Cesar Petri

Transformation of fruit trees. Useful breeding tool or continued future prospect

Regeneration and transformation systems using mature plant material of woody fruit species have to be achieved as a necessary requirement for the introduction of useful genes into specific cultivars and the rapid evaluation of resulting horticultural traits. Although the commercial production of transgenic annual crops is a reality, commercial genetically-engineered fruit trees are still far from common. In most woody fruit species, transformation and regeneration of commercial cultivars are not routine, generally being limited to a few genotypes or to seedlings. The future of genetic transformation as a tool for the breeding of fruit trees requires the development of genotype-independent procedures, based on the transformation of meristematic cells with high regeneration potential and/or the use of regeneration-promoting genes. The public concern with the introduction of antibiotic resistance into food and the restrictions due to new European laws that do not allow deliberate release of plants transformed with antibiotic-resistance genes highlight the development of methods that avoid the use of antibiotic-dependent selection or allow elimination of marker genes from the transformed plant as a research priority in coming years

Agrobacterium -mediated transformation of apricot ( Prunus armeniaca L.) leaf explants

A protocol for Agrobacterium-mediated stable transformation for scored, whole leaf explants of the apricot (Prunus armeniaca) cultivar Helena was developed. Regenerated shoots were selected using a two-step increased concentrations of paromomycin sulphate. Different factors affecting survival of transformed buds, including possible toxicity of green fluorescent protein (GFP) and time of exposure to high cytokine concentration in the regeneration medium, were examined. Transformation efficiency, based on PCR analysis of individual putative transformed shoots from independent lines was 5.6%, when optimal conditions for bud survival were provided. Southern blot analysis on four randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene. This is the first time that stable transformation of an apricot cultivar is reported and constitutes also one of the few reports on the transformation of Prunus cultivars.

Factors affecting gene transfer efficiency to apricot leaves during early Agrobacterium-mediated transformation steps

SUMMARY A procedure to transfer marker genes to apricot leaves has been developed by assessment of different factors affecting early Agrobacterium-mediated transformation steps. Three oncogenic strains (C58, Ach5 and A281) were equally efficient in inciting tumours in apricot seedlings, however two disarmed derivatives from those strains (C58/pMP90 and EHA105) differently transformed apricot leaves in vitro. Maximum transient gfp expression was found 7–9 d after infection and stable expression could be determined four weeks later. An optimized procedure, based on transformation efficiencies (measured as percentage of transformed explants and average gfp spots per transformed explant) and control of the Agrobacterium growth after co-culture, was established. It consisted of infecting leaves for 10 min in a bacterial suspension (cultured for 24 h in a simplified medium with 500 ¼M AS and diluted to about 107 cfu ml–1), with a co-culture time of 4 d in the presence of l00 ¼M acetosyringone. With these conditions all explants were transformed and more than 20 gfp spots per explant, as average, were recorded. Calli expressing gfp and resistant to 172 ¼M kanamycin have been obtained and maintained in culture for more than one year. Amplification of both transgenes was also verified by PCR and its integration by southern blot analysis. This is, to our knowledge, the first report on transformation of apricot leaves with an adult origin.

The effect of aminoglycoside antibiotics on the adventitious regeneration from apricot leaves and selection of nptII-transformed leaf tissues

The effect of different concentrations of the aminoglycoside antibiotics, geneticin, paromomycin and streptomycin on adventitious regeneration from leaf explants of apricot was tested to design an alternative procedure for selecting transgenic shoots. Streptomycin and paromomycin reduced shoot regeneration percentage with increasing concentration of antibiotics. Almost a complete inhibition of regeneration was reached when 20μM paromomycin was used, although up to 40 μM streptomycin was necessary to completely inhibit regeneration. Geneticin had a very toxic effect on apricot leaves and regeneration was inhibited at almost all concentrations tested. Addition of kanamycin hastened the development of adventitious buds although silver thiosulfate and not kanamycin was responsible for the observed increase in the consistency of the results from independent experiments. Kanamycin and paromomycin at the concentrations tested improved selection of transformed cells and resulted in a larger number of gfp-expressing regions. Paromomycin at 40 and 25.7 μM kanamycin improved proliferation of transformed tissues as compared with the other antibiotics used and non-selected controls.

Dual regulation of water retention and cell growth by a stress-associated protein ( SAP ) gene in Prunus

We have identified a gene (PpSAP1) of Prunus persica coding for a stress-associated protein (SAP) containing Zn-finger domains A20 and AN1. SAPs have been described as regulators of the abiotic stress response in plant species, emerging as potential candidates for improvement of stress tolerance in plants. PpSAP1 was highly expressed in leaves and dormant buds, being down-regulated before bud dormancy release. PpSAP1 expression was moderately induced by water stresses and heat in buds. In addition, it was found that PpSAP1 strongly interacts with polyubiquitin proteins in the yeast two-hybrid system. The overexpression of PpSAP1 in transgenic plum plants led to alterations in leaf shape and an increase of water retention under drought stress. Moreover, we established that leaf morphological alterations were concomitant with a reduced cell size and down-regulation of genes involved in cell growth, such as GROWTH-REGULATING FACTOR (GRF)1-like, TONOPLAST INTRINSIC PROTEIN (TIP)-like, and TARGET OF RAPAMYCIN (TOR)-like. Especially, the inverse expression pattern of PpSAP1 and TOR-like in transgenic plum and peach buds suggests a role of PpSAP1 in cell expansion through the regulation of TOR pathway.

