in this respect, bacterial contamination is responsible for considerable losses at each step of the micropropagation process, and also for the final consumers of the products (e.g. the greenhouse and nursery industries). Moreover, the increasing applications of micropropagated material as sources of reputedly pathogen-free status is placed in jeopardy since phytopathogenic bacteria can be found among these contaminating micro-organisms [2].
A conspicuous endogenous maize (Zea mays L.) β‐glucuronidase (GUS) activity was observed in histochemical assays of non‐transformed maize kernels, confounding the use of Escherichia coli gusA as a reporter gene. Appearance of the endogenous activity was developmentally dependent and highly tissue‐specific, being localized to the upper pedicel (basal maternal kernel) tissues where the black layer forms in the latter stages of kernel development. Pedicel homogenates exhibited GUS activity using either p‐nitrophenyl‐β‐D‐glucuronide or 4‐methylumbelliferyl‐β‐D‐glucuronide (MUG) as substrates. Pedicel GUS was apparently not the result of endophyte contamination of enzyme isolates since no endophytes could be cultured. The MUG‐based activity had a pH optimum of 4 to 5 and was separable into two isoforms by anion exchange chromatography with Km values for MUG of 2.2 and 2.7 µM for the early‐ and late‐eluting forms, respectively. The pedicel GUS isoforms had very similar characteristics: native Mr of approximately 32000, stimulation by assay at 60°C, inhibition at high ionic strength or in the presence of EDTA and relative insensitivity to the E. coli GUS inhibitor saccharic acid‐1,4‐lactone. Only the early‐eluting form, however, was capable of hydrolyzing the histocbemical GUS substrate 5‐bromo‐4‐chloro‐3‐indoyl‐β‐D‐glucuronide. Neither isoform exhibited antifungal activity against Fusarium moniliforme. In contrast to the in vitro activity, pedicel endogenous GUS measured histochemically was completely inhibited by saccharic acid‐1,4‐lactone, unaffected by EDTA and greatly decreased by incubation at elevated assay temperature. A modification of the standard histochemical GUS assay allowed complete suppression of endogenous GUS activity while enhancing E. coli‐derived GUS activity in kernels transiently expressing the gusA gene. Possible roles of these endogenous GUS activities within the black layer region of the kernel pedicel are proposed.
Verticordia grandis is an Australian native plant of the Myrtaceae family much prized for its display of bright red flowers. This paper describes a system for the transformation and regeneration of transgenic Verticordia grandis. The susceptibility of V. grandis to Agrobacterium rhizogenes was shown by simultaneous wounding and inoculation of the stems of shoot explants with 4 wild-type strains of A. rhizogenes. Shoots inoculated with 3 of the 4 strains developed abnormal tissues containing the opine (agropine or mannopine) characteristic of the bacterial strain used. A regeneration system for V. grandis was developed, using leaf discs excised from the petiole region of micropropagated shoots. These discs were used for transformation studies using two plasmid vectors in either the wild-type A. rhizogenes strains or a non-oncogenic A. tumifaciens strain, LBA4404. The plasmid vectors (pBI121 and pKiwi) contained chimeric kanamycin resistance genes, neomycin phosphotransferase II (NPTII) and the bacterial β-glucuronidase (GUS) uidA gene. Leaf discs were inoculated by wounding and selected for growth on kanamycin-containing medium. Regenerated shoots were transferred to root induction medium containing kanamycin and those plants which produced roots were regarded as potential transformants. These plants were assayed for GUS activity and transformation was confirmed by Southern DNA hybridisation and by PCR amplification of the GUS gene. These results represent the first report of transformation and subsequent regeneration of a plant from the economically important Myrtaceae.
