Non-transgenic Lines Research Articles

INHERITANCE OF RESISTANCE TO PStV IN TRANSGENIC PEANUTS CONTAINING cp PStV GENE

Inheritance of Resistance to PStV in Transgenic Peanuts Containing cp PStV Gene. We have obtained transgenic peanut lines containing coat protein gene of PStV. To get maximal use of the transgenic character in a breeding program, it is necessary that the transgene is also stably inherited and expressed. This experiment was conducted from June 2002 – January 2005 at Plant Molecular Biology Laboratory, Bogor Agricultural University, and Queensland Agricultural Biotechnology Center, The University of Queensland, Australia. The research aimed (1) to test whether PStV cp transgene was functional in progenies derived from crosses between transgenic peaanut plants containing PStV cp gene and non-transgenic ones and (2) to determine pattern of inheritance of resistance to PStV as a result of PStV cp gene action. Several crosses were made between trangenic peanut cv.Gajah resistant to PStV (T4 generation) and non-transgenic peanut line WS susceptible to PStV. The F1 and F2 populations were mechanically inoculated with PStV two weeks after planting. The experiment showed that all plants in the F1 population were less susceptible to PStV, suggesting that the transgene was partially dominant. Phenotipic segregation in F2 population was not Mendelian with the appearance of quick and slow recovery plants and the number of resistant plants being more than expected. However, the proportion of transgenic and non-transgenic plants followed 3:1 ratio, which was Mendelian.

Field evaluation of effects of transgenic cry1Ab/cry1Ac, cry1C and cry2A rice on Cnaphalocrocis medinalis and its arthropod predators

The impacts of transgenic Bt rice on target pests and their predators need to be clarified prior to the commercialization of Bt rice. In this study, the percentages of folded leaves of three transgenic Bt rice lines and non-transgenic parental rice line caused by Cnaphalocrocis medinalis were studied over two successive growing seasons. In addition, the population densities, relative abundance and population dynamics of C. medinalis and four species of its natural arthropod predators were investigated at three sites in China. The results showed that rice line significantly affected the percentages of folded leaves and population densities of C. medinalis larvae. Significantly higher percentages of folded leaves were observed on the non-transgenic rice compared with the three transgenic Bt rice on most sampling dates. Significantly higher densities of C. medinalis larvae and higher relative abundance of C. medinalis within phytophages were found on non-transgenic rice compared with three transgenic Bt rice at different sites across the study period. The population dynamics of C. medinalis larvae were significantly affected by rice line, rice line×sampling date, rice line×year, rice line×sampling date×year. However, there was little, if any, significant difference in the relative abundance, population density and population dynamics of the four arthropod predators between the three Bt rice lines and non-transgenic rice. The results of this study indicate that the Bt toxin in transgenic Bt rice can effectively suppress the occurrence of C. medinalis, but has no significant effects on the occurrence of the four predatory arthropod species.

Impacts of transgenic Bt cotton on a non-target pest, Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae), in northern China

Transgenic Bt ( Bacillus thuringiensis) cotton has been effectively used to control the cotton bollworm, Helicoverpa armigera (Hübner) in China. However, in recent years, following the wide commercialization of Bt cotton in northern China there have been frequent outbreaks of the non-target pest Apolygus lucorum (Meyer-Dür). To clarify how transgenic cotton contributes to these outbreaks, a four-year field investigation of population dynamics and laboratory life table studies were carried out from 2007 to 2010 to evaluate the impact of two transgenic cotton cultivars (SGK321 expressing Cry1Ac + CpTI and GK12 expressing Cry1Ac) and their corresponding parental non-transgenic lines (Shiyuan321 and Simian3) on A. lucorum. There were no significant differences in the population densities of A. lucorum found in Bt cotton and non-Bt cotton plots, whether one compared those that had received insecticide treatments or those that had not. However, population densities of A. lucorum were significantly lower in pesticide treated plots than in controls. Furthermore, there were no significant differences in the net reproductive rates, generation times or intrinsic rates of increase of A. lucorum when reared on either Bt or non-Bt cotton cultivars. These results suggest that Bt cotton has no direct positive or negative effects on the biology of A. lucorum, so the most logical explanation for the observed outbreaks is the decrease in pesticide applications following the commercial release of Bt cotton.

