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Fig. 3. A in Hairy root transformation of Brassica rapa with bacterial halogenase genes and regeneration to adult plants to modify production of indolic compounds
Fig. 3. A. All transgenic root lines were analyzed by the following experiments and the data are presented for selected lines transformed with the pMDC32+2xCaMV35S:pyrH:nosT construct (pyrH = 5-Cl-Trp-forming). Transformation of roots with A. rhizogenes was verified using gDNA and cDNA for a successful insertion and expression, respectively. A. Upper panel: Amplified rolB (423 bp) and rolC (626 bp) for three different lines (lanes 1–3) using gDNA. The virG gene (350 bp) was only detectable in the positive control (Ri-plasmid of A. rhizogenes) (lane +) "-" denotes a negative PCR control. Lower panel: Integration of full length hal gene (ca. 1.5 kb) using gDNA. Expression of full length hal gene (ca. 1.5 kb) using cDNA. "+": positive control (plasmid containing pyrH or the other hal genes), "-": negative PCR control, g: gDNA wild type, c: cDNA wild type, 1–3: three independent transgenic root culture lines with the pMDC32+2xCaMV35S:pyrH:nosT construct, "1-"-"3-": RT negative controls (containing no DNA). B. Western blot with the purified His-tagged proteins: 1: PyrH, 2: ThaI, 3: PrnA, a: PyrH synthesized in E. coli, b: positive control ThaI synthesized in E. coli, c: positive control PrnA synthesized in E. coli. Wild type protein as control did not show any signal (data not shown). C. Enzyme assay with the purified halogenase PyrH. The positive control is PyrH protein synthesized in bacteria. The negative control is purified protein from wild type root cultures. Since only for PyrH enzyme activity could be detected, the data for the other halogenases are shown in the supplement (Fig. S1). D. Production of chlorinated tryptophan (Cl-Trp) and indole-3-acetonitrile (ClIAN) in transgenic root lines. For each halogenase construct five independent lines were tested. Results for 5 lines per halogenase type with and without Histag are indicated by the numbers of lines with the respective metabolites. The detailed results for all individual lines are shown in the supplement (Fig. S2).
Fig. 6. A in Hairy root transformation of Brassica rapa with bacterial halogenase genes and regeneration to adult plants to modify production of indolic compounds
Fig. 6. A. Confirmation of i) that the regenerated plants contain still the rol genes and ii) the integration of the hal gene into the genome and its transcription into cDNA (1: regenerated plants from wild type root cultures; 2: BrRP-pyrHHIS.6; 3: A. rhizogenes plasmid; 4: negative PCR control; 5: positive control - hal amplification from plasmid; a: cDNA, b: cDNA "no template control; genomic DNA. B. Western blot of His-tagged halogenase (PyrH, Thal, PrnA: purified enzymes from overexpressing E. coli strain as positive controls; BrRP-HR = WT, regenerated plants from wild type root cultures; BrRP-S: regenerated plants from Chinese cabbage seedlings; BrRP-pyrH, -thal, -prnA: regenerated plants from transgenic roots.). Always two different dilutions were applied. The gel strips were from the same gel, but due to large parts with samples without an immunosignal, the respective areas were cut out and are presented here. C. Relative amounts of chlorinated metabolites in the regenerated plants.
Fig. 2 in Hairy root transformation of Brassica rapa with bacterial halogenase genes and regeneration to adult plants to modify production of indolic compounds
Fig. 2. Expected indole metabolites and their interconversion (in black) that could be derived from tryptophan via the indole glucosinolate/indole phytoalexin pathway. It is indicated (in grey) that there are alternative pathways to IAA. The possible induction (dashed arrows) of chlorinated metabolites by abiotic and biotic stress factors, the latter also via the signaling molecules salicylic acid and jasmonic acid, is shown.
Fig. 1 in Hairy root transformation of Brassica rapa with bacterial halogenase genes and regeneration to adult plants to modify production of indolic compounds
Fig. 1. Experimental scheme showing the different types of plant materials generated. A. Mature wild type plants/seedlings; B. Wild type and transgenic root cultures; C. Regenerated sterile plants from wild type seedlings; D. Regenerated sterile plants from wild type and transgenic root cultures; E. Adult plants in soil from wild type cultures; F. Adult plants in soil from transgenic root cultures.
