Isolation of protoplasts from the placental cells of Lycopersicum pimpinellifolium Mill

Living protoplasts were isolated from the interplacental regions of Lycopersicum pimpinellifolium berries by the removal of the walls from cells in tissue slices treated for 1–2 h with 10 % pectinase in 0.5 M sucrose solution. Protoplasts thus isolated, then washed and transferred to microculture chambers for observation were invariably spherical. Each protoplast contains a nucleus, a number of chloroplasts of variable shape and a vacuole with smaller vacuoles contained therein. Phase contrast optics reveal cytoplasmic granules, the size of mitochondria, which serve to indicate such dynamic processes as cyclosis. Treatment with ox bile salt and sodium lauryl sulphate cause rapid disruption of the protoplasts producing subprotoplasts and isolated tonoplasts and serves to confirm the absence of rigid cell walls.     

Highly efficient multiplex editing: one-shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

Genome editing by RNA-guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron-optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene-free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T₁ and T₂, respectively). We developed novel counter-selection markers for N. benthamiana, most importantly Sl-FAST2, comparable to the well-established Arabidopsis seed fluorescence marker, and FCY-UPP, based on the production of toxic 5-fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY-UPP for selection of non-transgenic T₁, we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher-order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.     

Stone martens (<Emphasis Type="Italic">Martes foina</Emphasis>) and cars: investigation of a common human–wildlife conflict

Stone martens (Martes foina) commonly climb into car engine compartments, where they may tear up heat and noise insulation mats or bite into rubber or plastic components. This brings martens into conflict with humans. Although various hypotheses have been suggested as to why martens damage cars once inside them, it is not known what makes martens associate with cars in the first place. We radio-tracked 13 urban stone martens over a 2-year period in Luxembourg to determine the context in which martens visit parked cars at night. Martens associated with cars most frequently in spring and early summer, when their activity on roads was highest and when they systematically patrolled and scent-marked cars. Most visits to cars were of short duration. We suggest, therefore, that the main factor promoting marten–car contact is territorial behaviour, rather than either the thermal benefits to be gained from proximity to recently used engines or the need for a safe environment for resting, hiding or food consumption.