Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation

<p>Abstract</p> <p>Background</p> <p>The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently.</p> <p>Results</p> <p>We show here that chimeras are a very frequent phenomenon observed after regenerating transgenic plants. Spatial and temporal analyses of transformed tobacco and apricot plants with a quantitative, real-time PCR amplification of the neomycin phosphotransferase (<it>npt</it>II) transgene as well as of an internal control (β-<it>actin</it>), used to normalise the amount of target DNA at each reaction, allowed detection of chimeras at unexpected rates. The amount of the <it>npt</it>II transgene differed greatly along with the sub-cultivation period of these plants and was dependent on the localisation of the analysed leaves; being higher in roots and basal leaves, while in the apical leaves it remained at lower levels. These data demonstrate that, unlike the use of the <it>gus </it>marker gene, real-time PCR is a powerful tool for detection of chimeras. Although some authors have proposed a consistent, positive Southern analysis as an alternative methodology for monitoring the dissociation of chimeras, our data show that it does not provide enough proof of uniform transformation. In this work, however, real-time PCR was applied successfully to monitor the dissociation of chimeras in tobacco plants and apricot callus.</p> <p>Conclusions</p> <p>We have developed a rapid and reliable method to detect and estimate the level of chimeras in transgenic tobacco and apricot plants. This method can be extended to monitor the dissociation of chimeras and the recovery of uniformly-transformed plants.</p

Cytosolic ascorbate peroxidase and Cu, Zn-superoxide dismutase improve seed germination, plant growth, nutrient uptake and drought tolerance in tobacco

The effects of over-expression of two cytosolic antioxidant enzymes (Cu, Zn-SOD and/or APX) on plant nutrition, gas exchange, chlorophyll fluorescence, seed viability and germination in transgenic tobacco (Nicotiana tabacum cv. Xanthi) under deficit irrigation or salinity conditions were investigated. Three transgenic lines of tobacco were used in this study: line 17, harboring 2 copies of the cytosolic CuZn-SOD (cytsod) gene; line 51, with 2 copies of the cytosolic APX (cytapx) gene and line 39, harboring one copy of each gene. Over-expression of cytosolic antioxidants enzymes in tobacco plants resulted in a better growth performance that correlated with an improved photosynthetic capacity and nutrient uptake. Moreover, cytsod or cytapx genes promoted seed germination, and enhanced tolerance to mild water stress. In addition, this enhanced antioxidant capacity protected seeds from ageing during prolonged storage, and stimulated germination under salt stress conditions. These results suggest that cytosolic antioxidant transgenes are useful tools to improve drought tolerance, nutrient uptake and seed germination under stressful conditions.PDV acknowledges the CSIC and the Spanish Ministry of Economy and Competitiveness for his ‘Ramon y Cajal’ research contract, co-financed by FEDER funds. This work was supported by the Spanish Ministry of Economy and Competitiveness (Project CICYT BFU2009-07443) co-financed by FEDER funds, and the Spanish Ministry of Economy and Competitiveness (Project INIA, RTA2013-00026-C03-00).Peer reviewe

Enhancement of plant growth, acclimatization, salt stress tolerance and verticillium wilt disease resistance using plant growth-promoting rhizobacteria (PGPR) associated with plum trees (Prunus domestica)

Plants interact with a great variety of microorganisms that inhabit the rhizosphere playing critical roles in several aspects of plant growth and protection against abiotic and biotic diseases. In this study, we performed a screening of bacteria associated with the rhizosphere of Prunus domestica trees to identify bacterial strains with plant growth-promoting activity. Ten strains isolated from the rhizosphere of P. domestica showed multiple in vitro plant growth promoting rhizobacteria (PGPR) activity such as the production of indole acetic acid, hydrogen cyanide, ammonia, solubilization of phosphates and antifungal activity against Verticillium dalhiae and Fusarium oxysporum f.sp. melonis. In planta, they significantly increased the growth (stem length, number of leaflets, leaf area and root weight) and biochemical (nitrate reductase activity, proline and chlorophyll content) parameters of tomato, as well as the rate of seed germination. Two selected strains (Pr7 and Pr8) with higher antagonistic activity against V. dalhiae and F. oxysporum f.sp. melonis protected tomato plants against Verticillium wilt and salt stress. In addition, they enhanced acclimatization of Vitis vinifera cv. Pinot noir and the peach root stock GF305 from in vitro to the greenhouse. 16S rRNA sequencing identified strains Pr7 and Pr8 as Pseudomonas stutzeri and Bacillus toyonensis, respectively. Since these two PGPR inoculants exhibited multiple traits beneficial to the examined host plants, they may be applied in the development of safe, and effective seed treatments as an alternative to chemical fungicides and fertilization but also for successful acclimatization of micropropagated plants.Mohamed Faize was supported by funding from the ‘Ministère de l'Enseignement Supérieur, de la Recherche Scientifique de la Formation des Cadres’ (MERSFC, Morocco) within the framework of ARIMNet2 Projec

