Transgenic Progeny Research Articles

Ectopic expression of the Anthurium andraeanum (Hort.) R2R3-MYB genes AaMYB4 and AaMYB5 enhance the flower color in transgenic tobacco

Anthocyanin coloration is crucially important for ornamental traits in Anthurium andraeanum. However, the molecular mechanisms responsible for anthocyanin accumulation in this important ornamental plant are poorly understood. Here, we isolated and functionally characterized two R2R3-MYB transcription factor genes AaMYB4 and AaMYB5 from A. andraeanum. Analysis of amino acid sequences indicate that both AaMYB4 and AaMYB5 share homology with the C1 subgroup R2R3-MYBs from monocots. AaMYB4 and AaMYB5 proved to be nucleus-localized proteins. Spatial and temporal expression analysis of AaMYB4 and AaMYB5 suggested that AaMYB4 was mainly expressed in the spathe and young leaves, whereas AaMYB5 was mainly expressed in the young leaves and the spadix. Although the expression patterns of AaMYB4 and AaMYB5 did not correlate with anthocyanin accumulation in the spathe and spadix of cultivar Vitara and Topical, they were positively correlated with anthocyanin concentration in the developing leaves of ‘Tropical’ and were coordinately expressed with key anthocyanin biosynthesis pathway genes. The ectopic expression of AaMYB4 and AaMYB5 in tobacco triggered the accumulation of anthocyanins in the corolla limbs of T1 transgenic progeny, and induced the up-regulation of expression of the key enzyme genes in the anthocyanin biosynthesis pathway of tobacco. Overexpression of AaMYB5 in tobacco also markedly up-regulated the expression of the endogenous MYB regulator gene NtAn2 involved in anthocyanin biosynthesis. Our findings suggest that both AaMYB4 and AaMYB5 from A. andraeanum are involved in the regulation of anthocyanin biosynthesis and have potential value in the metabolic engineering of anthocyanin biosynthesis in plants. AaMYB4 and AaMYB5 are R2R3-MYB transcription factors from Anthurium andraeanum. Overexpression of the two genes in tobacco resulted in large anthocyanin accumulation in the corolla limbs.

Isolation, cloning and expression of CCA1 gene in transgenic progeny plants of Japonica rice exhibiting altered morphological traits.

Circadian clock genes holds tremendous potential for breeding crops better adapted to environmental fluctuations inherent to climate change. Endogenous TOC1 promoter and CCA1 gene from rice were isolated, cloned and mobilized into pCAMBIA1300 vectors and RNAi constructs A, B and C. Embryogenic calli of varying ages derived from mature seeds of Taipei 309 were employed for Agrobacterium-mediated genetic transformation generating T0, T1 and T2 independent transgenic lines were analyzed for over-expression and repression of CCA1 gene along with various morphological traits. Six hundred and thirty two T0 transgenic plants were generated from rice calli using constructs A, B and C. T0 progeny plants derived from constructs A, B and C did not show any considerable difference in morphological traits. T1and T2 progeny plants derived from construct A exhibited over-expression of CCA1 gene, on the contrary, progeny plants derived from RNAi constructs B and C exhibited repression of CCA1 gene in qRT-PCR analysis at different time points and showed rhythmicity peaking at dawn (6:00 AM) and lowest expression at 12:00 Noon. T1 and T2 progeny plants derived from construct A, namely, A-17 and A-45 exhibited reduced number of tillers/panicles (6–8), reduced thousand seed weight (10.1–16.6g), decreased seed length (4.98 to 6.58mm), decreased seed width (1.1–1.8mm) as compared to wild type plants. T1 and T2 progeny plants of construct B and C showed increased number of tillers/panicles (8–19), better seed yield (4.98–28.9g), increased thousand seed weight (15.6–29.03g), slightly increased seed length (5.7–7.43mm) and slightly increased seed width (1.7–2.98mm) as compared to wild type plants. Chlorophyll content in T1 and T2 progeny plants did not show any significant difference among the three constructs, however, rhythmicity was observed over the period of time in conjunction to CCA1 gene expression. Evidence has been presented which demonstrates that endogenous repression of CCA1 gene resulted in improved morphological traits: increased number of tillers/panicle, thousand seed weight, seed size; whereas, over-expression leads to diminution in morphological traits: decreased number of tillers/panicle, thousand seed weight, seed size as compared to the wild type in T1 and T2 progeny plants. This is first report of successful regulation of endogenous CCA1 gene under control of TOC1 promoter and its effect on improved growth vigor in Japonica rice.

