BackgroundAlthough customized endonucleases [transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs)] are known to be effective agents of mutagenesis in various host plants, newly designed endonuclease constructs require some pre-validation with respect to functionality before investing in the creation of stable transgenic plants.ResultsA simple, biolistics-based leaf epidermis transient expression test has been developed, based on reconstituting the translational reading frame of a mutated, non-functional yfp reporter gene. Quantification of mutation efficacy was made possible by co-bombarding the explant with a constitutive mCherry expression cassette, thereby allowing the ratio between the number of red and yellow fluorescing cells to serve as a metric for mutation efficiency. Challenging either stable mutant alleles of a compromised version of gfp in tobacco and barley or the barley MLO gene with TALENs/RGENs confirmed the capacity to induce site-directed mutations.ConclusionsA convenient procedure to assay the cleavage activity of customized endonucleases has been established. The system is independent of the endonuclease platform and operates in both di- and monocotyledonous hosts. It not only enables the validation of a TALEN/RGEN’s functionality prior to the creation of stable mutants, but also serves as a suitable tool to optimize the design of endonuclease constructs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0118-6) contains supplementary material, which is available to authorized users.

Glucosinolates (GSLs) evolved in Brassicaceae as chemical defenses against herbivores. The GSL content in plants is affected by both abiotic and biotic factors, but also depends on the genetic background of the plant. Since the bitter taste of GSLs can be unfavorable for both livestock and human consumption, several plant varieties with low GSL seed or leaf content have been bred. Due to their lower GSL levels, such varieties can be more susceptible to herbivore pests. However, low GSL varieties may quickly increase GSL levels upon herbivore feeding by activating GSL biosynthesis, hydrolysis, or transporter genes. To analyze differences in herbivore-induced GSL responses in relation to constitutive GSL levels, we selected four Brassica rapa varieties, containing either low or high root GSL levels. Plants were infested either with Delia radicum or Delia floralis larvae. The larvae of both root flies are specialists on Brassica plants. Root samples were collected after 3, 5, and 7 days. We compared the effect of root herbivore damage on the expression of GSL biosynthesis (CYP79A1, CYP83B2), transporter (GTR1A2, GTR2A2), and GSL hydrolysis genes (PEN2, TGG2) in roots of low and high GSL varieties in conjugation with their GSL levels. We found that roots of high GSL varieties contained higher levels of aliphatic, indole, and benzyl GSLs than low GSL varieties. Infestation with D. radicum larvae led to upregulation of indole GSL synthesis genes in low and high GSL varieties. High GSL varieties showed no or later responses than low varieties to D. floralis herbivory. Low GSL varieties additionally upregulated the GSL transporter gene expression. Low GSL varieties did not show a stronger herbivore-induced response than high GSL varieties, which indicates that there is no trade-off between constitutive and induced GSLs.

Customizable endonucleases are providing an effective tool for genome engineering. The resulting primary transgenic individuals (T0) are typically heterozygous and/or chimeric with respect to any mutations induced. To generate genetically fixed mutants, they are conventionally allowed to self-pollinate, a procedure which segregates individuals into mutant heterozygotes/homozygotes and wild types. The chances of recovering homozygous mutants among the progeny depend not only on meiotic segregation but also on the frequency of mutated germline cells in the chimeric mother plant. In Nicotiana species, the heritability of Cas9-induced mutations has not been demonstrated yet. RNA-guided Cas9 endonuclease-mediated mutagenesis was targeted to the green fluorescent protein (GFP) gene harbored by a transgenic tobacco line. Upon retransformation using a GFP-specific guide RNA/Cas9 construct, the T0 plants were allowed to either self-pollinate, or were propagated via regeneration from in vitro cultured embryogenic pollen which give rise to haploid/doubled haploid plants or from leaf explants that form plants vegetatively. Single or multiple mutations were detected in 80% of the T0 plants. About half of these mutations proved heritable via selfing. Regeneration from in vitro cultured embryogenic pollen allowed for homozygous mutants to be produced more efficiently than via sexual reproduction. Consequently, embryogenic pollen culture provides a convenient method to rapidly generate a variety of genetically fixed mutants following site-directed mutagenesis. The recovery of a mutation not found among sexually produced and analyzed progeny was shown to be achievable through vegetative plant propagation in vitro, which eventually resulted in heritability when the somatic clones were selfed. In addition, some in-frame mutations were associated with functional attenuation of the target gene rather than its full knock-out. The generation of mutants with compromised rather than abolished gene functionality holds promise for future approaches to the conclusive functional validation of genes which are indispensible for the plant.