Ultra‐low gossypol cottonseed: generational stability of the seed‐specific, RNAi‐mediated phenotype and resumption of terpenoid profile following seed germination

Cottonseed, containing 22.5% protein, remains an under-utilized and under-valued resource because of the presence of toxic gossypol. RNAi-knockdown of δ-cadinene synthase gene(s) was used to engineer plants that produced ultra-low gossypol cottonseed (ULGCS). In the original study, we observed that RNAi plants, a month or older, maintain normal complement of gossypol and related terpenoids in the roots, foliage, floral organs, and young bolls. However, the terpenoid levels and profile of the RNAi lines during the early stages of germination, under normal conditions and in response to pathogen exposure, had not been examined. Results obtained in this study show that during the early stages of seed germination/seedling growth, in both non-transgenic and RNAi lines, the tissues derived directly from bulk of the seed kernel (cotyledon and hypocotyl) synthesize little, if any new terpenoids. However, the growing root tissue and the emerging true leaves of RNAi seedlings showed normal, wild-type terpenoid levels. Biochemical and molecular analyses showed that pathogen-challenged parts of RNAi seedlings are capable of launching a terpenoid-based defence response. Nine different RNAi lines were monitored for five generations. The results show that, unlike the unstable nature of antisense-mediated low seed-gossypol phenotype, the RNAi-mediated ULGCS trait exhibited multi-generational stability.

Effects of elevated CO2 on the interspecific competition between two sympatric species of Aphis gossypii and Bemisia tabaci fed on transgenic Bt cotton

Abstract Effects of elevated CO2 (twice ambient vs. ambient) and Bt Cry1Ac transgene (Bt cotton cv. 33B vs. its nontransgenic parental line cv. DP5415) on the interspecific competition between two ecologically similar species of cotton aphid Aphis gossypii and whitefly biotype-Q Bemisia tabaci were studied in open-top chambers. The results indicated that elevated CO2 and Bt cotton both affected the population abundances of A. gossypii and biotype-Q B. tabaci when introduced solely (i.e., without interspecific competition) or two species coexisted (i.e., with interspecific competition). Compared with ambient CO2, elevated CO2 increased the population abundances of A. gossypii and biotype-Q B. tabaci as fed on Bt and nontransgenic cotton on 45 (i.e., seedling stage) and 60 (i.e., flowering stage) days after planting (DAP), but only significantly enhanced aphid abundance without interspecific competition on the 45-DAP nontransgenic cotton and 60-DAP Bt cotton, and significantly increased whitefly abundance with interspecific competition on the 45-DAP Bt cotton and 60-DAP nontransgenic cotton. In addition, compared with nontransgenic cotton at elevated CO2, Bt cotton significantly reduced biotype-Q B. tabaci abundances without and with interspecific competition during seedling and flowering stage, while only significantly decreasing A. gossypii abundances without interspecific competition during the seedling stage. When the two insect species coexisted, the proportions of biotype-Q B. tabaci were significantly higher than those of A. gossypii on Bt and nontransgenic cotton at the same CO2 levels, and elevated CO2 only significantly increased the percentages of biotype-Q B. tabaci and significantly reduced the proportions of A. gossypii on seedling and flowering nontransgenic cotton. Therefore, the effects of elevated CO2 were favorable for biotype-Q B. tabaci to out-compete A. gossypii under the predicted global climate change.

Sclerotinia Blight Resistance in Virginia-Type Peanut Transformed with a Barley Oxalate Oxidase Gene

Transgenic peanut lines expressing oxalate oxidase, a novel enzyme to peanut, were evaluated for resistance to Sclerotinia blight in naturally infested fields over a 5-year period. Area under the disease progress curve (AUDPC) for transgenic lines in single rows planted with seed from single-plant selections averaged 78, 83, and 90% lower than nontransgenic parents in 2004, 2005, and 2006, respectively. In addition, AUDPC in 14 transgenic lines planted with bulked seed in two-row plots averaged 81% lower compared with nontransgenic parents in 2005 and 86% lower in 16 transgenic lines in 2006. Six transgenic lines yielded 488 to 1,260 kg/ha greater than nontransgenic parents in 2005, and 10 lines yielded 537 to 2,490 kg/ha greater in 2006. Fluazinam (0.58 kg a.i./ha) fungicide sprays in 2008 and 2009 reduced AUDPC in transgenic and nontransgenic lines but AUDPC was lowest in transgenic lines. Without fluazinam, yields of transgenic lines averaged 1,133 to 1,578 kg/ha greater than nontransgenic lines in 2008 and 1,670 to 2,755 kg/ha greater in 2009. These results demonstrated that the insertion of barley oxalate oxidase in peanut conveyed a high level of resistance to Sclerotinia blight, and negated the need for costly fungicide sprays.

