Salicylic-Acid-Induced Self-excision of the Marker Gene <Emphasis Type="Italic">npt</Emphasis>II from Transgenic Tomato Using the Cre–<Emphasis Type="Italic">loxP</Emphasis> System

The presence of antibiotic-resistant genes in genetically engineered crops together with the target gene has generated a number of environmental and consumer concerns. In order to alleviate public concerns over the safety of food derived from transgenic crops, marker gene elimination is desirable. Marker-free transgenic tomato plants were obtained by using a salicylic-acid-regulated Cre–loxP-mediated site-specific DNA recombination system in which the selectable marker neomycin phosphotransferase nptII and cre genes were flanked by two directly oriented loxP sites. Upon induction by salicylic acid, the cre gene produced a recombinase that eliminated sequences encoding nptII and cre genes, sandwiched by two loxP sites from the tomato genome. Regenerant plants with the Cre–loxP system were obtained by selection on kanamycin media and polymerase chain reaction (PCR) screening. Transgenic plants were screened for excision by PCR using nptII, cre, and PR-1a promoter primers following treatment with salicylic acid. The footprint of the excision was determined by sequencing the T-DNA borders after a perfect recombination event. The excision efficiency was 38.7%. A new plant transformation vector, pBLNSC (Genbank accession number EU327497), was developed, containing six cloning sites and the self-excision system. This provided an effective approach to eliminate the selectable marker gene from transgenic tomato, thus expediting public acceptance of genetically modified tomato.     

Safety assessment of leaf curl virus resistant tomato developed using viral derived sequences

Genetic engineering of food crops has significantly influenced the agricultural productivity over the past two decades. It has proved a valuable tool, offering crops with higher yields, improved nutritional quality, resistance against pesticides, herbicides and tolerance against abiotic stresses. However, the safety assessment of genetically engineered (GE) crops is prerequisite before introduction into human food chain. The present study was aimed to assess the toxicity and allergenicity of leaf curl virus resistant GE tomato compared to its wild-type species. Balb/c mice fed with genetically engineered or wild-type tomato did not show significant differences in growth, body weight (P > 0.05) and food consumption when compared with control mice. Values for serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase, urea and cholesterol were comparable in GE and wild-type tomato fed mice. Mice immunized with GE or wild-type tomato extract showed low IgE response. Lung histology of ovalbumin fed mice showed bronchoconstriction with eosinophilic infiltration whereas GE or wild-type tomato showed no cellular infiltration with normal airways. Genetically engineered and wild-type tomato sensitized mice demonstrated similar IL-4 release in splenic cell culture supernatant. GE and wild tomato extract on ELISA showed comparable IgE binding (P > 0.05) with food allergic patients’ sera. In conclusion, genetically engineered tomato showed no toxicity in mice and allergenicity is similar to the wild-type tomato.     

Expression of Rice Glutamate Decarboxylase in <Emphasis Type="Italic">Bifidobacterium Longum</Emphasis> Enhances γ-Aminobutyric Acid Production

Bifidobacteria are important for the production of fermented dairy products and probiotic formulas but have a low capacity for γ-aminobutyric acid (GABA) production. To develop a Bifidobacterium strain with an enhanced GABA production, we transformed Bifidobacterium longum with a rice glutamate decarboxylase (OsGADC⁻) gene by electroporation. When the transformed strain was cultured in medium containing monosodium glutamate, the amount of GABA increased significantly compared with those of untransformed Bifidobacterium. Thus, by introducing a plant derived GAD gene, a Bifidobacterium strain has been genetically engineered to produce high levels of GABA from glutamate.     

