Nicoletta Ferradini

Effect of leaf excision time and age, BA concentration and dark treatments on in vitro shoot regeneration of M.26 apple rootstock

SUMMARYThe effect of benzyladenine (BA) concentrations both during the last proliferating subculture before regeneration (10-222 and, 444 µM) and during organogenesis (11.1 and 22.2 µM), leaf excis...

Non-antibiotic, efficient selection for alfalfa genetic engineering

A selectable marker gene (SMG), usually conferring resistance to an antibiotic or herbicide, is generally introduced into the plant cells with the gene(s) for the trait of interest to allow only the cells that have integrated and express the foreign sequences to regenerate into a plant. The availability of several SMGs for each plant species is useful for both basic and applied research to combine several genes of interest in the same plant. A selection system based on gabaculine (3-amino-2,3-dihydrobenzoic acid) as the selective substance and the bacterial hemL gene [encoding a mutant for of the enzyme glutamate 1-semialdehyde aminotransferase (GSA-AT)] as the SMG was previously used for genetic transformation of tobacco. The hemL gene is a good candidate for a safe SMG, because GSA-AT is present in all plants and is likely involved in one metabolic step only, so that unintended effects of its overexpression in plants are not probable. In this work, we have compared this new selection system with the conventional, kanamycin-based system for alfalfa Agrobacterium-mediated transformation. The hemL and NptII genes were placed together into a T-DNA under the control of identical promoters and terminators. We show that the gabaculine-based system is more efficient than the conventional, kanamycin-based system. The inheritance of hemL was Mendelian, and no obvious phenotypic effect of its expression was observed.

Population Structure of Barley Landrace Populations and Gene-Flow with Modern Varieties

Landraces are heterogeneous plant varieties that are reproduced by farmers as populations that are subject to both artificial and natural selection. Landraces are distinguished by farmers due to their specific traits, and different farmers often grow different populations of the same landrace. We used simple sequence repeats (SSRs) to analyse 12 barley landrace populations from Sardinia from two collections spanning 10 years. We analysed the population structure, and compared the population diversity of the landraces that were collected at field level (population). We used a representative pool of barley varieties for diversity comparisons and to analyse the effects of gene flow from modern varieties. We found that the Sardinian landraces are a distinct gene pool from those of both two-row and six-row barley varieties. There is also a low, but significant, mean level and population-dependent level of introgression from the modern varieties into the Sardinian landraces. Moreover, we show that the Sardinian landraces have the same level of gene diversity as the representative sample of modern commercial varieties grown in Italy in the last decades, even within population level. Thus, these populations represent crucial sources of germplasm that will be useful for crop improvement and for population genomics studies and association mapping, to identify genes, loci and genome regions responsible for adaptive variations. Our data also suggest that landraces are a source of valuable germplasm for sustainable agriculture in the context of future climate change, and that in-situ conservation strategies based on farmer use can preserve the genetic identity of landraces while allowing adaptation to local environments.

Assessment of simple marker-free genetic transformation techniques in alfalfa.

Methods to avoid the presence of selectable marker genes (SMG) in transgenic plants are available but not implemented in many crop species. We assessed the efficiency of simple marker-free Agrobacterium-mediated transformation techniques in alfalfa: regeneration without selection, or marker-less, and co-transformation with two vectors, one containing the SMG and one containing a non-selected gene. To easily estimate the efficiency of marker-less transformation, the nptII and the GUS markers were used as non-selected genes. After Agrobacterium treatment, somatic embryos were regenerated without selection. The percentage of transgenic embryos was determined by a second cycle of regeneration using the embryos as starting material, in the presence of kanamycin, by PCR screening of T1 progenies, and by the GUS test. In two experiments, from 0 to 1.7% of the somatic embryos were transgenic. Co-transformation was performed with two vectors, one with the hemL SMG and one with the unselected nptII gene, each carried by a different culture of Agrobacterium. Only 15 putative co-transformed plants were regenerated from two experiments, with an average co-transformation percentage of 3.7. Southern blot hybridizations and/or T1 progeny segregation were used to confirm transgene integration, and qPCR was also used to estimate the T-DNA copy number. In the T1 progenies obtained by crossing with a non-transgenic pollinator, marker-free segregants were obtained. Both marker-free approaches showed very low efficiency.

