Expression of multiple insecticidal genes confers broad resistance against a range of different rice pests

We report the simultaneous introduction of three insecticidal genes (the Bt genes cry1Ac and cry2A, and the snowdrop lectin gene gna) into commercially important indica rice varieties M7 and Basmati 370, by particle bombardment. Transgenic plants expressed Cry1Ac, Cry2A and GNA at different levels, either singly or in combination at 0.03–1%, 0.01–0.5% and 0.01–2.5% of total soluble protein, respectively. The transgenes showed stable transmission and expression, and R₁ transgenic plants provided significant (p<0.01) protection against three of the most important insect pests of rice: rice leaf folder (Cnaphalocrocis medinalis), yellow stemborer (Scirpophaga incertulas) and brown planthopper (Nilaparvata lugens). The triple transformants showed significantly (p<0.05) higher resistance to these insects than plants expressing single transgenes. Bioassays using the triple-transgenic plants showed 100% eradication of the rice leaf folder and yellow stem borer, and 25% reduction in the survival of the brown planthopper. The greatest reduction in insect survival, and the greatest reduction in plant damage, occurred in plants expressing all three transgenes. This approach maximises the utility of gene transfer technology to introduce combinations of genes whose products disrupt different biochemical or physiological processes in the same insect, providing a multi-mechanism defence.     

Production of transgenic pineapple (Ananas cosmos (L.) Merr.) plants via adventitious bud regeneration

A new protocol for the production of transgenic pineapple plants was developed. Adventitious buds were induced directly from Agrobacterium-infected leaf bases and stem discs of in vitro plants, bypassing the establishment of callus cultures. Non-chimeric transgenic plants were obtained by multiple subculturing of primary transformants under increasing levels of selection. A total of 42 independent transgenic lines were produced from two cultivars with two different constructs: one containing a modified rice cystatin gene (Oc-IΔD86) and the other with the anti-sense gene to pineapple aminocyclopropane synthase (ACS). GUS histochemical staining provided the first evidence of the non-chimeric nature of the transformed plants. Their non-chimeric nature was further demonstrated by PCR analyses of the DNA extracted from individual leaves of a primary transformed plant and also from multiple plants propagated from a single transformation event. Southern hybridization confirmed random integration patterns of transgenes in the independent lines. For the Oc-IΔD86 gene, the expression at the mRNA level was detected via RT-PCR and its translation was detected by protein blot. Agronomic evaluation and bioassays of the transgenic plants will further validate the utility of this new tool for pineapple improvement.     

Transgenic indica Rice Expressing ns-LTP-Like Protein Shows Enhanced Resistance to Both Fungal and Bacterial Pathogens

Antimicrobial peptides (AMPs) from plant seeds, known to inhibit pathogen growth have a great potential in developing transgenic plants resistant to disease. Some of the nonspecific-lipid transfer proteins (ns-LTP) that facilitate in vitro transport of lipids, show antimicrobial activity in vitro. Rice seeds also contain ns-LTPs; however, these genes are expressed weakly in seedlings. We have transformed Pusa Basmati 1, an elite indica rice cultivar, with the gene for Ace-AMP1 from Allium cepa, coding for an effective antimicrobial protein homologous to ns-LTPs. The gene for Ace-AMP1 was cloned under an inducible rice phenylalanine ammonia-lyase (PAL) or a constitutive maize ubiquitin (UbI) promoter. Ace-AMP1 was expressed in transgenic lines and secreted in the apoplastic space. Protein extracts from leaves of transgenic plants inhibited three major rice pathogens, Magnaporthe grisea, Rhizoctonia solani and Xanthomonas oryzae, in vitro. Enhanced resistance against these pathogens was observed in in planta assays, and the degree of resistance correlating with the levels of Ace-AMP1 with an average increase in resistance to blast, sheath blight, and bacterial leaf blight disease by 86%, 67%, and 82%, respectively. Importantly, transgenic rice plants, with stable integration and expression of Ace-AMP1, retained their agronomic characteristics while displaying enhanced resistance to both fungal and bacterial pathogens.     