Bacterial β-glucuronidase is often introduced into plants as a reporter gene fused to constitutive or inducible promoters. However, the presence of both endogenous inhibitors of GUS activity and endogenous GUS enzymes in transgenic plants could lead to an underestimation of GUS. In this paper, a decrease of the Vm values and a greater affinity (Km) of the GUS enzyme for its substrate (p-NPG) has been recorded when increasing amounts of protein from untransformed tobacco cells has been added to the pure β-glucuronidase. The observed inhibition is not competitive and can be completely removed when the tobacco extracts are passed through Sephadex G-25 spin columns prior to the assays. After such a treatment, the activity of E. coli GUS in transgenic tobacco cells (constitutive or inducible systems) was stimulated by a factor 1.2 or 2 for p-NPG or 4-MUG substrates, respectively. This method was also effective in suppressing the endogenous GUS or GUS-like activity which can interfere with the activity originating from the introduced GUS gene.
Isolates of different endophytic bacteria were recovered from surface-disinfected seeds obtained from commercial companies, plants in the field and tissue culture. The bacteria were isolated from seeds after stringent surfacedisinfection.Pseudomonas fluorescens (isolate no. 14) from bean inhibited growth of all fungi tested and was fluorescent on King B medium.Bacillus cereus fromSinapis (isolate no. 65) inhibited growth ofRhizoctonia solani, Pythium ultimum andSclerotium rolfsii and also exhibited chitinase activity.Bacillus subtilis from onion tissue culture (isolate no. 72) inhibitedR. solani andP. ultimum growth.B. cereus from cauliflower (isolate no. 78) inhibited growth ofR. solani. B. pumilus from sunflower (isolate no. 85) inhibited growth ofR. solani andS. rolfsii. B. cereus (isolate no. 65) was introduced into cotton, and by using radioactive labelling we found that it was present for 16 days in the root-stem junction. It is most likely that these bacteria were still found 72 days after their introduction in the root and stem, at levels of 2.8·105 and 5·104 cfu g−1 fresh weight, respectively, when selective medium was used. There was no difference between control and treated plants in their height or in the fresh weight of roots, stems and leaves.
When cotton seedlings were inoculated withB. cereus (isolate no. 65),B. subtilis (isolate no. 72) orB. pumilus (isolate no. 85), disease incidence caused byRhizoctonia solani was reduced in the greenhouse by 51%, 46% and 56%, respectively. In bean seedlings inoculated withB. subtilis (isolate no. 72),B. cereus (isolate no. 78) orB. pumilus (isolate no. 65), disease incidence caused bySclerotium rolfsii was reduced by 72%, 79% and 26%, respectively, as compared to control. In both cotton and bean seedlings, these endophytes reduced the disease index more than 50%. These results indicate that endophytic bacteria can survive inside cotton plants and are efficient agents for biological control against plant pathogens under greenhouse conditions.
Patterns in the localization of compounds, metabolic processes, and regulatory machineries at the cellular and subcellular levels are studied by scientific disciplines, called cytochemistry (biochemistry of the cell, cellular topochemistry) or histochemistry, if the tissue level is concerned. In order to reach these goals, a whole range of procedures have been developed, which may be divided in two groups: the in vitro techniques, based on homogenate fractionation and the in situ techniques, realized on sections. Only in situ techniques are considered in this chapter (1–5).
Transformation of tomato (Lycopersicon esculentum Mill.) was carried out using disarmed Agrobacterium tumefaciens strain EHA 105 harboring a binary vector pBIG-HYG-bspA. The plasmid contains the bspA (boiling stable protein of aspen) gene under the control of a CaMV35S promoter and nopaline synthase (NOS) terminator, hygromycin phosphotransferase gene (hpt) driven by nopaline synthase promoter and polyadenylation signal of Agrobacterium gene7 as terminator and a promoterless gus gene. Very strong β-glucuronidase (GUS) expression was observed in transformed tomato plants but never in non-transformed
(control). Since GUS expression was observed only in transformed plants, the possibility of the presence of endogenous GUS
enzymes was ruled out. Possibility of false GUS positives was also ruled out because the GUS positive explants reacted positively
to polymerase chain reaction (PCR) and PCR-Southern tests carried out for the presence of bspA gene, which indicated the integration of T-DNA in tomato genome. The promoterless GUS expression was hypothesized either
due to leaky NOS termination signal of bspA gene or due to different cryptic promoters of plant origin. It was concluded that GUS expression was observed in the putative
transgenics either due to the read through mechanism by the strong CaMV35S promoter or due to several cryptic promoters driving
the gus gene in different transgenic lines.