A point mutation in the Medicago sativa GSA gene provides a novel, efficient, selectable marker for plant genetic engineering.

Bacterial selectable marker genes (SMG) conferring antibiotic resistance are valuable tools in plant genetic engineering, but public concern and regulatory requirements have stimulated the development of alternative selection systems. We have previously demonstrated that a mutated Synechococcus elongatus HemL gene encoding glutamate 1-semialdehyde aminotransferase (GSA) is an efficient SMG in alfalfa. In fact, GSA is irreversibly inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid), but the mutated enzyme is gabaculine insensitive. With the aim to develop a plant derived SMG, we cloned and sequenced the Medicago sativa GSA cDNA and reproduced one of the two mutations associated with gabaculine resistance in Synechococcus, a transversion resulting in a methionine to isoleucine (M→I) substitution. This mutated gene was assessed as a SMG in tobacco and alfalfa Agrobacterium transformation, in comparison with the wild type gene. In tobacco, about 43% of the leaf explants produced green shoots, whereas in alfalfa 47% of the explants produced green embryos in the presence of 30 μM gabaculine when the M→I GSA was introduced. Escapes were absent in tobacco and only 6% in alfalfa. No effect on the plant phenotype was noticed. We propose this new SMG as a widely acceptable alternative to those currently used.

Occurrence of a number of enzymes involved in either gluconeogenesis or other processes in the pericarp of three cultivars of grape (Vitis vinifera L.) during development.

It is uncertain whether the enzymes pyruvate orthophosphate dikinase (PPDK) or isocitrate lyase (ICL) are present in the pericarp of grape, in which they could function in gluconeogenesis. The occurrence of these and other enzymes was investigated in the pericarp of three cultivars of grape (Vitis vinifera L.). In particular, the abundance of the enzymes aldolase, glutamine synthase (GS), acid invertase, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPC), PPDK and ICL were determined during the development of the pericarp of the cultivars Cabernet Sauvignon, Chardonnay and Zibibbo. PPDK and ICL were not detected at any stage of development. Each of the other enzymes showed different changes in abundance during development. However, for a given enzyme its changes in abundance were similar in each cultivar. In the ripe pericarp of Cabernet Sauvignon, PEPC, cytosolic GS and aldolase were equally distributed between the vasculature and parenchyma cells of the flesh and skin. The absence or very low abundance of PPDK provides strong evidence that any gluconeogenesis from malate utilises phosphoenolpyruvate carboxykinase (PEPCK). The absence or very low abundance of ICL in the pericarp precludes any gluconeogenesis from ethanol.

Influence of growth regulators and light on in vitro shoot regeneration in M.26 apple roostock

SummaryThe effects of different BA and IBA concentrations and dark/light combinations, applied during both the last proliferating subculture (LPS) and the regeneration phase (RP), on shoot regeneration from leaves were evaluated in M.26 apple rootstock. A positive influence on caulogenesis was found with a low cytokinin/auxin ratio in the medium during the LPS. The increase in IBA concentration from 0.49 μM to 4.92 μM in the LPS, along with the absence or the use of a low cytokinin concentration (0.89 μM) in the medium, enhanced the subsequent shoot regeneration from leaves. During the RP, a concentration of 22.2 μM BA gave the best caulogenesis results. During both the LPS and the RP, high IBA concentrations were able to replace the combined effect of a low IBA concentration and dark treatment; this could indicate that dark treatments interact with the auxin metabolism in the leaf caulogenesis response. Auxin application could not reproduce all the effects of dark treatments, suggesting that dark also af...