Rice plants expressing the moss sodium pumping ATPase PpENA1 maintain greater biomass production under salt stress

High cytosolic concentrations of Na+ inhibit plant growth and development. To maintain low cytosolic concentrations of Na+ , higher plants use membrane-bound transporters that drive the efflux of Na+ or partition Na+ ions from the cytosol, either to the extracellular compartment or into the vacuole. Bryophytes also use an energy-dependent Na+ pumping ATPase, not found in higher plants, to efflux Na+ . To investigate whether this transporter can increase the salt tolerance of crop plants, Oryza sativa has been transformed with the Physcomitrella patens Na+ pumping ATPase (PpENA1). When grown in solutions containing 50 mm NaCl, plants constitutively expressing the PpENA1 gene are more salt tolerant and produce greater biomass than controls. Transgenics and controls accumulate similar amounts of Na+ in leaf and root tissues under stress, which indicates that the observed tolerance is not because of Na+ exclusion. Moreover, inductively coupled plasma analysis reveals that the concentration of other ions in the transformants and the controls is similar. The transgenic lines are developmentally normal and fertile, and the transgene expression levels remain stable in subsequent generations. GFP reporter fusions, which do not alter the ability of PpENA1 to complement a salt-sensitive yeast mutant, indicate that when it is expressed in plant tissues, the PpENA1 protein is located in the plasma membrane. PpENA1 peptides are found in plasma membrane fractions supporting the plasma membrane targeting. The results of this study demonstrate the utility of PpENA1 as a potential tool for engineering salinity tolerance in important crop species.     

Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides

Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.     

Over-expression of a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene improves salt tolerance in an upland rice

To develop a salt-tolerant upland rice cultivar (Oryza sativa L.), OsNHX1, a vacuolar-type Na⁺/H⁺ antiporter gene from rice was transferred into the genome of an upland rice cultivar (IRAT109), using an Agrobacterium-mediated method. Seven independent transgenic calli lines were identified by polymerase chain reaction (PCR) analysis. These 35S::OsNHX1 transgenic plants displayed a little accelerated growth during seedling stage but showed delayed flowering time and a slight growth retardation phenotype during late vegetative stage, suggesting that the OsNHX1 has a novel function in plant development. Northern and western blot analyses showed that the expression levels of OsNHX1 mRNA and protein in the leaves of three independent transgenic plant lines were significantly higher than in the leaves of wild type (WT) plants. T₂ generation plants exhibited increased salt tolerance, showing delayed appearance and development of damage or death caused by salt stress, as well as improved recovery upon removal from this condition. Several physiological traits, such as increased Na⁺ content, and decreased osmotic potential in transgenic plants grown in high saline concentrations, further indicated that the transgenic plants had enhanced salt tolerance. Our results suggest the potential use of these transgenic plants for further agricultural applications in saline soil.     

Development of transgenic rice pure lines with enhanced resistance to rice brown planthopper

Summary Mature seed-derived callus from an elite Chinese japonica rice cv. Ewan 5 was cotransformed with two plasmids, pWRG1515 and pRSSGNAl, containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snowdrop (Galanthus nivalis) lectin gene (gna) via particle bombardment. Thirty-five independent transgenic rice plants were regenerated from 177 bombarded calluses. Eighty-three percent of the transgenic plants contained all three genes, as revealed by Southern blot analysis. Western blot analysis revealed that 23 out of 29 gna-containing transgenic plants expressed Galanthus nivalis agglutinin (GNA) (79%) at various levels, with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of all three transgenes (gna, hpt and gusA) in the R2 progeny. Amongst the R2 generation two independent homozygous lines were identified that expressed all three transgenes. Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to rice brown planthopper (Nilaparvata lugens, BPH) by decreasing the survival, overall fecundity of BPH, retarding development, and decreasing the feeding of BPH. These BPH-resistant lines have been incorporated into a rice insect resistance breeding program. This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred enhanced resistance to BPH.     

Field evaluation and risk assessment of transgenic indica basmati rice

We report the first field trial of different transgenic lines of Indica Basmati rice (B-370) expressing cry1Ac and cry2A genes. Different transgenic lines were grown under field conditions for two consecutive years, according to RCBD and Split Plot Design respectively. All the biosafety measures were taken into consideration. Sixty neonate larvae of yellow stem borer were artificially infested into each plant in three installments. Data was recorded in terms of dead hearts and white heads at vegetative and flowering stage respectively. Transgenic lines exhibited inherent ability to protect rice plants from target insects (p<0.01). Natural infestations of rice skipper and rice leaf folder were also observed and transgenic plants were statistically superior to their untransformed counterparts. Green house whole plant bioassays were done by infesting two 2^nd instar larvae of rice leaf folder per tiller. Transgenics were 96% more resistant than untransformed control plants. The presence of cry genes was observed with Dot blot, PCR and Southern blot analysis, while ELISA and Western blot analysis confirmed the expression of Cry proteins. All lines expressed higher level of Cry proteins when compared with commercially released cultivars of Bt cotton, maize and potato. It was also observed that although toxin titer substantially decreased with increasing age of the plants, it remained well within the limits to kill the target insects. Morphological studies showed significant variation for days to maturity, plant height and panicle length. Cooking qualities of seeds harvested from these lines were compared with the untransformed control. The transgenic lines had no effect on non-target insects (insects belonging to orders other than diptera and lepidoptera) and germination of three local varieties of wheat. Chances of gene spread were calculated at a level of 0.18% cross pollination in experimental lines.     

