Production of transgenic barrel medic (Medicago truncatula Gaernt.) using the ipt-type MAT vector system and impairment of Recombinase-mediated excision events

Expression of the uidA reporter gene was tested in transformation experiments of barrel medic (Medicago truncatula Gaertn.) with the ipt-type control vectors pIPT5, pIPT10 and pIPT20 and distinct in vitro culture conditions. The highest GUS expression levels were obtained with the pIPT10 construct carrying the ipt gene under the control of the native ipt promoter and using kanamycin as selective agent. The ipt-shooty transformants, characterized by the absence of both rooting ability and apical dominance associated with vitrification, were easily identified by visual selection. Using only the ipt gene as selectable marker, we obtained a stable transformation frequency of 9.8% with pIPT10 construct. The ipt-type MAT vector pEXM2 was then used to monitor the excision events mediated by the yeast Recombinase and the consequent production of ipt marker-free transgenic plants. Transgenic ipt-shooty lines were recovered at a frequency of 7.9% in the absence of kanamycin-based selection. The ipt-shooty phenotype was maintained in all the transgenic lines and no reversion to the normal phenotype occurred. PCR analysis revealed the presence of the ‘hit and run’ cassette in the genome of all the regenerated ipt-shooty lines while RT-PCR experiments confirmed the expression of the R gene, encoding the yeast Recombinase. A detailed molecular investigation, carried out to verify the integrity of the RS sites, revealed that these regions were intact in most cases. Our results with barrel medic suggest that the MAT system must be carefully evaluated and discussed on a case by case basis. L. Scaramelli, A. Balestrazzi and M. Confalonieri have contributed equally to this work.     

PCD hallmarks in cell suspension cultures of white poplar (Populus alba L., cv. “Villafranca”) challenged with cadmium

Abstract

The response to oxidative stress was investigated in suspension cultures of Populus alba L. “Villafranca” exposed to cadmium. Cell death was demonstrated by Evans Blue staining. Although DNA laddering was not detected, the nuclear morphology evaluated by DAPI revealed irregularly stained granular nuclei derived from chromatin condensation, a programmed cell death hallmark.     

Nuclease-producing bacteria in soil cultivated with herbicide resistant transgenic white poplars

This study was carried out using soil cultivated, under greenhouse conditions, with transgenic white poplars expressing thebar gene for tolerance to the Basta® herbicide. The occurrence of extracellular nucleolytic activity was monitored in soil samples collected at four different times over a 26-month period. The fraction of nuclease producing bacteria (NPB) ranged from 62.5 to 100% of the total culturable bacterial population. The DNA-methyl green plate assay allowed to distinguish five groups of bacteria showing increasing levels of extracellular DNase activity. The NPB isolates were classified by 16S rDNA sequence analysis as members of theBacillus, Brevibacillus, Microbacterium, Pseudomonas andStenotrophomonas genera. For each genus, NPB isolates were cultured in liquid medium and the nucleolytic activity during different growth phases was monitored. Production of extracellular nucleases was observed only during the mid-exponential growth phase of theBrevibaccillus Microbacterium andStenotrophomonas isolates, while no activity was evidenced for isolates classified within theBacillus andPseudomonas genera.     

DNA extraction from soil: comparison of different methods using spore-forming bacteria and theswrAA gene as indicators

Soil microcosms seeded with spores of a tracer organism (Bacillus subtilis strain PB5332) were used to test five different DNA extraction protocols hereby indicated as A, B, C, D and E. The representativity of DNA samples obtained from each procedure was evaluated by PCR amplification of theswrAA gene, unique to PB5332 strain, followed by Southern hybridization with a gene-specific probe. A significant improvement of DNA extraction from spores was obtained using grinding under liquid N2 associated with sodium-dodecyl sulphate (SDS)-based lysis in presence of 1% hexadecyltrimethylammonium bromide (CTAB; protocol C). The same procedure was tested on soil samples from two distinct greenhouse trials carried out with genetically modified white poplars (Populus alba L) expressing theStSy gene for resveratrol production and thebar gene for Basta® tolerance, respectively. The representativity of DNA samples recovered from the greenhouse soil was assessed using three spore-forming bacteria (SFB) as tracer organisms. The tracers (SFB-1, SFB-2 and SFB-3) were previously isolated from the same trials classified as members of the genusBacillus. All the tested DNA samples produced the expected amplification products, indicating the presence at the soil level of the tracers and confirming the reliability of the optimized DNA extraction protocol.     

