Non-transformed Control Plants Research Articles

Cytokinin deficiency confers enhanced tolerance to mild, but decreased tolerance to severe salinity stress in in vitro grown potato.

Cytokinin (CK) is a plant hormone that plays crucial roles in regulating plant growth and development. CK-deficient plants are widely used as model systems for investigating the numerous physiological roles of CK. Since it was previously shown that transgenic or mutant CK-deficient Arabidopsis and Centaurium plants show superior tolerance to salinity, we examined the tolerance of three CK-deficient potato lines overexpressing the Arabidopsis thaliana CYTOKININ OXIDASE/DEHYDROGENASE2 (AtCKX2) gene to 50 mM, 100 mM, 150 mM, and 200 mM NaCl applied in vitro. Quantification of visible salinity injury, rooting and acclimatization efficiency, shoot growth, water saturation deficit, and chlorophyll content confirmed that the CK-deficient potato plants were more tolerant to low (50 mM) and moderate (100 mM) NaCl concentrations, but exhibited increased sensitivity to severe salinity stress (150 and 200 mM NaCl) compared to non-transformed control plants. These findings were corroborated by the data distribution patterns according to principal component analysis. Quantification of the activity of superoxide dismutases, peroxidases, and catalases revealed an impaired ability of AtCKX2-transgenic lines to upregulate the activity of antioxidant enzymes in response to salinity, which might contribute to the enhanced sensitivity of these potato lines to severe salt stress. Our results add complexity to the existing knowledge on the regulation of salinity tolerance by CK, as we show for the first time that CK-deficient plants can exhibit reduced rather than increased tolerance to severe salt stress.

CRISPR/Cas9 editing of Downy mildew resistant 6 (DMR6-1) in grapevine leads to reduced susceptibility to Plasmopara viticola.

Downy mildew of grapevine (Vitis vinifera), caused by the oomycete Plasmopara viticola, is an important disease that is present in cultivation areas worldwide, and using resistant varieties provides an environmentally friendly alternative to fungicides. DOWNY MILDEW RESISTANT 6 (DMR6) from Arabidopsis is a negative regulator of plant immunity and its loss of function confers resistance to downy mildew. In grapevine, DMR6 is present in two copies, named VvDMR6-1 and VvDMR6-2. Here, we describe the editing of VvDMR6-1 in embryogenic calli using CRISPR/Cas9 and the regeneration of the edited plants. All edited plants were found to be biallelic and chimeric, and whilst they all showed reduced growth compared with non-transformed control plants, they also had reduced susceptibility to P. viticola. Comparison between mock-inoculated genotypes showed that all edited lines presented higher levels of salicylic acid than controls, and lines subjected to transformation presented higher levels of cis-resveratrol than controls. Our results identify VvDMR6-1 as a promising target for breeding grapevine cultivars with improved resistance to downy mildew.

Overexpression of banana GDP-L-galactose phosphorylase (GGP) modulates the biosynthesis of ascorbic acid in Arabidopsis thaliana

l-Ascorbic acid (AsA) is a potent antioxidant and essential micronutrient for the growth and development of plants and animals. AsA is predominantly synthesized by the Smirnoff-Wheeler (SW) pathway in plants where the GDP-L-galactose phosphorylase (GGP) gene encodes the rate-limiting step. In the present study, AsA was estimated in twelve banana cultivars, where Nendran carried the highest (17.2 mg/100 g) amount of AsA in ripe fruit pulp. Five GGP genes were identified from the banana genome database, and they were located at chromosome 6 (4 MaGGPs) and chromosome 10 (1 MaGGP). Based on in-silico analysis, three potential MaGGP genes were isolated from the cultivar Nendran and subsequently overexpressed in Arabidopsis thaliana. Significant enhancement in AsA (1.52 to 2.20 fold) level was noted in the leaves of all three MaGGPs overexpressing lines as compared to non-transformed control plants. Among all, MaGGP2 emerged as a potential candidate for AsA biofortification in plants. Further, the complementation assay of Arabidopsis thaliana vtc-5-1 and vtc-5-2 mutants with MaGGP genes overcome the AsA deficiency that showed improved plant growth as compared to non-transformed control plants. This study lends strong affirmation towards development of AsA biofortified plants, particularly the staples that sustain the personages in developing countries.

