High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences

Abstract

Transformation of potato plastids is limited by low transformation frequencies and low transgene expression in tubers. In order to improve the transformation efficiency, we modified the regeneration procedure and prepared novel vectors containing potato flanking sequences for transgene integration by homologous recombination in the Large Single Copy region of the plastome. Vector delivery was performed by the biolistic approach. By using the improved regeneration procedure and the potato flanking sequences, we regenerated about one shoot every bombardment. This efficiency corresponds to 15-18-fold improvement compared to previous results with potato and is comparable to that usually achieved with tobacco. Further, we tested five promoters and terminators, and four 5 0 -UTRs, to increase the expression of the gfp transgene in tubers. In leaves, accumulation of GFP to about 4% of total soluble protein (TSP) was obtained with the strong promoter of the rrn operon, a synthetic rbcL-derived 5 0 -UTR and the bacterial rrnB terminator. GFP protein was detected in tubers of plants transformed with only four constructs out of eleven. Best results (up to approximately 0.02% TSP) were achieved with the rrn promoter and rbcL 5 0 -UTR construct, described above, and another containing the same terminator, but with the promoter and 5 0 -UTR from the plastid clpP gene. The results obtained suggest the potential use of clpP as source of novel regulatory sequences in constructs aiming to express transgenes in amyloplasts and other non-green plastids. Furthermore, they represent a significant advancement of the plastid transformation technology in potato, of relevance to its implementation in potato breeding and biotechnology.

Transplastomic plants for innovations in agriculture. A review

“…Non-antibiotic, native plant genes that offer dominant and portable selectable markers like ASA2 (Barone et al 2009) or confer additional agricultural advantage to the transformed crops such as BADH conferring salt or drought tolerance to transformed crops Kumar et al 2004a) may be good alternatives to antibiotic selection markers, and a few examples have also shown improved and in some cases inducible Lössl et al 2005;Buhot et al 2006;Tungsuchat et al 2006;Verhounig et al 2010) transgene expression in different organs and plastid types (e.g., Valkov et al 2011;Zhang et al 2012;Caroca et al 2013). A recently developed positive selection protocol utilizes the Agrobacterium tumefaciens isopentenyl transferase (ipt) gene involved in early stage of cytokinin biosynthesis and allows cell proliferation and differentiation into shoots without the use of exogenous cytokinin in the selection medium (Dunne et al 2014).…”

Unsolved problems in plastid transformation

“…The importance of the extent of sequence similarity between sequences involved in homologous recombination to obtain high transformation efficiency has been recently demonstrated in several species. 8,9 The extent of sequence divergence can influence the frequency of homologous recombination, and thus the integration efficiency, as well as the functionality of recombinant coding sequences, likely affecting the regeneration of viable plants. Plastid transformation efficiency in potato has been recently improved using novel vectors containing potato flanking sequences (Fig.…”

“…1). 9 Due to sequence variability in different regions, proper sequence analysis before attempting to use vectors developed in non-target species is recommended. 10 In some species, an additional limitation to plastid transformation is the lack of efficient selection/regeneration protocols.…”

“…12 Recently, several regulatory sequences that could increase the expression of transgenes in tomato fruit chromoplasts and potato tuber amyloplasts have been identified. 9,13,14 Furthermore, Zhang et al 12 developed a quantitative phenotypic method suitable to identify the in vivo activity of cis-acting elements conferring high levels of geneexpression in non-green plastids. The assay described is based on the use of the nptII gene as a visual reporter of gene-activity in non-green plastids and on the analysis of root length upon seedling growth in the presence of the plastid translational inhibitor kanamycin.…”

Efficient Plastid Transformation in Arabidopsis

“…The plastid genome of higher plants encodes about 100 genes, the products of which assemble with approximately 3,000 nucleus-encoded proteins to form the plastid transcription and translation machinery and carry out complex metabolic functions, including photosynthesis and fatty acid and amino acid biosynthesis. Plastid transformation is routine only in tobacco (Nicotiana tabacum; Svab et al, 1990;Svab and Maliga, 1993), but reproducible protocols for plastid transformation also have been described in tomato (Solanum lycopersicum; Ruf et al, 2001), potato (Solanum tuberosum; Valkov et al, 2011), lettuce (Lactuca sativa; Kanamoto et al, 2006;Ruhlman et al, 2010), and soybean (Glycine max; Dufourmantel et al, 2004). Still, the technology is available in only a relatively small number of crops.…”