Yams (Dioscorea spp., Dioscoreaceae), grown either for their starchy tubers or medicinal properties, are important crops in the tropics and
subtropics. Yams broaden the food base and provide food security and income to over 300 million people. They are vegetatively
propagated and comprise both diploid and polyploid species. Despite their economic and socio-cultural importance, very little
is known about the genetics and genomics of yams due to research neglect and several biological constraints. Consequently,
conventional breeding efforts have been severely hampered. Research to unravel the apparent complexity of the yam genome will
have far-reaching implications for genetic improvement of this important tuber crop. Nevertheless, progress has been made
recently towards understanding Dioscorea phylogeny and phylogenetic relationships within the genus. Also, improved molecular technologies have been developed for
genome analysis, including germplasm characterization, cytogenetics, genetic mapping and tagging, and functional genomics.
Genetic linkage maps have been constructed for D. rotundata and D. alata, and quantitative trait loci associated with resistance to Yam mosaic virus in D. rotundata and anthracnose (Colletotrichum gloeosporioides) in D. alata have been identified. In addition, candidate random amplified polymorphic DNA markers associated with major genes controlling
resistance to Yam mosaic virus and anthracnose have been identified. These markers could be converted to sequencecharacterized amplified regions and used
formarker-assisted selection for resistance to diseases. An initial cDNA library has been constructed to develop expressed
sequence tags for gene discovery and as a source of additional molecular markers. Genetic engineering offers a powerful tool,
complementing conventional breeding approaches, for yam improvement. Methods for yam transformation, including in vitro plant
regeneration, gene delivery, selection of transformed tissues, and recovery of transgenic plants have been developed but still
need improvements. This chapter reviews advances made in yam molecular marker development for genome analysis, phylogeny,
molecular cytogenetics, characterization of genetic diversity, genetic mapping and tagging, and progress in functional genomics.
The true yams belong to the monocotyledonous genus Dioscorea containing 600 species, many of which are of economic significance in the tropical and sub-tropical regions of the world as food or as sources of the steroid precursor diosgenin. Selected cultivars have been vegetatively propagated over centuries and, as a consequence, systemic pathogens such as viruses, bacteria and fungi have accumulated. When cultured in vitro from nodal segment and shoot tip explants of vigorously growing vines, most Dioscorea produce clean healthy shoots which can be multiplied satisfactorily using standard microcutting techniques [1]. Plantlets derived from yam apical meristems can also yield planting materials free of yam viruses [2]. It has been our experience, however, that apparently healthy yam shoot cultures can support a surprisingly high level of exopolysaccharide-embedded surface microflora in the absence of obvious signs of microbial contamination. Surface contaminants were first recognised during a series of SEM studies on the development of conidiospores of Colletotrichum gloeosporioides (the causal agent of anthracnose disease) on leaf surfaces of different yam species [3]. It was only when cultures were shipped by air mail (i.e. in the dark under fluctuating temperature conditions) or when shoot cultures were allowed to age for several months before subculturing, did conspicuous signs of bacterial growth on tissue culture media begin to appear at the bases of shoot cultures. Also when yam shoot tissues were used as sources of explants for cell [4] or protoplast [5] culture studies the presence of covert bacteria contaminants in two key food yams, Dioscorea alata and Dioscorea cayenensis [6,7], were discovered. Reported here are the results of our studies over a 10-year period on the natures of microbial contamination in tissue, cell and protoplast cultures of Dioscorea spp. using electron microscopy, tissue print immuno-blotting and PCR techniques.
Two regenerative alfalfa genotypes were transformed withAgrobacterium tumefaciens with binary vectors containing the coding sequences for ß-glucuronidase (GUS) and npt II (kanamycin resistance). The regenerative
genotypes and their transgenic populations were agronomically inferior, and one was a somaclonal variant for flower color.