Extending Omega 3/6 Fatty Acid Pathway in Arabidopsis thaliana using Microalgal Gene Δ6Des for Stearidonic acid and Dihomo-γ-linolenic acid Production

• Microalgal gene Δ6Des was cloned in a binary vector pCAMBIA1303. • F 1 transgenic plants having Δ6Des were viable at a concentration of 25 mg/L of hygromycin medium • Transgenic Arabidopsis thaliana showed novel ω3/6 fatty acids SDA (0.58%) and DGLA (25. 34%) To enhance the quality and quantity of oils in plant systems, specific candidate genes such as desaturase and elongase that can extend the biosynthetic pathways of very long chain poly unsaturated fatty acids (VLCPUFAs) are essential. In general, plant systems are capable of synthesizing essential fatty acids (EFAs). However, incorporation of a preliminary gene, Δ6Desaturase ( Δ6Des ), in the fatty acid biosynthetic pathway will elongate the pathway further to produce successive fatty acids. In this study, Δ6Des gene extracted from the marine microalga Isochrysis sp. was used to extend the omega (ω) 3/6 fatty acid biosynthetic pathway in a model plant Arabidopsis thaliana using Agrobacterium -mediated genetic transformation. Hygromycin-resistant plants were (25 mg/L) used for GUS and GFP assays and for molecular confirmation. GUS assay showed an intense blue color in F 1 transgenic A. thaliana plant tissues grown in the selection medium, which confirmed the presence of GUS gene in the T-DNA region, whereas non-transformed plant tissues such as leaf or stem did not show any blue color. GFP analysis also showed a green fluorescent color in tissues such as leaf, stem, and floral parts of F1 transgenic A. thaliana plants, which confirmed the presence of T-DNA. PCR and Southern blotting analyses also clearly showed the presence of Δ6Des in the T-DNA region of the F 1 transgenic plants. The fatty acid profile of F 1 transgenic plants showed the production of new fatty acids such as stearidonic acid (SDA-0.58%) and dihomo-γ-linolenic acid (DGLA-25.34%) that were not observed in non-transformed control plants. All these results showed the synthesis of new fatty acids in the transgenic plant, which proved that PUFA biosynthetic pathway can be extended in oilseed plants if specific genes are introduced through genetic transformation. This is the first study to report the productions of new fatty acids like SDA and DGLA in A. thaliana using Δ6Des gene from marine microalga Isochrysis sp.

Knockout of Tobacco Homologs of Arabidopsis Multi-Antibiotic Resistance 1 Gene Confers a Limited Resistance to Aminoglycoside Antibiotics.

To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the Arabidopsis Multi-Antibiotic Resistance 1 (MAR1)/RTS3 gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient Agrobacterium-mediated gene transfer. A Cas9 gene was introduced into Nicotiana tabacum and Nicotiana sylvestris together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of N. tabacum MAR1 homologs (NtMAR1hs). All three sgRNAs directed the introduction of InDels into NtMAR1hs, as demonstrated by CAPS and amplicon sequencing analyses, albeit with varying efficiency. Leaves of regenerated transformant shoots were evaluated for aminoglycoside resistance on shoot-induction media containing different aminoglycoside antibiotics. All transformants tested were as sensitive to those antibiotics as non-transformed control plants, regardless of the mutation rates in NtMAR1hs. The NtMAR1hs–knockout seedlings of the T1 generation showed limited aminoglycoside resistance but failed to form shoots when cultured on shoot-induction media containing kanamycin. The results suggest that, like Arabidopsis MAR1, NtMAR1hs have a role in plants’ sensitivity to aminoglycoside antibiotics, and that tobacco has some additional functional homologs.

