Journal: Plant Biotechnology Journal

Article Title: Abatement of microfibre pollution and detoxification of textile dye – Indigo by engineered plant enzymes

doi: 10.1111/pbi.13942

Figure Lengend Snippet: Chloroplast transformation vectors, site‐specific transgene integration and maternal inheritance of laccase and lignin peroxidase genes. (a) Schematic representation of chloroplast trn l/ trn A spacer region of untransformed wild‐type (UT‐WT) tobacco (b) chloroplast vectors of laccase (Lac) and lignin peroxidase (LiP) genes. Solid arrowhead with dotted line represents an expected DNA fragment size of ~4.0 kb from UT‐WT, and ~7.2, ~6.7 and ~6.6 kb from Ao Lac and Pc LiP transplastomic lines after digestion with Sma I restriction enzyme. A ~0.8 kb Bam HI and Bgl II restriction enzyme digested DNA fragment was used as probe for Southern blot analysis. Map is not in scale. (c) Seedlings from a homoplasmic transplastomic line (green, left) and untransformed wild‐type (UT‐WT, bleached white, right) germinated on 500 mg/L spectinomycin plate showing maternal inheritance. (d–f) Confirmation of integration of ligninase genes into the tobacco chloroplast genome. (d) Amplification of ~1.6 kb fragment using 16SrRNA _F (3P)/ aadA _R (3M). Amplification of ~1.1 kb (e) and (f) ~4.2 kb fragments using PpsbA _F (DV9)/ TpsbA _R (DV13) and aadA _F (5P)/ trnA _R (2M) from Cp‐LiP ( Pc LiP) respectively. Lanes M1 and M2 are DNA molecular weight ladders (NEB 1 kb ladder and Invitrogen 1 kb ladder plus, respectively). Lane 1 is negative control. Lane 2 is UT‐WT. Lanes 3 to 8 are six independent Cp‐LiP transplastomic lines.

Article Snippet: The pUC‐Ct vector plasmid DNA was digested with Bam HI and Bgl II restriction enzymes (NEB) to obtain 0.81 kb chloroplast flanking sequence probe as shown in Figure and described in the Verma et al . ( ).

Techniques: Transformation Assay, Southern Blot, Amplification, Molecular Weight, Negative Control

Journal: Plant Biotechnology Journal

Article Title: Abatement of microfibre pollution and detoxification of textile dye – Indigo by engineered plant enzymes

doi: 10.1111/pbi.13942

Figure Lengend Snippet: Evaluation of homoplasmy of transplastomic Cp‐laccase ( Ao Lac) and Cp‐LiP ( Pc LiP) plants by Southern blot analyses. Southern blot hybridization profile of Sma I restriction enzyme‐digested DNA isolated from leaves of Cp‐laccase, Cp‐LiP transplastomic and untransformed WT plants after hybridization with the probe. Presence of a single ~7.2 kb and ~6.7 kb band in five independent (lanes 1–5) lines of Cp‐laccase (a) and Cp‐LiP (b) transplastomic plants confirms stable integration and homoplasmic status, whereas existence of a single ~4.0 kb is expected WT (lane WT) plant (see Figure for chloroplast vector maps). Membrane after DNA transfer was hybridized with ~0.8 kb Bam HI and Bgl II restriction enzyme‐digested DNA fragment as the probe for Southern blot analysis.

Article Snippet: The pUC‐Ct vector plasmid DNA was digested with Bam HI and Bgl II restriction enzymes (NEB) to obtain 0.81 kb chloroplast flanking sequence probe as shown in Figure and described in the Verma et al . ( ).

Techniques: Southern Blot, Hybridization, Isolation, Plasmid Preparation