Agrobacterium-mediated transformation of two Serbian potato cultivars (Solanum tuberosum L. cv. Dragacevka and cv. Jelica)

An efficient protocol for Agrobacterium-mediated transformation of Serbian potato cultivars Dragaevka and Jelica, enabling the introduction of oryzacystatin genes OCI and OCII, was established. Starting with leaf explants, a two-stage transformation protocol combining procedures of Webb and Wenzler provided high shoot regeneration efficiency: 84 - 89% for Dragaevka cultivar and 60 - 68% for Jelica cultivar as compared to 76 - 86% for Desiree, the most frequently used cultivar in transformation experiments. PCR analysis of a small sample of putative transformants showed a nptII integration frequency of 90.9, 76.9 and 86.4% for Dragaevka, Jelica and Desiree, respectively. Regeneration and transformation efficiency was strongly genotype-dependent

Effect of activated charcoal, abscisic acid and polyethylene glycol on maturation, germination and conversion of Aesculus hippocastanum androgenic embryos

The influence of activated charcoal (AC), abscisic acid (ABA) and polyethylene glycol (PEG) on the maturation and conversion of horse chestnut (Aesculus hippocastanum L.) androgenic embryos weretested. Androgenic embryos originating from microspores and anther culture were maturated over 90 days. Androgenic embryos on media containing PEG (50 g l-1), in combination with AC (1 g l-1) showed arapid development of embryos in the cotyledonary stage and lowered percentage of abnormal structures. The best results of androgenic microspore embryo germination were observed on media supplemented with AC alone (99%) and in combination with PEG (100%). Also, the greatest number of androgenic microspore plants (18%) and androgenic anther plants (12%) were formed on media enriched with 1 % AC. Lowest germination percentages of 37 and 39% in microspore culture and 33 and38% in anther culture were obtained on maturation media with ABA 20 mg l-1 alone and in combination with AC 1g l-1. Flow cytometric analysis showed that most of the androgenic embryos were haploid, corresponding to their microspore origin, while half of these became diploid after maturation for 90 days. All regenerants originating from microspore culture were haploid immediately after germination, but only 10% embryos re ained haploidity after 3 years subculturing, while 10.5% were diploid, 73.5% tetraploid and 6% octaploid on hormone-free medium. Unlike those from anther culture, after 3 years of subculturing on hormone-free medium, there were no haploid regenerant from anther culture, while8.5% were diploid, 81% tetraploid and 10.5% octaploid

Hypericum perforatum plant cells reduce Agrobacterium viability during co-cultivation

Plant recalcitrance is the major barrier in developing Agrobacterium-mediated transformation protocols for several important plant species. Despite the substantial knowledge of T-DNA transfer process, very little is known about the factors leading to the plant recalcitrance. Here, we analyzed the basis of Hypericum perforatum L. (HP) recalcitrance to Agrobacterium-mediated transformation using cell suspension culture. When challenged with Agrobacterium, HP cells swiftly produced an intense oxidative burst, a typical reaction of plant defense. Agrobacterium viability started to decline and reached 99% mortality within 12 h, while the plant cells did not suffer apoptotic process. This is the first evidence showing that the reduction of Agrobacterium viability during co-cultivation with recalcitrant plant cells can affect transformation

Hypericum sp.: essential oil composition and biological activities

Phytochemical composition of Hypericum genus has been investigated for many years. In the recent past, studies on the essential oils (EO) of this genus have been progressing and many of them have reported interesting biological activities. Variations in the EO composition of Hypericum species influenced by seasonal variation, geographic distribution, phenological cycle and type of the organ in which EO are produced and/or accumulated have also been reported. Although many reviews attributed to the characterization as well as biological activities of H. perforatum crude extracts have been published, no review has been published on the EO composition and biological activities of Hypericum species until recently (Crockett in Nat Prod Commun 5(9):1493–1506, 2010; Bertoli et al. in Global Sci Books 5:29–47, 2011). In this article, we summarize and update information regarding the composition and biological activities of Hypericum species EO. Based on experimental work carried out in our laboratory we also mention possible biotechnology approaches envisaging EO improvement of some species of the genus.Fundação para a Ciência e a Tecnologia (FCT) - project PTDC/AGR AAM/70418/2006, SFRH/BD/ 13283/2003

In vitro propagation of Gentiana dinarica Beck.

Gentiana dinarica Beck, rare and endangered species of Balkan Dinaric alps, was in vitro propagated (micropropagated) from axillary buds of plants collected at Mt. Tara, Serbia. G. dinarica preferred MS to WPM medium, with optimal shoot multiplication on MS medium with 3% sucrose, 1.0 mg l(-1) BA and 0.1 mg l(-1) NAA. Rooting was not clearly separated from shoot multiplication since BA did not completely inhibit root initiation. Spontaneous rooting on plant growth regulator-free medium occurred in some 30% of shoot explants. Rooting was stimulated mostly by decreased mineral salt nutrition and a medium with 0.5 MS salts, 2% sucrose and 0.5-1.0 mg l(-1) IBA was considered to be optimal for rooting. Rooted plantlets were successfully acclimated and further cultured in peat-based substrate.Serbian Ministry of Education and Science [173015

In vitro propagation of Gentiana dinarica Beck.

