Razvoj transgene otpornosti šećerne repe na virus nekrotičnog žutila nerava repe (BNYVV)

Fragments of viral cDNA containing the coat protein gene of beet necrotic yellow vein virus were cloned in plant transformation vector pCAMBIA3301M with the bar gene as selectable marker. Vector pC3301MCPL carrying coat protein gene with leader sequence, and pC3301MCPS with coat protein gene, were used in Agrobacterium - mediated transformation of sugar beet. The transformation method used was based on the fact that sugar beet develops axillary shoots in in vitro conditions, when placed on media with citokinins. Since this ability is not genotype or ploidy dependant it is widely used for sugar beet vegetative multiplication. Sterile seedlings, with removed cotyledons and lower half of hypocotyl, were used as starting material. After transformation ex-plants were put on micropropagation medium with cephotaxime and phosphinotricyn (ppt), where axillary shoots started to develop. Since concentration of ppt was not selective enough, after two subcultivations it was increased twofold. Only one sample, transformed with pC3301MCPS preserved morphogenetic potential for micropropagatio, and it was tested for presence of COS fragment and bar gene bz PCR with soecific primers.Fragmenti virusne cDNK sa genom za protein omotača virusa nekrotičnog žutila nerava repe su klonirani u vektor za transformaciju biljaka pCAM-BIA3301M koji je sadržao bar gen kao selektivni marker. Vektori pC3301MCPL, sa genom za protein omotača virusa i njegovom lider sekvencom, i pC3301MCPS, sa genom za protein omotača, su korišćeni u tramsformaciji repe pomoću Agrobacterium-a. Metod transformacije se zasniva na sposobnosti repe da u uslovima in vitro razvije aksilarne pupoljke na podlozi sa citokininima. Pošto ova sposobnost ne zavisi od genotipa ili od nivoa plodnosti, postala je standardni metod za vegetativno umnožavanje repe. Kao početni materijal su korišćeni sterilni ponići kojima su odstranjeni kotiledoni i donja polovina hipokotila. Nakon transformacije eskplantati su postavljeni na selektivnu podlogu za mikropropagaciju sa cefotaksimom i fosfinotricinom (ppt) gde je došlo do razvoja bočnih pupoljaka. Po.sto koncentracija fosfinotricina nije bila dovoljno selektivna, ona je nakon dve subkultivacije dvostruko povećana. Samo je jedan uzorak, transformisan vektorom pC3301MCPS, nakon dve subkultivacije sačuvao mofrogenetski potencijal za mikropropagaciju, i bio testiran na prisustvo CPS fragmenta i bar gena PCR reakcijom sa specifičnim prajmerima

Genetic control of flower petal number in Rosa x Damascena Mill f. trigintipetala

We studied the petal number trait in a population obtained after self-pollination of R. x damascena f. trigintipetala following analysis of molecular markers which have previously been mapped near the major dominant locus Blfo/d6 determining this trait in other rose species including R. multiflora and R. hybrida. The results showed that the same genetic mechanism, which determines the petal number trait in R. multiflora and R. hybrida also controls the trait in R. x damascena f. trigintipetala and is related to the dominant effect of a single copy allele in the tetraploid genome of this species. We also analyzed the expression of several flower homeotic genes including R. x damascena APETALA1/FUL-like (paleo AP1 type), R. x damascena euAPETALA 3 (euAP3 line) and R. x damascena AGAMOUS in early stage flower buds corresponding to plants with double and simple flowers. The obtained results showed that only R. x damascena AGAMOUS was differentially expressed between the samples of double and simple flowers, its relative expression being upregulated 3.5-fold in simple flowers. We further cloned and sequenced the four genomic clones of R. x damascena AGAMOUS and studied the potential additive effect of this gene by analysing the segregation of its four alleles in the population of self-pollinated R. x damascena. Analysis of variance of the data for petal number and allele segregation did not show a statistically significant effect of any allele configuration of the AGAMOUS gene on the petal number trait in R. x damascena f. trigintipetala

Assessment of Public Awareness of the Forestry Sector: Biodiversity, Certification and Ecosystem Services in Velingrad Municipality

The purpose of this study was to survey the current public awareness in Velingrad Municipality in terms of biodiversity preservation, certification and ecosystem services in the regional forestry sector. The answers to the questions related to the biodiversity and ecosystem services showed good aware- ness and a very positive attitude of respondents regarding the need for conservation and sustainable use of biodiversity as well as the improvement of ecosystem services. According to the conducted surveys, the awareness concerning the certification and standards in the local forestry sector as well as the regional NATURA 2000 protected sites was still weak or lacking

Identification of QTL controlling the ratio of linalool to linalyl acetate in the flowers of Lavandula angustifolia Mill var. Hemus

