Centaurium erythraea, used as medical plant from the earliest times, is an immense depot of quite rare bioactive compounds. Secoiridoids (sweroside, swertiamarin, and gentiopicrin) and xanthones (methylbellidifolin and decussatin) are predominant bioactive compounds in C. erythraea. The present study aims at providing new insights into how the content of these bioactive principles can be related with plant ploidy level by characterizing possible differences in their biosynthesis and accumulation between diploid and tetraploid genotypes from both spatial and temporal aspects. In general, shoots are determined as the major site of secoiridoids' and xanthones' accumulation, whose ratio vary during the development. Genes involved in iridoid and xanthone metabolic pathways were found to be coordinately regulated at the transcriptional level both during the development and among organs. Biosynthetic gene expression levels were found highly correlated with the content of major compounds from these two classes. Diversification in chemical profiles between tetraploid and diploid genotypes may result from the expression difference between homologous loci correspondent to several key biosynthetic genes, which trigger changes in the two metabolic routes. Thus, enhanced expression of genes coding for geraniol synthase (GES), 8-hydroxygeraniol oxidoreductase (8HGO), and 7-deoxyloganic acid hydrolase (7DLH2) is strongly associated with intensive production of iridoids. Interestingly, transcript levels of beta-glucosidase (CebGLU), a candidate to catalyze the first step in the secoiridoid catabolism, is significantly positively correlated with the content of major secoiridoids. Elevated expression of genes coding for benzophenone synthase (BS) and 3-hydroxybenzoate:CoA ligase (3HBL) appear to account for enhanced production of hexa-substituted xanthones. Regarding content of iridoids and xanthones, a diploid genotype appeared to be more productive than a tetraploid genotype under controlled in vitro conditions, therewithal displaying significantly higher biomass.

The predominantly allogamous Broad-leaved Helleborines (Epipactis helleborine) and the predominantly autogamous Narrow-lipped Helleborines (Epipactis leptochila) represent two taxonomically complex groups with great morphological diversity and close genetic relationships at the subspecies level. They are influenced by environment, clinal variability, hybridization between allogamous taxa, and in particularly by speciation through transitions from allogamy to autogamy. We analysed floral traits, genome size and performed molecular analyses (Internal transcribed spacer region, ITS; Amplified Fragment Length Polymorphism, AFLP) of several individuals of E. helleborine and E. leptochila aggregate from different geographical regions in Slovenia, focusing on intermediate traits of E. helleborine subsp. moratoria, E. helleborine subsp. leutei and E. leptochila subsp. neglecta. In order to successfully distinguish studied taxa, the main attention should be paid to the shape of the epichil and the ovary, as well as to the size ratio of sepals and petals. Among the microfloral characters, the shape and size of the lower margin of the stigma and staminodes are found to be supportive. E. neglecta clearly shows intermediate floral characteristics between E. leptochila and E. leutei, suggesting that the transition to autogamy evolved E. neglecta as an intermediate stage in the speciation process, coinciding with the colonisation of forest sites with lower light intensity. Analysis of genome size revealed significant differences, with E. neglecta having the greatest value. Using molecular markers, we were able to determine two main ribotype clusters within the E. helleborine group, which were also determined by AFLP analyses. E. leutei and E. neglecta formed two clearly defined subclusters within the E. leptochila lineage. The results of the presented study suggest that combining molecular markers and genome size with information on how variation in floral morphology is distributed, using multivariate analyses, is the most reliable way to gain improved insight into the relationships among the taxa analysed.

