pLsLF-CTB-Fx3Pris

Protocol tips

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	Bombardment was done using the Bio-Rad PDS-1000/He gene gun as described previously (Verma et al., 2008). Sterile tobacco leaves were placed abaxial side up, whereas sterile lettuce leaves were placed abaxial side down on Murashige and Skoog (MS) medium and were bombarded under sterile conditions using pLDutr-CTB-Fx3Pris or pLsLF-CTB-Fx3Pris coated with gold particles prepared according to (Kumar and Daniell, 2004).

Protocol tips

Bombardment was done using the Bio-Rad PDS-1000/He gene gun as described previously (Verma et al., 2008). Sterile tobacco leaves were placed abaxial side up, whereas sterile lettuce leaves were placed abaxial side down on Murashige and Skoog (MS) medium and were bombarded under sterile conditions using pLDutr-CTB-Fx3Pris or pLsLF-CTB-Fx3Pris coated with gold particles prepared according to (Kumar and Daniell, 2004).

Publication protocol

Bombardment was done using the Bio-Rad PDS-1000/He gene gun as described previously (Verma et al., 2008). Sterile tobacco leaves were placed abaxial side up, whereas sterile lettuce leaves were placed abaxial side down on Murashige and Skoog (MS) medium and were bombarded under sterile conditions using pLDutr-CTB-Fx3Pris or pLsLF-CTB-Fx3Pris coated with gold particles prepared according to (Kumar and Daniell, 2004). Bombarded leaf pieces were incubated in the dark for 2 days at room temperature, and then leaf segments were transferred to regeneration media of plants (RMOP) containing 500 mg/L spectinomycin for tobacco or regeneration media of lettuce (RMOL) containing 100 mg/L spectinomycin (Ruhlman et al., 2010). The appearance of green shoots was seen after 4– 6 weeks. These shoots were allowed to grow, and the leaves were cut and subsequently transferred to RMOP or RMOL containing 500 mg/L or 100 mg/L spectinomycin, respectively, for the second round of selection. After new shoots formed, these were transferred for the third round of selection to MS containing 500 mg/L spectinomycin for tobacco or 100 mg/L spectinomycin for lettuce and allowed to establish roots. Plants were then transferred to the greenhouse.

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