Assessment of the salt tolerance and environmental biosafety of Eucalyptus camaldulensis harboring a mangrin transgene.

Increasing soil salinization of arable land has a major impact on the global ecosystem. One approach to increase the usable global forest area is to develop transgenic trees with higher tolerance to conditions of salt stress. An allene oxide cyclase homolog, mangrin, contains a core protein domain that enhances the salt tolerance of its host. We utilized this feature to develop improved salt-tolerant eucalyptus trees, by using transgenic Eucalyptus camaldulensis carrying the mangrin gene as a model. Since the Japanese government requires an environmental biosafety assessment for the surrounding biosphere, we performed experiments on trees grown in a special netted-house. This study examined the transgenic E. camaldulensis carrying the mangrin gene to assess the feasibility of using these transformants, and assessed their salt tolerance and environmental biosafety. We found that seven of 36 transgenic genotypes had significantly higher salt tolerance than non-transformants, and more importantly, that these plants had no significant impact on environmental biosafety. These results suggest that introduction of the mangrin gene may be one approach to safely enhance salt tolerance in genetically modified Eucalyptus species, and that the transformants have no apparent risks in terms of environmental biosafety. Thus, this study provides valuable information regarding the use of transgenic trees in situ.

Agrobacterium-mediated transformation of Eucalyptus globulus using explants with shoot apex with introduction of bacterial choline oxidase gene to enhance salt tolerance.

Eucalyptus globulus is one of the most economically important plantation hardwoods for paper making. However, its low transformation frequency has prevented genetic engineering of this species with useful genes. We found the hypocotyl section with a shoot apex has the highest regeneration ability among another hypocotyl sections, and have developed an efficient Agrobacterium-mediated transformation method using these materials. We then introduced a salt tolerance gene, namely a bacterial choline oxidase gene (codA) with a GUS reporter gene, into E. globulus. The highest frequency of transgenic shoot regeneration from hypocotyls with shoot apex was 7.4% and the average frequency in four experiments was 4.0%, 12-fold higher than that from hypocotyls without shoot apex. Using about 10,000 explants, over 250 regenerated buds were confirmed as transformants by GUS analysis. Southern blot analysis of 100 elongated shoots confirmed successful generation of stable transformants. Accumulation of glycinebetaine was investigated in 44 selected transgenic lines, which showed 1- to 12-fold higher glycinebetaine levels than non-transgenic controls. Rooting of 16 transgenic lines was successful using a photoautotrophic method under enrichment with 1,000 ppm CO(2). The transgenic whole plantlets were transplanted into potting soil and grown normally in a growth room. They showed salt tolerance to 300 mM NaCl. The points of our system are using explants with shoot apex as materials, inhibiting the elongation of the apex on the selection medium, and regenerating transgenic buds from the side opposite to the apex. This approach may also solve transformation problems in other important plants.

Monoterpene engineering in a woody plant Eucalyptus camaldulensis using a limonene synthase cDNA.

Metabolic engineering aimed at monoterpene production has become an intensive research topic in recent years, although most studies have been limited to herbal plants including model plants such as Arabidopsis. The genus Eucalyptus includes commercially important woody plants in terms of essential oil production and the pulp industry. This study attempted to modify the production of monoterpenes, which are major components of Eucalyptus essential oil, by introducing two expression constructs containing Perilla frutescens limonene synthase (PFLS) cDNA, whose gene products were designed to be localized in either the plastid or cytosol, into Eucalyptus camaldulensis. The expression of the plastid-type and cytosol-type PFLS cDNA in transgenic E. camaldulensis was confirmed by real-time polymerase chain reaction (PCR). Gas chromatography with a flame ionization detector analyses of leaf extracts revealed that the plastidic and cytosolic expression of PFLS yielded 2.6- and 4.5-times more limonene than that accumulated in wild-type E. camaldulensis, respectively, while the ectopic expression of PFLS had only a small effect on the emission of limonene from the leaves of E. camaldulensis. Surprisingly, the high level of PFLS in Eucalyptus was accompanied by a synergistic increase in the production of 1,8-cineole and alpha-pinene, two major components of Eucalyptus monoterpenes. This genetic engineering of monoterpenes demonstrated a new potential for molecular breeding in woody plants.

