Transfer of transformed Lesquerella fendleri (Gray) Wats. chloroplasts into Orychophragmus violaceus (L.) O.E. Schulz by protoplast fusion

Abstract

Asymmetric intergeneric hybrid plants were obtained through protoplast fusion between Orychophragmus violaceus (L.) O.E. Schulz and Lesquerella fendleri (Gray) Wats. The latter carried chloroplasts transformed with the fused aadA16gfp gene construct, conferring streptomycin–spectinomycin resistance and UV-induced green fluorescence. The somatic hybrids were selected using the properties of spectinomycin-induced plastid defects in “albino” O. violaceus plants (chloroplast recipient) combined with the γ-irradiation-induced inactivation of nuclei in plastid donor L. fendleri. The morphology and esterase isozyme pattern of the hybrid plant as well as the results of the PCR analysis of internal transcribed spacer of nuclear ribosomal DNA proved that the regenerated hybrids carried O. violaceus nuclei, while PCR amplification of the atpB–rbcL spacer and aadA16gfp gene fragments confirmed the presence of the transformed L. fendleri chloroplasts in these plants. Expression of the fused aadA16gfp gene construct was confirmed by sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis and the resistance of the obtained plants to both streptomycin and spectinomycin.