Aseptic Rearing and Infection with Gut Bacteria Improve the Fitness of Transgenic Diamondback Moth, Plutella xylostella.

Mass insect rearing can have a range of applications, for example in biological control of pests. The competitive fitness of released insects is extremely important in a number of applications. Here, we investigated how to improve the fitness of a transgenic diamondback moth, which has shown variation in mating ability when reared in different insectaries. Specifically we tested whether infection with a gut bacteria, Enterobacter cloacae, and aseptic rearing of larvae could improve insect growth and male performance. All larvae were readily infected with E. cloacae. Under aseptic rearing, pupal weights were reduced and there was a marginal reduction in larval survival. However, aseptic rearing substantially improved the fitness of transgenic males. In addition, under aseptic rearing, inoculation with E. cloacae increased pupal weights and male fitness, increasing the proportion of transgenic progeny from 20% to 30% relative to uninfected insects. Aseptic conditions may improve the fitness of transgenic males by excluding microbial contaminants, while symbiont inoculation could further improve fitness by providing additional protection against infection, or by normalizing insect physiology. The simple innovation of incorporating antibiotic into diet, and inoculating insects with symbiotic bacteria that are resistant to that antibiotic, could provide a readily transferable tool for other insect rearing systems.

The effects of omega-3 fatty acid deficiency during development on oxidative fatty acid degradation during maturity in a mouse model of Alzheimer's disease

Metabolic conditions during brain development may have long-term consequences on brain metabolism, thereby influencing the risk of neurodegenerative disease in later life. To ascertain the long-term consequences of omega-3 (ω3) fatty acid deficiency during brain development on oxidative fatty acid degradation in the brain and the development of Alzheimer-like pathology, wild-type (WT) female mice were fed diets that were either replete or deficient in ω3 fatty acids for 5 weeks. These females were then mated with hemizygous 5xFAD male transgenic (TG) mouse models of Alzheimer's disease, and the progeny were continued on diets that were either ω3-replete or ω3-deficient. When the progeny were 6 months of age, they received radiolabeled arachidonic acid (ARA) by intracerebroventricular injection. Five days after these injections, the brains were harvested and oxidative degradation of the radiolabeled ARA was characterized. Among the progeny of female mice on an ω3-replete diet, TG progeny had lower PSD-95 expression and higher oxidative ARA degradation than WT progeny. Progeny on an ω3-deficient diet, however, had no significant differences in PSD-95 expression between TG and WT mice, or in the extent of ARA degradation. In TG mice, an ω3-deficient diet reduced oxidative ARA degradation to a greater extent than in WT mice. The reductions in oxidative ARA degradation occurred even if the progeny of female mice on an ω3-deficient diet resumed an ω3-replete diet immediately on weaning. These results demonstrate that dietary ω3 fatty acid deficiency during development can cause long-term changes in the expression of a synaptic marker and long-term reductions in the rate of ARA degradation in the WT brain, which are not completely alleviated by an ω3-replete diet after weaning. The elimination of differences between TG and WT mice by an ω3-deficient diet suggests that mechanisms regulating PSD-95 expression and the oxidative degradation of ARA are related and that the timing of dietary ω3 intake during development may influence Alzheimer's disease–related pathological changes later in life.

Different genetic mechanisms mediate spontaneous versus UVR-induced malignant melanoma.