Containment of a genetically engineered microorganism during a field bioremediation application

A field release of a genetically engineered microorganism was performed at the Field Lysimeter Site on the Oak Ridge Reservation. Six large lysimeters were filled with soil that had been contaminated with a mixture of naphthalene, phenanthrene, and anthracene. A genetically engineered bacterial strain, Pseudomonas fluorescens HK44, was sprayed onto the surface of the soil during soil loading. This strain contains a fusion between the lux genes of Vibrio fischeri and the promoter for the lower pathway of naphthalene degradation, enabling the strain to become bioluminescent when it is degrading naphthalene. Release of the bacteria outside the lysimeters was monitored, using selective agar plates and one-stage Anderson air samplers. Although approximately 10¹⁴ bacteria were sprayed during the loading process, escape was only detected sporadically; the highest incidence of bacterial escape was found when the relative humidity and wind speed were low. Received: 6 March 1998 /thinsp;Received revision: 16 September 1998 / Accepted: 16 October 1998     

Comparison of physical and chemical characters of tomato fruits grown from plants received from seeds or obtained in vitro

Results of experiments concerning comparison of tomato fruit properties which were collected from plants obtained in three manners are described. Control plants were received from seeds. Remaining plants were derived in vitro from leaf and shoot fragments on MS medium supplemented with IAA 0.2 mg·l⁻¹ and BA 2 mg·l⁻¹ (Górecka and Krzyżanowska 1991, Górecka et al. 1994) or with Fari’s et al. (1992) method of obtaining plants by decapitation of sterile seedlings and culture on MS medium without hormones. Evaluation of physical and chemical fruit characters was performed. In the spring experiment the biggest diameter (72 mm), weight (154 g) and volumne (151 ml) were characterized to fruits from plants obtained in vitro on MS medium with IAA and BA. Also fruits from plants received by Fari’s methods were significantly superior in these characters over fruits from the control plants. The fruits from the plants obtained in vitro on MS medium with IAA and BA had highest sugar content (2.95 % f.wt.) and fruits from in vitro plants after Fari’s method contained highest vit.C-13.4 mg·100 g⁻¹ f.wt (significant differences in comparison to control fruits). In other characters fruits from in vitro did not differ as compared to control ones. In the autumn experiments significant differences among fruit groups and characters evaluated were not stated. Generally, yield quality was poorer in the all autumn treatments.     

Near isogenic lines of soybeans as tools to identify nodulation specificmutants of Bradyrhizobium elkanii

The dominant allele Rj4 in soybean interdicts or restricts the nodulation of plants by certain strains of bacteria, most of which are classified as Bradyrhizobium elkanii, while the recessive allele permits normal nodulation with the same strains. The near isogenic lines BARC-2 (Rj4) and BARC-3 (rj4) are calculated to be 99.95% identical in their nuclear DNA, but differ specifically in the allele present at the Rj4 locus. These lines were used to identify spontaneous mutants of the Rj4-restricted Bradyrhizobium elkanii strain USDA 61 Nal^r that had the ability to effectively nodulate plants of the Rj4 genotype. Of the eight rare nodules found on roots of soybean plants of the Rj4 genotype inoculated with the genetically marked strain USDA 61 Nal^r, four were identified as containing mutants with the ability to overcome the effects of the Rj4 allele.     

Prospecting the utility of a PMI/mannose selection system for the recovery of transgenic sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. hybrid) plants

For the first time, the phosphomannose isomerase (PMI, EC 5.3.1.8)/mannose-based “positive” selection system has been used to obtain genetically engineered sugarcane (Saccharum spp. hybrid var. CP72-2086) plants. Transgenic lines of sugarcane were obtained following biolistic transformation of embryogenic callus with an untranslatable sugarcane mosaic virus (SCMV) strain E coat protein (CP) gene and the Escherichia coli PMI gene manA, as the selectable marker gene. Postbombardment, transgenic callus was selectively proliferated on modified MS medium containing 13.6 μM 2,4-D, 20 g l⁻¹ sucrose and 3 g l⁻¹ mannose. Plant regeneration was obtained on MS basal medium with 2.5 μM TDZ under similar selection conditions, and the regenerants rooted on MS basal medium with 19.7 μM IBA, 20 g l⁻¹ sucrose, and 1.5 g l⁻¹ mannose. An increase in mannose concentration from permissive (1.5 g l⁻¹) to selective (3 g l⁻¹) conditions after 3 weeks improved the overall transformation efficiency by reducing the number of selection escapes. Thirty-four vigorously growing putative transgenic plants were successfully transplanted into the greenhouse. PCR and Southern blot analyses showed that 19 plants were manA-positive and 15 plants were CP-positive, while 13 independent transgenics contained both transgenes. Expression of manA in the transgenic plants was evaluated using a chlorophenol red assay and enzymatic analysis.     

