Agrobacterium tumefaciens mediated transformation of the aquatic carnivorous plant Utricularia gibba.

BACKGROUND: The genus Utricularia belongs to Lentibulariaceae, the largest family of carnivorous plants, which includes terrestrial, epiphytic and aquatic species. The development of specialized structures that evolved for carnivory is a feature of this genus that has been of great interest to biologists since Darwin's early studies. Utricularia gibba is itself an aquatic plant with sophisticated bladder traps having one of the most complex suction mechanisms for trapping prey. However, the molecular characterization of the mechanisms that regulate trap development and the biophysical processes involved in prey trapping are still largely unknown due to the lack of a simple and reproducible gene transfer system. RESULTS: Here, we report the establishment of a simple, fast and reproducible protocol for genetic transformation of U. gibba based on the T-DNA of Agrobacterium tumefaciens. An in vitro selection system using Phosphinotricin as a selective agent was established for U. gibba. Plant transformation was confirmed by histochemical GUS assays and PCR and qRT-PCR analyses. We report on the expression pattern of the 35S promoter and of the promoter of a trap-specific ribonuclease gene in transgenic U. gibba plants. CONCLUSIONS: The genetic transformation protocol reported here is an effective method for studying developmental biology and functional genomics of this genus of carnivorous plants and advances the utility of U. gibba as a model system to study developmental processes involved in trap formation.

Photosynthesis-associated gene families: differences in response to tissue-specific and environmental factors.

The endogenous small subunit of the ribulose-1,5-bisphosphate carboxylase gene rbcS and the light-harvesting chlorophyll a/b-binding protein gene (LHCP) of pea are expressed in a light-inducible manner and are active mainly in green chloroplast-containing tissue. Chimeric genes under control of the 5'-flanking sequences of the rbcS ss3.6 or LHCP AB80 genes from pea were used to study the factors relating to the issue-specific and lightinducible expression of these nuclear-encoded genes in transgenic tobacco plants. The results show that plastid development plays a crucial role in the activation of expression of these chimeric genes. Particular members of each of the above gene families respond differently to tissue-specific and environmental factors. Furthermore, the light-inducible expression directed by the 5'-flanking sequence of ss3.6 rbcSgene is not exclusively mediated by phytochrome, but probably is controlledin large part by another photoreceptor.

Aluminum tolerance in transgenic plants by alteration of citrate synthesis.

Aluminum when in soluble form, as found in acidic soils that comprise about 40 percent of the world's arable land, is toxic to many crops. Organic acid excretion has been correlated with aluminum tolerance in higher plants. Overproduction of citrate was shown to result in aluminum tolerance in transgenic tobacco (Nicotiana tabacum) and papaya (Carica papaya) plants.

Introduction of genetic material into plant cells.

The tumor-inducing (Ti) plasmid of the soil microorganism Agrobacterium tumefaciens is the agent of crown gall disease in dicotyledonous plants. The Ti plasmid contains two regions that are essential for the production of transformed cells. One of these regions, termed transfer DNA, induces tumor formation and is found in all established plant tumor lines; the other, termed the virulence region, is essential for the formation but not the maintenance of tumors. Transfer DNA, which transfers to the plant genomes in a somewhat predictable manner, can be increased in size by the insertion of foreign DNA without its transferring ability being affected. The tumor-causing genes can be removed so that they no longer interfere with normal plant growth and differentiation. This modified Ti plasmid can thus be used as a vector for the transfer of foreign genes into plants.

Enhanced phosphorus uptake in transgenic tobacco plants that overproduce citrate.

Phosphorus (P) is one of the most important nutrients limiting agricultural production worldwide. In acid and alkaline soils, which make up over 70% of the world's arable land, P forms insoluble compounds that are not available for plant use. To reduce P deficiencies and ensure plant productivity, nearly 30 million tons of P fertilizer are applied every year. Up to 80% of the applied P fertilizer is lost because it becomes immobile and unavailable for plant uptake. Therefore, the development of novel plant varieties more efficient in the use of P represents the best alternative to reduce the use of P fertilizers and achieve a more sustainable agriculture. We show here that the ability to use insoluble P compounds can be significantly enhanced by engineering plants to produce more organic acids. Our results show that when compared to the controls, citrate-overproducing plants yield more leaf and fruit biomass when grown under P-limiting conditions and require less P fertilizer to achieve optimal growth.