Characterization of the lentil landrace Santo Stefano di Sessanio from Abruzzo, Italy

In the world lentil is grown on more than 3 million hectares and is one of the most important, low-cost, food source of protein. In Italy lentil has been cultivated since ancient times, but in the last decades its cultivation has been confined to marginal areas, small islands and hilly, mountainous areas of central and southern Italy. Local varieties are still common and are often greatly appreciated for their taste and cooking qualities. Several accessions from the Santo Stefano di Sessanio area, Abruzzo Region, were collected and phenotypically and genotypically characterized in order to look for the existing variability within and between populations. Image analysis of seeds was also used. Populations grown in Santo Stefano di Sessanio and in the neighbouring area basically share most of their characteristics. However, some of the accessions anonymously gathered from the local market were shown to be different from those collected from farmers. The paper reports and discusses how this local product needs be characterized and promoted in order to avoid fraud that could negatively affect the local economy and put valuable, adapted, genetic resources at risk of erosion.

Expression of an evolved engineered variant of a bacterial glycine oxidase leads to glyphosate resistance in alfalfa

The main strategy for resistance to the herbicide glyphosate in plants is the overexpression of an herbicide insensitive, bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). A glyphosate resistance strategy based on the ability to degrade the herbicide can be useful to reduce glyphosate phytotoxicity to the crops. Here we present the characterization of glyphosate resistance in transgenic alfalfa (Medicago sativa L.) expressing a plant-optimized variant of glycine oxidase (GO) from Bacillus subtilis, evolved in vitro by a protein engineering approach to efficiently degrade glyphosate. Two constructs were used, one with (GO(TP+)) and one without (GO(TP-)) the pea rbcS plastid transit peptide. Molecular and biochemical analyses confirmed the stable integration of the transgene and the correct localization of the plastid-imported GO protein. Transgenic alfalfa plants were tested for glyphosate resistance both in vitro and in vivo. Two GO(TP+) lines showed moderate resistance to the herbicide in both conditions. Optimization of expression of this GO variant may allow to attain sufficient field resistance to glyphosate herbicides, thus providing a resistance strategy based on herbicide degradation.

Characterization and phylogenetic analysis of ancient Italian landraces of pear

Pear is one of the oldest fruit tree crops and the third most important temperate fruit species. Its domestication took place independently in the Far East (China) and in the Caucasus region. While the origin of Eastern Asian cultivars is clear, that of European cultivars is still in doubt. Italy has a wealth of local varieties and genetic resources safeguarded by several public and private collections to face the erosion caused by the introduction of improved varieties in specialized orchards. The objectives of the present study were: (i) to characterize the existing germplasm through nuclear (SSR) and (ii) to clarify the genetic divergence between local and cultivated populations through chloroplast DNA (cpDNA) markers in order to provide insights into phylogenetic relationships of Pyrus spp. For this reason, 95 entries from five different germplasm collections, including nine European, Mediterranean and Eastern Asian species, were analyzed, and the intergenic accD-psaI sequences were compared to the worldwide distributed dataset encompassing a total of 298 sequences from 26 different Pyrus species. The nine nuclear SSRs were able to identify a total of 179 alleles, with a loci polymorphism P = 0.89. Most of the variation (97%) was found within groups. Five accessions from different sources were confirmed to be the same. Eight out of 20 accessions of unknown origin were identified, and six synonyms were detected. Locus NH030a was found to be monomorphic in all the cultivated accessions and in reference species interfertile with P. communis, leading to hypothesize selection pressures for adaptation to cultivation. The cpDNA sequences of the 95 accessions were represented by 14 haplotypes, six of which (derived from P. communis, P. cossonii and P. ussuriensis) are recorded here for the first time and may suggest the ancient origin of some local varieties. The network analysis of the 298 cpDNA sequences allowed two different haplogroups, Eastern and Western Eurasia, to be defined, supporting recent views of a clear division between Occidental and Oriental species. By combining the results from nuclear and uniparental markers, it was possible to better define many unknown accessions.