Hybrid vigor has been demonstrated in the early 20th century after hybrid corn was invented. Procedures to breed hybrid cultivar are more complex than breeding open-pollinated cultivars or pure lines. Hybrid cultivars are most often based on the crossing of two true breeding lines that should genetically complement each other. This complementarity of genotypes of genetically different parental lines in the F-1 hybrid often results in a considerable improvement of e.g. growth characteristics, yield or adaptation to environmental stresses as compared to the individual parental lines and non-hybrid cultivars. Numerous methods have been proposed, the most common is based on development of large numbers of inbred lines originated form heterozygous parents by either self pollination or doubled haploid induction. Inbred lines are then tested for hybrid vigor often in two steps, by which first "general combining ability" and then "specific combining ability" is tested to identify a pair that expresses optimal characteristics. This standard procedure allows only a very limited number of line to line crosses since it is laborious and long lasting. Here we are proposing a new method that allows much higher number of line to line crosses as previously known. Our invention relates to innovative method of testing combining ability of inbred lines, which is based on genotyping of each genetically diverse inbred line followed by allowing the plants of the donor lines to intercross to obtain F-1 hybrid progeny. In the next season progeny is phenotyped on individual basis. For superior individuals parental lines are revealed by paternity testing, which due to homozygosity of lines and previous genotyping allows identifying both parental lines. By this method a much larger number of line to line testing can be achieved thus overcoming major bottleneck in breeding hybrid cultivars. Details of the protocol and its utilization will be discussed.

A correction to this paper has been published: https://doi.org/10.1038/s41587-020-00800-8

Some wild, morphologically diverse taxa of the genus Iris in the broad Alpine-Dinaric area have never been explored molecularly, and/or have ambiguous systematic status. The main aims of our research were to perform a molecular study of critical Iris taxa from that area (especially a narrow endemic accepted species I. adriatica, for which we also analysed genome size) and to explore the contribution of eight microsatellites and highly variable chloroplast DNA (ndhJ, rpoC1) markers to the understanding of the Iris taxa taxonomy and phylogeny. Both the microsatellite-based UPGMA and plastid markers-based maximum likelihood analysis discriminated three main clusters in the set of 32 analysed samples, which correspond well to the lower taxonomic categories of the genus, and support separate status of ambiguous regional taxa (e.g., I. sibirica subsp. erirrhiza, I. x croatica and I. x rotschildii). The first molecular data on I. adriatica revealed its genome size (2C = 12.639 ± 0.202 pg) and indicated the existence of ecotypes. For future molecular characterisation of the genus we recommend the utilisation of microsatellite markers supplemented with a combination of plastid markers.

EMBO Reports (2019) e48036 The cultivation of genetically modified (GM) crops in the EU remains a highly polemic issue. The only GM crop event that is currently authorised is the insect‐resistant maize “MON810”. The GM potato variety “Amflora” with improved tuber starch composition was approved for cultivation in 2010, but later withdrawn. One of the main reasons that not more GM crops are authorised for cultivation is a regulatory gridlock with a recurring inability to reach a qualified majority in the designated committee for either approval or rejection [1]. Several EU member states experience domestic pressure against adoption of GM crops [2], [3], [4]. The European Commission (EC) therefore developed legislation—the Directive EU 2015/412, adopted by the European Parliament (EP) in 2015—to give member states the possibility to restrict or prohibit cultivation of authorised GM crops in their territory (opt‐out mechanism). The request to exclude a particular GM event from cultivation may be communicated to the EC after risk assessment, or after authorisation provided that the restriction is in conformity with the EU law, reasoned, proportional, non‐discriminatory and based on compelling grounds (Article 26b(3) of Directive 2015/412). To date, 17 member states and two autonomous regions have used this possibility. In parallel, the EC also proposed an analogous mechanism for the import of GM food and …

The present study was undertaken to evaluate efficiency of callogenesis and regeneration by somatic embryogenesis of the subendemic Iris species Iris illyrica from Croatia and to select highly regenerative donor plants/genotypes. Leaf base segments were used as explants. Callogenesis and somatic embryogenesis were induced on MS media supplemented with: (1) 4.52 micromole 2,4-dichlorophenoxyacetic acid (2,4-D) + 4.83 micromole 1-naphthaleneacetic acid (NAA) + 0.46 micromole kinetin (Kin); (2) 4.52 micromole 2,4-D + 4.6 micromole Kin; (3) 13.4 micromole NAA + 2.3 micromole Kin. Transfer of embryogenic calli onto hormone-free medium enabled the development of mature somatic embryos. Frequency of callogenesis was influenced by the donor plant. Among 15 donor plants tested, 3 of them exhibited high regeneration capability and produced 87 regenerants. Seven morphological traits were observed in order to assess phenotypic variability of flowering regenerants. Regenerants with higher values of fall and standard width and length show potential for further breeding of new varieties of Illyrian iris. Flowering and non-flowering regenerants had the same ploidy level as donor plants. Also, the nuclear DNA content of this species was estimated for the first time using flow cytometry (2C = 12.936 ± 0.038 pg).