Genetic variation conferring resistance and susceptibility to carcinogen-induced tumorigenesis is frequently studied in mice. We have now turned this idea to melanoma using the collaborative cross (CC), a resource of mouse strains designed to discover genes for complex diseases. We studied melanoma-prone transgenic progeny across seventy CC genetic backgrounds. We mapped a strong quantitative trait locus for rapid onset spontaneous melanoma onset to Prkdc, a gene involved in detection and repair of DNA damage. In contrast, rapid onset UVR-induced melanoma was linked to the ribosomal subunit gene Rrp15. Ribosome biogenesis was upregulated in skin shortly after UVR exposure. Mechanistically, variation in the 'usual suspects' by which UVR may exacerbate melanoma, defective DNA repair, melanocyte proliferation, or inflammatory cell infiltration, did not explain melanoma susceptibility or resistance across the CC. Instead, events occurring soon after exposure, such as dysregulation of ribosome function, which alters many aspects of cellular metabolism, may be important.

The Current Status and Development of Insect-Resistant Genetically Engineered Poplar in China.

Poplar is one of the main afforestation tree species in China, and the use of a single, or only a few, clones with low genetic diversity in poplar plantations has led to increasing problems with insect pests. The use of genetic engineering to cultivate insect-resistant poplar varieties has become a hot topic. Over the past 20 years, there have been remarkable achievements in this area. To date, nearly 22 insect-resistant poplar varieties have been created and approved for small-scale field testing, environmental release, or pilot-scale production. Here, we comprehensively review the development of insect-resistant genetically modified (GM) poplars in China. This review mostly addresses issues surrounding the regulation and commercialization of Bt poplar in China, the various insecticidal genes used, the effects of transgenic poplars on insects, toxic protein expression, multigene transformation, the stability of insect resistance, and biosafety. The efficacy of GM poplars for pest control differed among different transgenic poplar clones, larval instars, and insect species. The Bt protein analysis revealed that the expression level of Cry3A was significantly higher than that of Cry1Ac. Temporal and spatial studies of Bt protein showed that its expression varied with the developmental stage and tissue. The inheritance and expression of the exogenous gene were reviewed in transgenic hybrid poplar progeny lines and grafted sections. Biosafety issues, in terms of transgene stability and the effects on soil microorganisms, natural enemies of insects, and arthropod communities are also discussed.

Feral rice from introgression of weedy rice genes into transgenic herbicide-resistant hybrid-rice progeny

Pollen-mediated transgenic flow of herbicide resistance occurs bidirectionally between transgenic cultivated rice and weedy rice. The potential risk of weedy traits introgressing into hybrid rice has been underestimated and is poorly understood. In this study, two glufosinate-resistant transgenic rice varieties, hybrid rice (F1), and their succeeding generations (F2-F4) were planted for 3 years in field plots free of weedy rice adjacent to experimental weedy-rice fields. Weedy-rice-like (feral) plants that were both glufosinate-resistant and had red-pericarp seed were initially found only among the F3 generations of the two glufosinate-resistant transgenic hybrid cultivars. The composite fitness (an index based on eight productivity and weediness traits) of the feral progeny was significantly higher than that of the glufosinate-resistant transgenic hybrid (the original female parent of the feral progeny) under monoculture common garden conditions. The hybrid rice progeny segregated into individuals of variable height and extended flowering. The hybrid rice F2 generations had higher outcrossing rates by pollen reception (0.96-1.65%) than their progenitors (0.07-0.98%). The results show that herbicide-resistant weedy rice can rapidly arise by pollen-mediated gene flow from weedy to transgenic hybrid rice, and their segregating pollen-receptive progeny pose a greater agro-ecological risk than transgenic varieties. The safety assessment and management regulations for transgenic hybrid rice should take into account the risk of bidirectional gene flow.

Development of transgenic tobacco lines with pyramided insect resistant genes.