Proteomic analysis of stress-related proteins in transgenic broccoli harboring a gene for cytokinin production during postharvest senescence

Our previous study revealed a cytokinin-related retardation of post-harvest floret yellowing in transgenic broccoli ( Brassica oleracea var. italica) that harbored the bacterial isopentenyltransferase ( ipt) gene. We aimed to investigate the underlining mechanism of this delayed post-harvest senescence. We used 2D electrophoresis and liquid chromatography–electrospray ionization–mass spectrometry/mass spectrometry for a proteomics analysis of heads of ipt-transgenic and non-transgenic inbred lines of broccoli at harvest and after four days post-harvest storage. At harvest, we found an accumulation of stress-responsive proteins involved in maintenance of protein folding (putative protein disulfide isomerase, peptidyl-prolyl cis–trans isomerase and chaperonins), scavenging of reactive oxygen species (Mn superoxide dismutase), and stress protection [myrosinase-binding protein, jasmonate inducible protein, dynamin-like protein, NADH dehydrogenase (ubiquinone) Fe-S protein 1 and stress-inducible tetratricopeptide repeat-containing protein]. After four days’ post-harvest storage of non-transgenic broccoli florets, the levels of proteins involved in protein folding and carbon fixation were decreased, which indicates cellular degradation and a change in metabolism toward senescence. In addition, staining for antioxidant enzyme activity of non-transgenic plants after post-harvest storage revealed a marked decrease in activity of Fe-superoxide dismutase and ascorbate peroxidase. Thus, the accumulation of stress-responsive proteins and antioxidant enzyme activity in ipt-transgenic broccoli are most likely associated with retardation of post-harvest senescence.

Agronomic Traits of Transgenic Soybean Overexpressing TaDREB3

Dongnong50 was overexpressing TaDREB3, a stress-tolerance gene, and transgenic progenies with TaDREB3 showing stable heredity characters were obtained in this study. The seeds of T4 generation were sowed on saline land of which salt concentration was 0.19% and pH was 8.43, and agronomic traits including growth period, plant height, numbers of main nodes, branching numbers, pods per plant, numbers of seeds per plant, and weight of 100-seed were collected and comparing to control plants Dongnong50 were made. The impacts of TaDREB3 gene on agronomic traits of soybeans under high concentration of salt were discussed. The results showed that the growth period was not altered in transgenic line with TaDREB3 under high concentration of saline and alkali, the extremely significant or significant increase in the numbers of main nodes, pods per plant and in branching numbers and other agronomic traits in transgenic lines in comparing with non-transgenic lines was an indicator of their resistance to saline-alkali stress.

Molecular and cytogenetic assessment of transgenic hot peppers resistant to Cucumber mosaic Virus

In this study, we have analyzed Cucumber mosaic virus (CMV)-resistant transgenic hot pepper (Capsicum annuum) plants harboring coat protein gene (CMVP0-CP) of the virus as a framework for molecular and cytogenetic assessment. PCR, Southern blot, RT-PCR, and Western blot techniques were applied to identify the existence and expression of the transgene in two independent transgenic hot pepper events in T4 generation, H15 and B20. Fluorescence in situ hybridization (FISH) was also conducted to detect physical location of the transgene on the chromosome of the plants. The amplified products of the inserted genes, 35S promoter with CMVP0-CP, CMVP0-CP, and NPT II were obtained from the genomic DNA of the transgenic hot pepper lines by PCR. A single band, indicative of a single copy insertion, was observed by Southern blot analysis. Transcription of the transgene was confirmed from RT-PCR fragments CMVP0-CP and NPT II genes in transgenic plants. Translated protein of the target gene, CMV-CP, was detected in the transgenic hot pepper events but not in nontransgenic control hot pepper lines by Western blot analysis. FISH analysis of transgenic line H15 revealed that the transgene was physically located at the sub-terminal portion of the long arm of chromosome d where the 5S rDNA locus is positioned. Conclusively, we observed that transgenes were maintained stably in T4 generation of transgenic hot peppers via cytogenetic investigation as well as molecular detection. These results can be useful in future studies conducted to evaluate breeding techniques and to assess genetically modified hot peppers.

Production of transgenic canine embryos using interspecies somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry 'foreign' DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8-16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8-16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine-bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models.

