Genetic transformation of Vitis vinifera via organogenesis

BACKGROUND: Efficient transformation and regeneration methods are a priority for successful application of genetic engineering to vegetative propagated plants such as grape. The current methods for the production of transgenic grape plants are based on Agrobacterium-mediated transformation followed by regeneration from embryogenic callus. However, grape embryogenic calli are laborious to establish and the phenotype of the regenerated plants can be altered. RESULTS: Transgenic grape plants (V. vinifera, table-grape cultivars Silcora and Thompson Seedless) were produced using a method based on regeneration via organogenesis. In vitro proliferating shoots were cultured in the presence of increasing concentrations of N(6)-benzyl adenine. The apical dome of the shoot was removed at each transplantation which, after three months, produced meristematic bulk tissue characterized by a strong capacity to differentiate adventitious shoots. Slices prepared from the meristematic bulk were used for Agrobacterium-mediated transformation of grape plants with the gene DefH9-iaaM. After rooting on kanamycin containing media and greenhouse acclimatization, transgenic plants were transferred to the field. At the end of the first year of field cultivation, DefH9-iaaM grape plants were phenotypically homogeneous and did not show any morphological alterations in vegetative growth. The expression of DefH9-iaaM gene was detected in transgenic flower buds of both cultivars. CONCLUSIONS: The phenotypic homogeneity of the regenerated plants highlights the validity of this method for both propagation and genetic transformation of table grape cultivars. Expression of the DefH9-iaaM gene takes place in young flower buds of transgenic plants from both grape cultivars

Expression profile analysis of early fruit development in iaaM-parthenocarpic tomato plants

<p>Abstract</p> <p>Background</p> <p>Fruit normally develops from the ovary after pollination and fertilization. However, the ovary can also generate seedless fruit without fertilization by parthenocarpy. Parthenocarpic fruit development has been obtained in tomato (<it>Solanum lycopersicum</it>) by genetic modification using auxin-synthesising gene(s) (<it>DefH9-iaaM</it>; <it>DefH9-RI-iaaM</it>) expressed specifically in the placenta and ovules.</p> <p>Findings</p> <p>We have performed a cDNA Amplified Fragment Length Polymorphism (cDNA-AFLP) analysis on pre-anthesis tomato flower buds (0.5 cm long) collected from <it>DefH9-iaaM </it>and <it>DefH9-RI-iaaM </it>parthenocarpic and wild-type plants, with the aim to identify genes involved in very early phases of tomato fruit development. We detected 212 transcripts differentially expressed in auxin-ipersynthesising pre-anthesis flower buds, 65 of them (31%) have unknown function. Several differentially expressed genes show homology to genes involved in protein trafficking and protein degradation via proteasome. These processes are crucial for auxin cellular transport and signaling, respectively.</p> <p>Conclusion</p> <p>The data presented might contribute to elucidate the molecular basis of the fruiting process and to develop new methods to confer parthenocarpy to species of agronomic interest. In a recently published work, we have demonstrated that one of the genes identified in this screening, corresponding to #109 cDNA clone, regulates auxin-dependent fruit initiation and its suppression causes parthenocarpic fruit development in tomato.</p

Auxin and nitric oxide control indeterminate nodule formation

<p>Abstract</p> <p>Background</p> <p>Rhizobia symbionts elicit root nodule formation in leguminous plants. Nodule development requires local accumulation of auxin. Both plants and rhizobia synthesise auxin. We have addressed the effects of bacterial auxin (IAA) on nodulation by using <it>Sinorhizobium meliloti </it>and <it>Rhizobium leguminosarum </it>bacteria genetically engineered for increased auxin synthesis.</p> <p>Results</p> <p>IAA-overproducing <it>S. meliloti </it>increased nodulation in <it>Medicago </it>species, whilst the increased auxin synthesis of <it>R. leguminosarum </it>had no effect on nodulation in <it>Phaseolus vulgaris</it>, a legume bearing determinate nodules. Indeterminate legumes (<it>Medicago </it>species) bearing IAA-overproducing nodules showed an enhanced lateral root development, a process known to be regulated by both IAA and nitric oxide (NO). Higher NO levels were detected in indeterminate nodules of <it>Medicago </it>plants formed by the IAA-overproducing rhizobia. The specific NO scavenger cPTIO markedly reduced nodulation induced by wild type and IAA-overproducing strains.</p> <p>Conclusion</p> <p>The data hereby presented demonstrate that auxin synthesised by rhizobia and nitric oxide positively affect indeterminate nodule formation and, together with the observation of increased expression of an auxin efflux carrier in roots bearing nodules with higher IAA and NO content, support a model of nodule formation that involves auxin transport regulation and NO synthesis.</p

