The endophytic microbiota of Citrus limon is transmitted from seed to shoot highlighting differences of bacterial and fungal community structures

Citrus limon (L.) Burm. F. is an important evergreen fruit crop whose rhizosphere and phyllosphere microbiota have been characterized, while seed microbiota is still unknown. Bacterial and fungal endophytes were isolated from C. limon surface-sterilized seeds. The isolated fungi—belonging to Aspergillus, Quambalaria and Bjerkandera genera—and bacteria—belonging to Staphylococcus genus—were characterized for indoleacetic acid production and phosphate solubilization. Next Generation Sequencing based approaches were then used to characterize the endophytic bacterial and fungal microbiota structures of surface-sterilized C. limon seeds and of shoots obtained under aseptic conditions from in vitro growing seedlings regenerated from surface-sterilized seeds. This analysis highlighted that Cutibacterium and Acinetobacter were the most abundant bacterial genera in both seeds and shoots, while Cladosporium and Debaryomyces were the most abundant fungal genera in seeds and shoots, respectively. The localization of bacterial endophytes in seed and shoot tissues was revealed by Fluorescence In Situ Hybridization coupled with Confocal Laser Scanning Microscopy revealing vascular bundle colonization. Thus, these results highlighted for the first time the structures of endophytic microbiota of C. limon seeds and the transmission to shoots, corroborating the idea of a vertical transmission of plant microbiota and suggesting its crucial role in seed germination and plant development

Micropropagation and in vitro culture of Pyrethrum [Chrysanthemum cinerariifolium (Trev.) Vis.]

Pyrethrum (Chrysanthemum cinerariifolium (Trev.) Vis. = Tanacetum cineariaefolium (Trev.) Schultz-Bip.) is a perennial herbaceous plant belonging to the family Asteraceae, native to Albania and the area of former Yugoslavia. It is the only species in the genus Tanacetum having an agronomic importance, although the genus consists of several species producing similar types of bioactive metabolites. The species is grown in order to obtain the insecticidal compounds collectively termed pyrethrins, which are found primarily in the flower heads. In this work we discuss the results found from a worldwide literature review about the micropropagation techniques followed on Pyrethrum, the in vitro culture conditions, and the ex vitro establishment trials under Mediterranean environmental conditions. Many technical problems concerning the propagation of the species seem to have been solved, and detailed protocols are available for an easy and fast propagation by seeds, vegetative splits, stem cuttings (rooted under mist or not), and tissue culture. The first attempts to introduce its cultivation into the semi-arid Mediterranean environments have brought to satisfactory results, and the species may be suggested as a valuable opportunity for the development strategies in new Mediterranean farming systems

Citrus rootstock breeding: response of four allotetraploid somatic hybrids to Citrus tristeza virus induced infections

Four allotetraploid somatic hybrids of citrus, with potential for rootstock improvement, have been evaluated for their response to Citrus tristeza virus (CTV) infection. CTV is the most important viral pathogen affecting citrus production worldwide. Somatic combinations of \u2018Milam\u2019 lemon (Citrus jambhiri Lush.) + Sour orange (C. aurantium L Osb.), Calamondin (C. madurensis Lour.) + \u2018Keen\u2019 sour orange (C. aurantium L.), Calamondin + \u2018Femminello\u2018 lemon (C. limon L. Burm. F.) and Cleopatra mandarin (C. reshni Hort. ex Tan.) + \u2018Femminello\u2019 lemon, were studied. Plants were grafted with CTV-infected BValencia^ sweet orange budwood. Two different CTV strains collected in Sicily, considered as Bmild^ and Bsevere^, were used to inoculate candidate rootstocks. The goal of this work is to select a rootstock alternative to CTV susceptible Sour orange, still the prevalent rootstock in the Mediterranean basin. DAS-ELISA and realtime PCR assays confirmed a decreased level of viral replication in tested somatic hybrids, as compared to the susceptible genotypes sour orange and Citrus alemow. The Calamondin+\u2018Keen\u2019 sour orange genotype did not support any replication in either CTV strain. Somatic hybridization is confirmed to be an effective tool to obtain functionally new rootstocks. Our results can be considered a starting point to open new approaches for the Mediterranean citrus industry. Indeed, the four somatic hybrids have been propagated for agronomical multisite evaluation trials to further assess if their horticultural performance, fruit holding capacity, and soil adaptation are adequate to replace sour orange

BIOTECHNOLOGICAL APPROACHES TO THE GENETIC IMPROVEMENT OF CHRYSANTHEMUM CINERARIAEFOLIUM L.