Production and evaluation of transgenic sweet orange ( Citrus sinensis Osbeck) containing bivalent antibacterial peptide genes ( Shiva A and Cecropin B) via a novel Agrobacterium-mediated transformation of mature axillary buds

Transgenic plants of Jincheng orange ( Citrus sinensis Osbeck) and Newhall navel orange ( C. sinensis Osbeck) containing antibacterial peptide genes Shiva A and Cecropin B were successfully obtained by a novel Agrobacterium tumefaciens-mediated transformation of the mature axillary buds. PCR and Southern blot analysis of the transgenic plants verified that the Shiva A and Cecropin B genes were integrated into the citrus genome. The transgenic plants began to blossom and bear fruit in the 2nd year after grafting on trifoliate orange ( Poncirus trifoliata Raf) rootstock in greenhouse. Water-soluble extracts from transgenic citrus leaves exhibit in vitro suppressive effects on Xanthomonas axonopodis pv. citri, suggesting that the expressed products of Shiva A and Cecropin B in citrus retain their native antibacterial activities. Artificial inoculation in greenhouse and open field further indicates significantly increased resistance of transgenic plants to X. axonopodis pv. citri when compared with non-transgenic lines. No significant difference was found in the content of total soluble solids, total acidity, reduced sugar content and other fruit characteristics between transgenic and non-transgenic plants. In this present study, 11 transgenic lines were obtained from 40 transgenic lines, showing enhanced resistance to citrus canker disease.

Enhancement of artemisinin content by constitutive expression of the HMG-CoA reductase gene in high-yielding strain of Artemisia annua L.

Artemisinin is effective against both chloroquine-resistant and -sensitive strains of Plasmodium species. However, the low yield of artemisinin from cultivated and wild plants is a serious limitation to the commercialization of this drug. Optimization of artemisinin yield either in vivo or in vitro is therefore highly desirable. To this end, we have overexpressed the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) gene (hmgr) from Catharanthusroseus L. in Artemisia annua L. and analyzed its influence on artemisinin content. PCR and Southern blot analyses revealed that the transgenic plants showed stable integration of the foreign hmgr gene. The reverse transcriptase-PCR results suggested that the hmgr was expressed at the transcriptional level in transgenic lines of Artemisia annua L., while the high-performance liquid chromatography analysis showed that artemisinin content was significantly increased in a number of the transgenic lines. Artemisinin content in one of the A. annua transgenic lines was 38.9% higher than that in non-transgenic plants, and HMGR enzyme activity in transgenic A. annua L. was also higher than that in the non-transgenic lines.

Ectopic Expression of AtCIPK23 Enhances Tolerance Against Low-K+ Stress in Transgenic Potato

Potato (Solanum tuberosum L.) has a relatively high requirement for potassium (K+). In the face of the declining of potato yield and quality as a result of reduction of soil K+ content, it is necessary to determine how to improve the tolerance of potatoes to low-K+ stresses. The protein kinase AtCIPK23 is essential for K+ uptake, and its overexpression significantly enhances K+ uptake and tolerance to low-K+ in Arabidopsis. In this research, AtCIPK23 was transformed into an E3 potato cultivar successfully. In low-K+ conditions, the transgenic potato lines had lower levels of leaf chlorosis, higher K+ uptake rate, increased dry weight and K+ content. The non-transgenic lines displayed reduced stature while the transgenic lines exhibited sustained growth. These results indicate that ectopic expression of AtCIPK23 increases low-K+ tolerance in potato. This study demonstrated the potential use of the transgenic approach to increase potato production in low-K+ fields.

Plant natural variability may affect safety assessment data

Before market introduction, genetic engineered (GE) food products, like any other novel food product, are subjected to extensive assessment of their potential effects on human health. In recent years, a number of profiling technologies have been explored aiming to increase the probability of detecting any unpredictable unintended effect and, consequently improving the efficiency of GE food safety assessment. These techniques still present limitations associated with the interpretation of the observed differences with respect to their biological relevance and toxicological significance. In order to address this issue, in this study, we have performed 2D-gel electrophoresis of five different ears of five different MON810 maize plants and of other five of the non-transgenic near-isogenic line. We have also performed 2D-gel electrophoresis of the pool of the five protein extractions of MON810 and control lines. We have notice that, in this example, the exclusive use of data from 2D-electrophoresed pooled samples, to compare these two lines, would be insufficient for an adequate safety evaluation. We conclude that, when using “omics” technologies, it is extremely important to eliminate all potential differences due to factors not related to the ones under study, and to understand the role of natural plant-to-plant variability in the encountered differences.

