Hairy root induction and plant regeneration of <Emphasis Type="Italic">Rehmannia glutinosa</Emphasis> Libosch. f. <Emphasis Type="Italic">hueichingensis</Emphasis> Hsiao via <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation

The objective of this research was to establish an efficient system of genetic transformation and plant regeneration from hairy roots by infecting the leaf sections and stem segments of in vitro Rehmannia glutinosa Libosch. f. hueichingensis Hsiao plantlets. Hairy roots were induced from them after co-culturing with Agrobacterium rhizogenes strain 15834 at a frequency of 32 and 29.4%, respectively. The calluses were induced from hairy roots on half-strength Murashige and Skoog medium containing 0.2 mg/l kinetin and 3.0 mg/l benzyladenine at a frequency of 100%, from which transgenic shoots and plantlets were developed. Transgenic plantlets did not have differences in morphology except the shortened internodes and an increase in adventitious root formation compared to wild-type plants. PCR and Southern-blot analyses confirmed that rolB gene of TL-DNA was inserted in the genome of transformed hairy roots and plantlets. RT-PCR analysis and opine paper electrophoresis revealed that rolB gene was expressed in the transformed hairy roots and plantlets. Conclusively, transgenic hairy roots and transgenic plants of Rehmannia glutinosa Libosch. f. hueichingensis Hsiao were developed for the first time. This text was submitted by the authors in English. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 2, pp. 247–255.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Resveratrol Synthase Transgene Expression and Accumulation of Resveratrol Glycoside in <Emphasis Type="Italic">Rehmannia glutinosa</Emphasis>

Rehmannia glutinosa L. is an important medicinal crop in Asian countries and contains trace amount of resveratrol compounds. To increase production of the compounds, we attempted ectopic expression of peanut resveratrol synthase gene (AhRS3) in R. glutinosa. The AhRS3 sequence that encompassed the open reading frame, including a 312 bp-long intron present between the 59th and 60th codon, was driven by the CaMV35S promoter and introduced into R. glutinosa via Agrobacterium-mediated transformation of leaf explants. The transgenic plants with one to three copies of AhRS3 transgene showed normal growth and development. The transgene was expressed constitutively in the leaf, root and flower at similar levels. Transgene expression in the leaf resulted in the production of new compounds identified as resveratrol and 3′-H-resveratrol-3-O-β-d-glucoside (R-gluc) through nuclear magnetic resonance spectroscopy and mass spectrometry. R-gluc accumulated predominantly and its content in the leaf of the 11 transgenic lines ranged from 22 to 116μg/gFW. The contents of resveratrol compounds in the transgenic plants were further increased by cold, UV, ethylene, and paraquat treatments, and were positively associated with the levels of AhRS3 mRNA levels. The R-gluc isolated from the transgenic plants exhibited antioxidant activity equivalent to one-third of resveratrol. Transgenic plants were highly resistant to Fusarium oxysporum infection. The results indicate that the ectopic expression of AhRS3 in R. glutinosa results in the production of R-gluc and resveratrol at hundreds of times higher levels than in peanut seed. The increased production of resveratrol compounds from R. glutinosa, which show diverse benefits for human and plant health, could provide a new opportunity for the improvement of R. glutinosa products.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Modelling the pollen season start in <Emphasis Type="Italic">Corylus avellana</Emphasis> and <Emphasis Type="Italic">Alnus glutinosa</Emphasis>

Hazel (Corylus avellana L.) and black alder (Alnus glutinosa (L.) Gaertn.) are important sources of airborne pollen and represent an allergen threat during the flowering period. Researches on airborne pollen concentrations in both species are useful in allergology, as well as for fruit production for hazel. The aims of the present study were: (1) to investigate the relationships between environmental conditions and the airborne pollen concentration of hazel and black alder during the flowering period by correlation and multiple regression analysis and (2) to predict the pollen season start (PSS) by using a sequential model, in order to obtain a helpful tool in allergology and hazel cultivation. In this study, the applied method defines the pollen season as the period in which 90 % of the total season’s catch occurred, using a data set of 18 years (1996–2014). The relationships between daily meteorological parameters (temperature, humidity, rainfall and wind speed) during the 14-day period that precedes the PSS and the PSS of hazel and black alder (day of the year) were investigated. The results showed that mean temperature and the number of rainy days before the PSS are the main factors influencing PSS for both taxa. Moreover, the chilling and heat needed to break dormancy were estimated in order to predict the PSS of both species. Different years and different thresholds of temperature and chill days were used to calibrate and validate the model.