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Background . Genetic modification of plants is one of the promising strategies to increase their resistance to insect pests. The development of metabolic or RNA interference systems for plant protection requires appropriate models of host-insect interactions. Nicotiana tabacum L. is a classical model plant used in molecular and metabolic engineering. We consider tobacco as a model for developing protective strategies against Colorado potato beetle ( Leptinotarsa decemlineata Say, CPB). Normally, tobacco is toxic for CPB due to high content of nicotine and related alkaloids in leaves. Modification of the tobacco genome could provide tobacco genotypes with altered metabolism suitable for CPB feeding. It is known that different mutations in Berberine Bridge-Like (BBL) genes cause different alterations in tobacco leaf alkaloid levels. In the current study, the Cas9/gRNA system targeting members of the BBL gene family of tobacco was used to create a line which can serve as a diet for CPB. Results . In order to obtain tobacco with modified alkaloid content, two gRNAs matching target sequences in six BBL genes were selected. Each gRNA was cloned into a gRNA/Cas9 generic vector. The created constructs were mixed and used for biolistic transformation of tobacco leaf explants together with the pBI121 plasmid harboring the kanamycin resistance gene nptII and the reporter E.coli betaglucuronidase (GUS) gene. Regenerants were selected on 100 mg/l of kanamycin and checked for transgene presence by histochemical GUS-assay. Unexpectedly, the regenerated plants displayed a variety of adverse phenotypic effects including different degree of growth and rooting inhibition, early flowering, increased number of internodes, changes in leaf shape, fusion of flowers, longostyly, and partial sterility. Only one from seven obtained calli produced a population of regenerated plants without severe phenotypic abnormalities. The NtaBBL5-14 line of clonally propagated plants was selected from this population and used for a CPB feeding experiment. It was shown that CPB larvae consume the leaves of NtaBBL5-14 line ten times more efficiently than the leaves of control plants (97±0.5% vs. 9±3% in 24 h respectively). Conclusion . The NtaBBL5-14 tobacco line is suitable for CPB feeding and can be further used as a model for studies in plant-pest interaction. The modification of other genes regulating nicotine metabolism can be a promising strategy to obtain tobacco plants edible for CPB with less pleiotropic effects.

The ability to generate (doubled) haploid plants significantly accelerates the crop breeding process. Haploids have been induced mainly through the generation of plants from cultivated gametophic (haploid) cells and tissues, i.e., in vitro haploid technologies, or through the selective loss of a parental chromosome set upon inter- or intraspecific hybridization. Here, we focus our review on the mechanisms responsible for the in vivo formation of haploids in the context of inter- and intraspecific hybridization. The application of a modified CENH3 for uniparental genome elimination, the IG1 system used for paternal as well as the BBM-like and the patatin-like phospholipase essential for maternal haploidy induction are discussed in detail.

Many plant genomes display high levels of repetitive sequences. The assembly of these complex genomes using short high-throughput sequence reads is still a challenging task. Underestimation or disregard of repeat complexity in these datasets can easily misguide downstream analysis. Detection of repetitive regions by k-mer counting methods has proved to be reliable. Easy-to-use applications utilizing k-mer counting are in high demand, especially in the domain of plants. We present Kmasker plants, a tool that uses k-mer count information as an assistant throughout the analytical workflow of genome data that is provided as a command-line and web-based solution. Beside its core competence to screen and mask repetitive sequences, we have integrated features that enable comparative studies between different cultivars or closely related species and methods that estimate target specificity of guide RNAs for application of site-directed mutagenesis using Cas9 endonuclease. In addition, we have set up a web service for Kmasker plants that maintains pre-computed indices for 10 of the economically most important cultivated plants. Source code for Kmasker plants has been made publically available at https://github.com/tschmutzer/kmasker. The web service is accessible at https://kmasker.ipk-gatersleben.de.