Functional Analogues of Salicylic Acid and Their Use in Crop Protection

Functional analogues of salicylic acid are able to activate plant defense responses and provide attractive alternatives to conventional biocidal agrochemicals. However, there are many problems that growers must consider during their use in crop protection, including incomplete disease reduction and the fitness cost for plants. High-throughput screening methods of chemical libraries allowed the identification of new compounds that do not affect plant growth, and whose mechanisms of action are based on priming of plant defenses, rather than on their direct activation. Some of these new compounds may also contribute to the discovery of unknown components of the plant immune system.This work was supported by the University Chouaib Doukkali, El Jadida, Morocco

Application of Ascophyllum nodosum-Based Soluble Extract on Micropropagation and Regeneration of Nicotiana benthamiana and Prunus domestica

In the present study, the effect of a commercial extract of the seaweed Ascophyllum nodosum on in vitro micropropagation, shoot regeneration, and rhizoghenesis were studied in Nicotiana benthamiana and Prunus domestica. Results showed that the MS medium supplemented with various concentrations of the Ascophyllum extract (5, 10, 50, and 100 mg L−1) significantly enhanced the number of regenerated buds from N. benthamiana leaf discs to the conventional MS regenerating medium. Increases ranged from 3.5 to 6.5 times higher than the control. The effect of the Ascophyllum extract on N. benthamiana micropropagation was assessed through the measurement of some plant growth parameters. Results showed that the extract alone could not replace the micropropagation medium since shoot length, shoot diameter, root length, and leaf area were significantly reduced. However, its combination with a half-strength MS medium enhanced these parameters. Its effect was also evaluated on regeneration from plum hypocotyl slices. When added to the shoot regeneration medium without any plant growth regulators, the Ascophyllum extract alone could induce shoot regeneration. However, the percentage of bud regeneration and number of regenerated buds were lower than with the conventional shoot regeneration medium containing complete growth regulators. In contrast, the Ascophyllum extract drastically promoted rhizogenesis from plum hypocotyl slices. These results pave the way for the possible use of A. nodosum extracts in in vitro mass propagation of higher plants

Ectopic expression of cytosolic superoxide dismutase and ascorbate peroxidase leads to salt stress tolerance in transgenic plums

To fortify the antioxidant capacity of plum plants, genes encoding cytosolic antioxidants ascorbate peroxidase (cytapx) and Cu/Zn-superoxide dismutase (cytsod) were genetically engineered in these plants. Transgenic plum plants expressing the cytsod and/or cytapx genes in cytosol have been generated under the control of the CaMV35S promoter. High levels of cytsod and cytapx gene transcripts suggested that the transgenes were constitutively and functionally expressed. We examined the potential functions of cytSOD and cytAPX in in vitro plum plants against salt stress (100 mm NaCl). Several transgenic plantlets expressing cytsod and/or cytapx showed an enhanced tolerance to salt stress, mainly lines C5-5 and J8-1 (expressing several copies of sod and apx, respectively). Transformation as well as NaCl treatments influenced the antioxidative metabolism of plum plantlets, including enzymatic and nonenzymatic antioxidants. Transgenic plantlets exhibited higher contents of nonenzymatic antioxidants glutathione and ascorbate than nontransformed control, which correlated with lower accumulation of hydrogen peroxide. Overall, our results suggest that transformation of plum plants with genes encoding antioxidant enzymes enhances the tolerance to salinity.This work was supported by the Spanish Ministry of Economy and Competitiveness (Project CICYT BFU2009-07443) cofinanced by FEDER funds. PDV acknowledges the CSIC and the Spanish Ministry of Economy and Competitiveness for his ‘Ramon y Cajal’ research contract, cofinanced by FEDER funds. GBE and CP thank CSIC for their ‘JAE-pre’ and ‘JAE-doc’ fellowships.Peer reviewe

Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought stress

In order to understand the role of cytosolic antioxidant enzymes in drought stress protection, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) (EC 1.15.1.1) or ascorbate peroxidase (cytapx) (EC 1.11.1.1) alone, or in combination, were produced and tested for tolerance against mild water stress. The results showed that the simultaneous overexpression of Cu/Znsod and apx or at least apx in the cytosol of transgenic tobacco plants alleviates, to some extent, the damage produced by wáter stress conditions. This was correlated with higher water use efficiency and better photosynthetic rates. In general, oxidative stress parameters, such as lipid peroxidation, electrolyte leakage, and H2O2 levels, were higher in non-transformed plants than in transgenic lines, suggesting that, at the least, overexpression of cytapx protects tobacco membranes from water stress. In these conditions, the activity of other antioxidant enzymes was induced in transgenic lines at the subcellular level. Moreover, an increase in the activity of some antioxidant enzymes was also observed in the chloroplast of transgenic plants overexpressing cytsod and/or cytapx. These results suggest the positive influence of cytosolic antioxidant metabolism on the chloroplast and underline the complexity of the regulation network of plant antioxidant defences during drought stress.This work was supported by BIOCARM BIO-AGR07/ 04-0011, CICYT AGL2006-01743/AGR, and CICYT BFU2009-07443. MF was supported by a ‘Ramo´n & Cajal’ contract from the Spanish Ministry of Education. GBE thanks the CSIC for his JAE research fellowship. MJCM thanks the Spanish Ministry of Science and Education for her FPI research fellowship.Peer reviewe

Effective transfer of plum pox virus resistance from transgenic plum rootstocks to apricot scions. Supplementary Material

Supplementary Figure 1. Apricot grafted onto a transgenic plum rootstock. (A) Apricot bud grafted sprouting after artificial winter. (B) Heavily infected apricot scion grafted onto the transgenic susceptible plum line St5’-7 and (C) healthy apricot grafted onto the resistant plum rootstocks. (D) Detail of an apricot grafted plant (ag) after trimming the plum rootstock (pr) where can be seen the point of chip-budding infection (cb). Supplementary Figure 2. Chip budding technique used to infect apricot scions with sharka. A cut in the apricot scion (1) allows the introduction of a piece of bark from an infected GF305 new growth (2) within the apricot scion (3). Grafting is wrapped with parafilm to avoid desiccation (4) and it is maintained until it dries out and falls (5). Supplementary Figure 3. Schematic representation of the h-UTR/P1 T-DNA showing the position of primers used in this study. Supplementary Figure 4. Electrophoretic analysis of PCR products for PPV detection. L: DNA ladder 100 pb (New England Biolabs, Cat. no. N3231L). Lanes 1-10: apricot scion grafted onto different St5’ transgenic rootstocks. Lane 1 and 2: onto St5’-7 plants. Lanes 3 and 4: onto St5’-6 plants. Lanes 5-7 onto St5’-1 plants. Lanes 5-7 onto St5’-9 plants. Lane 10 H2OPeer reviewe

Protective effect of symmetrical N‐heterocyclic 1,3,4‐oxadiazole and 1,3,4‐thiadiazole derivatives against Pepino mosaic virus of tomato

International audienceAbstract Pepino mosaic virus (PepMV) is a highly infectious potexvirus, which has presently become a major pathogen for tomato crops worldwide. Since there is no effective method control for PepMV, strict preventive hygienic measures and cross‐protection have been established. However, the use of mild isolates to protect against aggressive isolates provides opportunities for interaction with other microorganisms, which under certain conditions may worsen disease symptoms. Thus, alternative control methods are needed. In this study we aimed at the development of chemical control against PepMV based on the use of heterocyclic compounds including symmetrical 2,5‐disubstituted 1,3,4‐oxadiazoles, symmetrical 2,5‐disubstituted 1,3,4‐thiadiazole and dihydrotetrazine derivatives. Results showed that spray application of three 1,3,4‐oxadiazole derivatives (OH‐Oxa, CH 3 ‐Oxa, NO 2 ‐Oxa) and of the derivative of 1,3,4‐thiadiazole (OH‐Thia) resulted in reducing the main symptoms of PepMV in tomato leaves, conversely to the dihydrotetrazine carboxylic acid. Double‐antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) revealed that PepMV was not detected from tomato plants pre‐treated with the derivatives conferring elevated protection. Moreover, the protective ability of OH‐Oxa, CH 3 ‐Oxa, NO 2 ‐Oxa and OH‐Thia was closely related to the enhancement of the activity of antioxidant enzymes upon infection with PepMV