Insect pests are among the major constraints rendering drastic decreases in crop yield. The expression of stacked insecticidal genes in crops can lead to resistance durability and can delay the development of resistance in target insect pests. The present study was designed to introduce an insect resistance trait in locally cultivated Turkish tobacco cultivars (Basma and Nail) with pyramided insecticidal genes. Agrobacterium strain LBA4404 harboring plasmid pKGH4 with cry1Ac and cry2A genes under the control of 35S promoter was used to infect leaf discs of both cultivars; plasmid also contained uidA within the T-DNA region for earlier screening of putative transformants. The overall transformation efficiency was calculated as 30.7% and 18.8% in Basma and Nail, respectively. PCR results confirmed the integration of cry1Ac, cry2A, uidA, and nptII genes in 40 plants of Basma and 16 plants of Nail. ELISA results showed variation in expression of cry1Ac protein among transgenic plants varying from 0.017 to 0.607 µg/g of fresh tissue. Bioassay results with potato tuber moth (Phthorimea operculella Zeller) showed significant mortality of the targeted pest on primary transformants. Furthermore, T1 transgenic progeny exhibited the inheritance of T-DNA in Mendelian as well as non-Mendelian fashion. The results revealed that lines can serve as a source of germplasm in tobacco breeding programs.

The current status and development of insect-resistant genetically engineered poplar in China

Poplar is one of the main afforestation tree species in China, and the use of a single, or only a few, clones with low genetic diversity in poplar plantations has led to increasing problems with insect pests. The use of genetic engineering to cultivate insect-resistant poplar varieties has become a hot topic. Over the past 20 years, there have been remarkable achievements in this area. To date, nearly 22 insect-resistant poplar varieties have been created and approved for small-scale field testing, environmental release, or pilot-scale production. Here, we comprehensively review the development of insect-resistant genetically modified (GM) poplars in China. This review mostly addresses issues surrounding the regulation and commercialization of Bt poplar in China, the various insecticidal genes used, the effects of transgenic poplars on insects, toxic protein expression, multigene transformation, the stability of insect resistance, and biosafety. The efficacy of GM poplars for pest control differed among different transgenic poplar clones, larval instars, and insect species. The Bt protein analysis revealed that the expression level of Cry3A was significantly higher than that of Cry1Ac. Temporal and spatial studies of Bt protein showed that its expression varied with the developmental stage and tissue. The inheritance and expression of the exogenous gene were reviewed in transgenic hybrid poplar progeny lines and grafted sections. Biosafety issues, in terms of transgene stability and the effects on soil microorganisms, natural enemies of insects, and arthropod communities are also discussed.

Non-plastidial expression of a synthetic insect geranyl pyrophosphate synthase effectively increases tobacco plant biomass

Designing effective synthetic genes of interest is a fundamental step in plant synthetic biology for biomass. Geranyl pyrophosphate (diphosphate) synthase (GPPS) catalyzes a bottleneck step toward terpenoid metabolism. We previously designed and synthesized a plant (Arabidopsis thaliana)-insect (Myzus persicae, Mp) GPPS- human influenza hemagglutinin (HA) cDNA, namely PTP-MpGPPS-HA (or PTP-sMpGPPS-HA, s: synthetic), to localize the protein in plastids and improve plant biomass. To better understand the effects of different subcellular localizations on plant performance, herein we report PTP-sMpGPPS-HA re-design to synthesize a new MpGPPS-HA cDNA, namely sMpGPPS-HA, to express a non-plastidial sMpGPPS-HA protein. The sMpGPPS-HA cDNA driven by a 2 × S 35S promoter was introduced into Nicotiana tabacum Xanthi. PTP-MpGPPS-HA and PMDC84 vector transgenic plants were also generated as positive and negative controls, respectively. Eighteen to twenty transgenic T0 lines were generated for each sMpGPPS-HA, PTP-sMpGPPS-HA, and PMDC84. Transcriptional genotyping analysis demonstrated the expression of sMpGPPS-HA in transgenic plants. Confocal microscopy analysis of transgenic progeny demonstrated the non-plastidial localization of sMpGPPS-HA. Growth of T1 transgenic and wild-type control plants showed that the expression of sMpGPPS-HA effectively increased plant height by 50–80%, leaf numbers and sizes, and dry biomass by 60–80%. Calculation of the vegetative growth rates showed that the expression of sMpGPPS-HA increased plant height each week. Moreover, sMpGPPS-HA expression promoted early flowering and reduced leaf carotenoid levels. In conclusion, non-plastidial expression of the novel sMpGPPS-HA was effective for improving tobacco growth and biomass. Our data indicate that research examining different subcellular localizations facilitates a better understanding of in planta functions of proteins encoded by synthetic cDNAs.

