CRISPR-Cas Technology in Plant Science

CRISPR-Cas technology has raised considerable interest among plant scientists, both in basic science and in plant breeding. Presently, the generation of random mutations at a predetermined site of the genome is well mastered, just like the targeted insertion of transgenes, although both remain restricted to species or genotypes amenable for plant transformation. On the other hand, true genome editing, i.e. the deliberate replacement of one or several nucleotides of the genome in a predetermined fashion, is limited to some rather particular examples that generally concern genes allowing positive selection, for example tolerance to herbicides. Therefore, further technological developments are necessary to fully exploit the potential of genome editing in enlarging the gene pool beyond the natural variability available in a given species. In principle, the technology can be applied to any quality related, agronomical or ecological trait, under the condition of upstream knowledge on the genes to be targeted and the precise modifications necessary to improve alleles. Published proof of concepts concern a wide range of agronomical traits, the most frequent being disease resistance, herbicide tolerance and the biochemical composition of harvested products. The regulatory status of the plants obtained by CRISPR-Cas technology raises numerous questions, in particular with regard to the plants that carry in their genomes the punctual modifications caused by the presence of the Cas9 nuclease but not the nuclease itself. Without clarification by the competent authorities, CRISPR-Cas technology would continue to be a powerful tool in functional genomics, but its potential in plant breeding would remain untapped.     

System for quasi‐continuous simultaneous measurement of oxygen diffusion rate and redox potential in soil

Oxygen diffusion rate (ODR) and redox potential (E_H) are quantitative indices representing oxygen availability and redox status in soils, which is valuable information for better understanding causes and effects of soil aeration. Because these indices are spatially and temporally highly variable, continuous measurements and adequate numbers of repetitions are essential for accurate in situ monitoring. Here, we present a new, fully automated recording system for in situ measurements where ODR and E_H are measured at the same platinum electrode. The conflict between electrode polarization for ODR and the resulting biased E_H readings is solved by reducing the polarization time and introducing a recovery interval between two consecutive measurement cycles. The shorter polarization time ensures accurate E_H readings. It also results in moderately overestimated ODR readings, but this can be corrected before data analysis. The recovery interval restricts temporal resolution of the E_H‐ODR data pairs to 8 h. We illustrate the use of the system with measurements in a field experiment in Zürich, Switzerland. ODR curves at different depths ran roughly parallel to the corresponding curves of O₂ concentration in soil air but ODR was much more sensitive to precipitation. Low ODR was a necessary but not a sufficient condition for declining E_H. E_H ran parallel to O₂ concentration in soil air rather than to ODR. The fully automated system allows for time series of replicate measurements in multifactorial field studies with reasonable labor requirements. It may be particularly suitable for studies examining the effects of soil tillage, compaction, and irrigation, where structure‐related soil properties such as porosity, gas permeability, and soil aeration play a dominant role.     

Comparative proteomic analysis of mushroom cell wall proteins among the different developmental stages of Pleurotus tuber-regium

Cell wall proteins (CWPs) play a vital role in the development of the different morphological stages including mycelium, fruiting body, and sclerotium in mushrooms which are important human food sources. Using fractionation by detergents and reducing agents, mushroom cell wall fractions from the different developmental stages of Pleurotus tuber-regium (PTR) were prepared. Using one-dimensional gel electrophoresis coupled with LC-MS, there were 103, 91, and 48 noncovalently linked CWPs identified in the cell wall fractions of the PTR mycelium, fruiting body, and sclerotium, respectively. Comparing the CWPs in these cell wall fractions, 19 of them were in common, among which 17 belonged to the functional categories of translation, ribosomal structure, and biogenesis. This is the first study to provide important biochemical insights into the different developmental stages of PTR mediated by CWPs, and the identified CWPs helped to explain the morphological changes of PTR mushrooms during cultivation.     

Fecal steroid analysis for evaluating ovarian function in the greater kudu (Tragelaphus strepsiceros) and lesser kudu (Tragelaphus imberbis)

Seasonal reproductive-endocrine norms have not been described for the genus Tragelaphus, which consists of seven species of African antelope. Longitudinal patterns of progesterone metabolite excretion were assessed by radioimmunoassays in fecal samples collected noninvasively (three to seven samples per week) from greater kudu (Tragelaphus strepsiceros, n = 4) and lesser kudu (Tragelaphus imberbis, n = 4). Progesterone metabolite excretion patterns revealed seasonal estrous cycles in both species, and discrimination of pregnant versus nonpregnant females was achieved in lesser kudu. These data reveal the value of fecal progesterone metabolites for establishing reproductive-endocrine norms in both zoo-maintained and free-living antelopes of the genus Tragelaphus.     

Hair coat contamination with zoonotic helminth eggs of farm and pet dogs and foxes

Infections of dogs with Toxocara canis and Echinococcus multilocularis pose an infection-risk particularly for contact persons. We examined specimens of hair coat and faeces of 124 farm dogs, 118 household dogs, 49 kennel dogs, 15 puppies from two litters, and 46 red foxes. Microscopically identified eggs of Toxocara or taeniids were further investigated by species-specific PCRs. In farm dogs, eggs of E. multilocularis or T. canis were identified in each 2.4% of faecal samples, eggs of T. cati (gastrointestinal passage) in 7.3%, respectively. Household dogs excreted eggs of T. canis (0.8%) and of T. cati (2.5%). In kennel dogs, eggs of T. canis (4.1%), but not of T. cati were detectable. Coat samples contaminated with eggs of Toxocara spp. were found from farm dogs (5.6%), household dogs (1.7%) and kennel dogs (2.0%). Taeniid eggs were isolated from the coat samples from only two farm dogs (1.6%); a molecular species determination was not achieved. In six intrauterinely infected puppies, Toxocara-eggs were found in 17/38 samples taken within six weeks. No intact Toxocara eggs could be isolated from the coat of nine puppies from a second litter 13 days after deworming. Of the 46 red foxes investigated (dissection and faecal samples) 13 (28.3%) were infected with E. multilocularis and 20 (43.5%) with Toxocara. Eggs of taeniids and Toxocara were found in 13% (in three cases confirmed as E. multilocularis) and 21.7%, respectively, of the coat samples. None of the retrieved Toxocara eggs in the coat samples were embryonated. Thus, an infection of humans through the transmission of E. multilocularis eggs after direct contact with dogs or foxes is conceivable, whereas a corresponding infection risk by Toxocara eggs must be critically challenged.     

Molecular detection of BHV-1 in artificially inoculated semen and in the semen of a latently infected bull treated with dexamethasone

Two polymerase chain reaction (PCR) assays specific for glycoprotein B (gB) and glycoprotein E (gE) gene detection, respectively, were adopted for the detection of bovine herpesvirus-1 (BHV-1) in naturally infected bulls. The methods were tested on bovine semen artificially inoculated with BHV-1 and were compared with an optimised virus isolation method. Raw and extended semen samples were diluted in minimal essential medium (MEM) and spiked with equal dose of BHV-1. The extended semen was found to be more toxic for the cells than the raw semen, while the viral DNA could be detected by the PCR method in all tested dilutions of raw and extended semen samples. The sensitivity of both methods was compared also for BHV-1 detection in semen, nasal swabs and leucocytes of a seropositive bull in a different time period after virus reactivation with dexamethasone treatment. The sensitivity of virus detection by the PCR method was equivalent to that of virus isolation in cell culture. However, PCR was shown to be faster and easier to perform and may be a good alternative to virus isolation especially when bovine semen has to be screened for BHV-1 prior to artificial insemination.