Highly Efficient CRISPR/Cas9 Mediated Gene Editing in Ocimum basilicum 'FT Italiko' to Induce Resistance to Peronospora belbahrii.

Ocimum basilicum (sweet basil) is an economically important aromatic herb; in Italy, approximately 1000 ha of "Genovese-type" basil are grown annually in greenhouses and open fields and are subjected to Downy Mildew (DM) disease, caused by Peronospora belbahrii, leading to huge crop losses. Mutation of the Susceptibility (S) gene DMR6 (Downy Mildew Resistant 6) has been proven to confer a broad-spectrum resistance to DM. In this work, an effective Genome Editing (GE) approach mediated by CRISPR/Cas9 in O. basilicum 'Italiko', the élite cultivar used to produce "Pesto Genovese D.O.P", was developed. A highly efficient genetic transformation method mediated by A. tumefaciens has been optimized from cotyledonary nodes, obtaining 82.2% of regenerated shoots, 84.6% of which resulted in Cas9+ plants. Eleven T0 lines presented different type of mutations in ObDMR6; 60% were indel frameshift mutations with knock-out of ObDMR6 of 'FT Italiko'. Analysis of six T1 transgene-free seedlings revealed that the mutations of T0 plants were inherited and segregated. Based on infection trials conducted on T0 plants, clone 22B showed a very low percentage of disease incidence after 14 days post infection. The aromatic profile of all in vitro edited plants was also reported; all of them showed oxygenated monoterpenes as the major fraction.

Characterization and Antioxidant Activity of Essential Oil of Four Sympatric Orchid Species.

The volatile fractions from fresh inflorescences of naturally growing orchids Anacamptis coriophora (L.) R. M. Bateman, Pridgeon & M. W. Chase subsp. fragrans (Pollini), Anacamptis pyramidalis (L.) R. Ophrys holosericea (Burm.) Greuter and Serapias vomeracea (Burm. f.) B. were isolated by steam distillation and analyzed by GC/FID and GC/MS. Saturated hydrocarbons were quantified as the major constituents of the volatile fraction (47.87-81.57% of the total essential oil), of which long-chain monounsaturated hydrocarbons accounted from 9.20% to 32.04% of the total essential oil. Double bond position in linear alkenes was highlighted by dimethyl disulfide derivatization and MS fragmentation. Aldehydes (from 3.45 to 18.18% of the total essential oil), alcohols (from 0.19% to 13.48%), terpenes (from 0.98 to 2.50%) and acids (0.30 to 2.57%) were also detected. These volatiles compounds may represent a particular feature of these plant species, playing a critical role in the interaction with pollinators. DPPH assay evaluating the antioxidant activity of the essential oils was carried out, showing a dose-dependent antioxidant activity.

Experimentally induced subclinical mastitis: are lipopolysaccharide and lipoteichoic acid eliciting similar pain responses?

BACKGROUND: Pain accompanying mastitis has gained attention recently as a relevant welfare compromising aspect of disease. Adequate pain recognition and therapy are necessary in the context of a modern and ethically acceptable dairy care. For research purposes mastitis is often induced by intramammary infusion of immunogenic bacterial cell wall components. Lipopolysaccharide (LPS) from Escherichia coli and lipoteichoic acid (LTA) from Staphylococcus aureus are commonly administered to this end. While the immune response to specific immunogenic components has been well characterized, not much is known about their role on the expression of pain indicators. The aim of this study was to trial the effects of an intramammary challenge of LTA or LPS on the degree of pain and discomfort as indicated by both physiological and behavioral variables in cows. The hypothesis was that a similar degree of pain can be identified in LTA as well as in LPS induced mastitis. RESULTS: On the challenge day, compared to pre-challenge, total pain index increased for all treatment groups (LPS; LTA and control), the LPS group having significantly higher values than the control group (P = 0.01). Similarly, pain visual analogue scale (VAS) increased significantly in all cows following treatment on the challenge day. Furthermore, compared to baseline, higher VAS were found 3, 4 and 5 h after the challenge in cows of the LPS group (P3h, 4h < 0.001 and P5h = 0.001) and 7 h after the challenge in cows of the LTA group (P7h = 0.002). In the control group, VAS was higher 5 h after the challenge (P5h = 0.001). On the challenge day, udder edema was higher in the LPS than in the control group (P = 0.007). Furthermore, 4 h after the challenge, milk cortisol was significantly higher than at baseline in the LPS group (P < 0.001). CONCLUSIONS: When administered at equipotent doses targeting a standard somatic cell count increase, intramammary LPS seems to be accompanied by a higher degree of pain and discomfort than LTA, as suggested by the modifications of the outcome variables total pain index, VAS, udder edema and milk cortisol.