Overexpression of sugarcane SoTUA gene enhances cold tolerance in transgenic sugarcane

AbstractAlpha (α)–tubulin (TUA) is a primary unit of cytoskeleton microtubules. In recent years, more frequent frosts have caused considerable yield loss in sugarcane (Saccharum spp. hybrid) production. Temperature is one of the most influential abiotic factors affecting the microtubule polymerization state in plants. In this research, we developed overexpressing SoTUA transgenic sugarcane lines from the cold‐susceptible cultivar ROC22. The transgene was confirmed by polymerase chain reaction (PCR) and quantificational real‐time polymerase chain reaction (qRT–PCR) analysis, while the relative expression of SoTUA protein was verified by Western blot. Compared with the nontransformed control plants (wild type [WT]), the transgenic lines showed higher relative protein expression than the WT at 25 °C, which indicates the overexpression of SoTUA protein. The contents of soluble protein and sugar and the activity of peroxidase (POD) were higher while the content of maleicdialdehyde was lower in the transgenic plants than the WT plants under the chilling treatment. Meanwhile, the expression levels of cold‐defense related genes (P5CS, WRKY, Cu/Zn–SOD, and POD1) were higher in the transgenic lines than in the WT. These results suggest that the SoTUA protein plays an important role in protecting plants during chilling stress. This positive effect is not affected by transgenic plant generations.

Morphophysiological alterations in transgenic rice lines expressing PPDK and ME genes from the C4 model Setaria italica

C4 plants are superior to C3 plants in terms of productivity and limited photorespiration. PPDK (pyruvate orthophosphate dikinase) and NADP-ME (NADP-dependent malic enzyme) are two important photosynthetic C4-specific enzymes present in the mesophyll cells of C4 plants. To evaluate the effect of C4 enzymes in rice, we developed transgenic rice lines by separately introducing Setaria italica PPDK [SiPPDK] and S. italica ME [SiME] gene constructs under the control of the green tissue-specific maize PPDK promoter. Rice plant lines for both constructs were screened using the polymerase chain reaction (PCR), Southern hybridization, and expression analysis. The best transgenic plant lines for each case were selected for physiological and biochemical characterization. The results from qRT-PCR and enzyme activity analysis revealed higher expression and activity of both PPDK and NADP-ME genes compared with the nontransformed and empty-vector-transformed plants. The average photosynthetic efficiency of transgenic plant lines carrying the PPDK and NADP-ME genes increased by 18% and 12%, respectively, and was positively correlated with the increased accumulation of photosynthetic pigment. The decrease in Fv/Fm, increased electron transport rate (ETR), and increased photochemical quenching (qP) compared with nontransformed control plants suggest that transgenic rice plants transferred more absorbed light energy to photochemical reactions than wild-type plants. SiME-transgenic plants displayed reduced leaf malate content and superior performance under water deficit conditions. Interestingly, the transgenic plants showed yield enhancement by exhibiting increased plant height, panicle length, panicle weight and thousand grain weight. Overall, the exogenous foxtail millet C4 gene PPDK enhanced photosynthesis and yield to a greater extent than NADP-ME.