Expression of a transgene exchanged by the recombinase-mediated cassette exchange (RMCE) method in plants.

We developed a site-directed integration (SDI) system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined target locus by recombinase-mediated cassette exchange (RMCE). We produced site-specific transgenic tobacco plants from four target lines and examined expression of the transgene in T1 site-specific transgenic tobacco plants, which were obtained by backcrossing. We found that site-specific transgenic plants from the same target lines showed approximately the same level of expression of the transgene. Moreover, we demonstrated that site-specific transgenic plants showed much less variability of transgene expression than random-integration transgenic plants. Interestingly, transgenes in the same direction at the same target locus showed the same level of activity, but transgenes in different directions showed different levels of activity. The expression levels of transgene did not correlate with those of the target gene. Our results showed that the SDI system could benefit the precise comparisons between different gene constructs, the characterization of different chromosomal regions and the cost-effective screening of reliable transgenic plants.

Genetically modified rice seeds accumulating GLP-1 analogue stimulate insulin secretion from a mouse pancreatic beta-cell line.

Glucagon-like peptide-1 (7-36) amide (GLP-1) is the most potent physiological insulinotropic hormone in humans. We produced large amounts of a GLP-1 analogue, [Ser8, Gln26, Asp34]-GLP-1, which is resistant to trypsin-digestion, as part of a chimeric rice seed storage protein, a 26 kDa globulin, in genetically modified rice seeds. Junction sites between GLP-1 analogue and globulin were replaced by tryptic cleavage sites. The highest level of GLP-1 analogue accumulation was approximately 20-50 microg per seed. We found that GLP-1 analogue derived from trypsin-digested genetically modified rice seeds stimulated insulin secretion from a mouse pancreatic beta-cell line, MIN6.

Agrobacterium-mediated RMCE approach for gene replacement.

We describe the site-directed integration (SDI) system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined target locus by recombinase-mediated cassette exchange (RMCE). The system requires the selection of a transformed line with an integrated copy of a target cassette, and subsequent introduction of an exchange vector. The target cassette contains the npt and cod genes between oppositely orientated recognition sites (RS). The exchange vector T-DNA possesses an exchange cassette containing the gene of interest and a selectable marker gene, such as hpt, between oppositely orientated (inner) RS. Adjacent to the exchange cassette are ipt and recombinase (R) genes and an additional (outer) RS. The recombinase catalyses double-crossover between target RS and exchange inner RS to replace the integrated target cassette with the introduced exchange cassette. Transgenic plants that contain randomly integrated copies of the exchange vector T-DNA show an abnormal phenotype as a result of the overproduction of cytokinin from ipt gene expression. The recombinase can also act on the directly orientated outer RS to remove such randomly integrated copies. The system resulted in single-copy exchange into the target site only in regenerated tobacco at a frequency of 1%-3% per treated explant, or 4%-9% per regenerated line of normal phenotype. Thus, transgenic plants with only an exchanged copy can be efficiently accumulated and selected. Here, we show that the SDI system can efficiently replace the target cassettes with the exchange cassettes in a heterozygous or homozygous condition. The SDI system may be useful for precise comparisons of different gene constructs, the characterization of different chromosomal regions and the cost-effective screening of reliable transgenic plants.

Elimination of marker genes from transgenic plants using MAT vector systems.

We have developed an efficient system (Multi-Auto-Transformation [MAT] vectors) for the removal of marker genes and to increase the regeneration frequency of transgenic crops without using antibiotic selection, reducing their possible environmental impact. The MAT vector system is designed to use the oncogenes (ipt, iaaM/H, rol) of Agrobacterium, which control the endogenous levels of plant hormones and the cell responses to plant growth regulators, to differentiate transgenic cells, and to select marker-free transgenic plants. The oncogenes are combined with the site-specific recombination system (R/RS). At transformation, the oncogenes regenerate transgenic plants and then are removed by the R/RS system to generate marker-free transgenic plants. The choice of a promoter for the oncogenes and the recombinase (R) gene, the state of plant materials and the tissue culture conditions greatly affect efficiency of both the regeneration of transgenic plants and the generation of marker-free plants. We have evaluated these conditions in several plant species to increase their generation efficiency. This chapter describes our transformation protocols using MAT vectors.

