<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of mature <Emphasis Type="Italic">Prunus serotina</Emphasis> (black cherry) and regeneration of transgenic shoots

A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced for 12 h with 200 μM acetosyringone for vir gene induction before leaf explant inoculation. Explants were co-cultured for 3 days, and then cultured on woody plant medium supplemented with 9.08 μM thidiazuron, 1.07 μM napthaleneacetic acid, 60 μM silver thiosulphate, 3% sucrose, plus 200 mg l⁻¹ timentin in darkness for 3 weeks. Regenerating shoots were selected 27 days after initial co-culture, on Murashige and Skoog medium with 3% sucrose, 8.88 μM 6-benzylaminopurine, 0.49 μM indole-3-butyric acid, 0.29 μM gibberellic acid, 200 mg l⁻¹ timentin, and 30 mg l⁻¹ kanamycin for five subcultures. After 5–6 months of selection, transformation efficiencies were determined, based on polymerase chain reaction (PCR) analysis of individual putative transformed shoots relative to the initial number of leaf explants tested. The transformation efficiency was 1.2%. Southern blot analysis of three out of four PCR-positive shoots confirmed the presence of the neomycin phosphotransferase and AG genes. Transgenic shoots were rooted (37.5%), but some shoot tips and leaves deteriorated or died, making acclimatization of rooted transgenic plants difficult. This transformation, regeneration, and rooting protocol for developing transgenic black cherry will continue to be evaluated in future experiments, in order to optimize the system for several mature black cherry genotypes.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of gypsophila (<Emphasis Type="Italic">Gypsophila paniculata</Emphasis> L.)

As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility of this species’ genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium inoculation of highly regenerative stem segments. The transformation procedure employs stem explants derived from GA₃-pretreated mother plants and a two-step selection scheme. The GA₃ treatment was crucial for obtaining high gene-transfer frequencies (75–90% GUS-expressing explants out of total inoculated explants), as shown using three different gypsophila varieties. An overall transformation efficiency of five GUS-expressing shoots per 100 stem explants was demonstrated for cv. Arbel. The applicability of the transformation system to gypsophila was further reinforced by the generation of transgenic plants expressing Agrobacterium rhizogenes rolC driven by a CaMV 35S promoter. Transgenic gypsophila plantlets exhibited extensive rooting and branching, traits that could be beneficial to the ornamental industry.     

<Emphasis Type="Italic">WUS</Emphasis> and <Emphasis Type="Italic">STM</Emphasis>-based reporter genes for studying meristem development in poplar

We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5′ flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50–60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common in other organs, including in leaf veins (40 and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems as explants showed that expression was detectable prior to visible shoot development, starting 3–15 days after explants were placed onto callus inducing medium. A minority of WUS and STM events also showed expression in the cambium, phloem, or xylem of regenerated, greenhouse grown plants undergoing secondary growth. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Both paralogs of poplar STM were down-regulated threefold to sixfold during early callus initiation. We identified 15–35 copies of cytokinin response regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the events recovered may be useful for studying the process of primary and secondary meristem development, including treatments intended to stimulate meristem development to promote clonal propagation and genetic transformation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

New species of <Emphasis Type="Italic">Chalybea</Emphasis> and <Emphasis Type="Italic">Huilaea</Emphasis> (Melastomataceae)

One new species each is proposed in Chalybea and Huilaea (Melastomataceae: Blakeeae). Chalybea peruviana has elliptic, 5-plinerved leaves with entire, revolute margins, inflorescences with 33–39 flowers, and is endemic to Peru. Huilaea calyptrata has inflorescences with 15–17, irregularly calyptrate flowers, anthers with a warty connective in the shape of an inverted hand fan, and is endemic to Ecuador. A key to the eight species of Huilaea is provided.     