Application of GMOs in the U.S.: EPA research & regulatory considerations related to soil systems

During the last 20 years recombinant biotechnology has resulted in the development of organisms with unique genetic compositions, some of which are for intentional release to the environment. While concerns have been raised that such organisms may be capable of inducing transient unintended environmental effects, longer-term perturbations to soil processes and non-target species effects have yet to be demonstrated. In parallel with the growth of the commercial biotechnology industry has come a significant growth in regulatory review processes intended to evaluate the risks of these GMO products. Under the Toxic Substances Control Act (TSCA), certain new microbial products that undergo pre-manufacture review are examined for human and environmental risks using data and other information received in accordance with the U.S. Environmental Protection Agency’s (EPA’s) “Points to Consider” guidance document. In the risk assessment process, carried out under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and the Federal Food, Drug and Cosmetic Act (FFDCA) authorities, EPA evaluates both microbial pesticide products and plants with pesticidal properties to determine if Federal safety standards are met. For all pesticide products, including genetically engineered pesticides, EPA receives testing of product composition and chemical properties, human health effects, environmental effects on non-target pests, and the fate of the pesticide in the environment. The EPA’s Office of Research and Development supports risk assessment research related to such GMO products. This paper focuses on relevant EPA research and regulatory examples related to soil effects considerations for GMOs.     

A novel dichloromethane-degrading <Emphasis Type="Italic">Lysinibacillus sphaericus</Emphasis> strain wh22 and its degradative plasmid

Dichloromethane (DCM)-degrading bacterium strain wh22 (GenBank accession number FJ418643) was isolated and identified as Lysinibacillus sphaericus based on standard morphological and physiological properties, cellular fatty acid composition, mole percent guanine–cytosine content, and nucleotide sequence analysis of enzymatically amplified 16S ribosomal deoxyribonucleic acid. The strain also grew on many other halocarbons found in the waste gases of industrial effluents, such as 1,2-dichloroethane, chlorobromomethane, methylene bromide, 1,1,1-trichloroethane, trichloroethylene, and hexachlorobenzene. The strain harbored a novel degradative plasmid, pRC11 (48.8 kb). The genes coding for the metabolism of DCM were found to be plasmid-borne, and a physical map of the plasmid has been established. The purified plasmid was transformed to dcm ⁻ Escherichia coli DH5 at a rate of 1.65 × 10⁵. The transformed cells were able to grow on DCM at a concentration of 5–16 mM and can be further used as an excellent source for genetic manipulations leading to the construction of genetically modified microbial strains or genetically engineered microorganisms.     

Aerial Dispersal and Epiphytic Survival of Pseudomonas syringae during a Pretest for the Release of Genetically Engineered Strains into the Environment

Prospective experimental field evaluation of genetically engineered microorganisms, such as microbial pest control agents, raises issues of how to properly ascertain their fate and survival in the environment. Field trials with recombinant organisms must reflect requirements for sampling and monitoring. Field trials were conducted at Tulelake, Calif., to monitor the numbers of viable cells of a nonrecombinant strain of Pseudomonas syringae that entered the atmosphere and landed on plants and soil during and after an aerosol spray application. An exponential decrease in numbers of viable cells deposited at increasing distances from three sprayed plots was observed. The relative rate of survival of cells sprayed directly on plants was more than 10 times higher than that of cells dispersed through the air to similar adjacent plants. Results are being used to gain experience with the characteristics of a release site that influence containment or dispersal and to develop appropriate sampling methodologies for evaluating survival and dispersal characteristics of genetically engineered bacteria released into the environment. The ability to make predictions about microbial dispersal and survival will reduce the uncertainties associated with environmental releases of recombinant organisms.     