Light-regulated overexpression of an Arabidopsis phytochrome A gene in rice alters plant architecture and increases grain yield

The phytochromes are a family of red/far-red light absorbing photoreceptors that control plant developmental and metabolic processes in response to changes in the light environment. We report here the overexpression of Arabidopsis thaliana PHYTOCHROME A (PHYA) gene in a commercially important indica rice variety (Oryza sativa L. Pusa Basmati-1). The expression of the transgene was driven by the light-regulated and tissue-specific rice rbcS promoter. Several independent homozygous sixth generation (T₅) transgenic lines were characterized and shown to accumulate relatively high levels of PHYA protein in the light. Under both far-red and red light, PHYA-overexpressing lines showed inhibition of the coleoptile extension in comparison to non-transgenic seedlings. Furthermore, compared with non-transgenic rice plants, mature transgenic plants showed significant reduction in plant height, internode length and internode diameter (including differences in cell size and number), and produced an increased number of panicles per plant. Under greenhouse conditions, rice grain yield was 6–21% higher in three PHYA-overexpressing lines than in non-transgenic plants. These results demonstrate the potential of manipulating light signal-transduction pathways to minimize the problems of lodging in basmati/aromatic rice and to enhance grain productivity.     

Development of transgenic finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.) resistant to leaf blast disease

Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R₀ and R₁ progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R₁ lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation. Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study reporting the introduction of rice chitinase gene into finger millet for leaf blast resistance.     

The 3′-untranslated region of rice glutelin GluB-1 affects accumulation of heterologous protein in transgenic rice

We compared the effect of the rice storage protein glutelin B-1 (GluB-1) terminator with the nopaline synthase (Nos) terminator on the accumulation of the modified house dust mite allergen mDer f 2 driven by the maize ubiquitin promoter in transgenic rice. Accumulation of mDer f 2 in transgenic seed and leaf using the GluB-1 terminator was greater than when using the Nos terminator construct. The mDer f 2 mRNA containing the GluB-1 3′UTR was processed and polyadenylated at the same sites as the native GluB-1 mRNA in the seeds but diverged in leaves of the transgenic plants. In contrast, the poly(A) sites of mDer f 2 containing Nos 3′UTR were more divergent in both seed and leaf. These results suggest that GluB-1 3′UTR functions as a faithful terminator and that termination at the specific sites may play an important role in mRNA stability and/or translatability, resulting in higher levels of protein accumulation.     

The −689/+197 region of the maize protease inhibitor gene directs high level,wound-inducible expression of the cry1B gene which protects transgenic rice plants from stemborer attack

To investigate the activity of the regulatory region of the maize (Zea mays L.) proteinase inhibitor (mpi) gene, we transferred into rice (Oryza sativa L.) plants the –689/+197 (C1) fragment of the mpi genomic clone fused to either theuidA gene or a synthetic Bacillus thuringiensis cry1B gene. Although uidA and cry1B encode very different proteins consistent results were obtained from their respective histochemical and fluorometric and immunoblot detections in T₃ transgenic rice lines. In response to mechanical wounding, a 4–5 fold increase in GUS activity and a Cry1B accumulation reaching 0.1–0.2% of total soluble proteins were observed from basal and undetectable levels respectively in leaf tissue. The establishment of the time-course of wound response in both systems revealed a maximum induction level 12–16 h after treatment. From both systems we also deduced that the C1 region is not active in pollen and seed endosperm. Three independent transformation events expressing cry1B under the control of the C1 region exhibited protection against striped stem borer damage and showed 100% mortality of second instar larvae 8 days after release. These results illustrate the first evidence that wound-inducible expression of a Bacillus thuringiensis endotoxin gene affords full protection to transgenic rice plants.     