Simplified methodology for large scale isolation of homozygous transgenic lines of lettuce

Background: Lettuce is a globally important leafy vegetable and a model plant for biotechnology due to its adaptability to tissue culture and stable genetic transformation. Lettuce is also crucial for functional genomics research in the Asteraceae which includes species of great agronomical importance. The development of transgenic events implies the production of a large number of shoots that must be differentiated between transgenic and non-transgenic through the activity of the selective agent, being kanamycin the most popular. Results: In this work we adjusted the selection conditions of transgenic seedlings to avoid any escapes, finding that threshold concentration of kanamycin was 75 mg/L. To monitor the selection system, we studied the morphological response of transgenic and non-transgenic seedlings in presence of kanamycin to look for a visual morphological marker. Several traits like shoot length, primary root length, number of leaves, fresh weight, and appearance of the aerial part and development of lateral roots were affected in non-transgenic seedlings after 30 d of culture in selective media. However, only lateral root development showed an early, qualitative and reliable association with nptII presence, as corroborated by PCR detection. Applied in successive transgenic progenies, this method of selection combined with morphological follow-up allowed selecting the homozygous presence of nptII gene in 100% of the analyzed plants from T2 to T5. Conclusions: This protocol allows a simplified scaling-up of the production of multiple homozygous transgenic progeny lines in the early generations avoiding expensive and time-consuming molecular assays.

A High-Throughput Standard PCR-Based Genotyping Method for Determining Transgene Zygosity in Segregating Plant Populations.

In crop research programs that implement transgene-based strategies for trait improvement it is necessary to distinguish between transgene homozygous and hemizygous individuals in segregating populations. Direct methods for determining transgene zygosity are technically challenging, expensive, and require specialized equipment. In this report, we describe a standard PCR-based protocol coupled with capillary electrophoresis that can identify transgene homozygous and hemizygous individuals in a segregating population without knowledge of transgene insertion site. PCR primers were designed to amplify conserved T-DNA segments of the 35S promoter, OCS terminator, and NPTII kanamycin resistance gene in the pHellsgate-8 RNAi construct for the Gossypium hirsutum phytochrome A1 gene. Using an optimized multiplexed reaction mixture and an amplification program of only 10 cycles we could discriminate between transgene homozygous and hemizygous cotton control DNA samples based on PCR product peak characteristics gathered by capillary electrophoresis. The protocol was refined by evaluating segregating transgenic progeny from nine BC1S1 populations derived from crosses between the transgenic cotton parent ‘E-1-7-6’ and other cotton cultivars. OCS PCR product peak height and peak area, normalized by amplification of the native cotton gene GhUBC1, revealed clear bimodal distributions of OCS product characteristics for each BC1S1 population indicating the presence of homozygous and hemizygous clusters which was further confirmed via K-means clustering. BC1S1 plants identified as homozygous or hemizygous were self-fertilized to produce BC1S2 progeny. For the homozygous class, 19/20 BC1S2 families confirmed the homozygous BC1S1 prediction while 21/21 BC1S2 families confirmed the hemizygous prediction of the original parent. This relatively simple protocol provides a reliable, rapid, and high-throughput way of evaluating segregating transgenic populations using methods and equipment common to crop molecular breeding labs.

