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 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

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

Interaction with gravitropism, reversibility and lateral movements of phototropically stimulated potato shoots

Phototropic (PT) and gravitropic (GT) bending are the two major tropic movements that determine the spatial position of potato shoots. We studied PT bending of potato plantlets grown under long-day photoperiods in several prearranged position setups providing different interactions with the GT response. Starting with the standard PT stimulation setup composed of unilateral irradiation of vertically positioned shoots, experiments were also done in antagonistic and synergistic setups and in treatments with horizontal displacement of the light source. In the standard setup, PT bending suppressed the GT bending, which could occur only if the PT stimulation was cancelled. The antagonistic position, with phototropism and gravitropism attempting to bend shoots in opposite directions, showed phototropism and gravitropism as independent bending events with the outcome varying throughout the day reflecting diurnal changes in the competence of individual tropic components. Whilst gravitropism was constant, phototropism had a marked daily fluctuation of its magnitude with a prominent morning maximum starting an hour after the dawn in the growth room and lasting for the next 6 h. When phototropism and gravitropism were aligned in a synergistic position, stimulating shoot bending in the same direction, there was little quantitative addition of their individual effects. The long period of morning PT bending maximum enabled multiple PT bending events to be conducted in succession, each one preceded by a separate lag phase. Studies of secondary PT events showed that potato plantlets can follow and adjust their shoot position in response to both vertical and horizontal movements of a light source. PT bending was reversible, since the 180° horizontal change of a blue light (BL) source position resulted in reversal of bending direction after a 20-min-long lag phase

Gentiana dinarica Beck. hairy root cultures and evaluation of factors affecting growth and xanthone production

The induction and establishment of hairy root cultures of Gentiana dinarica using two strains of Agrobacterium rhizogenes (A4M70GUS and 15834/PI) is reported for the first time. Strain 15834/PI had higher induction rate of hairy roots (32.15 \%) than strain A4M70GUS (6.12 \%). Transgenic nature of the roots was confirmed by GUS assay and PCR analysis. Two clones per strain (A4M70GUS-D and -I, and 15834/PI-2 and -3) with marked differences in general morphology and growth rate were further studied. The methanol extracts of hairy root clones were analyzed for xanthones content using HPLC method. The effects of the type of carbohydrate source (sucrose, fructose or glucose) at different concentrations on the growth parameters (growth index, dry weight, fresh/dry weight ratio), phenolic and xanthone production, and free radical scavenging activity of the transgenic clones were evaluated. Statistical two level factorial design was used to define optimal conditions for growth and successful secondary metabolite production in G. dinarica hairy root clones. The results showed that clones A4M70GUS-D and 15834/PI-3 were the superior ones. These two clones had the highest dry weight on 116.8 mM sucrose, producing up to threefold higher amounts of total phenolics and norswertianin-1-O-primeveroside than other clones, untransformed roots and roots of wild-grown plants.Ministry of Education, Science and Technological Development of the Republic of Serbia {[}173015