Carbenicillin

The CD3-967/pBIN20-Golgi mCherry marker construct contains Agrobacterium rhizogenes strain (Arqua-1) was propagated on YEB agar plate supplemented with 100 mg L⁻¹ kanamycin at 28 °C. For more information about this construct see reference [67 ]. Hairy root transformation was performed as described [68 (link)]. Briefly, Arabidopsis thaliana Col-0 plants expressing 35S::cCRK1-GFP construct were propagated on ½ MS media as indicated in Section 4.1. Five days old plants were used for transformation. CD3-967/pBIN20 containing Arqua-1 strain was inoculated into 10 mL liquid YEB supplemented with 100 mg L⁻¹ kanamycin and rotated overnight at 28 °C in a shaker (200 rpm). Agrobacteria were centrifuged and resuspend in ½ MS liquid medium. Arabidopsis sp. plants were immersed in this solution for 2–5 min. After two days co-incubation in dark, plants were transferred onto ½ MS agar plates with 200 mg L⁻¹ cefotaxime (Duchefa, Haarlem, The Netherlands) and 200 mg L⁻¹ carbenicillin (Duchefa, Haarlem, The Netherlands) and cultured at 22 °C. After three to four weeks, the newly formed hairy roots were used for microscopy studies.

Eleven cassava genotypes representative of the varieties cultivated by farmers in Ghana (Afisiafi, Ankra, ADI 001, Bosomnsia, Dagarti, IFAD, Megyewontem, Nkabom, Santum, Tomfa, and Tuaka) were obtained from the in vitro cassava library of the Biotechnology and Nuclear Agriculture Research Institute (BNARI), Ghana. The model cultivar, 60444 was obtained from the in vitro germplasm collection of the Plant Biotechnology Laboratory at ETH Zurich. All plant material was maintained in vitro on cassava basic medium (CBM) media containing Murashige and Skoog (MS) basal salts with vitamins (Duchefa Biochemie, RV Haarlem, Netherlands), 20 g L⁻¹ sucrose (Roth, Switzerland), 2 mM CuSO₄ (Sigma-Aldrich, Munich, Germany), 3 g L⁻¹ Gelrite (Duchefa Biochemie, RV Haarlem, Netherlands), and 50 μg ml⁻¹ carbenicillin (Duchefa Biochemie, RV Haarlem, Netherlands) at 28°C, 16/8 h light/dark regime in growth cabinets.

TUB2 PCR products, obtained as described above from 17 samples, and showing single nucleotide polymorphisms (SNPs) in their chromatograms, were cloned into the pJET1.2/blunt vector to determine whether these are mixtures of two or three different TUB2 genotypes. The IGS PCR product obtained from a single sample, K10/Cs610, was also cloned in the same way because its chromatograms showed double peaks at the nucleotide positions that distinguish genotypes A and B. Cloning was conducted using the CloneJET PCR Cloning Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Competent Escherichia coli cells were prepared as described by Inoue et al. [33 (link)] and transformed with heat shock at 42 °C for 1 min. Suspensions were spread onto lysis broth medium containing 100 mg/mL carbenicillin (Duchefa Biochemie). Positive clones were selected by removing a part of the transformed bacterial colonies with sterile toothpicks and adding bacteria directly to the PCR mixtures. For these amplifications, primers provided with the kit were used, and the recommended PCR protocol was followed. Up to 10 clones per sample were sequenced with pJET1.2 Forward and reverse sequencing primers were from LGC Genomics GmbH. PCR products resulting from colony-based amplifications of seven samples were also included in CAPS analyses with XmiI, as described below.

Teicoplanin, vancomycin, oritavancin, and chloramphenicol (Sigma-Aldrich, Germany); dalbavancin (MedChemExpress, Sweden); MA79 (Csávás et al., 2015 (link)), ERJ390 (Pintér et al., 2009 (link)), and SZZS-12 (Szucs et al., 2017 (link)); carbenicillin, gentamicin and erythromycin (Duchefa Biochemie, Netherland); vancomycin BODIPY-FL conjugate (Thermo Fisher Scientific, Germany) and fluorescently labeled Teicoplanin (Vimberg et al., 2019 (link)).

The A. rhizogenes mediated A. sinicus transformation was carried out as described previously (82 (link)) with minor modifications. Briefly, the A. rhizogenes K599 line containing overexpression plasmids (pUB-AsGLP1 and empty pUB-GFP) or RNAi plasmids (empty pUB-RNAi, pUB-5′RNAi and pUB-3′RNAi) were cultured in 50 mL of LB medium until the OD₆₀₀ reached ~1.0. Sterilized 5-day-old seedlings were cut in the middle of the hypocotyl and incubated in bacterial suspension culture for 10 min. After blotting with autoclaved filter paper, the seedlings were placed on Murashige and Skoog basal medium and grown under 16-h/8-h light/darkness at 24°C/20°C, respectively. Three days later, the explants were transferred to fresh Murashige and Skoog medium with 500 mg L⁻¹ carbenicillin (Duchefa) and 30 mg L⁻¹ kanamycin (Duchefa) and grown for 10 more days until hairy roots developed from hypocotyls. The plants harboring positive transgenic hairy roots identified by using GFP were transferred to pots for phenotype analysis. For M. truncatula, the transfection was performed as described previously (83 (link)). The method for hairy root transformation of M. truncatula differed from that of A. sinicus. In order to obtain the transformed M. truncatula containing the pUB-MtGLPx, the operational process was performed according to the protocol of M. truncatula handbook (90 ).