Inheritance and expression stability of exogenous genes in insect-resistant transgenic poplar

To understand the genetic and expression stability of transgenic insect-resistant poplar 741, this study compared the experimental plantations of transgenic insect-resistant poplar 741 lines (pb1, pb6, pb11, pb17, and pb29) with non-transgenic poplar 741, P. tomentosa Carr.f.yixianensis (poplar 84 K) and transgenic hybrid progeny lines cultured from immature embryos. The insect resistance and growth stability of transgenic poplar 741 were investigated by detecting exogenous genes by polymerase chain reaction (PCR), measuring the diameter at breast height (DBH) and volume growth, and performing insect-resistance tests against Clostera anachoreta and Hyphantria cunea. The inheritance and expression of the exogenous gene was also examined in transgenic hybrid progeny lines. The results revealed that the exogenous gene was stable, remaining stable in 8–10-year-old transgenic poplar 741 trees. No significant difference was found between the height of 10-year-old transgenic poplar 741 and non-transgenic poplar 741 in the experimental plantations in Baoding, China. The DBH and volume growth of pb17 was significantly greater than that of pb29 and pb11. The 8-year-old transgenic poplar 741 pb29 grown in Zhuozhou showed no significant difference from poplar 741 in terms of height growth, DBH, and volume. From 1999 to 2013, pb29-fed larvae (C. anachoreta larvae and H. cunea) exhibited stable mortality rates >79%. Likewise, pb11-fed larvae showed stable mortality rates (C. anachoreta larvae had mortality rates >75%, and H. cunea larvae exhibited rates >80%). pb17 conferred low insect-resistant stability, showing mortality rates that varied from 28.2 to 99.27% in C. anachoreta and H. cunea larvae. Among the hybrid progeny lines acquired by hybridization of pb1, pb29, and pb11 with 84 K poplar, the ratios of PCR-positive to PCR-negative lines for the BtCry1Ac gene were 1.31, 1.15, and 0.86, respectively. X 2 tests showed that the ratio was consistent with the Mendelian law of 1:1 segregation controlled by an allele pair. The hybrid progeny of pb6 × 84 K had a segregation ratio of 3:1. The nptII gene followed the same segregation rule as Cry1Ac. The transgenic hybrid progeny that contained Cry1Ac gene exhibited the same insect resistance as the parent plants.

Heritable genetic transformation of Strongyloides stercoralis by microinjection of plasmid DNA constructs into the male germline

Heretofore, transgenesis in the parasitic nematode genus Strongyloides has relied on microinjecting transgene constructs into gonadal syncytia of free-living females. We now report transgenesis in Strongyloides stercoralis by microinjecting constructs into the syncytial testes of free-living males. Crosses of individual males microinjected with a construct encoding GFP with cohorts of 12 non-injected females produced a mean of 7.28±2.09 transgenic progeny. Progeny of males and females microinjected with distinct reporter constructs comprised 2.6%±0.7% of individuals expressing both paternal and maternal transgenes. Implications of this finding for deployment of CRISPR/Cas9 mutagenesis in Strongyloides spp. are discussed.

Pollen-mediated gene flow from transgenic to non-transgenic switchgrass (Panicum virgatum L.) in the field