The first intron of the Arabidopsis thaliana gene coding for elongation factor 1 beta contains an enhancer-like element.

Genomic and cDNA clones coding for elongation factor-1 beta (eEF-1 beta) from Arabidopsis thaliana (At) were isolated and characterized. eEF-1 beta was found to be encoded by a single-copy At gene. Chimeric genes fusing the promoter and the 5' untranslated region of the At eEF-1 beta gene to the gus reporter gene were constructed and used to study the expression of this gene in transgenic tobacco plants. Interestingly, it was found that the first intron of this gene is required for high levels of expression. Experiments using chimeric promoters showed that an enhancer-like element is present in the first intron of At eEF-1 beta. Gel-shift assays were used to demonstrate that this intron is specifically bound by putative transcription factors present in nuclear protein extracts.

Isolation and characterization of genes encoding chaperonin 60 beta from Arabidopsis thaliana.

Chaperonins (Cpn) are implicated in the folding and assembly of multimeric proteins in plastids and mitochondria of eukaryotes and in prokaryotes. Plastid Cpn is composed of two different polypeptides termed Cpn60 alpha and Cpn60 beta. We have isolated cDNA and genomic clones encoding Cpn60 beta from Arabidopsis thaliana. The steady-state level of the cpn60 beta mRNAs is higher in etiolated leaves and sucrose-treated plants as compared to control leaves. The A. thaliana cpn60 beta gene family consists of at least three different coding units. It was confirmed that Cpn beta-encoding genes have a high level of conservation among plants.

Characterization of a rice sucrose-phosphate synthase-encoding gene.

A rice genomic clone (sps1) coding for sucrose phosphate synthase (SPS) was isolated and sequenced. Rice sps1 contains 13 exons and 12 introns, an unusually long 366-bp leader region with a highly organized primary structure and a promoter region with no obvious homology with eukaryotic promoter consensus sequences. Southern blot analysis showed that SPS is encoded by a single-copy gene in the rice genome. Comparison of the rice, maize, potato and spinach SPS deduced amino acid (aa) sequences showed that these enzymes have a well conserved region comprising their first 700 aa, and a variable C-terminal region. Analysis of rice sps1 expression showed that mRNA levels change during leaf development. SPS activity and mRNA were undetectable in roots.

Transgenic plants of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. ex Steud., from microprojectile bombardment of highly chlorophyllous embryogenic cells.

For the first time, the production of transgenic plants of the forage grass blue grama, Bouteloua gracilis [H.B.K.] Lag. ex Steud., is reported. Transgenic plants containing a gus Colon, two colons nptll fusion driven by a double CaMV35S promoter were obtained by microprojectile bombardment of the highly chlorophyllous embryogenic cell line 'TIANSJ98'. Transformed B. gracilis cell lines resisted a lethal concentration of 160 mg/l of kanamycin for at least 8 months. Chlorophyll development and growth rate were used as useful criteria for discriminating transformed from non-transformed clones. Stable integration of the transgene in the blue grama genome was demonstrated by PCR and Southern-hybridization analysis. Expression of the NPTll protein in transgenic plants grown under greenhouse conditions was confirmed indirectly by spraying kanamycin (150-250 mg/l) on plant foliage, and directly by ELISA immunological tests. Control plants sprayed with kanamycin showed foliar necrosis and reduced growth (tillering) compared to plants containing the transgene. NPTll was found in transgenic plants in levels ranging between 12.6 and 29.6 ng/mg FW of cells, as determined by ELISA reactions.

Agrobacterium-mediated transformation of Amaranthus hypochondriacus: light- and tissue-specific expression of a pea chlorophyll a/b-binding protein promoter.

