Co-transformation of indica rice protoplasts with gusA and neo genes

Protoplasts of the indica rice (Oryza sativa L.) variety, IR54, were transiently transformed with the gusA gene and stably transformed with both the neo and gusA genes. We show that PEG-mediated co-transformation of protoplasts with two genes on separate plasmids coupled with selection on kanamycin is an effective way of transferring foreign gene(s) into the indica rice genome. The efficiency of co-transformation was generally 20–30%, i.e. the frequency of kanamycin-resistant calli having both the neo and gusA active genes. Southern blot analysis using a probe for gusA indicated integration of several copies of the gene, often as head to tail tandem repeats.Abbreviations GUS ß-glucuronidase - PEG polyethlene glycol - PCV packed cell volume     

Inheritance and segregation of exogenous genes in transgenic cotton

Three transgenic cotton varieties (lines) were chosen for the study of inheritance and segregation of foreign Bt (Bacillus thuringiensis toxin) andtfdA genes in cotton. The transformed cotton varieties CCRI 30 and NewCott 33B expressing the BtcryIA gene, and cotton line TFD expressing thetfdA gene were crossed with CCRI 19, CCRI 12 and Lumian 6. The results confirm inheritance and segregation of (i) the exogenous Bt gene in transgenic CCRI 30 and NewCott 33B, governing resistance to bollworm, and (ii) the exogenoustfdA gene in transgenic TFD, governing resistance to the herbicide 2,4-D. Both resistance characters were governed by a single dominant nuclear gene, and were not affected by cytoplasm. Our data support the conclusion that foreign traits encoded by single genes are inherited and expressed in Mendelian fashion in cotton. Our results also indicate that a practical backcross breeding program could be used to develop cotton cultivars combining one or more resistance traits from foreign and native gene sources.     

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.     

外源基因在转基因动物中遗传和表达的稳定性

转基因技术经过近半个世纪的发展,已成为当今生物技术研究的热点。近10多年来,与核移植技术的结合,转基因效率大大提高,携带有不同外源基因的不同种类的转基因动物迅速增加。但是,成功获得转基因动物并不是转基因动物研究的最终目的,如何利用转基因技术为人类的需求服务才是科研人员始终面对的课题。在畜牧生产领域,通过转基因技术培育家畜新品种是转基因技术应用的重要体现,在我国这方面已经引起了广泛关注。但迄今为止,外源基因在转基因动物中遗传和表达的稳定性仍然是亟待解决的问题,究其原因,这主要与位置效应、外源基因的表观遗传学修饰和遗传效率相关,文章结合目前的研究进展和本实验室的研究结果,从这3方面阐述其作用机制,期望为转基因动物遗传育种向产业化的迈进提供一定的理论探讨。     

Activity of a maize ubiquitin promoter in transgenic rice

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.     

Stable co-transformation of maize protoplasts with gusA and neo genes

An efficient co-transformation protocol using polyethylene glycol was developed for Zea mays L. (cv. A188 × BMS) protoplasts isolated from suspension culture cells. Co-transformation was accomplished by using plasmid constructions containing -glucuronidase (gusA) or neomycin phosphotransferase (neo) gene coding sequences; both were under control of the CaMV 35S promoter. Protoplast culture and transformation conditions were optimized to assure efficient recovery of transformed cells. The overall efficiency of transformation was 1 × 10^–4 (calculated per viable protoplast plated). Among kanamycin-resistant lines, 50% showed a high level of GUS activity (above one unit). Southern blot hybridization confirmed the presence of numerous gusA and neo coding sequences in the maize genome. In two analyzed lines, integrated sequences appeared to be organized in tandem head-to-tail repeats. Results also indicated that the integrated sequences were partially methylated.     

The impact of selection parameters on the phenotype and genotype of transgenic rice callus and plants

Transgenic rice embryogenic callus and plants were recovered from experiments involving electric discharge particle acceleration (Accell^TM technology). Critical parameters influencing successful delivery and stable integration of exogenous DNA into organized rice tissue had been identified previously. We report here on the effects of one selective agent (hygromycin B) on the phenotype and genotype of recovered callus and plants. The nature, timing and culture practices appeared to be more critical for the successful recovery of transgenic callus and plants than the level of selection used. By utilizing the procedures described in this report, transformation frequencies well in excess of 10% were obtained routinely in all varieties of rice tested. The combination of Accell^TM technology with a selectable and prolific regeneration culture system resulted in the development of a variety-independent and highly efficient method for the routine introduction of any gene into any rice variety.     

