Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance

Abstract

Salinity is one of the major factors that limits geographical distribution of plants and adversely affects crop productivity and quality. We report here high-level expression of betaine aldehyde dehydrogenase (BADH) in cultured cells, roots, and leaves of carrot (Daucus carota) via plastid genetic engineering. Homoplasmic transgenic plants exhibiting high levels of salt tolerance were regenerated from bombarded cell cultures via somatic embryogenesis. Transformation efficiency of carrot somatic embryos was very high, with one transgenic event per approximately seven bombarded plates under optimal conditions. In vitro transgenic carrot cells transformed with the badh transgene were visually green in color when compared to untransformed carrot cells, and this offered a visual selection for transgenic lines. BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7-fold more, and accumulated 50- to 54-fold more betaine (93-101 micromol g(-1) dry weight of beta-Ala betaine and Gly betaine) than untransformed cells grown in liquid medium containing 100 mm NaCl. Transgenic carrot plants expressing BADH grew in the presence of high concentrations of NaCl (up to 400 mm), the highest level of salt tolerance reported so far among genetically modified crop plants. BADH expression was 74.8% in non-green edible parts (carrots) containing chromoplasts, and 53% in proplastids of cultured cells when compared to chloroplasts (100%) in leaves. Demonstration of plastid transformation via somatic embryogenesis utilizing non-green tissues as recipients of foreign DNA for the first time overcomes two of the major obstacles in extending this technology to important crop plants.

Figure 1.

Physical map of the carrot chloroplast transformation vector pDD-Dc-aadA/badh. PCR primer landing sites and the probe used for Southern analysis are shown.

Figure 2.

Visual selection of green transgenic calli versus yellow nontransgenic carrot calli. A, Transformed. B, Untransformed. C and D, Heteroplasmic transgenic calli in the absence of a selection agent.

Figure 3.

Confirmation of transgene integration into the carrot plastid genome by PCR and Southern-blot analysis. A, PCR product (1.65 kb) from internal primers 3P (land on flanking sequence) and 3M (land on the aadA gene). B, PCR product (2.5 kb) from an external primer 16SF (land on the native chloroplast genome) and an internal primer 1M (land on the aadA gene). Lane 1, 1-kb DNA ladder; lane 2, untransformed; lanes 3 to 9, transgenic carrot cell lines. All primer landing sites are shown in Figure 1. C, Southern-blot analysis of untransformed and transformed carrot with the vector pDD-Dc-aadA/badh. Carrot genomic DNA (5 μg/lane) digested with AflIII and PvuII was hybridized with the 3.2-kb radioactive-labeled P³² DNA probe containing a 1.4-kb flanking sequence or a 1.8-kb aadA/badh sequence (see Fig. 1 for details). Lane 1, Untransformed plant; lanes 2 and 3, heteroplasmic transgenic plants; lanes 4 to 8, homoplasmic transgenic plants derived after repetitive subculture in liquid medium under spectinomycin selection.

Figure 4.

BADH enzyme activity and BADH expression in control and pDD-Dc-aadA/badh lines. A, A pDD-Dc-aadA/badh transgenic line shown with taproot and shoot. B, BADH activity in untransformed (U) and transformed (T) cell suspension, root, and leaf. Note: Mean and errors bars are the average of three replicates. C, Western blot using polyclonal anti-BADH serum. Antigenic peptides were detected using horseradish peroxidase-linked secondary antibody. Lanes 1 to 3, Untransformed cell culture, root, and leaf; lanes 4 to 6, transformed cell culture, root, and leaf.

Figure 5.

Effect of different salt concentrations on growth of untransformed and transformed cell lines with pDD-Dc-aadA/badh and study of betaine with ¹H-NMR spectra. Untransformed (A) and transformed (B) cell cultures grown on 100 mm NaCl. C, Stimulation of BADH activity in the presence of salt. Untransformed and transformed carrot cells in suspension cultures were placed on a shaker at 130 rpm for 2 weeks in liquid medium containing 0, 100, 200, and 300 mm NaCl. ¹H-NMR spectra (500 MHz) of extracts from untransformed (D and E) and transformed (F and G) carrot cell suspension cultures grown in the presence of 100 mm NaCl alone (D and F) or in combination with 4 mm choline (E and G). Purified samples were dissolved in D₂O and t-butanol (an internal standard). Integration of the singlet versus t-butanol was used for quantification of betaine. A dominant singlet of betaine is detected at 3.20 ppm.

Figure 6.

Effect of salt (100–500 mm NaCl) on untransformed (U) and transgenic (T) lines grown at different concentrations of NaCl. Plants were irrigated with water containing different concentrations of NaCl on alternate days for up to 4 weeks.