Expression of <Emphasis Type="Italic">Aprotinin</Emphasis> in Anther Causes Male Sterility in Tobacco var <Emphasis Type="Italic">Petit havana</Emphasis>

Expression of many proteinases has been documented during anther development. Although their roles are not completely understood, their inhibition could possibly result in impairment of anther development leading to male sterility. We proposed that such an impairment of anther development can be engineered in plants resulting in male sterile plants that can be used for hybrid seed production. Here, we report that anther-specific expression of Aprotinin gene (serine proteinase inhibitor) in tobacco has resulted in male sterility. Southern analysis and zymogram analysis confirmed the integration and expression of Aprotinin gene in the anthers of the transgenic plants. Transverse sections of anthers of transgenic male sterile plants showed damaged tapetum. The pollen germination in the transgenic plants ranged between 2% and 65% that confirmed the impairment in pollen production leading to male sterility and low seed yield. Thus, inhibition of serine proteinases that are expressed during anther development has resulted in impaired pollen production and male sterility, though the exact role of these proteinases in anther development still has to be elucidated.     

Ectopic expression of <Emphasis Type="Italic">MNX</Emphasis> gene from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> involved in auxin biosynthesis confers male sterility in transgenic cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) plants

A transgenic male sterile line of upland cotton was generated by the ectopic expression of the monooxygenase (MNX) gene from Arabidopsis thaliana via Agrobacterium-mediated transformation. The bacterium harbored a plasmid pBinplus carrying a 1.25-kb MNX coding sequence together with a GUS reporter gene; the former was driven by the MS2 promoter of a male sterility gene in Arabidopsis, and the latter was under the control of CaMV 35S promoter. Twenty-seven putative transgenic plants (T₀) were obtained, all of which showed GUS activity and positive signals of NPTII and MNX genes by PCR analysis, and also showed male sterility to some extent. It was further confirmed by Southern blotting that one copy of the NPTII and MNX gene was integrated in the genome of the plants which expressed male sterility to a higher degree. Northern blotting assay also demonstrated that the transgenes stably transcribed in the genome of the transgenic plants in F₄ generation. The male sterile plants usually display lower plant height, shortened internodes, shrunken anthers without pollen grains or with some abortive pollen grains, and unusual leaves with deeper multi-lobes. Microscope observations on the meiosis processes of pollen mother cells (PMCs) showed that the abortion of pollen grains mainly resulted from abnormalities of meiosis such as direct degeneration of PMCs, degenerations of dyad and tetrads, amitosis, lagging chromosomes, and the multi-polar segregations of chromosomes and so on. This study indicates a method of developing novel cotton male sterile materials for potential application in agriculture and for engineering of male sterility in other important crops.     

Temperature effect on the growth of <Emphasis Type="Italic">Buchnera</Emphasis> endosymbiont in <Emphasis Type="Italic">Aphis craccivora</Emphasis> (Hemiptera: Aphididae)

Effect of temperature on the growth of the primary endosymbiont Buchnera aphidicola in the cowpea aphid Aphis craccivora was studied by measuring quantitatively the copy number of 16S rDNA of this endosymbiont. A 1.5 kb segment of eubacterial 16S rDNA amplified by PCR from total DNA of Aphis craccivora was confirmed by RFLP analysis and sequence BLAST as that of Buchnera aphidicola. No secondary endosymbiont was detected in the aphid population studied. The relative levels of Buchnera ratio, quantified by real-time PCR, were higher in old nymphs than in young ones at temperatures between 10–30˚C, and this age-dependent difference was more pronounced at lower temperatures. Throughout the entire reproductive stage of Aphis craccivora, the relative levels of Buchnera ratio were higher at 10–25˚C than at 30˚C and 35˚C. A close relationship was found between these levels and the net reproductive rate (R ₀ ) of aphid, which was suppressed not only at 35˚C but also at 10˚C. The decoupling of Aphis craccivora and Buchnera response at low temperatures suggests that the cowpea aphid was more sensitive to low temperatures, while Buchnera was more sensitive to high temperatures.     