Enrichments of gene replacement events by Agrobacterium-mediated recombinase-mediated cassette exchange.

We report recombinase-mediated cassette exchange (RMCE), which can permit integration of transgenes into pre-defined chromosomal loci with no co-expressed marker gene by using Agrobacterium-mediated transformation. Transgenic tobacco plants which have a single copy of negative marker genes (codA) at target loci in heterozygous and homozygous conditions were used for gene exchange by the RMCE method. By negative selection, we were able to obtain five heterozygous and four homozygous transgenic plants in which the genes were exchanged from 64 leaf segments of heterozygous and homozygous target plants, respectively. Except for one transgenic plant with an extra copy, the other eight plants had only a single copy of marker-free transgenes, and no footprint of random integrated copies was detected in half of the eight plants. The RMCE re-transformation frequencies were calculated as 6.25 % per explant and were approximately the same as the average percentage of intact single-copy transformation events for standard tobacco Agrobacterium-mediated transformation.

Marker-free gene targeting by recombinase-mediated cassette exchange.

Repeated gene targeting by recombinase-mediated cassette exchange (RMCE) is an efficient tool for the study of gene function and regulation because of the high predictability and repeatability of gene expression. We have developed the site-directed integration (SDI) vector system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined chromosomal locus in the absence of any coexpressed selection marker gene (Nanto et al. Plant Biotechnol J 3:203-214, 2005; Nanto and Ebinuma Transgenic Res 17:337-344, 2008; Nanto et al. Plant Cell Rep 28:777-785, 2009; Ebinuma and Nanto (2009) Marker-free targeted transformation, in Molecular techniques in crop improvement (2nd Edition). (Jain, S. M. and Brar, D. S. eds.), Springer Netherlands, pp. 527-543; Ebinuma and Nanto in preparation). The SDI vector system consists of a target vector to introduce the target cassette and an exchange vector to reintroduce the exchange cassette for gene replacement. We describe the molecular design and experimental protocol that can efficiently enrich RMCE events through the removal of randomly integrated copies and select clean marker-free targeted transgenic plants by using a negative marker.

26-Week oral safety study in macaques for transgenic rice containing major human T-cell epitope peptides from Japanese cedar pollen allergens.

A study of repeated oral administration of transgenic rice containing a hybrid peptide of major human T-cell epitopes (7Crp) from Japanese cedar pollen allergens was carried out in cynomolgus macaques over 26 weeks. The monkeys were divided into three groups, each comprising three males and three females, administered a high dose of transgenic rice, a low dose of transgenic rice, or a high dose of the parental rice strain. The transgenic rice 7crp#10 and the parental nontransgenic control were polished, steamed, mashed, and prepared in water at 40% (w/v). Monkeys were orally administered a high or low dose of transgenic rice or the nontransgenic control by gavage every day. No adverse effects on general behavior or body weight of animals were observed during the study. Analysis of blood from monkeys administered for 26 weeks showed that, with few exceptions, there were no significant differences in hematological or biochemical values between them. Additionally, neither pathological symptoms nor histopathological abnormalities were observed. Thus, it was concluded that oral administration of transgenic rice containing T-cell epitopes from Japanese cedar pollen allergens has no adverse effects.

Marker-free site-specific integration plants.

Recently, site-specific recombination methods in plants have been developed to delete selection markers to produce marker-free transgenic plants or to integrate the transgene into a pre-determined genomic location to produce site-specific transgenic plants. However, these methods have been developed independently, and although the strategies of producing marker-free site-specific integration plants have been discussed, the concept has not been demonstrated. In the present study, we combined two approaches to site-specific recombination and demonstrated the concepts for removing the marker after site-specific integration for producing marker-free site-specific transgenic plants.

Production of trans-10, cis-12 conjugated linoleic acid in rice.