Fig. 5 in Hairy root transformation of Brassica rapa with bacterial halogenase genes and regeneration to adult plants to modify production of indolic compounds
Fig. 5. Left: Average number of regenerated shoots for 25 Brassica rapa "hairy root" lines (denoted therefore as BrHR ….) on 3 media compositions (n = 24). Medium A: GB5 medium containing 8 g l−1 phytoagar, 20 g l−1 sucrose and 10 mg l−1 6-BAP. Medium B: MS medium containing 8 g l−1 phytoagar, 30 g l−1 sucrose, 4 mg l−1 6-BAP, 4 mg l−1 AgNO3 and 3 mg l−1 NAA. Medium C: MS medium containing 8 g l−1 phytoagar, 30 g l−1 sucrose, 4 mg l−1 6-BAP, 4 mg l−1 AgNO3 and 0.5 mg l−1 NAA. Right: Shoot regeneration from B. rapa root cultures. Pieces from these root cultures were cut into pieces of approximately 1 cm2 and placed on semisolid agar (A). Regeneration of shoots was visible after 4 weeks of cultivation (B). Regenerated shoots were separated and transferred to fresh media (C). Shoot growth was often accompanied by growth of transformed/transgenic roots (D). Shoots of adequate biomass quality were subcultivated (E). Some B. rapa lines displayed a shortened life cycle after regeneration and began flowering (F).
Fig. 7 in Hairy root transformation of Brassica rapa with bacterial halogenase genes and regeneration to adult plants to modify production of indolic compounds
Fig. 7. Comparison of phenotypic traits for three groups of Brassica rapa grown in the greenhouse. WT – shoots from wild type plants grown from seeds (photo A); REG – regenerated shoots originated from transformed root cultures (photo B); HLR – regenerated shoots originated from transgenic roots transfected with bacterial hal genes (photo C). Significant differences between treatments are labeled as follows: 0 '***' 0.001 '**' 0.01 '*' 0.05 (with n = minimum of 20 individually potted plants). Leaves of in vitro shoots originated from seeds (left) or regenerated from root cultures (right) are shown.
Brassica Intake and Isothiocyanate Absorption
ClinicalTrials.gov study NCT02346812. IPD Sharing: Not stated. Countries: 1. Publications: 1.
Influence of Spices on Mixed Vegetable Intake Including Brassica Vegetables
ClinicalTrials.gov study NCT02012283. IPD Sharing: NO. Countries: 0. Publications: 0.
Genotyping-by-Sequencing data of weedy and domesticated Brassica rapa L.
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Data from: Genetic and environmental determinants of unreduced gamete production in Brassica napus, Sinapis arvensis and their hybrids
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Data from: Allelic variation of BnaC.TT2.a and its association with seed coat color and fatty acids in rapeseed (Brassica napus L.)
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The Evolutionary History of Wild, Domesticated, and Feral Brassica oleracea (Brassicaceae)
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Predation on sentinel prey increases with increasing latitude in Brassica-dominated agroecosystems
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Reprotoxic effects of the systemic insecticide fipronil on the butterfly Pieris brassicae
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Allelopathic effects of Brassica nigra in both its native and invaded ranges do not support the novel weapons hypothesis
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Data from: Temporal population genetic structure in the pollen pool for flowering time: a field experiment with Brassica rapa (Brassicaceae)
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Data from: Increased susceptibility to fungal disease accompanies adaptation to drought in Brassica rapa
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A diversity arrays technology developed high-density DArT markers as a tool for efficient and rapid genomic assessment in Ethiopian Mustard (Brassica carinata A. Braun)
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Data from: Testing weed risk assessment paradigms: intraspecific differences in performance and naturalisation risk outweigh interspecific differences in alien Brassica
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Aphid-induced phytochemicals in Brassica juncea (L.) Czern & Coss. afflicting host preference and bionomics of Lipaphis erysimi (Kaltenbach)
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