Phospholipid and triacylglycerol profiles modified by PLD suppression in soybean seed

Phospholipase D (PLD) is capable of hydrolyzing membrane phospholipids, producing phosphatidic acid. To alter phospholipid profiles in soybean seed, we attenuated PLD enzyme activity by an RNA interference construct using the partial sequence from a soybean PLDα gene. Two transgenic soybean lines were established by particle inflow gun (PIG) bombardment by co-bombarding with pSPLDi and pHG1 vectors. The lines were evaluated for the presence and expression of transgenes thoroughly through the T(4) generation. PLD-suppressed soybean lines were characterized by decreased PLDα enzyme activity and decreased PLDα protein both during seed development and in mature seeds. There was no change in total phospholipid amount; however, the PLD-attenuated transgenic soybean seed had higher levels of di18:2 (dilinoleoyl)-phosphatidylcholine (PC) and -phosphatidylethanolamine (PE) in seeds than the non-transgenic lines. The increased polyunsaturation was at the expense of PC and PE species containing monounsaturated or saturated fatty acids. In addition to increased unsaturation in the phospholipids, there was a decrease in unsaturation of the triacylglycerol (TAG) fraction of the soybean seeds. Considering recent evidence for the notion that desaturation of fatty acids occurs in the PC fraction and that the PC→DAG (diacylglycerol)→TAG pathway is the major route of TAG biosynthesis in developing soybean seed, the current data suggest that PLDα suppression slows the conversion of PC to TAG. This would be consistent with PLD playing a positive role in that conversion. The data indicate that soybean PLD attenuation is a potentially useful approach to altering properties of edible and industrial soybean lecithin.

High-efficiency transformation and selective tolerance against biotic and abiotic stress in mulberry, Morus indica cv. K2, by constitutive and inducible expression of tobacco osmotin

Osmotin and osmotin-like proteins are stress proteins belonging to the plant PR-5 group of proteins induced in several plant species in response to various types of biotic and abiotic stresses. We report here the overexpression of tobacco osmotin in transgenic mulberry plants under the control of a constitutive promoter (CaMV 35S) as well as a stress-inducible rd29A promoter. Southern analysis of the transgenic plants revealed the stable integration of the introduced genes in the transformants. Real-time PCR analysis provided evidence for the expression of osmotin in the transgenic plants under both the constitutive and stress-inducible promoters. Transgenic plants with the stress-inducible promoter were observed to better tolerate salt and drought stress than those with the constitutive promoter. Transgenic plants when subjected to simulated salinity and drought stress conditions showed better cellular membrane stability (CMS) and photosynthetic yield than non-transgenic plants under conditions of both salinity and drought stress. Proline levels were very high in transgenic plants with the constitutive promoter relative to those with the stress-inducible promoter. Fungal challenge undertaken with three fungal species known to cause serious losses to mulberry cultivation, namely, Fusarium pallidoroseum, Colletotrichum gloeosporioides and Colletotrichum dematium, revealed that transgenic plants with osmotin under control of the constitutive promoter had a better resistance than those with osmotin under the control of the stress-inducible promoter. Evaluation in next generation was undertaken by studying bud break in transgenic and non-transgenic plants under simulated drought (2% polyethylene glycol) and salt stress (200mM NaCl) conditions. The axillary buds of the selected transgenic lines had a better bud break percentage under stressed conditions than buds from non-transgenic mulberry lines. A biotic assay with Bombyx mori indicated that osmotin protein had no undesirable effect on silkworm rearing and feeding. We therefore conclude that 35S transgenic plants are better suited for both abiotic stress also biotic challenges (fungal), while the rd29A transgenic plants are more responsive to drought.

PYRAMIDING IMPORTANT DISEASE-RESISTANT CHARACTERS BY HYBRIDIZATION OF TRANSGENIC AND NON-TRANSGENIC PEANUTS (ARACHIS HYPOGAEA L.)