Modest calcium increase in tomatoes expressing a variant of <Emphasis Type="Italic">Arabidopsis</Emphasis> cation/H<Superscript>+</Superscript> antiporter

The over-expression of Arabidopsis CAX1 and CAX2 causes transgenic tomato plants to reveal severe Ca²⁺ deficiency-like symptoms such as tip-burn and/or blossom end rot, despite there being sufficient Ca²⁺ in each plant part. To correct the symptoms and to moderately enhance the calcium level, a worldwide vegetable tomato was genetically engineered using a modified Arabidopsis cation/H⁺ antiporter sCAX2A, a mutant form of Arabidopsis CAX2. Compared with the wild-type, the sCAX2A-expressing tomato plants demonstrated elevated Ca²⁺ levels in the fruits with almost no changes in the levels of Mn²⁺, Cu²⁺, and Fe²⁺. Moreover, expression of sCAX2A in tomato plants did not show any significant alterations in their morphological phenotypes. Unlike 35S::sCAX1 construct, sCAX2A antiporter gene driven by 35S promoter can be a valuable tool for enriching Ca²⁺ contents in the tomato fruit without additional accumulation of the undesirable cations.     

Chemical-induced autoexcision of selectable markers in elite tomato plants transformed with a gene conferring resistance to lepidopteran insects

Marker-free transgenic tomato plants harboring a synthetic Bacillus thuringiensis endotoxin gene, cryIAc, were obtained by using a chemically regulated, Cre/loxP-mediated site-specific DNA recombination system, in which the selectable marker neomycin phosphotransferase gene flanked by two directly oriented loxP sites was located between the cauliflower mosaic virus 35S promoter and a promoterless cryIAc. Upon induction by 2 μM β-estradiol, sequences encoding the selectable marker and cre sandwiched by two loxP sites were excised from the tomato genome, leading to activation of the downstream endotoxin gene cryIAc with high expression levels as shown by Northern blot and ELISA assay (250–790 ng g⁻¹ fresh wt) in T₁ generation. For transgenic line with single transgenic loci, 15% of T₁ progenies were revealed marker-free. This autoexcision strategy provides an effective approach to eliminate a selectable marker gene from transgenic tomato, thus expediting the public acceptance of genetically modified crop.     

High-efficiency phosphinothricin-based selection for alfalfa transformation

Despite the significant advantages of using herbicide resistance for selection of genetically engineered plants, alfalfa transformation has relied primarily on selection for antibiotic resistance. In the few studies reporting the use of resistance to the herbicide phosphinothricin (PPT), transformation efficiencies were low. The present investigation describes a PPT-based selection system for alfalfa transformation that uses the phosphinothricin acetyl-transferase (pat) gene as a selectable marker and 5.0 mg l⁻¹ of bialaphos as the selective agent. The method achieves transformation efficiencies, measured as the percentage of explants giving rise to one or more transformed plantlets, greater than 50%. These plantlets accumulated detectable amounts of PAT at levels varying from 2 to 1367 pg μg⁻¹ total protein. Transformed plants transferred to soil in the greenhouse were phenotypically normal and exhibited resistance to bialaphos leaf painting at 5 g l⁻¹ and applications of PPT equivalent to field-level use (0.5 kg ha⁻¹).     

Soybean response to nodulation by bradyrhizobia differing in rhizobitoxine phenotype