The interest in sustainable agriculture has increased the demand of plant-derived compounds which can be less toxic both to mammals and to the environment than the synthetic agrochemicals. Chrysanthemum cinerariaefolium L. (Asteraceae), commonly termed pyrethrum, is an economically important crop from highlands of tropical and subtropical regions of the world. It is grown for the extraction of pyrethrins, natural insect repellents of plant origin. Pyrethrins are a mixture of six compounds produced by esterification of two acids (chrysanthemic and pyrethric acid) with three mono-terpene-alcohols (pyrethrolone-5, jasmolone-3 and cinerolone-4). The principale source of pyrethrins are the dried flowers of Chrysanthemum cinerariaefolium. Thanks to the low toxicity to mammals and other warm blooded animals, pyrethrum is the only plant species whose metabolites are currently commercially exploited in insecticides, and its worldwide demand exceeds the supply. Asteraceae species are considered as recalcitrant to successful growth in in vitro condition. Recalcitrance in root or shoot formation or in regeneration are associated to endogenous bacterial contamination, hyperhydricity, and tissue browning, hence studies on in vitro culture of Chrysanthemum cinerariaefolium have been rather limited. The aim of this study was to establish highly reproducible in vitro regeneration systems for an efficient multiplication of Chrysanthemum cinerariaefolium, since the pointing out of an efficient regeneration system could play an important role for the industrial exploitation of this plant. Petiole explants and leaf segments were used for micropropagation, callus induction and protoplast isolation. Sterile seeds were used as plant material source. The ability of petiole cuttings to produce direct shoot buds varied depending upon the different media composition tested for the experiments. Growth rate of shoots as well as root induction from shoots have been periodically analyzed. The in vitro raised plantlets were acclimatized and transferred to greenhouse with 60% success. For protoplast isolation and culture, young leaves of in vitro grown plants were used as initial plant material. Different enzymatic combinations were tested to achieve the highest protoplast release. We were able to recover a reasonable number of viable protoplast for further manipulation at the ploidy level with the aim to enhance the biological activity of Chrysanthemum cinerariaefolium and for the regeneration of novel insecticidal plant germplasm

An integrated proteomic and metabolomic study to evaluate the effect of nucleus-cytoplasm interaction in a diploid citrus cybrid between sweet orange and lemon

Key message: Our results provide a comprehensive overview how the alloplasmic condition might lead to a significant improvement in citrus plant breeding, developing varieties more adaptable to a wide range of conditions. Abstract: Citrus cybrids resulting from somatic hybridization hold great potential in plant improvement. They represent effective products resulting from the transfer of organelle-encoded traits into cultivated varieties. In these cases, the plant coordinated array of physiological, biochemical, and molecular functions remains the result of integration among different signals, which derive from the compartmentalized genomes of nucleus, plastids and mitochondria. To dissect the effects of genome rearrangement into cybrids, a multidisciplinary study was conducted on a diploid cybrid (C2N), resulting from a breeding program aimed to improve interesting agronomical traits for lemon, the parental cultivars \u2018Valencia\u2019 sweet orange (V) and \u2018femminello\u2019 lemon (F), and the corresponding somatic allotetraploid hybrid (V + F). In particular, a differential proteomic analysis, based on 2D-DIGE and MS procedures, was carried out on leaf proteomes of C2N, V, F and V + F, using the C2N proteome as pivotal condition. This investigation revealed differentially represented protein patterns that can be associated with genome rearrangement and cell compartment interplay. Interestingly, most of the up-regulated proteins in the cybrid are involved in crucial biological processes such as photosynthesis, energy production and stress tolerance response. The cybrid differential proteome pattern was concomitant with a general increase of leaf gas exchange and content of volatile organic compounds, highlighting a stimulation of specific pathways that can be related to observed plant performances. Our results contribute to a better understanding how the alloplasmic condition might lead to a substantial improvement in plant breeding, opening new opportunities to develop varieties more adaptable to a wide range of conditions