AbstractThe content of linalyl acetate and the ratio of linalool to linalyl acetate (L/LA ratio) are one of the important parameters that determine the quality of lavender oil. The characterization of a segregating population derived from a self-pollinated Lavandula angustifolia var. Hemus resulted in the identification of a single quantitative trait locus controlling the L/LA ratio (L/LA-QTL) and located on chromosome 8 of the lavender reference genome. The L/LA data analysis demonstrated that plants homozygous for one of the L/LA-QTL alleles had significantly higher linalool content, lower linalyl acetate content and higher L/LA ratio, than the plants which were either heterozygous or homozygous for the other allele. No significant difference was observed for the sum of linalool and linalyl acetate content among these three groups of plants, suggesting that the identified L/LA-QTL is related to an enzyme conversion of linalool to linalyl acetate. The BLAST search revealed that the L/LA-QTL region included a sequence of the LiAAT4 gene of monoterpene acetyltransferase, considered as a candidate gene for the L/LA-QTL locus. Sequence analysis of the LiAAT4 gene of var. Hemus revealed the presence of two alleles differing in two nucleotides and predicted amino acid substitutions. The comparison of the allele configurations of SSR, SRAP and LiAAT4 loci and the L/LA ratio of plants showing recombination in the L/LA-QTL region provided further support that LiAAT4 is a candidate gene underlying the identified L/LA-QTL and controlling the L/LA ratio. The application of molecular markers for the identified L/LA-QTL is discussed

A Set of Highly Polymorphic Microsatellite Markers for Genetic Diversity Studies in the Genus <i>Origanum</i>

This study reports the development of a set of 20 highly polymorphic genomic SSR markers which can be used for both cultivar identification and genetic diversity studies in several Origanum species, including some of the most popular ones like Greek oregano (Origanum vulgare L. ssp. hirtum), common oregano (O. vulgare L. ssp. vulgare), and sweet marjoram (O. majorana L.). Analysis of the polymorphic information content (PIC) showed an average PIC value of 0.75 with a minimum of 0.41 and a maximum of 0.89, where 17 of the markers showed PIC values above 0.73. Comparative analysis of the genetic diversity of eight natural populations of Greek oregano in Bulgaria showed that six of the genomic SSR markers revealed significantly higher portions of genetic diversity in the populations, compared to 12 EST SSR markers used in our previous study. We also compared the performance of the same six genomic SSR markers with the results for eight SRAP primer combinations, which showed that SRAP markers captured more precisely the genetic structure in natural populations. The developed highly polymorphic genomic SSR markers can be successfully applied to evaluation of the genetic diversity in the genus Origanum, based on the expected and observed heterozygosity in the populations as well as for easy identification of breeding lines and cultivars based on unique SSR fingerprints

Indigenous Yeasts from Rose Oil Distillation Wastewater and Their Capacity for Biotransformation of Phenolics

The indigenous yeasts associated with the spontaneous fermentation of phenolic-rich rose oil distillation wastewater (RODW) generated after the industrial distillation of rose oil were studied. The ITS-rDNA sequence analysis of the samples collected from RODW fermented at semi-sterile conditions, a waste deposition lagoon and endophytic yeasts isolated from industrially cultivated Rosa damascena suggests that the spontaneous RODW fermentation is caused by yeasts from the genus Cyberlindnera found also as endophytes in the rose flowers. Phylogenetic analysis based on the nucleotide sequences of the translation elongation factor (TEF1α) and 18S- and 26S- rRNA genes further confirmed the taxonomic affiliation of the RODW yeast isolates with the genus Cyberlindnera. The RODW fermentation capacity of a selected set of indigenous yeast isolates was studied and compared with those of common yeast strains. The indigenous yeast isolates demonstrated a superior growth rate, resulting in a nearly double reduction in the phenolic content in the fermented RODW. The indigenous yeasts’ fermentation changed the RODW phenolics’ composition. The levels of some particular phenolic glycosides decreased through the depletion and fermentation of their sugar moiety. Hence, the relative abundance of the corresponding aglycons and other phenolic compounds increased. The capacity for the biotransformation of RODW phenolics by indigenous yeasts is discussed

Sequence-related amplified polymorphism (SRAP) markers, an efficient and affordable tool for evaluation genetic diversity in forest areas

This article describes testing the application of SRAP (sequence-related amplified polymorphism) markers for characterisation of a small set of plants of Quercus coccifera L and samples from 25 other forest tree species. The results suggest that SRAP markers could be used as an affordable and efficient tool for the characterisation of the genetic diversity in populations of tree species as a part of the characterisation of forest biodiversity and the related decision making and management

Identification of QTL controlling the ratio of linalool to linalyl acetate in the flowers of <i>Lavandula angustifolia</i> Mill var. Hemus