Engineering tolerance to CLCuD in transgenic Gossypium hirsutum cv. HS6 expressing Cotton leaf curl Multan virus-C4 intron hairpin

Cotton leaf curl disease (CLCuD), caused by begomoviruses in combination with betasatellite molecule, has adversely affected cotton industry of Indian subcontinent. To devise a CLCuD-control strategy, RNAi-mediated approach was followed in this study. Gossypium hirsutum cv. HS6 plants were transformed with intron-hairpin RNAi (ihpRNAi-C4) construct carrying silencing suppressor C4 gene of Cotton leaf curl Multan virus (CLCuMuV). Efficacy of the construct in imparting CLCuD resistance was evaluated in transgenic (T0, T1) cotton lines. Accumulation of CLCuMuV/betasatellite and attenuation of CLCuD symptoms in the transgenic lines were monitored at different times interval after virus inoculation. Northern hybridization revealed the expression of C4-gene derived siRNA. Expression of the ihpRNAi transcript was recorded higher in transgenic lines expressing siRNA which supposedly targeted the C4 gene. A significant delay in detection of virus as well as betasatellite was observed in the transgenic lines. At 30 days post inoculation (dpi), none of the lines tested positive. At 45 dpi, however, it could be detected in few lines having much lower titre as compared to non-transformed control plants. Notably, till 60 dpi, no significant progression of the virus/betasatellite DNA was observed and the plants did not exhibit any characteristic CLCuD symptoms. A tolerance phenomenon leading to escape of CLCuD symptoms in the transformed cotton was described.

Transgenic expression of Pinellia ternata agglutinin (PTA) and Arisaema heterophyllum agglutinin (AHA) in wheat confers resistance against the grain aphid, Sitobion miscanthi

The present study investigated the feeding behaviour and life-table parameters of the grain aphid Sitobion miscanthi in response to being fed on transgenic wheat lines expressing Pinellia ternata agglutinin (PTA) and Arisaema heterophyllum agglutinin (AHA), both of which originate from Chinese medicinal plants. The findings revealed that the feeding behaviour of S. miscanthi on transgenic wheat lines was negatively affected. The aphids that feed on the PTA and AHA transgenic wheat lines had longer total non-probing (np) periods than the aphids that feed on non-transformed lines but exhibited shorter durations of salivation (E1), phloem sap ingestion phase (E2) and sustained ingestion (E2 > 10 min). Moreover, aphids feeding on the 171 line, which expresses PTA, displayed a significant increase in the number of np patterns and time taken to initiate the first probe. Furthermore, the lifespan of S. miscanthi feeding on any of the wheat lines expressing PTA and AHA was found to reduce significantly. The maximum fecundity of the aphids on the AHA-expressing wheat lines was significantly lower than that of the control group. The net reproductive rate (R0) and intrinsic rate of increase (rm) were also significantly lower for the aphids feeding on the PTA and AHA transgenic wheat than for the aphids feeding on the non-transformed control plants. These findings indicate successful detection of resistance to S. miscanthi in the PTA- and AHA-expressing wheat lines, providing a new option for engineering protection against crop pests by expressing unique proteins that occur naturally in medicinal plants.

Introduction of the Nicotiana protein kinase (NPK1) gene by combining Agrobacterium-mediated transformation and recurrent somatic embryogenesis to enhance salt tolerance in cauliflower

Cauliflower is exposed to various biotic and abiotic stresses, including increased salinity due to the intensive irrigation of crops. Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules that play important roles in regulating innate immune responses in plants. Based on involvement of tobacco MAP kinase kinase kinase (NPK1) in stress response, the effect of the expression of NPK1 transgene to NaCl salt stress tolerance in cauliflower KFRM4 lines was studied. The Agrobacterium tumefaciens-mediated transformation protocol, using EHA101(pSHX004) vector harbouring the NPK1 and phosphinothricin N-acetyltransferase (bar) genes, the cyclic somatic embryogenesis regeneration pathway, the application of acetosyringone (AS) during co-cultivation and a delayed phosphinothricine (PPT) selection procedure provided sufficient transformation efficiency of 7.33% without escapes. PCR analysis indicated the integration of both NPK1 and bar transgenes in regenerated cauliflower lines. Transgenic cauliflower lines, exposed to NaCl stress in vitro, showed higher growth rates, greater ability to retain chlorophyll and carotenoids, and increased osmotic regulation capacity compared with non-transformed control plants. The tolerance level of transformed lines correlated with the level of NPK1 gene expression estimated by RT-qPCR, and the L2 line with the highest NPK1 expression displayed the greatest tolerance to NaCl stress. None of the obtained cauliflower transformed lines grown in greenhouses showed any morphological or yield differences compared with non-transformed plants. Furthermore, the expression of the bar gene facilitated the tolerance of transformed lines to the total herbicide PPT, applied at concentrations 2–3 times higher than those routinely used for weed control in the crop field. The results underlined that constitutively expressing NPK1 can significantly contribute to enhanced salt stress tolerance in cauliflower, suggesting that this could be a promising basis for the creation of new stress tolerance cruciferous vegetable lines.