The present study was undertaken to evaluate efficiency of callogenesis and regeneration by somatic embryogenesis of the subendemic Iris species Iris illyrica from Croatia and to select highly regenerative donor plants/genotypes. Leaf base segments were used as explants. Callogenesis and somatic embryogenesis were induced on MS media supplemented with: (1) 4.52 M 2,4-dichlorophenoxyacetic acid (2,4-D) + 4.83 M 1-naphthaleneacetic acid (NAA) + 0.46 M kinetin (Kin); (2) 4.52 M 2,4-D + 4.6 M Kin; (3) 13.4 M NAA + 2.3 M Kin. Transfer of embryogenic calli onto hormone-free medium enabled the development of mature somatic embryos. Frequency of callogenesis was influenced by the donor plant. Among 15 donor plants tested, 3 of them exhibited high regeneration capability and produced 87 regenerants. Seven morphological traits were observed in order to assess phenotypic variability of flowering regenerants. Regenerants with higher values of fall and standard width and length show potential for further breeding of new varieties of Illyrian iris. Flowering and non-flowering regenerants had the same ploidy level as donor plants. Also, the nuclear DNA content (2C = 12.936 ± 0.038 pg) of this species was estimated for the first time using flow cytometry.

Testing inbred lines for their combining ability is, due to high numbers of line to line testing needed for determination of hybrid performance, the most limiting factor in the F1 hybrid breeding procedure. We propose a novel method of F1 hybrid breeding that enables evaluation of large number of line to line crosses for their hybrid performance. Inbred lines (preferably doubled haploid - DH) are produced from heterozygous populations, genotyped and maintained. A group of lines is inter-pollinated randomly and their progeny examined. To identify elite F1 hybrids, these individual plants are selected by their superior phenotypic characteristics. Finally using paternity testing only of selected hybrids, the origin of paternal lines is revealed. To predict the number of F1 offspring needed in relation to the number of inbred lines being inter-pollinated, a mathematical formula was developed. For instance, using this formula for the inter-pollination of 60 distinct lines, the probability of obtaining all descendants of paternal-parent lines in a maternal-parent row represented at least once is achieved with 420 F1 plants in a row (p = 0.95). In a practical experiment with white cabbage, DH lines were produced using microspore culture; plants were grown to maturity and genotyped at eight polymorphic SSR loci. Two groups of lines (36 and 33 lines per group) were inter-pollinated by two methods, either using cage pollination with bumblebees or using open pollination in isolated field. A total of 9,858 F1 plants were planted and based on their phenotypic characteristics 213 were selected as elite phenotypes. 99 of them were genetically diverse and 5 of them were selected as super elite. Selected plants were analysed by the same SSR markers and the paternal origin of selected F1 plants was determined. Out of 213 selected elite plants 48 were reciprocals thus exhibiting power of selection based on single plant. We demonstrate that this new approach to hybrid development is efficient in white cabbage and we propose breeders to test it in various vegetable and crop species. Moreover, some other aspects of the proposed technique need to be tested and verified both for practical and economic criteria.

CRISPR/Cas9 is a versatile and highly efficient genome editing tool used in many different plant species. In the present study, we compared the two most commonly used transient expression methods for genome editing, protoplast transfection and infiltration of Agrobacterium tumefaciens, to develop a rapid and efficient validation protocol. Vectors designed to target four different sites in the cabbage genome (two of which were model target genes and two related to the centromere-specific histone H3 (CENH3) gene) were delivered to two red cabbage cultivars, ‘Huzaro F1’ and ‘Rebecca F1’. Targeted deep sequencing analysis showed that CRISPR/Cas9 vectors induced mutations in both cultivars at all target sites and revealed mutation rates of 1.27–11.95% for protoplast transfection and 0.07–14.42% for agroinfiltration. Our results demonstrate successful genome editing in cabbages with CRISPR/Cas9 by two different approaches for the rapid evaluation of genome editing efficiency. Comparison of two different transient transformation methods for the validation of sgRNA in red cabbage (B. oleracea var. capitata f. rubra).

Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.), a plant species endemic to the east Adriatic coast, is used worldwide for production of the organic insecticide, pyrethrin. Most studies concerning Dalmatian pyrethrum have focused on its morphological and biochemical traits relevant for breeding. However, little is known about the chromosomal evolution and genome organization of this species. Our study aims are to identify, classify, and characterize repetitive DNA in the T. cinerariifolium genome using clustering analysis of a low coverage genomic dataset. Repetitive DNA represents about 71.63% of the genome. T. cinerariifolium exhibits linked 5S and 35S rDNA configuration (L-type). FISH reveals amplification of interstitial telomeric repeats (ITRs) in T. cinerariifolium. Of the three newly identified satellite DNA families, TcSAT1 and TcSAT2 are located subterminally on most of T. cinerariifolium chromosomes, while TcSAT3 family is located intercalary within the longer arm of two chromosome pairs. FISH reveals high levels of polymorphism of the TcSAT1 and TcSAT2 sites by comparative screening of 28 individuals. TcSAT2 is more variable than TcSAT1 regarding the number and position of FISH signals. Altogether, our data highlights the dynamic nature of DNA sequences associated with subtelomeres in T. cinerariifolium and suggests that subtelomeres represent one of the most dynamic and rapidly evolving regions in eukaryotic genomes.

The CRISPR/Cas9 genome editing system has already proved its efficiency, versatility and simplicity in numerous applications in human, animal, microbe and plant cells. Together with the vast amount of genome and transcriptome databases available, it represents an enormous potential for plant breeding and research. Although most changes produced with CRISPR/Cas9 do not differ from naturally occurring mutations, the use of transgenesis during varietal development can still trigger GMO legislation in countries that rely on process-based regulation. Moreover, stable integration of DNA coding for genome-editing tools into plant genomes can result in insertional mutagenesis, while its prolonged expression can cause mutations in off-target sites. These pitfalls can be avoided with the delivery of preassembled ribonucleoprotein complexes (RNPs) composed of purified recombinant enzyme Cas9 and in vitro-transcribed or synthesized sgRNA. We therefore aimed to develop a DNA-free protocol for site-directed mutagenesis of three species of the genus Brassica (B. oleracea, B. napus, and B. rapa) with the use of RNPs. We chose cabbage, rapeseed and Chinese cabbage as species representatives and introduced RNPs into their protoplasts with PEG 4000. Four sgRNAs targeting two endogenous genes (the FRI and PDS genes, two sgRNAs per gene) were introduced into all three species. No mutations were detected after transfection of rapeseed protoplasts, while we obtained mutation frequencies of 0.09 to 2.25% and 1.15 to 24.51% in cabbage and Chinese cabbage, respectively. In both species, a positive correlation was displayed between the amount (7.5, 15, 30, and 60 μg) of Cas9 enzyme and sgRNA introduced and mutation frequency. Nucleotide changes (insertions and deletions) were detected 24 h after transfection and did not differ 72 h after transfection. They were species-, gene- and locus-dependent. In summary, we demonstrated the suitability of RNP transfection into B. oleracea and B. rapa protoplasts for high-efficiency indel induction of two endogenous genes. Due to the relatively high mutation frequencies detected (up to 24.51%), this study paves the way for regeneration of precisely mutated Brassica plants without the use of transgenesis.