Molecular evolution of the clustered <Emphasis Type="Italic">MIC</Emphasis>-<Emphasis Type="Italic">3</Emphasis> multigene family of <Emphasis Type="Italic">Gossypium</Emphasis> species

The Gossypium MIC-3 (Meloidogyne Induced Cotton-3) gene family is of great interest for molecular evolutionary studies because of its uniqueness to Gossypium species, multi-gene content, clustered localization, and root-knot nematode resistance-associated features. Molecular evolution of the MIC-3 gene family was studied in 15 tetraploid and diploid Gossypium genotypes that collectively represent seven phylogenetically distinct genomes. Synonymous (d_S) and non-synonymous (d_N) nucleotide substitution rates suggest that the second of the two exons of the MIC-3 genes has been under strong positive selection pressure, while the first exon has been under strong purifying selection to preserve function. Based on nucleotide substitution rates, we conclude that MIC-3 genes are evolving by a birth-and-death process and that a ‘gene amplification’ mechanism has helped to retain all duplicate copies, which best fits with the “bait and switch” model of R-gene evolution. The data indicate MIC-3 gene duplication events occurred at various rates, once per 1 million years (MY) in the allotetraploids, once per ~2 MY in the A/F genome clade, and once per ~8 MY in the D-genome clade. Variations in the MIC-3 gene family seem to reflect evolutionary selection for increased functional stability, while also expanding the capacity to develop novel “switch” pockets for responding to diverse pests and pathogens. Such evolutionary roles are congruent with the hypothesis that members of this unique resistance gene family provide fitness advantages in Gossypium.     

Effects of plant genotype and bacterial strain on <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">capitata</Emphasis>

Two inbred lines of Brassica oleracea L. var. capitata were transformed with two Agrobacterium tumefaciens strains harboring resistance to herbicide Basta: AGL1/pDM805 and LBA4404/pGKB5 (LB5-1). Inoculated cotyledons and hypocotyls provided equally good explants and manifested a high percentage of shoot regeneration on MS medium supplemented with 1 mg/l benzyladenine and 0.5 mg/l indole-3-butyric acid. The P₃₄I₅ genotype was superior to P₂₂I₅ in shoot regeneration (48.1 vs. 26.9%), multiplication, and acclimation in the greenhouse (76 vs. 40%). A. tumefaciens AGL1/pDM805 provided more regenerated shoots per explant, especially in the case of cotyledon explants, and the higher transformation rate (up to 35% vs. up to 12%) as compared to LB5-1. Putative transformants survived spraying with 10–30 mg/l phosphinothricin. Transformation was confirmed by GUS assay and PCR analysis in T0 and T1 generations. Published in Russian in Fiziologiya Rastenii, 2007, vol. 54, No. 5, pp. 738–743. The text was submitted by the authors in English.     

Biotransformation of eugenol by suspension cultures of transgenic crown galls of <Emphasis Type="Italic">Panax quinquefolium</Emphasis> and suspension cultures of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

The biocatalytic ability of transgenic crown galls of Panax quinquefolium was evaluated by using eugenol (1) as a substrate and suspension cultures of Nicotiana tabacum as control system. Three biotransformed products, namely: 2-methoxy-4-(2-propenyl)phenyl-O-β-d-glucopyranoside (2, 67.11%), 2-methoxy-4-(2-propenyl)phenyl-O-β-d-glucopyranosyl (6′ → 1″)-β-d-xylopyranoside (3, 2.85%) and methyl eugenol (4, 14.30%) were obtained after 5 days of administration of eugenol to the suspension cultures of transgenic crown galls of P. quinquefolium. In contrast, only one product, compound 2 (15.41%), was obtained in suspension cultures of N. tabacum after 5 days of incubation. The results indicated that the glycosylation ability of transgenic crown galls of P. quinquefolium was much higher than that of the cultured cells of N. tabacum.     