Introduction of the harpinXooc-encoding gene hrf2 in soybean enhances resistance against the oomycete pathogen Phytophthora sojae.

Phytophthora root and stem rot (PRR) caused by an oomycete pathogen Phytophthora sojae is one of the most devastating and widespread diseases throughout soybean-producing regions worldwide. The diversity and variability of P. sojae races make effective control of the pathogen challenging. Here, we introduced an elicitor of plant defense response, the harpinXooc-encoding hrf2 gene from the rice bacterial pathogen Xanthomonas oryzae pv. oryzicola into soybean and evaluated resistance to P. sojae infection. Molecular analysis confirmed the integration and expression of hrf2 in the transgenic soybean. After inoculation with P. sojae, non-transformed control (NC) plants exhibited typical PRR symptoms, including necrotic and wilting leaves, and plant death, whereas most of the transgenic plants showed slightly chlorotic leaves and developed normally. Through T3 to T5 generations, the transgenic events displayed milder disease symptoms and had higher survival rates compared to NC plants, indicating enhanced and stable resistance to P. sojae infection, whereas without P. sojae inoculation, no significant differences in agronomic traits were observed between the transgenic and non-transformed plants. Moreover, after inoculation with P. sojae, significant upregulation of a set of plant defense-related genes, including salicylic acid- and jasmonic acid-dependent and hypersensitive response-related genes was observed in the transgenic plants. Our results indicate that hrf2 expression in transgenic soybean significantly enhanced resistance to P. sojae by eliciting multiple defense responses mediated by different signaling pathways. The potential functional role of the hrf2 gene in plant defense against P. sojae and other pathogens makes it a promising tool for broadening disease resistance in soybean.

Enhanced resistance to sclerotinia stem rot in transgenic soybean that overexpresses a wheat oxalate oxidase.

Sclerotinia stem rot (SSR), caused by the oxalate-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, is one of the devastating diseases that causes significant yield loss in soybean (Glycine max). Until now, effective control of the pathogen is greatly limited by a lack of strong resistance in available commercial soybean cultivars. In this study, transgenic soybean plants overexpressing an oxalic acid (OA)-degrading oxalate oxidase gene OXO from wheat were generated and evaluated for their resistance to S. sclerotiorum. Integration and expression of the transgene were confirmed by Southern and western blot analyses. As compared with non-transformed (NT) control plants, the transgenic lines with increased oxalate oxidase activity displayed significantly reduced lesion sizes, i.e., by 58.71-82.73% reduction of lesion length in a detached stem assay (T3 and T4 generations) and 76.67-82.0% reduction of lesion area in a detached leaf assay (T4 generation). The transgenic plants also showed increased tolerance to the externally applied OA (60mM) relative to the NT controls. Consecutive resistance evaluation further confirmed an enhanced and stable resistance to S. sclerotiorum in the T3 and T4 transgenic lines. Similarly, decreased OA content and increased hydrogen peroxide (H2O2) levels were also observed in the transgenic leaves after S. sclerotiorum inoculation. Quantitative real-time polymerase chain reaction analysis revealed that the expression level of OXO reached a peak at 1h and 4h after inoculation with S. sclerotiorum. In parallel, a significant up-regulation of the hypersensitive response-related genes GmNPR1-1, GmNPR1-2, GmSGT1, and GmRAR occurred, eventually induced by increased release of H2O2 at the infection sites. Interestingly, other defense-related genes such as salicylic acid-dependent genes (GmPR1, GmPR2, GmPR3, GmPR5, GmPR12 and GmPAL), and ethylene/jasmonic acid-dependent genes (GmAOS, GmPPO) also exhibited higher expression levels in the transgenic plants than in the NT controls. Our results demonstrated that overexpression of OXO enhances SSR resistance by degrading OA secreted by S. sclerotiorum and increasing H2O2 levels, and eliciting defense responses mediated by multiple signaling pathways.