BackgroundSwitchgrass is C4 perennial grass species that is being developed as a cellulosic bioenergy feedstock. It is wind-pollinated and considered to be an obligate outcrosser. Genetic engineering has been used to alter cell walls for more facile bioprocessing and biofuel yield. Gene flow from transgenic cultivars would likely be of regulatory concern. In this study we investigated pollen-mediated gene flow from transgenic to nontransgenic switchgrass in a 3-year field experiment performed in Oliver Springs, Tennessee, U.S.A. using a modified Nelder wheel design. The planted area (0.6 ha) contained sexually compatible pollen source and pollen receptor switchgrass plants. One hundred clonal switchgrass ‘Alamo’ plants transgenic for an orange-fluorescent protein (OFP) and hygromycin resistance were used as the pollen source; whole plants, including pollen, were orange-fluorescent. To assess pollen movement, pollen traps were placed at 10 m intervals from the pollen-source plot in the four cardinal directions extending to 20 m, 30 m, 30 m, and 100 m to the north, south, west, and east, respectively. To assess pollination rates, nontransgenic ‘Alamo 2’ switchgrass clones were planted in pairs adjacent to pollen traps.ResultsIn the eastward direction there was a 98% decrease in OFP pollen grains from 10 to 100 m from the pollen-source plot (Poisson regression, F1,8 = 288.38, P < 0.0001). At the end of the second and third year, 1,820 F1 seeds were collected from pollen recipient-plots of which 962 (52.9%) germinated and analyzed for their transgenic status. Transgenic progeny production detected in each pollen-recipient plot decreased with increased distance from the edge of the transgenic plot (Poisson regression, F1,15 = 12.98, P < 0.003). The frequency of transgenic progeny detected in the eastward plots (the direction of the prevailing wind) ranged from 79.2% at 10 m to 9.3% at 100 m.ConclusionsIn these experiments we found transgenic pollen movement and hybridization rates to be inversely associated with distance. However, these data suggest pollen-mediated gene flow is likely to occur up to, at least, 100 m. This study gives baseline data useful to determine isolation distances and other management practices should transgenic switchgrass be grown commercially in relevant environments.

Diabetic phenotype of transgenic pigs introduced by dominant-negative mutant hepatocyte nuclear factor 1α

AimThe present study aimed to identify the characteristics of genetically modified pigs carrying a mutant human gene as a research model for diabetes and its complications. MethodsWe developed a transgenic cloned pig (founder, male) carrying a mutant gene, i.e., human HNF-1α (P291fsinsC), which is responsible for maturity-onset diabetes of the young type 3. Transgenic progeny obtained via the artificial insemination of wild type (WT) sows with the cryopreserved sperm derived from the founder pig was pathologically examined. ResultsThe transgenic progeny maintained a high blood glucose level (>200mg/dL). Additionally, the oral glucose tolerance test results showed that the recovery of blood glucose levels in the transgenic progeny was significantly delayed compared with that in the WT semi-siblings. Hypoplasia of the islets of Langerhans was confirmed by the histopathological image of the pancreas, based on the hyperglycemia noted in the progeny being ascribed to decreased insulin secretion. Retinal hemorrhage and cotton-wool spots, i.e., findings consistent with non-proliferative diabetic retinopathy, were detected, and these progressed over time. The histopathological image of the renal glomeruli showed a nodular lesion that is characteristic of diabetic nephropathy in humans. ConclusionsThese data demonstrated that the genetically modified pig that we developed is a promising model for research on diabetes and its complications.

Agrobacterium-Mediated Transformation of Wheat Using Immature Embryos.

Methods of the Agrobacterium-mediated transformation of bread wheat (Triticum aestivum L.) have been improved in recent years so that genetic engineering can be routinely used for functional genomics as well as for wheat breeding. In the protocol described here, immature embryos of the spring-type model genotype Bobwhite SH 98 26 have been used. Preculture and temperature pretreatment of these explants have led to the reproducible generation of transgenic plants at efficiencies between 5 and 15%. Whereas primary transgenic plants regenerated in vitro commonly show reduced fitness and fertility, no apparent variations with regard to morphology and grain set in their transgenic progeny as compared to wild-type counterparts were observed.

Caenorhabditis elegans Microinjection.

Microinjection is the most frequently used tool for genetic transformation of the nematode Caenorhabditis elegans, facilitating the transgenic expression of genes, genome editing by the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system, or transcription of dsRNA for RNA intereference (RNAi). Exogenous DNA is delivered into the developing oocytes in the germline of adult hermaphrodites, which then generate transgenic animals among their offspring. In this protocol, we describe the microinjection procedure and the subsequent selection of transgenic progeny.

