Construction of transgenic Ipomoea obscura that exhibits new reddish leaf and flower colors due to introduction of β-carotene ketolase and hydroxylase genes

Abstract

Ipomoea obscura, small white morning glory, is an ornamental plant belonging to the family Convolvulaceae, and cultivated worldwide. I. obscura generates white petals including a pale-yellow colored star-shaped center (flower vein). Its fully opened flowers were known to accumulate trace amounts of carotenoids such as β-carotene. In the present study, the embryogenic calli of I. obscura, were successfully produced through its immature embryo culture, and co-cultured with Agrobacterium tumefaciens carrying the β-carotene 4,4'-ketolase (crtW) and β-carotene 3,3'-hydroxylase (crtZ) genes for astaxanthin biosynthesis in addition to the isopentenyl diphosphate isomerase (idi) and hygromycin resistance genes. Transgenic plants, in which these four genes were introduced, were regenerated from the infected calli. They generated bronze (reddish green) leaves and novel petals that exhibited a color change from pale-yellow to pale-orange in the star-shaped center part. Especially, the color of their withered leaves changed drastically. HPLC-PDA-MS analysis showed that the expanded leaves of a transgenic line (T₀) produced astaxanthin (5.2% of total carotenoids), adonirubin (3.9%), canthaxanthin (3.8%), and 3-hydroxyechinenone (3.6%), which indicated that these ketocarotenoids corresponded to 16.5% of the total carotenoids produced there (530 µg g^-1 fresh weight). Furthermore, the altered traits of the transgenic plants were found to be inherited to their progenies by self-crossing.

Keywords: Ipomoea obscura; astaxanthin; embryogenic callus; transgenic plant.

Figure 1. Structure of the plasmid for plant transformation, pUTR-crtZWidi(pZH2B). Spec^r, the spectinomycin resistance gene; LB and RB, left and right borders; P_35S, the cauliflower mosaic virus 35S promoter; Hm^r, the hygromycin resistance gene; T_nos, the terminator of the nopaline synthase gene (nos); T_HSP, the terminator of the heat shock protein (HSP18.2) gene from Arabidopsis thaliana; DT2, double terminator that consists of T_HSP-T_nos; UTR, 5′-untranslated region of the alcohol dehydrogenase gene from tobacco (Nicotiana tabacum); tp, the transit peptide sequence from the pea RuBisCO small subunit gene; crtZ and crtW, the β-carotene 3,3′-hydroxylase and β-carotene 4,4′-ketolase genes, respectively, which code for the corresponding proteins from Brevundimonas sp. strain SD212; idi, the isopentenyl diphosphate (IPP) isomerase (type 2) gene that encodes the corresponding protein from Paracoccus sp. strain N81106.

Figure 2. Embryogenic callus formation and transgenic plant regeneration in I. obscura. (a) Embryogenic callus derived from an immature embryo. Bar represents 30 mm; (b) hygromycin-resistant somatic embryo formation from an Agrobacterium-infected embryogenic callus. Bar represents 2 mm; (c) raw expanded leaves of non-transgenic (left, Wild) and transgenic (right, T) plants (upper side). Aged leaves of non-transgenic (left, Wild) and transgenic (right, T) plants (lower side). Bar represents 20 mm; (d) flowers of non-transgenic (left, Wild) and transgenic line #21-1 (right, T) plants. Bar represents 20 mm.; (e) appearance of non-transgenic (left, Wild) and transgenic line #21-1 (right, T) plants. Bar represents 50 mm; (f) self progenies of non-transgenic (left, Wild) and transgenic line #21-1 (right, T) plants. Bar represents 50 mm.

Figure 3. PCR analysis for detection of the crtW and crtZ genes in three independent transgenic plant lines of Ipomoea obscura. Lane WT non-transgenic plant, lanes 1, 3, and 4 independent transgenic plants #21-1, -3 and -4, lane plasmid plasmid DNA.

Figure 4. HPLC chromatograms (at 450 nm) of extracts from the leaves of transgenic Ipomoea obscura #21-1 (T₀) plant (a) and I. obscura wild type plant (b). Chl, Chlorophylls; Neo, Neoxanthin; Ast, Astaxanthin; Vio, Violaxanthin; Lut, Lutein; Ado, Adonirubin; OH-Echi, 3-Hydroxyechinenone; cis-Lut, cis-Lutein; Can, Canthaxanthin; b-Car, β-Carotene.

Figure 5. Proposed carotenoid biosynthetic pathway in the leaves of transgenic ornamental Ipomoea obscura plants. Heavy blue arrows and narrow black arrows represent metabolic routes mediated by the introduced foreign genes and the endogenous genes, respectively.

Figure 6. PCR analysis for the detection of the crtW and crtZ genes in self cross progenies of transgenic plant line 1-1 of Ipomoea obscura. Lane WT non-transgenic plant, lanes 1, 2, 3, 5, 7 to 10 self-pollinated progenies, lane P plasmid DNA, lane M molecular size marker.