Pollen Grain Preservation and Fertility in Valuable Commercial Rose Cultivars.

In the cut flower market, traditional breeding is still the best way to achieve new rose cultivars. The geographical delocalization of cultivar constitution (generally made in Europe and North America) and plant cultivation (large areas in Africa and South America) represents a limit point for crossing and selection. Rose breeders often need to overcome geographical distances, resulting in asynchrony in flowering among crossing parents, by storing and sending pollen. Hence, a key aspect in breeding programs is linked to pollen availability and conservation, jointly with the identification of parameters related to pollen fertility. In this study we present the results of three different trials. In the first, pollen diameter and pollen viability were chosen as fertility predictors of 10 Rosa hybrida commercial cultivars. In the second trial, aliquots of dried pollen grains of six R. hybrida cultivar were stored under two different temperatures (freezer at T = -20 °C and deep freezer at T = -80 °C) and after a wide range of conservation period, their viability was measured. In the third trial, the effective fertilization capacity of frozen pollen of 19 pollen donor cultivars was evaluated during 2015 crossing breeding plan, performing 44 hybridizations and correlating the number of seeds and the ratio seeds/crossing, obtained by each cultivar, with in vitro pollen germination ability.

Combinations of dexmedetomidine and alfaxalone with butorphanol in cats: application of an innovative stepwise optimisation method to identify optimal clinical doses for intramuscular anaesthesia.

OBJECTIVES: The aim of this study was to optimise dexmedetomidine and alfaxalone dosing, for intramuscular administration with butorphanol, to perform minor surgeries in cats. METHODS: Initially, cats were assigned to one of five groups, each composed of six animals and receiving, in addition to 0.3 mg/kg butorphanol intramuscularly, one of the following: (A) 0.005 mg/kg dexmedetomidine, 2 mg/kg alfaxalone; (B) 0.008 mg/kg dexmedetomidine, 1.5 mg/kg alfaxalone; (C) 0.012 mg/kg dexmedetomidine, 1 mg/kg alfaxalone; (D) 0.005 mg/kg dexmedetomidine, 1 mg/kg alfaxalone; and (E) 0.012 mg/kg dexmedetomidine, 2 mg/kg alfaxalone. Thereafter, a modified 'direct search' method, conducted in a stepwise manner, was used to optimise drug dosing. The quality of anaesthesia was evaluated on the basis of composite scores (one for anaesthesia and one for recovery), visual analogue scales and the propofol requirement to suppress spontaneous movements. The medians or means of these variables were used to rank the treatments; 'unsatisfactory' and 'promising' combinations were identified to calculate, through the equation first described by Berenbaum in 1990, new dexmedetomidine and alfaxalone doses to be tested in the next step. At each step, five combinations (one new plus the best previous four) were tested. RESULTS: None of the tested combinations resulted in adverse effects. Four steps and 120 animals were necessary to identify the optimal drug combination (0.014 mg/kg dexmedetomidine, 2.5 mg/kg alfaxalone and 0.3 mg/kg butorphanol). CONCLUSIONS AND RELEVANCE: The investigated drug mixture, at the doses found with the optimisation method, is suitable for cats undergoing minor clinical procedures.

Exploring the molecular and chemical-physical aspects of low-dose irradiation using radio-tolerant plant cells.

