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Hairy root transformation system for common bean

Hairy root transformation system for common bean published in the paper named "Optimizing CRISPR/Cas9 gene editing in common bean (Phaseolus vulgaris L.) using a hairy root transformation system and in silico prediction models"

Fig. 4. A in Biotransformation of xenobiotics by hairy roots

Fig. 4. A Biotransformation of paracetamol into conjugates of glutathione to form a stable complex during phase II reaction stage B Biotransformation of paracetamol into conjugates of glucoside to form a stable complex which undergoes storage into lignin fraction of cell wall during phase III reaction by HRs of A. rusticana.

Fig. 5 in Biotransformation of xenobiotics by hairy roots

Fig. 5. Biotransformation of oxybenzone into conjugates of glucose and malonyl-glucoside during phase II reaction and further storage of malonyl conjugate in vacuole or cell wall during phase III reaction by HRs of A. rusticana.

Fig. 2 in Biotransformation of xenobiotics by hairy roots

Fig. 2. Biotransformation of phenols by hydroxylation, dehydrogenation, ortho-cleavage and oxidation reactions of Phase I reaction by HRs of H. annuus.

Fig. 7 in Biotransformation of xenobiotics by hairy roots

Fig. 7. Proposed biotransformation stages of TNT (2,4,6-trinitrotoluene) to 4-amino-2,6-dinitrotoluene and 2-amino-4,6-nitrotoluene by HRs of C. roseus.

Fig. 3 in Biotransformation of xenobiotics by hairy roots

Fig. 3. Biotransformation of 2, 4-dichlorophenol into 2-chloro-6-(2,4-dichlorophenoxy)-1,4-benzoquinone by HRs of N. tabacum during phase II reaction.

Fig. 6 in Biotransformation of xenobiotics by hairy roots

Fig. 6. Biotransformation of DDT to DDD, DDE, and DDMU during phase I reaction by HRs of B. juncea and C. intybus.

Fig. 8 in Biotransformation of xenobiotics by hairy roots

Fig. 8. Biotransformation of textile azo dyes: A) Reactive Red 198, B) Acid Red 114 by HRs of T. patula and I. carnea, respectively.

Fig. 4. A in Hairy root transformation of Brassica rapa with bacterial halogenase genes and regeneration to adult plants to modify production of indolic compounds

Fig. 4. A. Growth of wild type root cultures on different Trp derivatives as compared to growth on MS medium only. B. Correlation of the relative growth and the production of different Cl-Trp compounds.

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.

Fig. 7 in SmbHLH3 acts as a transcription repressor for both phenolic acids and tanshinone biosynthesis in Salvia miltiorrhiza hairy roots

Fig. 7. (A) Identification of positive transgenic hairy roots lines by PCR (A, 35S + SmbHLH3; B, hpt II; C, rol b; D, rol c). Numbers above represent individual transgenic lines and M represent DL2000 DNA marker. (B) The phenotypes of hairy roots. Hairy roots were cultured in 6,7-V liquid medium for 30 days before being photographed. (C) Relative quantitative analysis of SmbHLH3 expression in transgenic lines and control of S. miltiorrhiza hairy roots. Bars are means ± SD from three independent biological replicates. One-way ANOVA (followed by a Turkey comparison) was tested for significant differences among the means (indicated by different letters at P <0.01).

Fig. 5 in SmbHLH3 acts as a transcription repressor for both phenolic acids and tanshinone biosynthesis in Salvia miltiorrhiza hairy roots

Fig. 5. Subcellular localization of SmbHLH3 protein in onion epidermal cells. Fluorescence was observed using a confocal laser scanning microscope at 24 h after incubation. The pictures showed bright field (TD), green fluorescent field (GFP), DAPI and overlay of three fields (Merge). The numerical reading of red ruler is 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2 in SmbHLH3 acts as a transcription repressor for both phenolic acids and tanshinone biosynthesis in Salvia miltiorrhiza hairy roots

Fig. 2. Relative expression levels of phenolic acids biosynthetic pathway genes in transgenic hairy roots lines and the control. The results were analyzed using the comparative Ct method. The S. miltiorrhiza Actin gene was used as an internal control to normalize expression levels. The vertical bars show the SD values (n = 3). One-way ANOVA (followed by a Turkey comparison) was tested for significant differences among the means (indicated by different letters at P <0.01).

Fig. 3 in SmbHLH3 acts as a transcription repressor for both phenolic acids and tanshinone biosynthesis in Salvia miltiorrhiza hairy roots

Fig. 3. Relative expression levels of tanshinone biosynthesis pathway genes in transgenic hairy roots lines and the control. The vertical bars show the SD values (n = 3). One-way ANOVA (followed by a Turkey comparison) was tested for significant differences among the means (indicated by different letters at P <0.01).

Fig. 4 in SmbHLH3 acts as a transcription repressor for both phenolic acids and tanshinone biosynthesis in Salvia miltiorrhiza hairy roots

Fig. 4. Sequence analysis of SmbHLH3. (A) Deduced amino acid sequence of SmbHLH3. Predicted HLH domain was present in shaded area. (B) Phylogenetic analysis of SmbHLH3. A phylogenetic tree was constructed based on the amino acid sequences of SmbHLH3 and SibHLH3 (XP_011080018), EgbHLH3-like (XP_012836308.1), OebHLH3 (XP_022854140), StbHLH (XP_006352746), NsbHLH3 (XP_009803066), CbbHLH3 (PHT46573), AcbHLH3 (PSS14567), CabHLH3 (PHT80432), LnbHLH3- like (XP_019165044), OebHLH3-like (XP_022872401), CcbHLH3 (PHU16394), PabHLH3 (XP_021814813), AtbHLH3 (AT4G16430), AtbHLH13 (AAM10932), AtbHLH17 (AT2G46510) and all of the SmbHLHs that previously reported. These phylogenetic trees were constructed via MEGA6, using the neighbor-joining method with 500 bootstrap replicates.