Pds 1000 he biolistic gun

Young leaves from aseptically grown tobacco plants were bombarded with plasmid-coated 0.6 μm gold particles using a PDS1000He Biolistic gun (Bio-Rad). Primary spectinomycin-resistant lines were selected on regeneration medium containing 500 mg/L spectinomycin [25 (link)]. Spontaneous spectinomycin-resistant plants were eliminated by double selection on medium containing spectinomycin and streptomycin (500 mg/L each). For each transformation construct, several independent transplastomic lines were subjected to two to three additional rounds of regeneration on spectinomycin-containing medium to enrich the transplastome and select for homoplasmic tissue. Physical maps of the psbA gene area of the wild type (WT) and the transgenic (R238A, R238D, R238E, WT-aadA) tobacco lines are drawn in S5 Fig.

Lactuca sativa cv. Barkley plants were grown in vitro from surface sterilized seeds on solid MS medium (Murashige and Skoog, 1962) containing 20 g/L sucrose. Leaves from aseptically grown lettuce plants were bombarded with 0.6 μm gold‐microcarriers coated with plasmid DNA using a Bio‐Rad Biolistic PDS‐1000/He gun (Daniell, 1997; Svab and Maliga, 1993) Several independently transformed plant lines were subjected to three additional regeneration rounds on RMOP medium (Svab and Maliga, 1993; Verma et al., 2008) containing spectinomycin. Regenerated shoots were rooted on MS medium containing spectinomycin to maintain the selection pressure. Rooted, homoplastomic plants were transferred to soil and grown to maturity in the greenhouse under standard conditions. Inheritance assays on spectinomycin‐containing MS medium were performed with the harvested seeds.

ToMoV virus A and B components were mixed in a 1:1 ratio (1 μg of component A: ToMoVΔCP, ToMoV_ChII or ToMoV_SCEI and 1 μg of component B: ToMoV B) after their digestion from the pBS II SK vector with ApaI. Twenty-two day-old plants were treated with the virus mixture using the Biolistic® PDS-1000He gun (Bio Rad, Hercules, CA, USA) at low pressure (Carrillo-Tripp et al., 2006 (link)). The plants were then maintained for 60 days post-treatment (dpt) in a greenhouse at 25–30°C before inoculation with Cmm. This time period was chosen because ToMoV_ChII inoculated plants showed bleaching of all leaves by 60 days, which indicated that silencing had occurred. By 15 days post inoculation (dpi) with Cmm, symptoms of leaf wilting and necrosis were observed and recorded by scanning excised damaged leaves on a flat-bed scanner. A tif file was created and the number of pixels of damaged tissue was quantified by Scion Image (Scion Corporation, Frederick, MD, USA) (Wijekoon et al., 2008 (link)). Statistical analysis was based on T-test with unpaired data with Graph Pad Prism® V.5 (GraphPad, San Diego, CA, USA), and a statistically significant result was considered to be P < 0.01.