Stable Plastid Transformation in Nicotiana benthamiana

Abstract

Plastids from Nicotiana benthamiana were transformed with the vector for dicistronic expression of two genes-aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp)-in the plastids of Nicotiana tabacum. Transplastomic shoots exhibited green fluorescence under UV light. Transformation efficiencies were similar between species. Although the border sequence (trnI and trnA) for homologous recombination to transform the plastid genome of N. benthamiana was identical to that sequence of N. tabacum, the exception was a 9-bp addition in the intron of trnI. This indicated that the N. tabacum sequence used as a border region for recombination was sufficient to insert the foreign gene into the target site between the trnI and trnA of N. benthamiana with similar efficiency. Southern blot analysis detected the presence of aadA and gfp between trnI and trnA in the plastid genome of N. benthamiana. Northern and western blot analyses revealed high expression of gfp in the plastids from petals and leaves. Our results suggest that the plastid transformation system established here is applicable to investigations of the interactions between plastid and nucleus in N. benthamiana.

Stable chloroplast transformation of immature scutella and inflorescences in wheat (<italic>Triticum aestivum</italic> L.)

“…First, successful chloroplast transformation was reported in Chlamydomonas [12]. Then chloroplast transformation has been extended to an increasing number of dicotyledonous plants, including important plant bioreactor for pharmaceutical proteins: tobacco [7,[13][14][15]; the model plant species: Arabidopsis thaliana [16], petunia [17], and poplar tree [18]; species important in industrial economy: oilseed rape [19], Lesquerella fendleri [20], cotton [21], and sugar beet [22]; and edible crops: potato [23], tomato [24], carrot [25], soybean [26], lettuce [27,28], cauliflower [29], and cabbage [30]. Some preliminary studies have been made in chloroplast transformation of monocotyledonous species.…”

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

“…However, based on GFP protein accumulation in leaves, tubers and petals, it is clear that the relative performance of rrn and clpP regulatory sequences, and their potential applicability in plastid transformation approaches, vary with plastid types. In recent experiments in transplastomic N. benthamiana plants expressing the gfp gene under the control of the rrn promoter, decreasing transcript and protein expression levels were observed in leaves, petals and roots (Davarpanah et al 2009). The clpP 5 0 -UTR was previously used in combination with the rrn promoter to express the NPTII protein in tobacco leaves, but a mutant (chlorotic) phenotype was observed, even at low accumulation levels (Kuroda and Maliga 2002).…”

“…In contrast, no apparent reductions in transformation frequencies were observed in tobacco and tomato using homologous or homeologous N. tabacum and S. nigrum ptDNA sequences in transformation vectors (Kavanagh et al 1999;Horváth et al 2000;Nugent et al 2005;Nugent et al 2006), or in transformations of N. benthamiana plastids with tobacco-derived vectors (Davarpanah et al 2009), indicating that species-specific vectors are not always necessary. In the tobacco and tomato studies, it was demonstrated that integration of the cloned homeologous ptDNA sequences in the host plastome occurred by multiple recombination events in a similar region of the inverted repeats (IRs), that in comparison with ''Single Copy'' regions of the plastid genome, generally show a higher gene order conservation and a lower sequence divergence (Maier et al 1995).…”

“…The relative expression level of transgenes in tomato fruit chromoplasts, however, was higher with other proteins (Ruf et al 2001) and in any case sufficient to induce significant metabolic changes (Wurbs et al 2007;Apel and Bock 2009). Very low levels of mRNA and GFP protein were detected in roots of transplastomic Nicotiana benthamiana plants (less than 2% of that in the leaves), albeit expression levels up to 40-75% of those in leaf chloroplasts could be achieved in petal leucoplasts of the same plants or in chromoplasts of carrot taproots (Kumar et al 2004a;Davarpanah et al 2009). …”

“…A similar approach was adopted by Wurbs et al (2007), who used the atpI promoter containing both PEP and NEP signals, and the rps16 3 0 -UTR, to express carotenoidrelated genes in tomato fruits. Nevertheless, the short rrn promoter, containing only the sequences for PEP recognition, was successfully used to express transgenes in fruit chromoplasts of transplastomic tomato, and in petal leucoplasts and root amyloplasts of tobacco and Nicotiana benthamiana (Khan and Maliga 1999;Ruf et al 2001;Apel and Bock 2009;Davarpanah et al 2009).…”