Expression of self-complementary hairpin RNA under the control of the rolC promoter confers systemic disease resistance to plum pox virus without preventing local infection

BACKGROUND: Homology-dependent selective degradation of RNA, or post-transcriptional gene silencing (PTGS), is involved in several biological phenomena, including adaptative defense mechanisms against plant viruses. Small interfering RNAs mediate the selective degradation of target RNA by guiding a multicomponent RNAse. Expression of self-complementary hairpin RNAs within two complementary regions separated by an intron elicits PTGS with high efficiency. Plum pox virus (PPV) is the etiological agent of sharka disease in Drupaceae, although it can also be transmitted to herbaceous species (e.g. Nicotiana benthamiana). Once inside the plant, PPV is transmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The rolC promoter drives expression in phloem cells. RolC expression is absent in both epidermal and mesophyll cells. The aim of the present study was to confer systemic disease resistance without preventing local viral infection. RESULTS: In the ihprolC-PP197 gene (intron hair pin rolC PPV 197), a 197 bp sequence homologous to the PPV RNA genome (from base 134 to 330) was placed as two inverted repeats separated by the DNA sequence of the rolA intron. This hairpin construct is under the control of the rolC promoter.N. benthamiana plants transgenic for the ihprolC-PP197 gene contain siRNAs homologous to the 197 bp sequence. The transgenic progeny of ihprolC-PP197 plants are resistant to PPV systemic infection. Local infection is unaffected. Most (80%) transgenic plants are virus free and symptomless. Some plants (20%) contain virus in uninoculated apical leaves; however they show only mild symptoms of leaf mottling. PPV systemic resistance cosegregates with the ihprolC-PP197 transgene and was observed in progeny plants of all independent transgenic lines analyzed. SiRNAs of 23–25 nt homologous to the PPV sequence used in the ihprolC-PP197 construct were detected in transgenic plants before and after inoculation. Transitivity of siRNAs was observed in transgenic plants 6 weeks after viral inoculation. CONCLUSIONS: The ihprolC-PP197 transgene confers systemic resistance to PPV disease in N. benthamiana. Local infection is unaffected. This transgene and/or similar constructs could be used to confer PPV resistance to fruit trees where systemic disease causes economic damage

Editorial: Advances in genetic engineering strategies for fruit crop breeding, volume II

Breeding of fruit crops is a challenging process that must consider the need to preserve the characteristics of elite cultivars and the urgent need to obtain new cultivars with high resilience and high productivity. Changes in climate and the progressive limits on the use of agrochemicals may require the development of new genetic stocks in relatively short periods of time. Classical and innovative genetic engineering approaches may help achieve these goals. The second volume of this Research Topics aimed to present an update on the genetic tools available for the breeding of fruits crops for new traits. The articles of this Research Topic represent well the opportunities offered by genetic engineering to future fruit crop breeding

Evaluation of the Potential Use of a Collagen-Based Protein Hydrolysate as a Plant Multi-Stress Protectant

Protein hydrolysates (PHs) are a class of plant biostimulants used in the agricultural practice to improve crop performance. In this study, we have assessed the capacity of a commercial PH derived from bovine collagen to mitigate drought, hypoxic, and Fe deficiency stress in Zea mays. As for the drought and hypoxic stresses, hydroponically grown plants treated with the PH exhibited an increased growth and absorption area of the roots compared with those treated with inorganic nitrogen. In the case of Fe deficiency, plants supplied with the PH mixed with FeCl3 showed a faster recovery from deficiency compared to plants supplied with FeCl3 alone or with FeEDTA, resulting in higher SPAD values, a greater concentration of Fe in the leaves and modulation in the expression of genes related to Fe. Moreover, through the analysis of circular dichroism spectra, we assessed that the PH interacts with Fe in a dose-dependent manner. Various hypothesis about the mechanisms of action of the collagen-based PH as stress protectant particularly in Fe-deficiency, are discussed

The involvement of root-specific LTPs in the symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti

Lipid transfer proteins (LTPs) are small basic proteins that constitute a large family characterized by the ability to transfer phospholipids between a donor and an acceptor membrane and can have many different roles in vivo. Recently it has been demonstrated that MtN5, a non specific LTP (ns-LTP) classified as type III (Wang et al., 2012), is involved in the symbiotic interaction between legumes and rhizobia (Pii et al. 2009, Pii et al., 2012). MtN5 is a nod factor responsive gene expressed at a very early phase of rhizobial symbiosis in the epidermis and root hairs and later in primordia and nodules. There are evidences that MtN5 positively regulates the nodulation process. Interestingly, two other putative type III ns-LTPs (Medtr3g055250 and Medtr7g052640) have been identified in Medicago truncatula genome. The aim of this study is to shed light on the role of these ns-LTPs in the symbiotic interaction between M. truncatula and Sinorhizobium meliloti

Growth Stimulatory Effects and Genome-Wide Transcriptional Changes Produced by Protein Hydrolysates in Maize Seedlings

Protein hydrolysates are an emerging class of crop management products utilized for improving nutrient assimilation and mitigating crop stress. They generally consist of a mixture of peptides and free amino acids derived from the hydrolysis of plant or animal sources. The present work was aimed at studying the effects and the action mechanisms of a protein hydrolysate derived from animal residues on maize root growth and physiology in comparison with the effects induced by either free amino acids or inorganic N supply. The application of the protein hydrolysate caused a remarkable enhancement of root growth. In particular, in the protein hydrolysate-treated plants the length and surface area of lateral roots were about 7 and 1.5 times higher than in plants treated with inorganic N or free amino acids, respectively. The root growth promoting effect of the protein hydrolysate was associated with an increased root accumulation of K, Zn, Cu, and Mn when compared with inorganic N and amino acids treatments. A microarray analysis allowed to dissect the transcriptional changes induced by the different treatments demonstrating treatment-specific effects principally on cell wall organization, transport processes, stress responses and hormone metabolism

The DefH9-iaaM auxin-sinthesizing gene increases plant fecundity and fruit production in strawberry and raspberry

Background: The DefH9-iaaM gene fusion which is expressed specifically in placenta/ovules andpromotes auxin-synthesis confers parthenocarpic fruit development to eggplant, tomato andtobacco. Transgenic DefH9-iaaM eggplants and tomatoes show increased fruit production duemainly to an improved fruit set. However, the weight of the fruits is also frequently increased.Results: DefH9-iaaM strawberry and raspberry plants grown under standard cultivation conditionsshow a significant increase in fruit number and size and fruit yield. In all three Rosaceae speciestested, Fragaria vesca, Fragaria x ananassa and Rubus idaeus, DefH9-iaaM plants have an increasednumber of flowers per inflorescence and an increased number of inflorescences per plant. Thisresults in an increased number of fruits per plant. Moreover, the weight and size of transgenic fruitswas also increased. The increase in fruit yield was approximately 180% in cultivated strawberry,140% in wild strawberry, and 100% in raspberry. The DefH9-iaaM gene is expressed in the flowerbuds of all three species. The total IAA (auxin) content of young flower buds of strawberry andraspberry expressing the DefH9-iaaM gene is increased in comparison to untransformed flowerbuds. The DefH9-iaaM gene promotes parthenocarpy in emasculated flowers of both strawberryand raspberry.Conclusions: The DefH9-iaaM gene is expressed and biologically active in Rosaceae. The DefH9-iaaM gene can be used, under cultivation conditions that allow pollination and fertilization, toincrease fruit productivity significantly in Rosaceae species. The finding that the DefH9-iaaM auxinsynthesizinggene increases the number of inflorescences per plant and the number of flowers perinflorescence indicates that auxin plays a role in plant fecundity in these three perennial Rosaceaespecies

Seedless Fruit Production by Hormonal Regulation of Fruit Set

Seed and fruit development are intimately related processes controlled by internal signals and environmental cues. The absence of seeds is usually appreciated by consumers and producers because it increases fruit quality and fruit shelf-life. One method to produce seedless fruit is to develop plants able to produce fruits independently from pollination and fertilization of the ovules. The onset of fruit growth is under the control of phytohormones. Recent genomic studies have greatly contributed to elucidate the role of phytohormones in regulating fruit initiation, providing at the same time genetic methods for introducing seedlessness in horticultural plants