Animals and plants show different levels of radio-sensitivity, with safe dose values in the 0.001-1 and 1-100 Gy range, respectively. The increased radio-tolerance observed in plant cells might represent a valuable tool to investigate the events underlying the low dose (LD) response in the highly radio-sensitive animal cells. The use of radio-resistant plant systems would allow investigation of the LD effects using irradiation conditions that can be easily managed, without the technical constrains currently encountered with animal systems. The basal knowledge on the molecular mechanisms involved in the DNA damage response in plants is rapidly expanding, revealing common features with animal cells at the level of DNA damage sensing/repair, transduction pathways and antioxidant response. The present work provides a short update of the current literature dealing with the DNA damage response in animal and plant cells exposed to LD treatments (ionising radiation, particularly gamma ray) combined with the more recent advances in free radical research.

Single Cell Gel Electrophoresis (Comet) assay with plants: research on DNA repair and ecogenotoxicity testing.

Single Cell Gel Electrophoresis is currently used to investigate the cell response to genotoxic agents as well as to several biotic and abiotic stresses that lead to oxidative DNA damage. Different versions of Single Cell Gel Electrophoresis have been developed in order to expand the range of DNA lesions that can be detected and guidelines for their use in genetic toxicology have been provided. Applications of Single Cell Gel Electrophoresis in plants are still limited, compared to animal systems. This technique is now emerging as a useful tool in assessing the potential of higher plants as stable sensors in ecosystems and source of information on the genotoxic impact of dangerous pollutants. Another interesting application of Single Cell Gel Electrophoresis deals with Mutation Breeding or the combined use of irradiation and in vitro culture technique to enhance genetic variability in elite plant genotypes. SCGE, in combination with in situ detection of Reactive Oxygen Species (ROS) induced by γ-rays and expression analysis of both DNA repair and antioxidant genes, can be used to gather information on the radiosensitivity level of the target plant genotypes.

γ irradiation with different dose rates induces different DNA damage responses in Petunia x hybrida cells.

In plants, there is evidence that different dose rate exposures to gamma (γ) rays can cause different biological effects. The dynamics of DNA damage accumulation and molecular mechanisms that regulate recovery from radiation injury as a function of dose rate are poorly explored. To highlight dose-rate dependent differences in DNA damage, single cell gel electrophoresis was carried out on regenerating Petunia x hybrida leaf discs exposed to LDR (total dose 50 Gy, delivered at 0.33 Gy min(-1)) and HDR (total doses 50 and 100 Gy, delivered at 5.15 Gy min(-1)) γ-ray in the 0-24h time period after treatments. Significant fluctuations of double strand breaks and different repair capacities were observed between treatments in the 0-4h time period following irradiation. Dose-rate-dependent changes in the expression of the PhMT2 and PhAPX genes encoding a type 2 metallothionein and the cytosolic isoform of ascorbate peroxidase, respectively, were detected by Quantitative RealTime-Polymerase Chain Reaction. The PhMT2 and PhAPX genes were significantly up-regulated (3.0- and 0.7-fold) in response to HDR. The results are discussed in light of the potential practical applications of LDR-based treatments in mutation breeding.

Antimicrobial activity and phenolic content of natural site and micropropagated Limonium avei (De Not.) Brullo & Erben plant extracts.

This study reported the antimicrobial activity and phenolic content of natural site and micropropagated Limonium avei (De Not.) Brullo & Erben inflorescences. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ethanolic extracts were determined according to the Clinical Laboratory Standards Institute guidelines. Individual phenolic acids and flavonoids were detected by a high performance liquid chromatography (HPLC-DAD) method. The samples showed a comparable antimicrobial activity, although the natural site extract possessed the lower MIC values. The best activity was detected against Gram-positive bacteria, such as Staphylococcus aureus including methicillin resistant strains (MIC and MBC values ranging from 7.81 to 62.50 µg mL(-1) and from 500 to 2000 µg mL(-1) respectively). In contrast, a low activity was found on Gram-negative bacteria and Candida albicans. The HPLC-DAD analysis revealed ten phenolic acids and four flavonoids with a major amount of m-coumaric acid, naringin and quercetin in the natural site extract.

In vitro regeneration and multiplication of Passiflora hybrid "Guglielmo Betto".