Mature embryos of Amaranthus hypochondriacus (amaranth) were used to develop an in vitro culture system for plant regeneration and genetic transformation. Plants were regenerated from embryo-derived callus cultivated on Murashige and Skoog medium supplemented with 10 µM 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-2-methoxybenzoic acid and 10% coconut liquid endosperm. Transgenic plants were obtained by inoculation of mature embryo explants with a disarmed Agrobacterium strain containing the plasmid pGV2260(pEsc4), which carried the genes encoding neomycin phosphotransferase type II and ß-glucuronidase. The presence of transgenes in the genome of transformed amaranth plants and their progeny was demonstrated by Southern blot hybridization. Tissue specific and light-inducible expression directed by a pea chlorophyll a/b-binding protein promoter was observed in transgenic amaranth plants and their progeny.

Establishment, characterization and plant regeneration from highly chlorophyllous embryogenic cell cultures of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. ex Steud.

A finely dispersed, homogeneous and highly chlorophyllous cell suspension (TIANSJ98 cell line) was obtained from shoot apices of Bouteloua gracilis (H.B.K.) Lag. ex Steud. cultured on MPC medium containing MS salts supplemented with 2,4-D (1 mg/l), BAP (2 mg/l) and adenine (40 mg/l). When the TIANSJ98 cell line was grown in this medium with shaking at 180 rpm it had doubling times of 7.2 and 3.7 days in terms of fresh and dry weight, respectively. Total chlorophyll content in this cell culture ranged from 121.6 to 18.3 µg/g FW at 12 and 21 days following culture initiation. Plants regenerated from the TIANSJ98 cell line, via somatic embryogenesis, were grown to maturity and produced seeds. Although different cell culture systems have been described for cereals and grasses, to the best of our knowledge this is the first report of a highly chlorophyllous and regenerable cell suspension in Poaceae.

Expression of a bacterial phaseolotoxin-resistant ornithyl transcarbamylase in transgenic tobacco confers resistance to Pseudomonas syringae pv. phaseolicola.

Toxins have been shown to be an important virulence component for most pathovars of Pseudomonas syringae. Here we have examined the role of phaseolotoxin in the virulence mechanism of P. syringae pv. phaseolicola by producing transgenic tobacco plants that express a pathogen-derived toxin-resistant target enzyme. Such plants are insensitive to the toxin and less prone to infection by the pathogen.

Transgenic plants for tropical regions: some considerations about their development and their transfer to the small farmer.

Biotechnological applications, especially transgenic plants, probably hold the most promise in augmenting agricultural production in the first decades of the next millennium. However, the application of these technologies to the agriculture of tropical regions where the largest areas of low productivity are located, and where they are most needed, remains a major challenge. In this paper, some of the important issues that need to be considered to ensure that plant biotechnology is effectively transferred to the developing world are discussed.

Genetically engineered resistance to bacterial and fungal pathogens.

In the past 10 years, different strategies have been used to produce transgenic plants that are less susceptible to diseases caused by phytopathogenic fungi and bacteria. Genes from different organisms, including bacteria, fungi and plants, have been successfully used to develop these strategies. Some strategies have been shown to be effective against different pathogens, whereas others are specific to a single pathogen or even to a single pathovar or race of a given pathogen. In this review, we present the strategies that have been employed to produce transgenic plants less susceptible to bacterial and fungal diseases and which constitute an important area of plant biotechnology.

Experimental and theoretical definition of geminivirus origin of replication.

Geminiviruses are plant pathogens that replicate by a rolling-circle mechanism, analogous to that used by several prokaryotic ssDNA replicons. Recent reports provide important progress in understanding the structure and functioning of replication origin from these viruses. We have used these data to propose models for the initiation of replication in dicot- and monocot-infecting geminiviruses.

DNA methylation and polyamines in regulation of development of the fungus Mucor rouxii.

DNA from intact or spherically growing spores of Mucor rouxii is highly methylated, whereas DNA from germlings has low levels of methylation. DNA from spores incubated with hydroxyurea or 1,4-diaminobutanone is also highly methylated. The reversal of the effect of 1,4-diaminobutanone by azacytidine correlated with DNA hypomethylation. These data suggest that the change in growth pattern from spherical to polarized correlates with the degree of DNA methylation and that this, in turn, may be controlled by polyamine levels.