RNAi-mediated silencing of endogenous Vlnv gene confers stable reduction of cold-induced sweetening in potato (Solanum tuberosum L. cv. Désirée)

Potato tubers must be cold-stored to extend their shelf life and maintain an uninterrupted supply chain for food processors. However, a side-effect of low-temperature storage is manifested in terms of cold-induced sweetening (CIS) of potato tubers, which reduces the processing quality and the commercial value of the end-products. RNA interference (RNAi) technology, whereby transgene-derived small interfering RNAs can trigger the homology-based knockdown of cognate host genes and can initiate gene silencing, has been successfully applied in crop improvement through targeted gene knockout in host plants. In the current study, transgenic potato plants (Solanum tuberosum cv. Desiree) were generated, expressing a 300 bp hairpin loop nucleotide sequence targeting the potato vacuolar invertase gene (VInv), under the constitutive Cauliflower mosaic virus 35S promoter. Tubers collected from transgenic lines showed a significant reduction in reducing sugar content after 180 days of cold storage, without showing any measurable off-target effects on plant morphology and tuberization compared to non-transformed control plants. The cold-stored tubers were further assayed for chip color, which showed a fairly light colored quality in the samples originating from RNAi lines. Together with similar effects seen in previously published experiments involving other potato varieties, the Desiree results described here establish the efficacy of using RNAi for the successful reduction of CIS in potato tubers.

Development and Characterization of Transgenic Sugarcane with Insect Resistance and Herbicide Tolerance.

Genetically modified crops which had been commercial applied extensively majorly are the insect resistance and herbicide tolerance events. In this study, the Bt insecticidal gene Cry1Ab, the glyphosate-tolerant gene EPSPS, and the selection marker gene PMI were combined into a single transferred DNA fragment and introduced into sugarcane by Agrobacterium-mediated transformation. Thirty-three resistant plantlets were obtained after selection using a PMI/mannose selection system. Thirty of these resistant plantlets were PCR positive for the three target genes. Southern blot assay revealed that the copy number of the integrated fragment in the transformed plantlets varied from 1 to 7. ELISA analysis showed that 23 of the 33 resistant plantlets expressed Cry1Ab and EPSPS protein. Five single-copy and ELISA-positive transgenic lines were tested under laboratory and field conditions to determine their resistance to insects and herbicides, and also evaluated their agronomic characteristics and industrial traits. Results showed that larvae fed with fodder mixture containing stem tissues from single-copy transgenic lines were weak and small, moreover, pupation and eclosion were delayed significantly during voluntary feeding bioassays. None of transgenic sugarcane was destroyed by cane borer while more than 30% of wild type sugarcane was destroyed by cane borer. For herbicide resistance, the transgenic plantlets grew healthy even when treated with up to 0.5% roundup while wild type plantlets would die off when treated with 0.1% roundup. Thus demonstrate that these transgenic lines showed strong insect resistance and glyphosate tolerance under both laboratory and field conditions. But in the field most of the transgenic plants were shorter and more slender than non-transformed control plants. So they presented poor agronomic characteristics and industrial traits than non-transformed control plants. Thus, a considerable number of embryogenic calli should be infected to obtain transgenic lines with potential for commercial use.