The content of linalyl acetate and the ratio of linalool to linalyl acetate (L/LA ratio) are one of the important parameters that determine the quality of lavender oil. The characterization of a segregating population derived from a self-pollinated Lavandula angustifolia var. Hemus resulted in the identification of a single quantitative trait locus controlling the L/LA ratio (L/LA-QTL) and located on chromosome 8 of the lavender reference genome. The L/LA data analysis demonstrated that plants homozygous for one of the L/LA-QTL alleles had significantly higher linalool content, lower linalyl acetate content and higher L/LA ratio, than the plants which were either heterozygous or homozygous for the other allele. No significant difference was observed for the sum of linalool and linalyl acetate content among these three groups of plants, suggesting that the identified L/LA-QTL is related to an enzyme conversion of linalool to linalyl acetate. The BLAST search revealed that the L/LA-QTL region included a sequence of the LiAAT4 gene of monoterpene acetyltransferase, considered as a candidate gene for the L/LA-QTL locus. Sequence analysis of the LiAAT4 gene of var. Hemus revealed the presence of two alleles differing in two nucleotides and predicted amino acid substitutions. The comparison of the allele configurations of SSR, SRAP and LiAAT4 loci and the L/LA ratio of plants showing recombination in the L/LA-QTL region provided further support that LiAAT4 is a candidate gene underlying the identified L/LA-QTL and controlling the L/LA ratio. The application of molecular markers for the identified L/LA-QTL is discussed.</p

High cross-pollination rate of Greek oregano (O. vulgare ssp. hirtum) with Common oregano (O. vulgare ssp. vulgare) under open field conditions as revealed by microsatellite marker analysis

AbstractWe studied the mode of pollination in Greek oregano (Origanum vulgare ssp. hirtum) under both controlled and open pollination conditions. When grown indoors without the presence of insects, Greek oregano plants did not develop any seeds, indicating a low level of spontaneous self-pollination. Applying manual self-pollination under the same conditions resulted in only 16 seeds, of which only five were able to germinate. At the same time, a clonally propagated Greek oregano plant of the same genotype produced a rich set of over 300 seeds in open field conditions when the flowers were visited by insects in an area where no other Origanum species were observed. Analysis with SSR markers showed that over 70% of the seeds likely resulted from self-pollination, indicating that insect-mediated pollination is essential for the seed development. We further analyzed the cross-pollination of Greek oregano with Common oregano (O. vulgare ssp. vulgare) in open field conditions where the two subspecies were grown in close proximity. Applying SSR markers, we analyzed 83 plants obtained from seeds of three vegetatively propagated Greek oregano mother plants. Surprisingly, the results showed that all analyzed seedlings resulted from cross-pollination of Greek oregano with Common oregano, indicating that cross-pollination between the two subspecies can completely take over the self-pollination or cross-pollination between the Greek oregano plants. The possible impact of the observed high cross-pollination rate on the genetic origin of seeds of selected Greek oregano lines and varieties, as well as on the genetic diversity and structure of natural populations, is discussed

Tyrosinase inhibitory constituents from a polyphenol enriched fraction of rose oil distillation wastewater

During the water steam distillation process of rose flowers, the non-volatile phenolic compounds remain in the waste. We recently developed a strategy to separate rose oil distillation water (RODW) into a polyphenol depleted water fraction and a polyphenol enriched fraction (RF20-SP207). Bioassay-guided investigation of RF20-SP207 led to the isolation of quercetin, kaempferol and ellagic acid. Their structures were elucidated by spectroscopic analysis as well as by comparison with literature data. Tyrosinase inhibition studies were performed with RF20-SP207, fractions I–IV, and the isolated compounds of the most active fraction. RF20-SP207 strongly inhibited the enzyme with an IC50 of 0.41 μg/mL. From the tested fractions only fraction IV (IC50 = 5.81 μg/mL) exhibited strong anti-tyrosinase activities. Quercetin, kaempferol and ellagic acid were identified in fraction IV and inhibited mushroom tyrosinase with IC50 values of 4.2 μM, 5.5 μM and 5.2 μM, respectively, which is approximately 10 times more potent than that of the positive control kojic acid (56.1 μM). The inhibition kinetics, analyzed by Lineweaver–Burk plots, indicated that RF20-SP207 and fraction IV are uncompetitive inhibitors of tyrosinase when l-tyrosine is used as a substrate. A mixed inhibition was determined for ellagic acid, and a competitive inhibition for quercetin and kaempferol. In conclusion, the recovered polyphenol fraction RF20-SP207 from RODW was found to be a potent tyrosinase inhibitor. This value-added product could be used as an active ingredient in cosmetic products related to hyperpigmentation