Protocols leading to the development of doubled haploid (DH) lines by microspore culture are widely used in white cabbage (Brassica oleracea var. capitata L.), but efficiency varies according to the cultivar and induction procedure. Forty different genotypes consisting of F1 cultivars and their crosses with responsive doubled haploid lines were tested to evaluate the androgenic response. In total, 20,032 embryos were produced. On average, the haploid induction response of F1 cultivars was 7.0 embryos/Petri dish, but the average of these hybrids crossed to responsive DH lines was 26.6 embryos/Petri dish. In seven reciprocal crosses, a difference was observed in just one, meaning that the maternal effect probably has a minor influence on haploid embryogenesis in cabbage. Addition of 0.02% activated charcoal (AC) to the induction media increased embryo formation in several low-responsive genotypes, but its effect on embryo formation of high-responsive genotypes was predominantly negative, although larger embryos were formed on media containing AC than without AC. Further development into plantlets was tested by two procedures. Formed embryos were either transferred directly to regeneration medium or treated with abscisic acid and desiccated for 4 weeks. Regrowth and further development reached on average 15.5 and 57.6%, for the first and second procedures, respectively. Plantlets developed by direct transfer often exhibited abnormal development or hyperhydricity, unlike the desiccated embryos. Spontaneous diploidisation of embryos reached 42.5% in total and was not affected by AC added to the induction media.

Freshly released pollen of black elderberry (Sambucus nigra L.) was incubated under various culture conditions until germination was achieved. Optimal conditions for germination were determined and used for maturation of unicellular microspores in vitro. Staining with 5-diphenyltetrazolium bromide, propidium iodide and iodine potassium iodide was used to assess pollen viability, nuclear phase and maturation, respectively. The germination rate was highest when fresh pollen was agitated at 40 rpm in Petri dishes containing a liquid medium consisting of Brewbaker and Kwack salts, 15% (w/v) sucrose, 500 mg/l MES sodium salt, at pH 5.0; germination reached nearly 70% after only 1 h in culture. Under these conditions, and with addition of 200 mg/l glutamine, 260 mg/l cytidine and 500 mg/l uridine, uninucleate microspores developed into mature pollen at a 12% germination rate. Our report is the first demonstration of maturation of S. nigra microspores in vitro.

Plant breeders’ rights are undergoing dramatic changes due to changes in patent rights in terms of plant variety rights protection. Although differences in the interpretation of »breeder’s exemption«, termed research exemption in the 1991 UPOV, did exist in the past in some countries, allowing breeders to use protected varieties as parents in the creation of new varieties of plants, current developments brought about by patenting conventionally bred varieties with the European Patent Office (such as EP2140023B1) have opened new challenges. Legal restrictions on germplasm availability are therefore imposed on breeders while, at the same time, no practical information on how to distinguish protected from non-protected varieties is given. We propose here a novel approach that would solve this problem by the insertion of short DNA stretches (labels) into protected plant varieties by genetic transformation. This information will then be available to breeders by a simple and standardized procedure. We propose that such a procedure should consist of using a pair of universal primers that will generate a sequence in a PCR reaction, which can be read and translated into ordinary text by a computer application. To demonstrate the feasibility of such approach, we conducted a case study. Using the Agrobacterium tumefaciens transformation protocol, we inserted a stretch of DNA code into Nicotiana benthamiana. We also developed an on-line application that enables coding of any text message into DNA nucleotide code and, on sequencing, decoding it back into text. In the presented case study, a short command line coding the phrase »Hello world« was transformed into a DNA sequence that was inserted in the plant genome. The encoded message was reconstructed from the resulting T1 seedlings with 100 % accuracy. The feasibility and possible other applications of this approach are discussed.

Modern hybrid maize varieties dominated for several decades both in Slovenia and elsewhere in the world. The production is maintained only of few landrace populations, but some are maintained at Slovenian Gene Bank. Improvement of local varieties is possible by recurrent selection and may in the case of their specific characteristics deserve their cultivation for specific purposes. Presented experiments have been conducted to characterize selected old Slovenian accessions in two regions - in central Slovenia and in Bela Krajina. Yields of corn at both locations and both years were significantly depending on the genotype. 'Rdeča Bohinjka' and partially 'Rumena Bohinjka' and, similarly, the 'LJ 180' had a predominantly low yields but were also the earliest, while modern hybrid variety 'P 9074' and 'Ronaldinio KWS ' had the expected highest yield. Yield of 'P 9074' grown at the Laboratory field of the Biotechnical Faculty was almost twice higher than the second best accession 'Metliška Plut'. However it is this semi-flint accessions 'Metliška Plut' that proved very suitable at all four experiments and is recommended for further maintenance and varietal testing as it can serve as appropriate alternative in a less intensive production.