Transformation of four pathogenic Phytophthora spp by microprojectile bombardment on intact mycelia.

Phytophthora capsici, P. citricola, P. cinnamomi and P. citrophthora were transformed without the removal of cell walls by particle acceleration with plasmids containing the beta-glucuronidase gene and hygromycin B resistance. Transformants were detected by histochemical and fluorometric beta-glucuronidase assays and confirmed by Southern-blot hybridization. It was found that the promoter of a plant virus is functional in Phytophthora. In addition, a method was designed to visually identify homogeneous transformed colonies, derived from zoospores of transformed multinucleated Phytophthora mycelia, based on blue color development on plates containing X-Gluc.

Ancestral multipartite units in light-responsive plant promoters have structural features correlating with specific phototransduction pathways.

Regulation of plant gene transcription by light is mediated by multipartite cis-regulatory units. Previous attempts to identify structural features that are common to all light-responsive elements (LREs) have been unsuccessful. To address the question of what is needed to confer photoresponsiveness to a promoter, the upstream sequences from more than 110 light-regulated plant genes were analyzed by a new, phylogenetic-structural method. As a result, 30 distinct conserved DNA module arrays (CMAs) associated with light-responsive promoter regions were identified. Several of these CMAs have remained invariant throughout the evolutionary radiation of angiosperms and are conserved between homologous genes as well as between members of different gene families. The identified CMAs share a gene superfamily-specific core that correlates with the particular phytochrome-dependent transduction pathway that controls their expression, i.e. ACCTA(A/C)C(A/C) for the cGMP-dependent phenylpropanoid metabolism-associated genes, and GATA(A/T)GR for the Ca2+/calmodulin-dependent photosynthesis-associated nuclear genes. In addition to suggesting a general model for the functional and structural organization of LREs, the data obtained in this study indicate that angiosperm LREs probably evolved from complex cis-acting elements involved in regulatory processes other than photoregulation in gymnosperms.

The mannopine synthase promoter contains vectorial cis-regulatory elements that act as enhancers and silencers.

A 479-bp bi-directional promoter controls the expression of two genes (mas1' and mas2') that encode enzymes for the synthesis of the opine mannopine in plant tissues infected with Agrobacterium tumefaciens. This 5' regulatory region (mas promoter) contains all the cis-acting elements involved in mediating the complex regulatory properties of these genes in plants. Using different mas promoter regions fused to a minimal 35S promoter (35Sdelta108), we found that the regulatory properties of these divergent promoters result from the presence of orientation-dependent negative and positive regulatory regions. Some of these elements have the unusual property of acting as enhancers in one orientation and as silencers in the other. Using electrophoretic mobility shift analysis (EMSA), we showed that the functional mas promoter regions identified by fluorometric and histochemical assays for reporter gene activity in transgenic plants have the ability specifically to bind nuclear protein factors from Nicotiana tabacum, Phaseolus vulgaris, Solanum tuberosum, and Arabidopsis thaliana.

Genetic transformation of the plant pathogens Phytophthora capsici and Phytophthora parasitica.

Phytophthora capsici and P.parasitica were transformed to hygromycin B resistance using plasmids pCM54 and pHL1, which contain the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the Ustilago maydis heat shock hsp70 gene. Enzymes Driselase and Novozyme 234 were used to generate protoplasts which were then transformed following exposure to plasmid DNA and polyethylene glycol 6000. Transformation frequencies of over 500 transformants per micrograms of DNA per 1 x 10(6) protoplasts were obtained. Plasmid pCM54 appears to be transmitted in Phytophthora spp. as an extra-chromosomal element through replication, as shown by Southern blot hybridization and by the loss of plasmid methylation. In addition, transformed strains retained their capacity of infecting Serrano pepper seedlings and Mc. Intosh apple fruits, the host plants for P.capsici and P.parasitica, respectively.