Conjugated linoleic acid (CLA) has anti-carcinogenic and anti-atherosclerosis activity, and modulatory effects on the immune system and lipid metabolism. To produce a transgenic rice plant that can accumulate CLA, a linoleate isomerase gene that can convert linoleic acid to trans-10, cis-12 CLA was introduced and expressed under the control of seed-specific promoters from the oleosin and globulin genes. The fatty acid composition of the transgenic rice grain was analyzed by gas chromatography. Although there was no clear difference in the fatty acid composition between seeds from transformed versus untransformed plants, a peak of trans-10, cis-12 CLA methyl ester, which was not present in seeds from untransformed plants, was found in transformed plants. The trans-10, cis-12 CLA comprised an average of 1.3% (w/w) of the total fatty acids in seeds carrying the oleosin promoter in comparison to 0.01% (w/w) in seeds carrying the globulin promoter. In addition, approximately 70 and 28% of the total amount of the CLA isomer were present in the triacylglycerol and free fatty acid fractions, respectively. These results demonstrate the ability to produce fatty acid components of vegetable oils with novel physiological activities in crops.

Ntlim1, a PAL-box binding factor, controls promoter activity of the horseradish wound-inducible peroxidase gene.

To understand molecular mechanisms underlying wound-induced expression of plant peroxidase genes, the promoter of a horseradish C2 peroxidase (prxC2) gene was analyzed. We had previously isolated a tobacco nuclear protein, Ntlim1, as a trans factor binding to a PAL-box motif of the prxC2 promoter; however, the function of the Ntlim1 trans factor and the PAL-box motif in wound-responsive expression of the prxC2 gene remains unclear. Here, we found that the prxC2 promoter without the intact PAL-box motif failed to direct a normal level of both the basal and the wound-induced expression of beta-glucuronidase (GUS) reporter gene in transgenic tobacco plants, indicating that the PAL-box motif functions as an essential cis element of the prxC2 promoter. We also found that antisense expression of Ntlim1 in transgenic plants carrying the prxC2 promoter::GUS chimeric construct decreased not only the level of the basal and the wound-induced expression of the GUS reporter gene but also the extent of wound inducibility of the prxC2 promoter itself. This result indicates that Ntlim1 is required for the basal level of prxC2 promoter activity as well as its up-regulation under wound stress. Moreover, consistent with the results obtained in planta, result from super-shift assay indicates that the Ntlim1 binds to the PAL-box motif independently of wound stress.

Ectopic expression of a horseradish peroxidase enhances growth rate and increases oxidative stress resistance in hybrid aspen.

We previously demonstrated that overexpression of the horseradish (Armoracia rusticana) peroxidase prxC1a gene stimulated the growth rate of tobacco (Nicotiana tabacum) plants. Here, the cauliflower mosaic virus 35S::prxC1a construct was introduced into hybrid aspen (Populus sieboldii x Populus grandidentata). The growth rate of these transformed hybrid aspen plants was substantially increased under greenhouse conditions. The average stem length of transformed plants was 25% greater than that of control plants. There was no other obvious phenotypic difference between the transformed and control plants. Fast-growing transformed hybrid aspen showed high levels of expression of prxC1a and had elevated peroxidase activities toward guaiacol and ascorbate. However, there was no increase of the endogenous class I ascorbate peroxidase activities in the transformed plants by separate assay and activity staining of native polyacrylamide gel electrophoresis. Furthermore, calli derived from the transformed hybrid aspen grew faster than those from control plants and were resistant to the oxidative stress imposed by hydrogen peroxide. Therefore, enhanced peroxidase activity affects plant growth rate and oxidative stress resistance.

Single-step transformation for generating marker-free transgenic rice using the ipt-type MAT vector system.

The ipt-type MAT vector uses the ipt gene for regeneration of marker-free transgenic plants. However, it was pointed out that this system was not suitable for most economically important crops that regenerated through auxin-dependent embryogenesis. We report a single-step transformation system of rice using MAT vector. When we transformed scutellum tissues of 5 days pre-cultured rice seeds, marker-free transgenic rice plants directly regenerated from 25.5% infected scutellum tissues without forming ipt-intermediates within 4 weeks after an infection. Excision of the ipt gene caused the regeneration of marker-free transgenic rice plants through embryogenic tissues. Therefore, this system needs no selective agent and no sexual crossing for identification of transgenic plants not containing a selectable marker gene. This system is highly effective for generation of marker-free transgenic plants in economically important crops.