Species within the genus Centaurium readily hybridize and polyploid complexes are often seen in natural populations. We describe phytochemical profiles of newly discovered allohexaploid hybrid, here named Centaurium pannonicum, and its parental tetraploid species C. erythraea and rare C. littorale ssp. compressum. Our aim was to examine chemodiversity of these taxa in the area of Vojvodina (North Serbia) and to perform metabolomics search for chemical classifiers which would provide high resolution discrimination of parental and hybrid individuals. In sum, UHPLC-MS/MS Orbitrap metabolomics fingerprinting revealed seventy compounds in methanol extracts. Despite the lack of qualitative chemical novelty in hybrid plants, UHPLC-qqqMS targeted metabolomics approach, aimed at three secoiridoid compounds and seventeen phenolics, pointed to considerable differences in quantitative composition of these dominant compounds among the plant taxa studied. In addition to the difference in the ploidy levels, the hybrid taxon was well distinguished from both parental species based on metabolite profiles, and, for most individuals, positioned intermediately to the parental taxa in both PCA and hierarchical clustering. After optimizing and comparing several statistical learning methods, it was possible to narrow the number of taxonomic classifiers to five (three xanthones, one secoiridoid glycoside, and one phenolic acid), while increasing the differentiation resolution. The presented metabolomics approach will certainly, along with morphometrics and molecular genetics studies, have high impact on further elucidation of complex relationships among taxa within the genus Centaurium.

The aim of the present study was to investigate the effect of sucrose on shoot regeneration potential in Hypericum perforatum L. roots obtained by Agrobacterium rhizogenes transformation. The morphological evaluation of transgenic roots grown on media supplemented with sucrose (0.5, 1, 2, 4, 6 and 8 %) indicated that both genotype and sucrose concentration significantly affected root elongation and branching, as well as shoot regeneration. For two of five analyzed clones, lower sucrose concentrations (up to 2 %) led to intensive shoot regeneration, while the other three clones intensified shoot development only at elevated sucrose concentrations (4 %). For all clones, concentrations above 4 % had a deleterious effect on both root and shoot development. Genetic characterization of regenerated shoots revealed that all tested clones were diploid with an average of 0.670 ± 0.002 pg of DNA per nucleus, with no significant differences between transgenic and non-transformed plants and, according to PCR, with integrated A. rhizogenes rolA, -B, -C and -D genes. Real-time RT-PCR confirmed the expression of rolA, -B and -C, while expression of the rolD gene was not detected. Differences were detected in the absolute amounts of transcripts between analyzed clones, with the highest levels of expression for all three analyzed rol genes in a clone previously defined as having high root differentiation and less effective shoot regeneration potential. The observed variations in morphogenesis potential could be attributed to different levels of expression of integrated rolA, -B and -C genes; while sucrose additionally pointed out these trends.

The primary aim of this study was to establish a micropropagation procedure for the Slovenian frost-tolerant olive cultivar ‘Istrska belica’. Establishing an in vitro culture was very difficult due to constant contaminations, tissue browning and stunted shoot growth. A sterile shoot culture was finally achieved by washing with running tap water, immersing in a mixture of ascorbic and citric acid and sterilizing with 70% ethanol and dichloroisocyanuric acid. Shoot growth was optimal on DKW medium supplemented with 4 mg/L of 2iP. Even in optimized conditions, sporadic fungal outbursts occurred. Fungi were isolated and their taxonomic origin was determined by morphological observation and molecular identification. Based on BLAST queries in the NCBI database, five genera of fungi were identified: Cladosporium, Chaetomium, Preussia, Biscogniauxia and Sistotrema, the last three genera being isolated from olives for the first time. A detailed literature search was performed to provide data on previous reports of these genera in relation to their putative endophytic presence and their possible pathogenic status. This is the first study reporting the presence of endophytic fungi in olive tissue culture. The information provided in this work can be very useful for the optimization of micropropagation protocols of recalcitrant olive cultivars and can potentially improve field performance of nursery plants.