Robust RNAi-mediated resistance to infection of seven potyvirids in soybean expressing an intron hairpin NIb RNA.

Viral pathogens, such as soybean mosaic virus (SMV), are a major constraint in soybean production and often cause significant yield loss and quality deterioration. Engineering resistance by RNAi-mediated gene silencing is a powerful strategy for controlling viral diseases. In this study, a 248-bp inverted repeat of the replicase (nuclear inclusion b, NIb) gene was isolated from the SMV SC3 strain, driven by the leaf-specific rbcS2 promoter from Phaseolus vulgaris, and introduced into soybean. The transgenic lines had significantly lower average disease indices (ranging from 2.14 to 12.35) than did the non-transformed (NT) control plants in three consecutive generations, exhibiting a stable and significantly enhanced resistance to the SMV SC3 strain under field conditions. Furthermore, seed mottling did not occur in transgenic seeds, whereas the NT plants produced ~90% mottled seeds. Virus resistance spectrum screening showed that the greenhouse-grown transgenic lines exhibited robust resistance to five SMV strains (SC3, SC7, SC15, SC18, and a recombinant SMV), bean common mosaic virus, and watermelon mosaic virus. Nevertheless, no significantly enhanced resistance to bean pod mottle virus (BPMV, Comovirus) was observed in the transgenic lines relative to their NT counterparts. Consistent with the results of resistance evaluation, the accumulation of each potyvirid (but not of BPMV) was significantly inhibited in the transgenic plants relative to the NT controls as confirmed by quantitative real-time (qRT-PCR) and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). These results demonstrate that robust RNAi-mediated resistance to multiple potyvirids in soybean was conferred by expressing an intron hairpin SMV NIb RNA.

Biochemical composition of symplastic sap from sugarcane genetically modified to overproduce proline

Global interest in sugarcane has increased significantly in recent years because of its economic impact on sustainable energy production. The purpose of the present study was to evaluate changes in the concentrations of total sugars, amino acids, free proline, and total proteins by colorimetric analyses and nuclear magnetic resonance (NMR) to perform a metabolic profiling of a water-soluble fraction of symplastic sap in response to the constitutive expression of a mutant Δ1-pyrroline-5-carboxylate synthetase (P5CS) gene from Vigna aconitifolia. However, there was not a significant increase in the free proline content in the sap of transgenic plants compared to the non-transformed control plants. The most noticeable difference between the two genotypes was an almost two-fold increase in the accumulation of sucrose in the stem internodes of P5CS transgenic sugarcane plants. The results presented in this work showed that transgenic sugarcane plants with increased levels of free proline accumulates high soluble sugar content and, therefore, may represent a novel genotype for improving sugarcane cultivars.

Inducible expression of Bs2 R gene from Capsicum chacoense in sweet orange (Citrus sinensis L. Osbeck) confers enhanced resistance to citrus canker disease.

Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.

Metabolic engineering of apple by overexpression of the MdMyb10 gene.

Flavonoids are low-molecular-weight phenolic compounds that are widely distributed in the plant kingdom. They have different roles in plant resistance to biotic and abiotic stresses.The transcription factor gene MdMyb10 (Gene Bank: DQ267896) was introduced into two apple (Malus domestica Borkh.) cultivars i.e. ‘Holsteiner Cox (HC)’ and ‘Gala’ via Agrobacterium-mediated transformation. The regenerated shoots were selected on kanamycin containing media. The presence of additional MdMyb10 gene in putative shoots was confirmed by PCR, RT-PCR and Southern blotting. Expression level of introduced MdMyb10 gene was analyzed by quantitative real time PCR. The results confirmed a dramatic increase in overexpression of MdMyb10 in the transgenic plants, up to 1261 and 847-folds for cultivars Holsteiner Cox and Gala, respectively compared to non-transformed negative control plants.HPLC-MS was used to determine the levels of different flavonoid compounds in both non-transgenic and transgenic plants. In MdMyb10 ‘HC’ transgenic plants, some of the polyphenols analyzed were enhanced while others were reduced in comparison to their levels in the non-transgenic plants. On the other hand, all of the analyzed polyphenol classes were induced in MdMyb10 ‘Gala’ transgenic plants in comparison to their levels in the non-transgenic plants.In the present study, the flavonoid pathway was successfully modified in apple by overexpressing the MdMyb10 transcription factor to validate the hypothesis of increased effect on plant disease resistance.