Rhizobitoxine-producing (RT⁺) strains of Bradyrhizobium japonicum, differing in their abilities to induce foliar chlorosis with ‘Forrest’ soybean (Glycine max [L.] Merr.), were evaluated for effects on short term shoot productivity, nodulation, N₂ fixation, and nodule protein production under greenhouse conditions. Soybeans were singly inoculated with washed suspensions of (Group II) USDA strains 31, 46, 76, 94, 110, 123 or 130. Strains USDA 110 and USDA 123 (Group I/Ia) were included as RT-controls. The plants were cultured in the absence of combined N in horticultural-grade vermiculite for 49 days. Beginning 21 days after planting, plants were evaluated weekly for chlorophyll, leaf protein and biomass accumulation, nodular contents of leghemoglobin, soluble protein and RT, and total shoot N content. Rhizobitoxine was detected in nodules of all RT⁺ strains with the exception of USDA 31. However, only USDA 76 and USDA 94 produced both quantifiable concentrations of RT and symptoms of RT-induced chlorosis. Coincident with moderate to severe chlorosis were reductions in chlorophyll concentrations, shoot and nodule dry weight, leaf protein and total N₂ fixation. During extended periods of severe chlorosis, reductions in Lb and soluble nodular protein were observed. Based on carbon accumulation, all non-chlorotic treatments were statistically more productive than the chlorotic treatments. Similarly, non-chlorotic Group II treatments tended to fix less carbon relative to the RT-Group I/Ia controls, although these differences were not statistically significant. The results of this study suggest that, in the absence of discernable foliar chlorosis, the effect of RT⁺ (Group II) nodulation on short term soybean productivity is minimal. Published as Miscellaneous Paper No. 1439 of the Delaware Agricultural Experiment Station. Published as Miscellaneous Paper No. 1439 of the Delaware Agricultural Experiment Station.     

Phosphomannose isomerase: An efficient selectable marker for plant transformation

Summary Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of mannose 6-phosphate and fructose 6-phosphate. Plant cells lacking this enzyme are incapable of surviving on synthetic medium containing mannose as a carbon source. Maize, wheat and barley plants, genetically modified to express the Escherichia coli manA gene (pmi) under the control of a plant promoter, were able to survive selection on mannose-containing medium. Transformation frequencies averaged 45% for maize transformation via Biolistics^™ 35% for maize Agrobacterium-mediated transformation, 20% for wheat, 3% for barley, and 2% for watermelon transformation. Moreover, the frequencies exceeded those obtained for maize and wheat using the pat or bar gene with Basa^? selection. A preliminary safety assessment has been conducted for PMI. Purified PMI protein demonstrates no adverse, effects in an acute mouse toxicity test. Purified PMI protein was readily digested in simulated mammalian gastric and intestinal fluids. Plants derived from surgar beet and corn cells that had been genetically modified to express the E. coli manA gene were evaluated for biochemical changes in mannose-associated pathways. No detectable changes in glycoprotein profiles were detected in PMI-transformed plants as compared to nontransgenic controls. The yield and nutritional composition of grain from PMI-transformed corn plants compared to their non-transformed isogenic counterparts were also determined and no statistically significant differences were found. The inherent safety of a system based on simple sugar metabolism coupled with high transformation frequencies for monocots make pmi and ideal selectable marker for plant transformation.     

Steroidal glycoalkaloid content of potato,tomato and their somatic hybrids

Summary Analyses of leaves and ‘tubers’ from somatic hybrids of potato and tomato (‘pomato’ with plastids of potato, ‘topato’ with plastids of tomato) produced by fusion of protoplasts from liquid cultures of dihaploid potato and mesophyll of tomato revealed the presence of the two major potato glycoalkaloids (α-solanine and α-chaconine) as well as the tomato glycoalkaloid (αtomatine). The total alkaloid content of leaves was greater than that of ‘tubers’ and similar to levels in the foliage of parent plants. However, glycoalkaloids were more abundant in hybrid ‘tubers’ than in normal potato tubers by a factor of 5–15. In hybrid foliage, approximately 98% of the alkaloid present was of potato origin whereas in ‘tubers’ the reverse was the case, with tomatine comprising 60–70% of the total alkaloid. The similarities in alkaloid content and ratios between the pomato and the topato lines indicate that plastomes do not influence the biosynthesis and distribution of these alkaloids. The results indicate that major secondary metabolites may prove useful for assessing the hybrid nature of such plants.     