A transformation booster sequence (<Emphasis Type="Italic">TBS</Emphasis>) from <Emphasis Type="Italic">Petunia hybrida</Emphasis> functions as an enhancer-blocking insulator in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Several matrix-attachment regions (MARs) from animals have been shown to block interactions between an enhancer and promoter when situated between the two. Since a similar function for plant MARs has not been discerned, we tested the Zea mays ADH1 5′ MAR, Nicotiana tabacum Rb7 3′ MAR and a transformation booster sequence (TBS) MAR from Petunia hybrida for their ability to impede enhancer–promoter interactions in Arabidopsis thaliana. Stable transgenic lines containing vectors in which one of the three MAR elements or a 4 kb control sequence were interposed between the cauliflower mosaic virus 35S enhancer and a flower-specific AGAMOUS second intron-derived promoter (AGIP)::β-glucuronidase (GUS) fusion were assayed for GUS expression in vegetative tissues. We demonstrate that the TBS MAR element, but not the ADH1 or Rb7 MARs, is able to block interactions between the 35S enhancer and AGIP without compromising the function of either with elements from which they are not insulated. Accession numbers: TBS from Petunia hybrida cultivar V26, GenBank accession number EU864306.     

Expression of CoQ10-producing <Emphasis Type="Italic">ddsA</Emphasis> transgene by efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Panicum meyerianum</Emphasis>

Panicum meyerianum Nees is a wild relative of Panicum maximum Jacq. (guinea grass), which is an important warm-season forage grass and biomass crop. We investigated the conditions that maximized the transformation efficiency of P. meyerianum by Agrobacterium infection by monitoring the expression of the β-glucuronidase (GUS) gene. The highest activities of GUS in calli were achieved by the co-cultivation of plants with Agrobacterium at 28°C for 6 days. We transferred the ddsA gene, which encodes decaprenyl diphosphate synthase and is required for coenzyme Q10 (CoQ10) synthesis, into P. meyerianum by using our optimized co-cultivation procedure for transformation. We confirmed by PCR and DNA gel blot hybridization that all hygromycin-resistant plants retained stable insertion of the hpt and ddsA genes. We also demonstrated strong expression of S14:DdsA protein in the leaves of transgenic P. meyerianum. Furthermore, we showed that transgenic P. meyerianum produced CoQ10 at levels 11–20 times higher than that of non-transformants. By comparison, the CoQ9 level in transgenic plants was dramatically reduced. This is the first report of efficient Agrobacterium-mediated transfer of a foreign gene into the warm-season grass P. meyerianum.     

The ectopic expression of <Emphasis Type="Italic">PttKN1</Emphasis> gene causes pleiotropic alternation of morphology in transgenic carnation (<Emphasis Type="Italic">Dianthus caryophyllus</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Carnation (Dianthus caryophyllus L.) is one of the most important ornamental plants in the world. Though morphological modification of carnation is very important to its commercial value, there have been no relevant reports until now. PttKN1 (Populus tremula × Tremuoides knotted1), isolated from the vascular cambial region of hybrid aspen, is a novel member of KNOX gene family. In this paper, we transformed 35S:PttKN1 to carnation via Agrobacterium tumefaciens. All primary transformants subsequently obtained were placed into phenotypic categories and self-pollinated. A total of 32 T0 progeny with aberrant phenotypes were obtained. PCR assay proved the validity of these transgenic plants. Phenotypes of 32 35S:PttKN1 plants were distinct from those of wild-type plants, including: (1) modification of phyllotaxis (15/32): wild-type carnation was with typical opposite phyllotaxis, while transgenic plants displayed tricussate whorled and multiple-cussate whorled phyllotaxis. Irregular modification of phyllotaxis was also observed; (2) modification of stem (9/32): wild-type stems were round; however, some transgenic plants exhibited much thicker and flatter stem; (3) the whole transgenic plants of carnation (8/32) became dwarf. These morphological modifications of carnation indicate that we have successfully attained some novel lines of carnation. These lines can have potential practical applications. In conclusion, the selection of stably genetic lines is discussed.     

