pLD-CTB-GAA

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	Transformation of tobacco (Nicotiana tabacum cv. Petit Havana) chloroplasts with the expression vector pLD-CTB-GAA and PCR analysis of the transplastomic lines were performed according to the previously published protocols (Verma et al., 2008). The CTB-GAA transplastomic tobacco lines were selected and regenerated on spectinomycin (500 mg/L) selection media.

Protocol tips
Transformation of tobacco (Nicotiana tabacum cv. Petit Havana) chloroplasts with the expression vector pLD-CTB-GAA and PCR analysis of the transplastomic lines were performed according to the previously published protocols (Verma et al., 2008). The CTB-GAA transplastomic tobacco lines were selected and regenerated on spectinomycin (500 mg/L) selection media.

Publication protocol

Transformation of tobacco (Nicotiana tabacum cv. Petit Havana) chloroplasts with the expression vector pLD-CTB-GAA and PCR analysis of the transplastomic lines were performed according to the previously published protocols (Verma et al., 2008). The CTB-GAA transplastomic tobacco lines were selected and regenerated on spectinomycin (500 mg/L) selection media. Site-specific integration of the transgenes including the aadA selection cassette and the CTB-GAA expression cassette was first confirmed by PCR analysis with two primer sets 3P/3M and 5P/2M (Verma et al., 2008). The 3rd primer pair CTB-Fw (5′-CA TATGACACCTCAAAATATTACTGATT-3′) and GAA-Rv (the same primer used for overlap extension PCR) was also used to identify the integration of CTB-GAA expression cassette. Southern blot analysis was carried out essentially as described by Kumar and Daniell (2004). Total tobacco DNA (1 μg) was digested with AflIII, separated on a 0.8% agarose gel and then transferred to a nylon membrane. A 0.81-kb DNA probe was isolated from pUC-CT plasmid with BamHI and BglII digestion and labeled with 32P. After labeling, membrane hybridization was carried out by following the QUICK-HYB hybridization protocol (Stratagene, La Jolla, CA).

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