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 c-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.

Production and characterization of somatic hybrids between Solanum tuberosum L. and S. pinnatisectum Dun.

“…In recent years, wild potato species S. pinnatisectum (Greplová et al 2008;Polzerová et al 2011), S. cardiophyllum (Thieme et al 2010), S. 9 michoacanum (Szczerbakowa et al 2010), S. bulbocastanum (Greplová et al 2008) and S. tarnii (Thieme et al 2008) were used to transfer late blight resistance into cultivated potato through somatic hybridization. Protoplast fusion has also been succeeded in other crops like citrus (Grosser and Gmitter 2011), cotton (Sun et al 2011), sweet potato (Yang et al 2009), Brassicaceae (Ovcharenko et al 2011). In order to facilitate potato breeding against continuous threat of P. infestans, the introduction of new resistant clones is great concern in potato.…”

Engineering Plastid Genomes: Methods, Tools, and Applications in Basic Research and Biotechnology

“…Subsequent plant regeneration from fused protoplasts in the presence of the selection agent that kills nontransgenic chloroplasts gives rise to plants harboring the nuclear genome of the recalcitrant species and the chloroplast genome of the transformable species. Although proof-of-concept studies have demonstrated that this approach works (97,135,156), it is technically demanding and limited in applicability, because for species recalcitrant to plastid transformation, sufficiently efficient protoplast isolation, fusion, and regeneration protocols are usually not available.…”

Tri-parental protoplast fusion ofBrassicaspecies to produce somatic hybrids with high genetic and phenotypic variability

“…However, incompatibility between species coupled with low fertility in the F 1 hybrids severely limits the introgression of desirable traits (Wang et al 1983). Somatic hybridization via protoplast fusion makes it possible to bypass sexual-crossing barriers and facilitate transfer of desirable traits that are only present in the cytoplasm such as cytoplasmic male sterility (CMS) (Pelletier et al 1983;Liu et al 2005;Prakash et al 2009), transfer chloroplast (Ovcharenko et al 2011), and generate new unexpected alleles (Cardi and Earle, 1997). These techniques have been used for scion and rootstock breeding in citrus (Grosser and Gmitt 2011;Wang et al 2010) as well as for the introgression of genes from wild species into commercial cultivars in cotton (Sun et al 2011).…”