GUS was used as a dominant genetic marker in a model system for studying backcrossing to improve transgenic alfalfa. Agronomic
yield deficiencies and somaclonal changes were corrected by one to three backcrosses to cultivar genotypes, depending on the
vigor of the original transformant. Three backcrosses were considered optimal because progeny contain 94% cultivar germplasm
and could be used as parents of a new cultivar. Use of different cultivar genotypes each generation of backcrossing minimized
inbreeding and maximized the heterotic potential of backcross derivatives. The improvement of transgenic alfalfa by backcrossing
using a dominant marker required only as much time as the original transformation experiment.
The effects of timentin on shoot regeneration of tobacco (Nicotiana tabaccum) and Siberian elm (Ulmus pumila L.) and its use for the suppression of Agrobacterium tumefaciens in Agrobacterium-mediated genetic transformation were determined. Timentin is a mixture of ticarcillin and clavulanic acid, and at concentrations
of 200–500 mg/l with ratios of ticarcillin:clavulanic acid of 50:1 and 100:1, it had little effect on shoot regeneration of
tobacco or Siberian elm. Timentin was as effective in suppressing A. tumefaciens as carbenicillin and cefatoxime at concentrations commonly used in transformation. The disarmed A. tumefaciens strain LBA4404 in infected tobacco leaf tissues was visually undetectable after three subcultures on medium with 500 mg/l
of timentin and 250 mg/l carbenicillin. Timentin was stable in solid agar medium and remained effective for at least 70 days,
but was unstable when stored as a mixed stock solution or as separate ticarcillin and clavulanic acid stock solutions at –20°C
or –80°C freezer for 4 weeks. Timentin may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation.
Morphogenically competent cell clusters were induced from 3–4 mm-long root segments excised from in vitro shoot cultures of the greater yam (Dioscorea alata L. Oriental Lisbon)plated in a liquid modified Murashige and Skoog (1962) medium (PMS) supplemented with 1 mg l–1 2,4-dichlorophenoxy-acetic acid and 3% sucrose at 25C in the light. Embryogenic cell clusters released from subcortical regions of the root explants proliferated further into proembryonic cell masses which, when transferred to auxin-free medium, differentiated into somatic embryos. A proportion of the somatic embryos (as high as 80% in some experiments) converted to plantlets within two months after initial root segment culture. A description of the different stages of yam somatic embryogenesis is presented. Cultural factors which affected somatic embryo maturation in liquid medium to the greatest extent were embryogenic cell cluster density and gibberellic acid (GA3) level present in the medium. Optimal somatic embryo maturation occurred when the density of embryogenic clusters was adjusted to 2–4 per 5 ml PMS and when GA3 was present in conditioned PMS medium at a concentration of 1.5 mg l–1
Dioscorea zingiberensis Wright has been cultivated as a pharmaceutical crop for production of diosgenin, a precursor for synthesis of various important
steroid drugs. Because breeding of D.zingiberensis through sexual hybridization is difficult due to its unstable sexuality and differences in timing of flowering in male and
female plants, gene transfer approaches may play a vital role in its genetic improvement. In this study, the Agrobacterium tumefaciens-mediated transformation of D.zingiberensis was investigated with leaves and calli as explants. The results showed that both leaf segments and callus pieces were sensitive
to 30mg/l hygromycin and 50–60mg/l kanamycin, and using calli as explants and addition of acetosyringone (AS) in cocultivation
medium were crucial for successful transformation. We first immersed callus explants in A.tumefaciens cells for 30min and then transferred the explants onto a co-cultivation medium supplemented with 200μM AS for 3days. Three
days after, we cultured the infected explants on a selective medium containing 50mg/l kanamycin and 100mg/l timentin for
formation of kanamycin-resistant calli. After the kanamycin-resistant calli were produced, we transferred them onto fresh
selective medium for shoot induction. Finally, the kanamycin resistant shoots were rooted and the stable incorporation of
the transgene into the genome of D.zingiberensis plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The method reported here can be used to
produce transgenic D.zingiberensis plants in 5months and the transformation frequency is 24.8% based on the numbers of independent transgenic plants regenerated
from initial infected callus explants.