Inheritance of resistance against biotype 2 of the Asian rice gall midge, Orseolia oryzae

The inheritance of resistance in the rice cultivars Phalguna, ARC5984, ARC 5158, Veluthacheera, and T1477 to the Asian rice gall midge biotype 2 was studied under both natural and artificial infestation conditions against the susceptible cultivars Jaya and IR20. A single recessive gene in Veluthacheera and two recessive complementary genes in T1477 control resistance. Phalguna and ARC5984 possess a single dominant gene while ARC5158 has a single dominant and a single recessive gene for resistance. Allelism studies showed that genes for resistance in Veluthacheera and T1477 are allelic but non-allelic to the resistance genes in Phalguna and ARC5984, which are allelic to each other. Genes for resistance in ARC5158 are allelic to resistance genes of the other four donors. There was no cytoplasmic inhibition of resistance by the susceptible parents.     

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.     

Inheritance of a co-transferred foreign gene in the progenies of transgenic rice plants

The integration pattern and the inheritance of exogenous DNA in transgenic rice plants were analysed. Plasmid pCH (4.8 kb), that contains chimaeric cauliflower mosaic virus 35S promoter-hygromycin phosphotransferase structural gene, and plasmid pGP400 (7.2 kb), possessing oat phytochrome promoter and structural gene of bacterial -glucuronidase, were co-transferred into protoplasts of rice (Oryza sativa L.) plants via electroporation. Primary transformants (T₀ generation) and their progenies (T₁, T₂ and T₃) were selected by hygromycin B. Southern blot analysis of inserted genes in transgenic rice plants suggests the integration of an intact hygromycin phosphotransferase gene and non-functional DNA fragments into host genome. Co-inheritance of the hygromycin phosphotransferase gene and -glucuronidase gene was also observed. There were no significant differences in terms of the morphology and size of seeds between untransformed and transgenic plants (T₃ generation).     

Enhanced insect resistance in Thai rice varieties generated by particle bombardment

We used particle bombardment to transform two elite Thai rice varieties, Khao Dawk Mali 105 (KDML105) and Supanburi 60 (SP60), with the snowdrop lectin gene gna (Galanthus nivalis agglutinin). This gene confers resistance to sap-sucking insects such as the brown planthopper (BPH; Nilaparvata lugens), which is one of the most damaging pests of rice. Traditionally, KDML105 and SP60 have been regarded as recalcitrant to transformation, and this is the first account of successful gene transfer to these varieties. By molecular analysis, we confirmed the recovery of over thirty gna-transgenic lines. GNA protein expression was characterised by western blot analysis, and we achieved expression levels of up to 0.25% total soluble protein. GNA-producing R₁ transgenic plants were significantly more resistant to BPH than control plants (P<0.0001), with 37% and 42% reduction in nymphal survival for constitutive and phloem-specific expression, respectively. Transferring the gna gene to these superior rice varieties thus represents a major step forward for crop improvement in Thailand, and should help to reduce the damage caused by rice pests, and hence increase yields for this vital domestic and export market.     

Drought tolerance genes in rice

Quantitative trait loci (QTLs) for drought tolerance (DT) can be readily identified in available databases and in this paper, these QTLs were summarized in the form of a consensus map. An in silico strategy was then deployed to mine for candidate genes associated with DT QTLs using rice dbEST and rice genome databases. DT QTLs on rice chromosomes 1, 2, 4, 8, and 9 were selected to test the method. The result showed candidate genes associated with DT could be readily identified.     

Cloning and characterization of three genes encoding Qb-SNARE proteins in rice

Qb-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and function as important components of the vesicle trafficking machinery in eukaryotic cells. Here, we report three novel plant SNARE (NPSN) genes isolated from rice and named OsNPSN11, OsNPSN12 and OsNPSN13. They have about 70% nucleotide identity over their entire coding regions and similar genomic organization with ten exons and nine introns in each gene. Multiple alignment of deduced amino acid sequences indicate that the OsNPSNs proteins are homologous to AtNPSNs from Arabidopsis, containing a Qb-SNARE domain and a membrane-spanning domain in the C-terminal region. Semi-quantitative RT-PCR assays showed that the OsNPSNs were ubiquitously and differentially expressed in roots, culms, leaves, immature spikes and flowering spikes. The expression of OsNPSNs was significantly activated in rice seedlings treated with H₂O₂, but down-regulated under NaCl and PEG6000 stresses. Transient expression method in onion epidermal cells revealed that OsNPSNs were located in the plasma membrane. Transformed yeast cells with OsNPSNs had better growth rates than empty-vector transformants when cultured on either solid or liquid selective media containing various concentrations of H₂O₂, but more sensitive to NaCl and mannitol stresses. The 35S:OsNPSN11 transgenic tobacco also showed more tolerance to H₂O₂ and sensitivity to NaCl and mannitol than non-transgenic tobacco. These results indicate that OsNPSNs may be involved in different aspects of the signal transduction in plant and yeast responses to abiotic stresses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.