With more than 450 species, Passiflora is the most important genus of the family Passifloraceae. It comprises many species grown for their edible fruits, for their high ornamental value, and further for the therapeutic properties. With their striking exotic flowers, they are of particular interest for the floriculture market. With the aim of evaluating the in vitro propagation of an Italian ornamental hybrid, axillary tendrils of Passiflora "Guglielmo Betto" M. Vecchia (P. incarnata L. x P. tucumanensis L.) were sterilized and placed in vitro. Direct shoot regeneration was achieved from young tendrils cultivated on MS medium containing, either 4.43 microM 6-benzylaminopurine (BAP) and 11.41 microM indoleacetic acid (IAA), or 49.20 microM 6-gamma-gamma-dimethylallylaminopurine (2iP) and 2.68 microM alpha-naphthalene acetic acid (NAA), respectively. In vitro shoot multiplication, rooting, and regenerated plant acclimatization protocols were established.

Effect of the number of rol genes integrations on phenotypic variation in hairy root-derived Hypericum perforatum L. plants.

The extent of phenotypic variation of St. John's wort (Hypericum perforatum L.) plants transformed with wild agropine ATCC 15834 Agrobacterium rhizogenes plasmid was evaluated with respect to the number of rol genes integrations. The transfer of T(L)-DNA to plant explants during each transformation event was incomplete with different rolA, rolB, and rolC copy numbers. Along with typical features representing the hairy root syndrome, an altered size, number and density of dark and translucent glands, changes in ability to synthesize secondary metabolites, and reduced fertility were observed. The highest copy number of transferred rol genes resulted in weak expression of transgenic character and comparable quantitative parameters with the controls. Only 1 out of 11 transgenic clones was able to produce seed progeny and not more than 4 out of its 35 offsprings were positive for rolC gene integration. Sterility of the clones was due to retarded development of both gametophytes.

Hairy root cultures for secondary metabolites production.

Hairy roots (HRs) are differentiated cultures of transformed roots generated by the infection of wounded higher plants with Agrobacterium rhizogenes. This pathogen causes the HR disease leading to the neoplastic growth of roots that are characterized by high growth rate in hormone free media and genetic stability. HRs produce the same phytochemicals pattern of the corresponding wild type organ. High stability and productivity features allow the exploitation of HRs as valuable biotechnological tool for the production of plant secondary metabolites. In addition, several elicitation methods can be used to further enhance their accumulation in both small and large scale production. However, in the latter case, cultivation in bioreactors should be still optimized. HRs can be also utilised as biological farm for the production of recombinant proteins, hence holding additional potential for industrial use. HR technology has been strongly improved by increased knowledge of molecular mechanisms underlying their development. The present review summarizes updated aspects of the hairy root induction, genetics and metabolite production.

Agrobacterium rhizogenes-mediated transformation of Hypericum tomentosum L and Hypericum tetrapterum Fries.

This is the first evidence on successful Agrobacterium rhizogenes-mediated genetic transformation of two species from the genus Hypericum, H. tomentosum L. and H. tetrapterum Fries. Hairy root cultures were induced from root segments of both Hypericum species by two agropine wild-type strains of A. rhizogenes, ATCC 15834 and A4. The transgenic character of the hairy root cultures was proved by PCR amplification of the rolABCD genes. In some H. tetrapterum transgenic lines aux genes were detected as well.

Bioactive constituent production in St. John's Wort in vitro hairy roots. Regenerated plant lines.

A wild strain of Agrobacterium rhizogenes was used to regenerate twelve in vitro plant lines from different hairy roots of H. perforatum (St. John's Wort). The production of the main bioactive constituents was observed even though their yields varied in the different plant lines. Two lines were selected for the hyperoside production (4.9-4.6 mg/gdw) while nine were characterized by significant yields of chlorogenic acid (ranged from 0.47 to 1.09 mg/gdw). Furthermore, one out of twelve lines showed a 10-fold higher hypericin content (0.25 mg/gdw) than that reported for the in vitro shoots in the literature. Morphological and phytochemical features were determined in order to select H. perforatum genotypes enriched in valuable bioactive compounds.