Spore-forming bacteria in soil cultivated with GM white poplars: Isolation and characterization

The impact of transgenic white poplars (Populus alba L. cv. ‘Villafranca’) was assessed on the soil aerobic spore-forming bacteria (SFB). The genetically modified poplars, expressing either the StSy gene for resveratrol production or the bar gene for herbicide tolerance, were cultivated in greenhouse. The occurrence of SFB was monitored in soil samples collected at eight different timepoints over a two-year period. The total culturable bacterial population of the StSy and bar trials underwent significant seasonal fluctuations in the range of 10⁶−2.5 × 10⁸ CFU/g dry soil and of 10⁴−5 × 10⁸ CFU/g dry soil, respectively. Changes occurred also within the culturable SFB population with size varying at 10³−5 × 10⁴ CFU/g dry soil and 10²−2 × 10⁵ CFU/g dry soil in the StSy and bar trials, respectively. No significant differences in the size of the total and SFB culturable populations were observed when comparing each transgenic line with the nontransformed control line while seasonal shifts of soil bacterial populations were evident in both trials. The culturable SFB fraction included three isolates (SFB-1, SFB-2 and SFB-3) classified by 16S rDNA sequence analysis as members of the Bacillus genus. According to the reported data, cultivation of both herbicide-resistant and resveratrol-producing GM white poplars did not affect the culturable SFB population at the soil level.     

Recovery of useful traits from isolates inhabiting an agricultural soil cultivated with herbicide-resistant poplars

The aim of this study was to investigate the culturable bacteria living in soil cultivated with Basta-tolerant transgenic white poplars (Populus alba L. 'Villafranca'). Plate Count Agar medium containing phosphinothricin, the active component of Basta, was used to isolate the herbicide-resistant bacteria (HRB). No significant changes in the size of the soil microbial flora following herbicide treatment were observed. The characterization of HRB isolates by 16S rDNA-based taxonomy revealed a predominance of Pseudomonas and Bacillus species. The screening carried out on soil samples allowed for the recovery of isolates with useful properties for biotechnological and agronomical purposes, particularly in relation to root development. Among the tested isolates, only HRB-1b, HRB-1c, and HRB-7 showed remarkable swarming ability, a valuable trait supporting the beneficial plant-microbe interactions. HRB-1c was also characterized by consistent production of indoleacetic acid (17.8 +/- 0.09 microg x mL-1 x (OD600 unit)-1), and it was able to stimulate the in vitro growth of Villafranca explants. Since novel tools are constantly required to enhance productivity of perennial species and to expand their use for practical purposes, the availability of bacteria that support tree growth, such as the HRB-1c isolate, represents a significant advantage.     

Evaluation of MAT-vector system in white poplar (<Emphasis Type="Italic">Populus alba</Emphasis> L.) and production of <Emphasis Type="Italic">ipt</Emphasis> marker-free transgenic plants by ‘single-step transformation’

Genetic transformation of an elite white poplar genotype (Populus alba L., cv. ‘Villafranca’) was performed with MAT vectors carrying the ipt and rol genes from Agrobacterium spp. as morphological markers. The effects associated with the use of different gene promoters and distinct in vitro regeneration protocols were evaluated. Poplar plantlets showing abnormal ipt and rol phenotypes were produced only in the presence of exogenous growth regulators. The occurrence of abnormal ipt and rol phenotypes allowed the visual selection of transformants. The ipt-type MAT vector pEXM2 was used to monitor the activity of the yeast site-specific recombination R/RS system in the transformed white poplar cells. Results from these experiments demonstrated that recombinase-mediated excision events occurred during the early stages of in vitro culture, thus causing the direct production of ipt marker-free transgenic plants with normal phenotype at an estimated frequency of 36.4%. Beside this unexpected finding, transgenic ipt-shooty plants were obtained at a frequency of 63.6% and normal shoots were subsequently recovered after a prolonged period of in vitro culture. Although the transformation efficiency observed in this study, using both ipt and nptII genes as selection markers, was similar to that previously reported with standard vectors carrying only the nptII gene, the easy identification of ipt transformants, the early recombinase-mediated excision events and finally the relatively short time period required to produce ipt marker-free transgenic plants support for the choice of MAT vectors as a reliable strategy for the future production of marker-free GM poplars.