We have produced transgenic peanut lines carrying a coat protein gene of peanut stripe virus (PStV) and showing resistance to the virus. However, their susceptibility to leafspot disease caused by Cercospora sp. and their lower productivity compared to their genetic background cultivar make them commercially less undesireable. The objective of this research was to test whether crossing the transgenic peanut plants with a non-transgenic peanut line WS, which was resistant to leafspot disease and high-yielding, could produce progenies in F2 generation that were resistant to both PStV and leafspot disease as well as of higher yield campared to their transgenic progenitor. If this test was proven, pyramiding novel transgenic and non-transgenic characters in peanut plants by hybridization would probably be a routine procedure in the future. Crosses were made between transgenic peanut plants that were resistant to PStV and non-transgenic peanut line WS. F2 population was evaluated for resistance to PStV and leafspot disease. Number of filled pods, filled pod dry weight per plant, and dry weight of each pod were measured. Result of the experiment showed that some of the plants in F2 population exhibited resistant both to PStV and leafspot disease and produced higher number of filled pods, filled pod dry weight per plant, and dry weight of each pod compared to those produced by their transgenic parent plants.

Genome-wide gene responses in a transgenic rice line carrying the maize resistance gene Rxo1 to the rice bacterial streak pathogen, Xanthomonas oryzae pv. oryzicola

BackgroundNon-host resistance in rice to its bacterial pathogen, Xanthomonas oryzae pv. oryzicola (Xoc), mediated by a maize NBS-LRR type R gene, Rxo1 shows a typical hypersensitive reaction (HR) phenotype, but the molecular mechanism(s) underlying this type of non-host resistance remain largely unknown.ResultsA microarray experiment was performed to reveal the molecular mechanisms underlying HR of rice to Xoc mediated by Rxo1 using a pair of transgenic and non-transgenic rice lines. Our results indicated that Rxo1 appeared to function in the very early step of the interaction between rice and Xoc, and could specifically activate large numbers of genes involved in signaling pathways leading to HR and some basal defensive pathways such as SA and ET pathways. In the former case, Rxo1 appeared to differ from the typical host R genes in that it could lead to HR without activating NDR1. In the latter cases, Rxo1 was able to induce a unique group of WRKY TF genes and a large set of genes encoding PPR and RRM proteins that share the same G-box in their promoter regions with possible functions in post-transcriptional regulation.ConclusionsIn conclusion, Rxo1, like most host R genes, was able to trigger HR against Xoc in the heterologous rice plants by activating multiple defensive pathways related to HR, providing useful information on the evolution of plant resistance genes. Maize non-host resistance gene Rxo1 could trigger the pathogen-specific HR in heterologous rice, and ultimately leading to a localized programmed cell death which exhibits the characteristics consistent with those mediated by host resistance genes, but a number of genes encoding pentatricopeptide repeat and RNA recognition motif protein were found specifically up-regulated in the Rxo1 mediated disease resistance. These results add to our understanding the evolution of plant resistance genes.

Determining Sex and Screening for the Adventitious Presence of Transgenic Material in Carica papaya L. Seed Germplasm

Papaya ringspot virus (PRSV) is a devastating disease that has a detrimental impact on both commercial papaya production and Caricaceae germplasm conservation. In 1998, the PRSV coat protein transgenic line 55-1 and derived progeny were released to growers in Hawaii. The transgenic varieties have provided durable and practical control of the disease that have saved the papaya industry. However, like with transgenic crops throughout the world, there is public concern about the possibility of cross-contamination of these transgenic materials into nontransgenic lines. As the designated germplasm repository for Caricaceae, we are responsible for maintaining the genetic integrity of each accession. Therefore, we have developed a protocol using polymerase chain reaction for detection of the adventitious presence of the 55-1 transgene insertion event in both parental plants and their progeny seed populations. This protocol assures a 99.9% confidence level of obtaining seeds that are 99.5% transgene-free. The protocol developed in this study is not typical for most seed validation techniques because there is a higher than normal producer risk resulting from the potential of large numbers of seeds not meeting the stringent criteria. However, we believe this is necessary to ensure the genetic integrity of seeds stored in the repository.

10.1007/s11183-008-2014-z

The genetically engineered osmotin gene located in a binary vector was transferred by shotgun to an Iranian wheat cultivar (Triticum aestivum, cv. Marvdasht). Screening of transgenic plants was carried out by using both molecular and phenotypic approaches. Transgenic lines were studied for their ability to tolerate salinity stress at various concentrations of NaCl. Transgenic line of cv. Marvdasht exhibited a high ability to produce roots at the concentration of 250 mM NaCl. In this research, a resistant Iranian cv. Zarrin and nontransgenic lines of cv. Marvdasht were also compared for their root length under salinity condition. Results showed that transgenic Marvdasht lines are significantly more tolerant than nontransgenic ones and very similar to naturally resistant Zarrin in this aspect.