Plant roept hulptroepen tegen vraat

Maarten Jongsma van Plant Research International onderzoekt de verdedigingsstrategie die één van de meest geharnaste planten gebruikt tegen insecten. Die strategie blijkt complexer dan gedacht. De kennis biedt wellicht mogelijkheden om gewassen te beschermen tegen insectenvraat

A novel expression cassette for the efficient visual selection of transformed tissues in florists' chrysanthemum (Chrysanthemum morifolium Ramat.).

Constructs carrying visual reporter genes coupled with efficient promoters could facilitate the process of identification and selection of stable transformants in recalcitrant crops. Here, a novel construct utilizing a ribulose-1,5-bisphosphate carboxylase (RbcS) promoter combined with the green fluorescent protein (GFP) reporter gene to initiate very high expression of GFP in florist's chrysanthemum (Chrysanthemum morifolium Ramat.) was described. Based on this expression cassette, a new regeneration protocol using leaf discs as explants was developed for the Agrobacterium-mediated transformation of Chrysanthemum genotype ‘1581’, and a transformation efficiency of 7% was obtained. The expression of two different GFP constructs targeted to either cytosol or plastids was compared in transgenic lines. Both GFP constructs were expressed at such a high level that the green fluorescence dominated red fluorescence in the leaf tissues, allowing easy observation and microdissection of transformed tissues even without a GFP filter. Under normal light, plants with GFP targeted to plastids had a light green phenotype deriving from the high GFP expression. Quantitative reverse transcriptional PCR analysis showed that the plastid targeted construct with intron had significantly higher steady state transcript levels of GFP mRNA. This novel expression cassette may allow direct visual selection of transformed tissues independent of antibiotic selection in a wide range of plant specie

Shoot organogenesis in leaf explants of Hydrangea macrophylla ‘Hyd1’ and assessing genetic stability of regenerants using ISSR markers

For the first time, an in vitro regeneration protocol of Hydrangea macrophylla 'Hyd1' was developed. Effects of different plant growth regulators (PGRs) on shoot regeneration were investigated jointly with selecting optimal basal media and cefotaxime concentrations. The highest frequency of shoot organogenesis (100%) and mean number of shoots per explant (2.7) were found on Gamborg B5 basal medium supplemented with 2.25 mg/l 6-benzyladenine (BA), 0.1 mg/l Indole-3-butyric acid (IBA), 100 mg/l cefotaxime and 30 g/l sucrose solidified by 7 g/l agar. Regenerated shoots were rooted by culturing on perlite plus half strength liquid B5 basal medium with 0.5 mg/l NAA. Rooted plantlets were transplanted to the greenhouse with 100% survival rate. Genetic stability of 32 plantlets (one mother plant and 31 regenerants) was assessed by 44 ISSR markers. Out of 44 ISSR markers, ten markers produced clear, reproducible bands with a mean of 5.9 bands per marker. The in vitro regeneration protocol is potentially useful for the genetic transformation of Hydrangea macrophylla 'Hyd1'

Aphid resistance in florist's chrysanthemum (Chrysanthemum morifolium Ramat.) induced by sea anemone equistatin overexpression

Florist's chrysanthemum (Chrysanthemum morifolium Ramat.) belongs to the Asteraceae family and represents the second most important floricultural crop in the world. Most genotypes are sensitive to aphids and infestations can lower quality and cause transmission of viruses. The protease inhibitor Sea Anemone Equistatin (SAE) carries three domains responsible for the inhibition of both cysteine and aspartic proteases. Artificial diet bioassays showed that SAE is readily toxic when ingested by the pea aphid, Acyrthosiphon pisum, and the cotton aphid, Aphis gossypii. We transformed chrysanthemum genotype 1581 by Agrobacterium tumefaciens-mediated transformation with the SAE gene under the control of the chrysanthemum RbcS promoter to induce aphid resistance. Non-choice leaf disk and whole plant bioassays were carried out to analyze deleterious effects of SAE on population growth and survival of both Myzus persicae and A. gossypii. After 7 days, M. persicae populations on specific transgenic lines were up to 69% smaller relative to control populations in a whole plant bioassay. The mortality of cotton aphids was 11% on control lines and up to 32% on transgenic lines after 5 days. The results show that SAE may be a promising agent for the control of some aphid species in transgenic plants. Key words: Chrysanthemum morifolium, aphid resistance, RbcS promoter, sea anemone equistatin, agrobacterium transformation

Biosynthesis of Sesquiterpene Lactones in Pyrethrum (Tanacetum cinerariifolium)

The daisy-like flowers of pyrethrum (Tanacetum cinerariifolium) are used to extract pyrethrins, a botanical insecticide with a long history of safe and effective use. Pyrethrum flowers also contain other potential defense compounds, particularly sesquiterpene lactones (STLs), which represent problematic allergenic residues in the extracts that are removed by the pyrethrum industry. The STLs are stored in glandular trichomes present on the pyrethrum achenes, and have been shown to be active against herbivores, micro-organisms and in the below-ground competition with other plants. Despite these reported bioactivities and industrial significance, the biosynthetic origin of pyrethrum sesquiterpene lactones remains unknown. In the present study, we show that germacratrien-12-oic acid is most likely the central precursor for all sesquiterpene lactones present in pyrethrum. The formation of the lactone ring depends on the regio- (C6 or C8) and stereo-selective (a or ß) hydroxylation of germacratrien-12-oic acid. Candidate genes implicated in three committed steps leading from farnesyl diphosphate to STL and other oxygenated derivatives of germacratrien-12-oic acid were retrieved from a pyrethrum trichome EST library, cloned, and characterized in yeast and in planta. The diversity and distribution of sesquiterpene lactones in different tissues and the correlation with the expression of these genes are shown and discussed