ToMV-Resistant transgenic tomato as a material for the first field experiment of genetically engineered plants in Japan

Summary An F₁ hybrid betweenLycopersicon esculentum andL. peruvianum was transformed using a Ti-plasmid binary vector with a coat protein gene cDNA of an attenuated tomato mosaic virus (ToMV) strain L₁₁A which was expressible by the 35S promoter of cauliflower mosaic virus (CaMV). A transgenic plant which expressed the most resistance to ToMV was chosen as a material to be tested in a nonisolated greenhouse and in the field. This transgenic tomato plant was propagated by cutting. In the first test using an isolated greenhouse and in the second test conducted in an nonisolated greenhouse, no major morphologic and physiologic differences were found between the transgenic plants and the nontransgenic control plants. Also, there was no evidence that the transgenic plants produced any new hazardous substances. Both the transgenic and the nontransgenic plants were self-sterile, and crossing of the cultivated species with pollen of these plants produced few seeds. These features of the transgenic plants satisfied the requirements for a small scale field test. The field test of the transgenic plants are in progress. Presented in the Session-in-Depth “Field Test Requirements and Performance of Transgenic Plants” at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–20, 1991.     

Genetic transformation of the medicinal plant <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated method

A high-frequency and simple procedure for Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Salvia miltiorrhiza was developed. Leaf discs were pre-cultured on MS medium supplemented with 6.6 μmol l⁻¹ BAP and 0.5 μmol l⁻¹ NAA for one day, then co-cultured with A. tumefaciens strain EHA105 harboring the plasmid pCAMBIA 2301 for three days on the same medium. Regenerated buds were obtained on selection medium (co-culture medium supplemented with 60 mg l⁻¹ kanamycin and 200 mg l⁻¹ cefotaxime) after two cycles’ culture of 10 days each and then transferred to fresh MS medium with 60 mg l⁻¹ kanamycin for rooting. Fifteen days later, the rooted plantlets were obtained and then successfully transplanted to soil. The transgenic nature of the regenerated plants was confirmed by PCR, Southern hybridization analysis and GUS histochemical assay. Averagely, 1.1 independent verified transgenics per explant plated were obtained through this protocol. Adopting this procedure, positive transformed plants could be obtained within 2–3 months from mature seeds germination to transplant to soil, and more than 1,000 transgenic plants with several engineered constructs encoding different genes of interest were produced in our lab in the past two years.     

Whitefly control in cut gerbera: is it possible to control Trialeurodes vaporariorum with Encarsia formosa?

We investigated the impact of inundative releases of the parasitoid, Encarsia formosa Gahan (Hymenoptera: Aphelinidae), for control of greenhouse whitefly, Trialeurodes vaporariorum (Westwood), on cut gerbera (Gerbera jamesonii L.) under controlled greenhouse conditions. Experimental units consisted of ten plants covered and separated from other units by gauze tents. We assessed three release rates of the aphelinid parasitoid: a 7-week experiment with a standard release rate (10 m⁻²/14 days), and a subsequent 3-month trial with high (100 m⁻²/week) and very high (1,000 m⁻²/week) release rates. Experimental units without release of parasitoids served as control treatment. Gerbera plants were infested initially with 50–100 juvenile and 50–70 adult whiteflies in the first experiment, and in the second experiment with less than 50 juveniles per plant and 50–70 adults. Whitefly and parasitoid population density were assessed in weekly intervals using infestation and activity categories. Results show that parasitized whiteflies were present in all treatments within 2 weeks after initial release. Unfortunately, it was not possible to control whiteflies with standard release rates of E. formosa. Although parasitism rates slightly increased, the effect on whitefly populations was negligible. Large amounts of honeydew and growth of sooty mold fungi caused the termination of the first experiment. In a second experiment, E. formosa was tested at 10–100 times higher release densities. In contrast to the first experiment, whitefly densities increased steadily during the first 8 weeks, but remained constant until the end of the experiment in both treatments. Parasitism by E. formosa reached its maximum after 8 weeks. We discuss possible reasons for the low efficiency of E. formosa as a whitefly antagonist in greenhouse production of gerbera.