Behavioural and population responses to changing availability of <Emphasis Type="Italic">Artemia</Emphasis> prey by moulting black-necked grebes, <Emphasis Type="Italic">Podiceps nigricollis</Emphasis>

Chemical communication may inform about the location of prey, predators, co-specifics, and mate partners in zooplankton. In this study, we evaluated several life-history traits of the rotifer, Brachionus calyciflorus, exposed to conditioned media by a rotifer predator (Asplanchna brightwelli) and a cladocera competitor (Daphnia similis), quantifying population growth and life-table demography at two algal food levels (2.0 and 0.5 × 10⁶ cells ml⁻¹ of Chlorella pyrenoidosa). At both food levels, B. calyciflorus grown in predator-conditioned media had lower population abundance and slower population growth rate than controls. Conversely, the competitor-conditioned media treatments produced both higher rotifer population abundance and faster population growth rate than controls. Life-history parameters varied significantly depending on the presence of predator and competitor-conditioned media. The Asplanchna-conditioned media significantly decreased gross reproductive rate (GRR): 8–9 offsprings per female; net reproductive rate (R ₀): 6–7 offsprings per female; population growth rate (r): 0.34–0.37 day⁻¹; and increased generation time (T): 5.5–5.6 days. On the other hand, The Daphnia-conditioned media significantly increased the GRR (13–14 offsprings per female); net reproductive rate (8–9 offsprings per female); population growth rate (0.42–0.43 day⁻¹); and decreased generation time (4.9–5.0 days). However, the effects of food level on the life-history characteristic were not significant in both treatments. Maximum values of the population abundance and the population growth rate are significantly influenced by the predator densities and pre-culture time. This study suggests that rotifers use variable life-history strategies (low reproduction and high survivorship versus high reproduction and low survivorship) based on the presence of predators and competitors.     

Chitinases of <Emphasis Type="Italic">Coffea arabica</Emphasis> genotypes resistant to orange rust <Emphasis Type="Italic">Hemileia vastatrix</Emphasis>

Two Coffea arabica — Hemileia vastatrix incompatible interactions (I₁: coffee cv. Caturra — rust race VI and I₂: coffee cv S4 Agaro — rust race II) and a compatible interaction (coffee cv. Caturra — rust race II) were compared in relation to the infection process and chitinase activity. In the two incompatible interactions the fungus ceased growth in the early infection stages, while in the compatible interaction no fungus growth inhibition was observed. A high constitutive level of chitinase activity was detected in the intercellular fluid of healthy leaves. Upon infection, chitinase isoforms were more abundant in incompatible interactions than in the compatible interaction. Immunodetection showed that class I chitinases are particularly relevant in the incompatible interactions and might participate in the defence response of the coffee plants.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Lotus tenuis</Emphasis> and regeneration of transgenic lines

A protocol for the production of transgenic plants was developed for Lotus tenuis via Agrobacterium-mediated transformation of leaf segments. The explants were co-cultivated (for 3 days) with an A. tumefaciens strain harbouring either the binary vector pBi RD29A:oat arginine decarboxylase (ADC) or pBi RD29A:glucuronidase (GUS), which carries the neomycin phosphotransferase II (nptII) gene in the T-DNA region. Following co-cultivation, the explants were cultured in Murashige and Skoog medium supplemented with naphthalenacetic acid (NAA) and benzyladenine (BA) and containing kanamycin (30 μg ml⁻¹) and cefotaxime (400 μg ml⁻¹) for 45 days. The explants were subcultured several times (at 2-week intervals) to maintain the selection pressure during the entire period. About 40% of the explants inoculated with the pBiRD29:ADC strain produced eight to ten adventitious shoots per responsive explant through a direct system of regeneration, whereas 69% of the explants inoculated with the pBi RD29A:GUS strain produced 13–15 adventitious shoots per responsive explant. The selected transgenic lines were identified by PCR and Southern blot analysis. Three ADC transgenic lines were obtained from 30 infected explants, whereas 29 GUS transgenic lines were obtained from 160 explants, corresponding to a transformation efficiency of 10 and 18.1%, respectively. More than 90% of the in vitro plantlets were successfully transferred to the soil. The increase in the activity of arginine decarboxylase from stressed ADC- Lt19 lines was accompanied by a significant rise in the putrescine level. The GUS transgenic line driven by the RD29A promoter showed strong signals of osmotic stress in the leaves and stem tissues. All of the transgenic plants obtained exhibited the same phenotype as the untransformed controls under non-stress conditions, and the stability of the gene introduced into the cloned materials was established.