Key Success Factors of Innovation Projects of Vegetable Breeding Companies in China

The vegetable breeding industry is generally recognized as an innovation-driven industry. However, innovation is costly, time-consuming and uncertain. This study aims to identify the key success factors of innovation project performance of vegetable breeding companies (VBCs) in China. Based on empirical data that was collected from 53 innovation projects in 38 VBCs, it was found that integrative capabilities play an important role in the novelty and newness of the innovation to enhance product potential (superiority) and also in improving functional capabilities and in gaining market potential. Furthermore, market competition is a positive factor for inspiring innovation in the breeding industry

Kinetics of neuroendocrine differentiation in an androgen-dependent human prostate xenograft model

It was previously shown in the PC-295 xenograft that the number of chromogranin A (CgA)-positive neuroendocrine (NE) cells increased after androgen withdrawal. NE cells did not proliferate and differentiated from G0-phase-arrested cells. Here we further characterized NE differentiation, androgen receptor status, and apoptosis-associated Bcl-2 expression in the PC-295 model after androgen withdrawal to assess the origin of NE cells. PC-295 tumor volumes decreased by 50% in 4 days. Intraperitoneal bromodeoxyuridine (BrdU) incorporation and MIB-1 labeling decreased to 0%, and the apoptosis was maximal at day 4. Androgen receptor expression and prostate-specific antigen (PSA) serum levels decreased rapidly within 2 days. The number of NE cells increased 6-fold at day 4 and 30-fold at day 7. Five and ten percent of the CgA-positive cells were BrdU positive after continuous BrdU labeling for 2 and 4 days, respectively. However, no MIB-1 expression was observed in CgA-positive cells. NE cells expressed the regulated secretory pathway marker secretogranin III but were negative for androgen receptor and Bcl-2. Bcl-2 expression did increase in the non-NE tumor cells. In conclusion, androgen withdrawal leads to a rapid PC-295 tumor regression and a proliferation-independent induction of NE differentiation. The strictly androgen-independent NE cells that were still present after 21 days differentiated mainly from G0-phase-arrested cells

Metabolic engineering of terpenoid biosynthesis in plants

Metabolic engineering of terpenoids in plants is a fascinating research topic from two main perspectives. On the one hand, the various biological activities of these compounds make their engineering a new tool for improving a considerable number of traits in crops. These include for example enhanced disease resistance, weed control by producing allelopathic compounds, better pest management, production of medicinal compounds, increased value of ornamentals and fruit and improved pollination. On the other hand, the same plants altered in the profile of terpenoids and their precursor pools make a most important contribution to fundamental studies on terpenoid biosynthesis and its regulation. In this review we describe our recent results with terpenoid engineering, focusing on two terpenoid classes the monoterpenoids and sesquiterpenoids. The emerging picture is that engineering of these compounds and their derivatives in plant cells is feasible, although with some requirements and limitations. For example, in terpenoid engineering experiments crucial factors are the subcellular localisation of both the precursor pool and the introduced enzymes, the activity of endogenous plant enzymes which modify the introduced terpenoid skeleton, the costs of engineering in terms of effects on other pathways sharing the same precursor pool and the phytotoxicity of the introduced terpenoids. Finally, we will show that transgenic plants altered in their terpenoid profile exert novel biological activities on their environment, for example influencing insect behaviou

16 kDa Heat Shock Protein from Heat-Inactivated Mycobacterium tuberculosis Is a Homodimer – Suitability for Diagnostic Applications with Specific Llama VHH Monoclonals

Background: The 16 kDa heat shock protein (HSP) is an immuno-dominant antigen, used in diagnosis of infectious Mycobacterium tuberculosis (M.tb.) causing tuberculosis (TB). Its use in serum-based diagnostics is limited, but for the direct identification of M.tb. bacteria in sputum or cultures it may represent a useful tool. Recently, a broad set of twelve 16 kDa specific heavy chain llama antibodies (VHH) has been isolated, and their utility for diagnostic applications was explored. Methodology/Principal Findings: To identify the epitopes recognized by the nine (randomly selected from a set of twelve 16 kDa specific VHH antibodies) distinct VHH antibodies, 14 overlapping linear epitopes (each 20 amino acid long) were characterized using direct and sandwich ELISA techniques. Seven out of 14 epitopes were recognized by 8 out of 9 VHH antibodies. The two highest affinity binders B-F10 and A-23 were found to bind distinct epitopes. Sandwich ELISA and SPR experiments showed that only B-F10 was suitable as secondary antibody with both B-F10 and A-23 as anchoring antibodies. To explain this behavior, the epitopes were matched to the putative 3D structure model. Electrospray ionization time-of-flight mass spectrometry and size exclusion chromatography were used to determine the higher order conformation. A homodimer model best explained the differential immunological reactivity of A-23 and B-F10 against heat-treated M.tb. lysates. Conclusions/Significance: The concentrations of secreted antigens of M.tb. in sputum are too low for immunological detection and existing kits are only used for identifying M.tb. in cultures. Here we describe how specific combinations of VHH domains could be used to detect the intracellular HSP antigen. Linked to methods of pre-concentrating M.tb. cells prior to lysis, HSP detection may enable the development of protein-based diagnostics of sputum samples and earlier diagnosis of diseases