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.Fil: Foschi, María Laura. Universidad Nacional de Cuyo. Facultad de Ciencias AgrariasFil: Martínez, Liliana. Universidad Nacional de Cuyo. Facultad de Ciencias AgrariasFil: Ponce, María T.. Universidad Nacional de Cuyo. Facultad de Ciencias AgrariasFil: Galmarini, Claudio. Universidad Nacional de Cuyo. Facultad de Ciencias AgrariasFil: Bohanec, Borut. University of Ljubljana, Jamnikarjeva. Biotechnical Faculty

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

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 losestomas

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

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

Cucumber: a model angiosperm for mitochondrial transformation

Abstract. Plants possess three major genomes, carried in the chloroplast, mitochondrion, and nucleus. The chloroplast genomes of higher plants tend to be of similar sizes and structure. In contrast both the nuclear and mitochondrial genomes show great size differences, even among closely related species. The largest plant mitochondrial genomes exist in the genus Cucumis at 1500 to 2300 kilobases, over 100 times the sizes of the yeast or human mitochondrial genomes. Biochemical and molecular analyses have established that the huge Cucumis mitochondrial genomes are due to extensive duplication of short repetitive DNA motifs. The organellar genomes of almost all organisms are maternally transmitted and few methods exist to manipulate these important genomes. Although chloroplast transformation has been achieved, no routine method exists to transform the mitochondrial genome of higher plants. A mitochondrial-transformation system for a higher plant would allow geneticists to use reverse genetics to study mitochondrial gene expression and to establish the efficacy of engineered mitochondrial genes for the genetic improvement of the mitochondrial genome. Cucumber possesses three unique attributes that make it a potential model system for mitochondrial transformation of a higher plant. Firstly, its mitochondria show paternal transmission. Secondly, microspores possess relatively few, huge mitochondria. Finally, there exists in cucumber unique mitochondrial mutations conditioning strongly mosaic (msc) phenotypes. The msc phenotypes appear after regeneration of plants from cell culture and sort with specific rearranged and deleted regions in the mitochondrial genome. These mitochondrial deletions may be a useful genetic tool to develop selectable markers for mitochondrial transformation of higher plants

The Repetitive DNA Composition in the Natural Pesticide Producer Tanacetum cinerariifolium: Interindividual Variation of Subtelomeric Tandem Repeats

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

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