Evaluation of a grapevine-derived reporter gene system for precision breeding of Vitis

A grapevine-derived VvMybA1 transcription factor was evaluated for its efficiency as a reporter gene by comparing it with existing reporter genes, the green fluorescent protein (GFP) and β-glucuronidase (GUS). Embryogenic cultures of Vitis cultivars Thompson Seedless and Bronx Seedless were initiated from leaves of in vitro grown micropropagation cultures and somatic embryos were used as explants for Agrobacterium-mediated transformation. Transient and stable expression of the MybA1 gene was characterized by intense red pigmentation in co-cultivated explants, callus tissues and secondary embryos lines compared to GFP and GUS that exhibited green fluorescence and blue coloration following a substrate assay. No differences were observed in transient gene expression frequencies between the MybA1 gene and GUS among the two compared reporter genes. Visual levels of stable gene expression were higher in GFP and GUS expressing cultures compared to MybA1 expressing cultures. The presence of the inserted genes and their expression in regenerated plant lines was confirmed by PCR and RT-PCR. Embryo and plant lines expressing the MybA1 gene accumulated varying levels of anthocyanin pigment in plant tissues and organs, and were characterized by slower growth compared to plant lines expressing GFP and GUS. Scanning electron microscopy analyses revealed a significant change in abaxial and adaxial leaf epidermal cells of MybA1-expressing plant lines compared to those expressing GFP and GUS, and non-transformed control plants. The study demonstrated the utility of the VvMybA1 transcription factor as a reliable reporter gene for identification of gene insertion events in cell culture and regeneration of modified plants.

Transgenic Sugar Beet Expressing a Bacterial Mannitol-1-Phosphate Dehydrogenase (mtlD) Gene Shows Enhanced Resistance to Fungal Pathogens

The E. coli mtlD gene encoding mannitol-1-phosphate dehydrogenase has been shown to be involved in the plant response to abiotic stresses such as salinity, drought and chilling. Nevertheless, in this study transgenic sugar beet lines containing the mtlD gene under control of a stress-inducible rd29A promoter were evaluated for their potential of resistance against three fungi including Alternaria alternata, Botrytis cinerea and Cercospora beticola. Southern blot analysis revealed the low copy number (1-3 copies) integration of the transgene in the transgenic lines. Expression profiling by semi-quantitative-RT-PCR analysis showed different levels of cold-inducible expression of mtlD in independent T1 transformants. Mannitol content quantified by HPLC analysis ranged from 1.8 to 3.7 µmol g−1 dry weight in different transgenic lines. Detached leaf bioassay showed that lower transgene expression levels in the transgenic line mt-LS4-32 under non-stressed conditions reduced the disease severity (DS) of A. alternata and B. cinerea by 49 to 87 %, respectively. At the whole-plant level, under non-stressed conditions, the transgenic line showed better performance to A. alternata with a delay of fungal symptom appearance. Following exposure to low temperature (4 °C), the transgenic line showed no fungal infection after 14 days of inoculation. Transgenic lines inoculated with C. beticola significantly (P < 0.001) showed higher resistance than the non-transformed control plants. DS in transgenic lines expressing mtlD gene was zero and 0.05 % compared to 25 % for the control. These results indicate that the mtlD gene can be used as a target to improve plant tolerance to both abiotic and biotic stresses.