In the past two decades, a great deal of information on the role of endophytic microorganisms in nature has been collected. The capability of colonizing internal host tissues has made endophytes valuable for agriculture as a tool to improve crop performance. In this review, we addressed the major topics concerning the control of insects-pests by endophytic microorganisms. Several examples of insect control are described, notably those involving the interactions between fungi and grazing grasses from temperate countries. The mechanisms by which endophytic fungi control insect attacks are listed and include toxin production as well as the influence of these compounds on plant and livestock and how their production may be affected by genetic and environmental conditions. The importance of endophytic entomopathogenic fungi for insect control is also addressed. As the literature has shown, there is a lack of information on endophytes from tropical hosts, which are more severely affected by pests and diseases. Having this in mind, we have included an updated and extensive literature in this review, concerning new findings from tropical plants, including the characterization of endophytic fungi and bacteria microbiota from several Amazon trees, citrus and medicinal plants among others.
A series of transposons are described which contain the gusA gene, encoding beta-glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive. The regulated promoters include the tac promoter which can be induced by IPTG, and nifH promoters which are symbiotically activated in legume nodules. One transposon contains gusA with a strong Shine-Dalgarno translation initiation context, but no promoter, and thus acts as a promoter-probe transposon. In addition, a gus operon deletion strain of Escherichia coli, and a transposon designed for use in chromosomal mapping using PFGE, are described. The GUS transposons are constructed in a mini-Tn5 system which can be transferred to Gram-negative bacteria by conjugation, and will form stable genomic insertions. Due to the absence of GUS activity in plants and many bacteria of economic importance, these transposons constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants, as well as for studies of the fundamental molecular basis of such interactions. The variety of assays available for GUS enable both quantitative assays and spatial localization of marked bacteria to be carried out.
Patatin is a family of lipid acyl hydrolases that accounts for 30 to 40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. To examine the regulation of the patatin genes, we constructed a chimeric gene containing 2.5 kb of 5' flanking sequence from the class I patatin genomic clone PS20 transcriptionally fused to β-glucuronidase (GUS) and introduced it into potato plants using an Agrobacterium tumefaciens Tiplasmid vector. While the chimeric gene was expressed at high levels in tubers and in stolons attached to developing tubers, it was not normally expressed in leaves, stems, roots, or in stolons before tuberizatization. However, the expression of the class I patatin-GUS construct was not "tuber-specific" since leaf and stem explants cultured on medium containing 300 to 400 mM sucrose showed GUS activity equal or greater than that of tubers. The sucrose induction of GUS activity in leaf and stem explants was accompanied by the accumulation of patatin protein and large amounts of starch, but not by the morphological changes that normally are associated with tuberization. In contrast, the GUS reporter gene under the control of the 35S promoter of cauliflower mosaic virus showed an essentially uniform pattern of expression in transgenic potato plants and was not induced by sucrose.
Fifty-two plant species, covering some Gymnosperms and all the key groups of Angiosperms, were chosen for surveying their intrinsic beta-glucuronidase-like activities. Histochemical (overnight incubation) and qualitative fluorometric (24 h incubation) assays indicated that, with few exceptions, such activities were detected in certain part(s) of the fruit walls, seed coats, endosperms or, especially, the embryos of the tested plants. Most of such activities in the excised immature embryos of soybean and string bean disappeared after one to a few days' in vitro culturing. Such activities in the intact mature seeds of these two species diminished also during germination process. The vegetative organs of seedlings/mature plants usually lack such activities. The enzyme(s) responsible for such activities was antigenically dissimilar to E. coli beta-glucuronidase.
Bacterial glucuronidase which hydrolyzes the glucuronides of estriol, pregnanediol, menthol and phenolphthalein liberates a large proportion of estrogen, ketosteroid and corticosteroid of human urine.