Gentiana dinarica Beck, rare and endangered species of Balkan Dinaric alps, was in vitro propagated (micropropagated) from axillary buds of plants collected at Mt. Tara, Serbia. G. dinarica preferred MS to WPM medium, with optimal shoot multiplication on MS medium with 3% sucrose, 1.0 mg l(-1) BA and 0.1 mg l(-1) NAA. Rooting was not clearly separated from shoot multiplication since BA did not completely inhibit root initiation. Spontaneous rooting on plant growth regulator-free medium occurred in some 30% of shoot explants. Rooting was stimulated mostly by decreased mineral salt nutrition and a medium with 0.5 MS salts, 2% sucrose and 0.5-1.0 mg l(-1) IBA was considered to be optimal for rooting. Rooted plantlets were successfully acclimated and further cultured in peat-based substrate.Serbian Ministry of Education and Science [173015

Quantitative determination of secoiridoid and xanthone glycosides of Gentiana dinarica Beck cultured in vitro

Gentiana dinarica Beck, native to the Balkan Dinaric Mountains, was established in vitro from axillary shoot buds. It was maintained in the form of shoot cultures on MS medium supplemented with 1.0 mg l(-1) 6-benzyladenine (BA) and 0.1 mg l(-1) alpha-naphthaleneacetic acid and excised root cultures were maintained on 1/2MS medium with 0.5 mg l(-1) indole-3-butyric acid (IBA). Shoot cultures, adventitious roots and excised root cultures were analysed by HPLC techniques for the presence of secoiridoids and xanthones. Gentiopicrin and swertiamarin, the dominant components of shoot cultures, could not be detected in root cultures. Xanthones were present in both shoot and root cultures with norswertianin-1-O-primeveroside as the dominant metabolite. The secoiridoid and xanthone content, although characteristic for certain plant organs, was dependent on the concentration of plant growth regulators (BA and IBA) added to the medium. BA in the shoot multiplication stage strongly increased the secondary metabolite (SEM) content of shoot cultures. IBA had little effect on SEM accumulation in shoots during rooting, while it moderately stimulated SEM accumulation in excised root cultures.Serbian Ministry of Education and Science [173015

Response of unbounded soil in scaled boundary finite-element method

The effects of different types and concentrations of sugars on root growth and xanthone production in root culture of Gentiana dinarica were investigated. The results showed that sucrose, glucose and fructose all supported root growth, and sucrose was superior in terms of growth index, dry mass and fresh/dry mass ratio then fructose or glucose at the same concentrations. However, considering equimolar concentration of sugars, their contribution to the root growth was similar. The HPLC analysis of roots indicated the presence of xanthone compounds, and the contents of norswertianin-1-O-primeveroside (1), norswertianin-1-O-glucoside (2), gentioside (3) and norswertianin (4) were evaluated. In all samples, norswertianin-1-O-primeveroside (1) was present in highest concentration, followed by norswertianin-1-O-glucoside (2), whereas gentioside (3) and norswertianin (4) were present in lower amounts. The production of xanthones was affected by both type and concentration of sugar. In general, roots growing in media supplemented with sucrose contained higher levels of xanthones. The amounts of xanthone primeveroses (1) and (3) increased with the increase of concentrations of all types of sugars, whereas higher sugar concentrations resulted in reduction of the contents of norswertianin-1-O-glucoside (2) and aglycone norswertianin (4). The roots were also evaluated regarding the content of total phenolics and higher accumulation of total phenolic compounds was observed in roots grown in fructose-containing medium. Antioxidant activity was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, and high correlation between total phenolic content and antiradical activity was observed (r = -0.83).Ministry of Education, Science and Tecnological Development of Serbia [173015

Quantitative determination of secoiridoid and xanthone glycosides of Gentiana dinarica Beck cultured in vitro

Gentiana dinarica Beck, native to the Balkan Dinaric Mountains, was established in vitro from axillary shoot buds. It was maintained in the form of shoot cultures on MS medium supplemented with 1.0 mg l(-1) 6-benzyladenine (BA) and 0.1 mg l(-1) alpha-naphthaleneacetic acid and excised root cultures were maintained on 1/2MS medium with 0.5 mg l(-1) indole-3-butyric acid (IBA). Shoot cultures, adventitious roots and excised root cultures were analysed by HPLC techniques for the presence of secoiridoids and xanthones. Gentiopicrin and swertiamarin, the dominant components of shoot cultures, could not be detected in root cultures. Xanthones were present in both shoot and root cultures with norswertianin-1-O-primeveroside as the dominant metabolite. The secoiridoid and xanthone content, although characteristic for certain plant organs, was dependent on the concentration of plant growth regulators (BA and IBA) added to the medium. BA in the shoot multiplication stage strongly increased the secondary metabolite (SEM) content of shoot cultures. IBA had little effect on SEM accumulation in shoots during rooting, while it moderately stimulated SEM accumulation in excised root cultures.Serbian Ministry of Education and Science [173015

Xanthone compounds in shoot cultures of Gentianella bulgarica

Shoot cultures of Gentianella bulgarica established from seedling epicotyls were maintained on MS medium supplemented with BA 0.2 mg l(-1) + NAA 0.1 mg l(-1). Cultures were prone to precocious flowering requiring the use of small shoot buds for multiplication purposes. The contents of three xanthone compounds identified as DGL, BGL, and DMB, in different plant material were determined by HPLC. The analysis revealed that the production of xanthones was affected by different concentrations of BA in medium. Shoot cultures grown at higher BA concentrations contained more DGL than material grown in nature. The concentrations of other two xanthones were lower in shoot cultures than in plants from nature. The radical scavenging activity of plant extracts and xanthones was investigated by DPPH test. Samples from plants grown in nature showed the highest activity (IC(50) = 0.26 mg ml(-1)), while the extracts of shoot cultures grown in media with higher concentrations of BA showed moderate activities (IC(50) from 1.6 to 4.4 mg ml(-1)).Ministry of Science and Technological Development of Serbia [173015