Effect of colchicine and amiprophos-methyl on the production of in vitro doubled haploid onion plants and correlation assessment between ploidy level and stomatal size = Efecto de la colchicina y del amiprofos-metil en la producción in vitro de plantas dihaploides de cebolla y determinación de la correlación entre el nivel de ploidía y tamaño de los estomas

Doubled haploid onion (Allium cepa L.) plants allow the production of completely homozygous lines for a later production of hybrids. The haploid plants are normally produced using in vitro gynogenesis. The obtained haploid plantlets must be treated with different agents for doubling chromosomes. It is necessary to adjust the concentration and the length of treatment of the doubling agent. In this case, the effect of 250 and 500 mg.L-1 colchicine and 15.2; 30 and 60 mg.L- 1 amiprophos-methyl during 24 and 48 h was assessed over the rate of onion haploid plantlets chromosome doubling. The best duplication treatment was 250 mg.L-1 colchicine for 48 h, which yielded 100% of doubled haploid plants. On the other hand, a positive correlation resulted from the ploidy level and stomatal size, and a negative correlation between the level of ploidy and stomatal density. Significant differences between the stomatal length, width and density in haploid and doubled haploid plantlets were observed. An economical and quick method to test ploidy level in onion plantlets is proposed through the measurement of stomatal size and density.La producción de plantas dihaploides de cebolla (Allium cepa L.), permite obtener líneas completamente homocigotas para luego producir híbridos. A través de la ginogénesis in vitro se ha logrado producir plantas haploides, las cuales deben ser sometidas a tratamientos de duplicación de sus cromosomas, siendo necesario ajustar la concentración del agente duplicador y la duración del tratamiento. En este trabajo se evaluó el efecto de la aplicación de 250 y 500 mg.L-1 de colchicina y 15,2; 30 y 60 mg.L-1 de amiprofos-metil durante 24 y 48 h, sobre la tasa de duplicación de cromosomas en plántulas haploides de cebolla. El mejor tratamiento de duplicación fue 250 mg.L- 1 de colchicina, durante 48 h, logrando un 100% de plantas dihaploides. Por otra parte, se obtuvo una correlación positiva entre el nivel de ploidía y el tamaño de estomas y una correlación negativa entre el nivel de ploidía y la densidad estomática, registrándose diferencias significativas entre los valores promedio de largo, ancho y densidad de estomas para plantas haploides y dihaploides. Se propone un método económico y rápido para verificar el nivel de ploidía de las plántulas de cebolla mediante la medición del tamaño y densidad estomática.EEA La ConsultaFil: Foschi, María Laura. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; ArgentinaFil: Martínez, Liliana Estela. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Ponce, María Teresa. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Galmarini, Claudio Romulo. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria La Consulta; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bohanec, Borut. University of Ljubljana. Biotechnical Faculty; Esloveni

Genetic characterization of Genista sericea Wulfen (Cytiseae - Fabaceae) as revealed by nuclear DNA content and ITS nrDNA region analysis

Genista sericea Wulfen, a northern Illyrian amphi-Adriatic species, presents a certain morphological variability. To clarify whether the genetic variations support the morphological differences among accessions of different geographic origin, analysis of nuclear DNA content and polymorphism of the internal transcribed spacer (ITS) dataset was studied. The variation in nuclear DNA content of G. sericea var. sericea and var. rigida is minimal (2.09 and 2.08 pg/nucleus respectively) and is correlated with equal chromosome numbers in both varieties. Intraspecific variability of the ITS region was studied on 13 accessions of G. sericea, 6 belonging to var. sericea and 7 to var. rigida. These accessions were analyzed in comparison to closely related species already studied. ITS sequences of G. sericea revealed large polymorphism and formed two main clusters. One cluster (6 accessions) comprehends var. sericea of northern Italy, Slovenia and northern Croatia; the other cluster (7 accessions) includes five accessions of var. rigida from southern Croatia and Montenegro and two from the Pollino massif (southern Italy). The later two accessions considerably differed from other accessions of var. rigida. This genetic analysis supports the previous assumptions, which subdivided G. sericea into at least two taxa. On the basis of the results presented, it is here suggested that the subdivision of G. sericea into var. sericea and var. rigida should be maintained

DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes

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

Supplementary data for article: Banjanac, T.; Dragićević, M.; Šiler, B.; Gašić, U.; Bohanec, B.; Nestorović Živković, J.; Trifunović, S.; Mišić, D. Chemodiversity of Two Closely Related Tetraploid Centaurium Species and Their Hexaploid Hybrid: Metabolomic Search for High-Resolution Taxonomic Classifiers. Phytochemistry 2017, 140, 27–44. https://doi.org/10.1016/j.phytochem.2017.04.005

Supplementary material for: [https://doi.org/10.1016/j.phytochem.2017.04.005]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2477

Supplementary data for article: Banjanac, T.; Dragićević, M.; Šiler, B.; Gašić, U.; Bohanec, B.; Nestorović Živković, J.; Trifunović, S.; Mišić, D. Chemodiversity of Two Closely Related Tetraploid Centaurium Species and Their Hexaploid Hybrid: Metabolomic Search for High-Resolution Taxonomic Classifiers. Phytochemistry 2017, 140, 27–44. https://doi.org/10.1016/j.phytochem.2017.04.005

Supplementary material for: [https://doi.org/10.1016/j.phytochem.2017.04.005]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2477

Plant regeneration and clonal fidelity assessment of subendemic species Iris illyrica Tomm. originated from Croatia

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)