Metabolomics and Biochemical Approaches Link Salicylic Acid Biosynthesis to Cyanogenesis in Peach Plants

Despite the long-established importance of salicylic acid (SA) in plant stress responses and other biological processes, its biosynthetic pathways have not been fully characterized. The proposed synthesis of SA originates from chorismate by two distinct pathways: the isochorismate and phenylalanine (Phe) ammonia-lyase (PAL) pathways. Cyanogenesis is the process related to the release of hydrogen cyanide from endogenous cyanogenic glycosides (CNglcs), and it has been linked to plant plasticity improvement. To date, however, no relationship has been suggested between the two pathways. In this work, by metabolomics and biochemical approaches (including the use of [13C]-labeled compounds), we provide strong evidences showing that CNglcs turnover is involved, at least in part, in SA biosynthesis in peach plants under control and stress conditions. The main CNglcs in peach are prunasin and amygdalin, with mandelonitrile (MD), synthesized from phenylalanine, controlling their turnover. In peach plants MD is the intermediary molecule of the suggested new SA biosynthetic pathway and CNglcs turnover, regulating the biosynthesis of both amygdalin and SA. MD-treated peach plants displayed increased SA levels via benzoic acid (one of the SA precursors within the PAL pathway). MD also provided partial protection against Plum pox virus infection in peach seedlings. Thus, we propose a third pathway, an alternative to the PAL pathway, for SA synthesis in peach plants.

Greenhouse evaluation confirms in vitro sharka resistance of genetically engineered h-UTR/P1 plum plants

Plum pox virus (PPV), a quarantine virus, is the causal agent of sharka, the most devastating viral disease of stone fruits. The aim of this study was to produce several transgenic plum lines resistant to PPV, set up an efficient and reliable in vitro PPV resistant test and compare it with the standard greenhouse evaluation. Transgenic plants with the hairpin h-UTR/P1 construct were produced after infection of hypocotyls from mature European plum seeds with Agrobacterium tumefaciens. Ten transgenic lines were in vitro grafted onto PPV-D infected ‘GF305’ peach and evaluated for PPV presence by RT-PCR. PPV was detected in all non-transgenic grafts whereas in seven transgenic lines it was never found or disappeared with time. PPV-resistant transgenic lines were rooted, acclimatized and evaluated with the standard procedures under greenhouse conditions. Our data indicates that greenhouse and in vitro results agreed. The proposed in vitro evaluation method could be used to screen sharka resistant plum lines providing a fast, reliable and contained methodology.

The effect of abiotic and biotic stress on the salicylic acid biosynthetic pathway from mandelonitrile in peach

Highlight We show that the recently suggested third pathway for SA biosynthesis from mandelonitrile in peach is also functional under both abiotic and biotic stress conditions. Abstract Salicylic acid (SA) plays a central role in plant responses to environmental stresses via the SA-mediated regulation of many metabolic and molecular processes. In a recent study, we suggested a third pathway for SA biosynthesis from mandelonitrile (MD) in peach plants. This pathway is alternative to the phenylalanine ammonia-lyase pathway and links SA biosynthesis and cyanogenesis. In the present work, we show that this new SA biosynthetic pathway is also functional under abiotic (salt) and biotic (Plum pox virus infection) stress conditions, although the contribution of this pathway to the SA pool does not seem to be important under such conditions. Treating peach plants with MD not only affected the SA content, but it also had a pleiotropic effect on abscisic acid and jasmonic acid levels, two well-known stress related hormones, as well as on the H2O2-related antioxidant activities. Furthermore, MD improved plant performance under the stressful conditions, probably via the activation of different signaling pathways. We have thus proven that SA is not limited to biotic stress responses, but that it also plays a role in the response to abiotic stress in peach, although the physiological functions of this new SA biosynthetic pathway from MD remain to be elucidated. Abbreviations ABA abcisic acid APX ascorbate peroxidase BA benzoic acid CAT catalase CNglcs cyanogenic glycosides MD mandelonitrile NPR1 non-expressor of pathogenesis-related gene PAL phenylalanine ammonia-lyase Phe phenylalanine POX peroxidase PPV Plum pox virus SA salicylic acid SOD superoxide dismutase TRX thioredoxinsSalicylic acid (SA) plays a central role in plant responses to environmental stresses via the SA-mediated regulation of many metabolic and molecular processes. In a recent study, we suggested a third pathway for SA biosynthesis from mandelonitrile (MD) in peach plants. This pathway is alternative to the phenylalanine ammonia-lyase pathway and links SA biosynthesis and cyanogenesis. In the present work, we show that this new SA biosynthetic pathway is also functional under abiotic (salt) and biotic (Plum pox virus infection) stress conditions, although the contribution of this pathway to the SA pool does not seem to be important under such conditions. Treating peach plants with MD not only affected the SA content, but it also had a pleiotropic effect on abscisic acid and jasmonic acid levels, two well-known stress related hormones, as well as on the H2O2-related antioxidant activities. Furthermore, MD improved plant performance under the stressful conditions, probably via the activation of different signaling pathways. We have thus proven that SA is not limited to biotic stress responses, but that it also plays a role in the response to abiotic stress in peach, although the physiological functions of this new SA biosynthetic pathway from MD remain to be elucidated.