Transgenic barley expressing a fungal xylanase gene in the endosperm of the developing grains

The feasibility of producing plant cell wall polysaccharide-hydrolysing feed enzymes in the endosperm of barley grain was investigated. The coding region of a modified xylanase gene (xynA) from the rumen fungus, Neocallimastix patriciarum, linked with an endosperm-specific promoter from cereal storage protein genes was introduced into barley by Agrobacterium-mediated transformation. Twenty-four independently transformed barley lines with the xylanase gene were produced and analysed. The fungal xylanase was produced in the developing endosperm under the control of either the rice glutelin B-1 (GluB-1) or barley B1 hordein (Hor2-4) promoter. The rice GluB-1 promoter provided an apparently higher expression level of recombinant proteins in barley grain than the barley Hor2-4 promoter in both transient and stable expression experiments. In particular, the mean value for the fungal xylanase activity driven by the GluB-1 promoter in the mature grains of transgenic barley was more than twice that with the Hor2-4 promoter. Expression of the xylanase transgene under these endosperm-specific promoters was not observed in the leaf, stem and root tissues. Accumulation of the fungal xylanase in the developing grains of transgenic barley followed the pattern of storage protein deposition. The xylanase was stably maintained in the grain during grain maturation and desiccation and post-harvest storage. These results indicate that the cereal grain expression system may provide an economic means for large scale production of feed enzymes in the future.     

A simple,rapid and systematic method for the developed GM rice analysis

We generated 383 independent transgenic lines that contained the PsGPD (Glyceraldehyde-3-Phosphate Dehydrogenase), ArCspA (Cold Shock Protein), BrTSR15 (Triple Stress Resistance 15) and BrTSR53 (Triple Stress Resistance 53) genes under the control of a constitutive (CaMV 35S) promoter to generate genetically modified (GM) rice. TaqMan copy number assay was performed to determine the copy numbers of inserted T-DNA. Flanking sequence tags (FSTs) were isolated from 203 single copy T-DNA lines of transgenic plants, and their sequences were mapped to the rice chromosomes. Of the 157 flanking sequence tags that were isolated from single copy lines, transgenes were found to be integrated into genic regions in 58 lines (36 %), whereas 97 lines (62 %) contained transgene insertions in intergenic regions. Approximately 27 putative homozygous lines were obtained through multi-generations of planting, resistance screening and TaqMan copy number assays. To investigate the transgene expression patterns, quantitative real-time PCR analysis was performed using total RNA from leaf tissue of homozygous T1 plants with a single copy and an intergenic insertion of T-DNA. The mRNA expression levels of the examined transgenic rice were significantly increased in all transgenic plants. In addition, myc-tagged 35S:BrTSR15 and 35S:BrTSR53 transgenic plants displayed higher levels of transgene protein. Using numerical data for the mass production of transgenic plants can reduce the time required to obtain a genetically modified plant. Moreover, the duration, cost, and efforts required for transformation can be deliberately predicted. These results may be useful for the large-scale production of transgenic plants or T-DNA inserted rice mutants.     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.     

Resveratrol Synthase Transgene Expression and Accumulation of Resveratrol Glycoside in <Emphasis Type="Italic">Rehmannia glutinosa</Emphasis>

Rehmannia glutinosa L. is an important medicinal crop in Asian countries and contains trace amount of resveratrol compounds. To increase production of the compounds, we attempted ectopic expression of peanut resveratrol synthase gene (AhRS3) in R. glutinosa. The AhRS3 sequence that encompassed the open reading frame, including a 312 bp-long intron present between the 59th and 60th codon, was driven by the CaMV35S promoter and introduced into R. glutinosa via Agrobacterium-mediated transformation of leaf explants. The transgenic plants with one to three copies of AhRS3 transgene showed normal growth and development. The transgene was expressed constitutively in the leaf, root and flower at similar levels. Transgene expression in the leaf resulted in the production of new compounds identified as resveratrol and 3′-H-resveratrol-3-O-β-d-glucoside (R-gluc) through nuclear magnetic resonance spectroscopy and mass spectrometry. R-gluc accumulated predominantly and its content in the leaf of the 11 transgenic lines ranged from 22 to 116μg/gFW. The contents of resveratrol compounds in the transgenic plants were further increased by cold, UV, ethylene, and paraquat treatments, and were positively associated with the levels of AhRS3 mRNA levels. The R-gluc isolated from the transgenic plants exhibited antioxidant activity equivalent to one-third of resveratrol. Transgenic plants were highly resistant to Fusarium oxysporum infection. The results indicate that the ectopic expression of AhRS3 in R. glutinosa results in the production of R-gluc and resveratrol at hundreds of times higher levels than in peanut seed. The increased production of resveratrol compounds from R. glutinosa, which show diverse benefits for human and plant health, could provide a new opportunity for the improvement of R. glutinosa products.