Expression of a rice GLP in Medicago truncatula exerting pleiotropic effects on resistance against Fusarium oxysporum through enhancing FeSOD-like activity

To evaluate the effectiveness of a germin-like protein (GLP) in legumes against the serious soil-borne pathogen Fusarium oxysporum f. sp. lentis, an Oryza sativa root-expressed GLP (OsRGLP1) was expressed in the model legume Medicago truncatula using the recombinant vector pCOsRGLP1. The transgene was highly expressed in M. truncatula transformed lines as assessed by RT-qPCR. Consistent with the active status of the transgene there was an elevated accumulation of H2O2 in transformed progeny. Enzymatic characterization of T1 transgenic progeny showed increased superoxide dismutase (SOD) activity. The additional SOD activity in transgenic lines was insensitive to potassium cyanide and sensitive to H2O2 indicating its resemblance to FeSOD. The effectiveness of the OsRGLP1 gene was tested by monitoring the root disease after infection of wild-type and transgenic lines. Wild-type plants were greatly affected by the pathogen infection showing a percent disease index value of 50 compared to 10–18 for the transgenic lines. The tolerance of the transgenic lines leads to recovery in fresh weight and pod production to an almost normal level. Analysis of defense-related genes downstream of hydrogen peroxide (H2O2) in transgenic plants showed induction of salicylic acid and jasmonate signaling pathways and increased expression of some pathogenesis-related-1 (PR-1) genes and a plant defensin gene. Overall, the findings suggest that OsRGLP1 provides protection against the fungal pathogen F. oxysporum that may involve the direct influence of H2O2 on signaling pathways leading to the activation of defense-related genes.

Association of Increased Grain Iron and Zinc Concentrations with Agro-morphological Traits of Biofortified Rice.

Biofortification of rice (Oryza sativa L.) with micronutrients is widely recognized as a sustainable strategy to alleviate human iron (Fe) and zinc (Zn) deficiencies in developing countries where rice is the staple food. Constitutive overexpression of the rice nicotianamine synthase (OsNAS) genes has been successfully implemented to increase Fe and Zn concentrations in unpolished and polished rice grain. Intensive research is now needed to couple this high-micronutrient trait with high grain yields. We investigated associations of increased grain Fe and Zn concentrations with agro-morphological traits of backcross twice second filial (BC2F2) transgenic progeny carrying OsNAS1 or OsNAS2 overexpression constructs under indica/japonica and japonica/japonica genetic backgrounds. Thirteen agro-morphological traits were evaluated in BC2F2 transgenic progeny grown under hydroponic conditions. Concentrations of eight mineral nutrients (Fe, Zn, copper, manganese, calcium, magnesium, potassium, and phosphorus) in roots, stems/sheaths, non-flag leaves, flag leaves, panicles, and grain were also determined. A distance-based linear model (DistLM) was utilized to extract plant tissue nutrient predictors accounting for the largest variation in agro-morphological traits differing between transgenic and non-transgenic progeny. Overall, the BC2F2 transgenic progeny contained up to 148% higher Fe and 336% higher Zn concentrations in unpolished grain compared to non-transgenic progeny. However, unpolished grain concentrations surpassing 23 μg Fe g-1 and 40 μg Zn g-1 in BC2F2 indica/japonica progeny, and 36 μg Fe g-1 and 56 μg Zn g1 in BC2F2 japonica/japonica progeny, were associated with significant reductions in grain yield. DistLM analyses identified grain-Zn and panicle-magnesium as the primary nutrient predictors associated with grain yield reductions in the indica/japonica and japonica/japonica background, respectively. We subsequently produced polished grain from high-yield BC2F2 transgenic progeny carrying either the OsNAS1 or OsNAS2 overexpression constructs. The OsNAS2 overexpressing progeny had higher percentages of Fe and Zn in polished rice grain compared to the OsNAS1 overexpressing progeny. Results from this study demonstrate that genetic background has a major effect on the development of Fe and Zn biofortified rice. Moreover, our study shows that high-yielding rice lines with Fe and Zn biofortified polished grain can be developed by OsNAS2 overexpression and monitoring for Zn overaccumulation in the grain.