Genetic Modification of the Soybean to Enhance the β-Carotene Content through Seed-Specific Expression

<div><p>The carotenoid biosynthetic pathway was genetically manipulated using the recombinant <em>PAC</em> (<em>Phytoene synthase-2A-Carotene desaturase</em>) gene in Korean soybean (<em>Glycine max</em> L. cv. Kwangan). The <em>PAC</em> gene was linked to either the β-conglycinin (β) or CaMV-35S (35S) promoter to generate <em>β-PAC</em> and <em>35S-PAC</em> constructs, respectively. A total of 37 transgenic lines (19 for <em>β-PAC</em> and 18 for <em>35S-PAC</em>) were obtained through <em>Agrobacterium</em>-mediated transformation using the modified half-seed method. The multi-copy insertion of the transgene was determined by genomic Southern blot analysis. Four lines for <em>β-PAC</em> were selected by visual inspection to confirm an orange endosperm, which was not found in the seeds of the <em>35S-PAC</em> lines. The strong expression of <em>PAC</em> gene was detected in the seeds of the <em>β-PAC</em> lines and in the leaves of the <em>35S-PAC</em> lines by RT-PCR and qRT-PCR analyses, suggesting that these two different promoters function distinctively. HPLC analysis of the seeds and leaves of the T₂ generation plants revealed that the best line among the <em>β-PAC</em> transgenic seeds accumulated 146 µg/g of total carotenoids (approximately 62-fold higher than non-transgenic seeds), of which 112 µg/g (77%) was β-carotene. In contrast, the level and composition of the leaf carotenoids showed little difference between transgenic and non-transgenic soybean plants. We have therefore demonstrated the production of a high β-carotene soybean through the seed-specific overexpression of two carotenoid biosynthetic genes, <em>Capsicum</em> phytoene synthase and <em>Pantoea</em> carotene desaturase. This nutritional enhancement of soybean seeds through the elevation of the provitamin A content to produce biofortified food may have practical health benefits in the future in both humans and livestock.</p> </div

Tocopherol and phytosterol composition in the seeds of <i>β-PAC</i> and <i>35S-PAC</i> transgenic soybean plants.

<p>(a) Tocopherols. (b) phytosterols. The individual compositions in representative lines for <i>β-PAC</i> (line 16) and <i>35S-PAC</i> (line 6) are shown. Values (µg/g dry weight) are the mean of three replicates. Error bars represent the standard deviations.</p

DPPH radical scavenging activity of <i>β-PAC</i> and <i>35S-PAC</i> transgenic soybean seeds.

<p>The values of the DPPH are the mean of three determinations ± standard deviation (SD). Different letters represent significant (<i>P</i><0.05) differences between means according to ANOVA combined with Duncan’s multiple-range test.</p

Photographs of <i>β-PAC</i> and <i>35S-PAC</i> transgenic soybean seeds.

<p>(a) T₁ seeds. (b) T₂ seeds. (c) Cross sections of T₂ seeds. NT, non-transgenic plants; EV, empty vector-transgenic plants.</p

Reverse-transcriptase PCR (upper) and quantitative real-time (bottom) analyses of <i>β-PAC</i> and <i>35S-PAC</i> transgenic soybean lines.

<p>(a) Seeds. (b) Leaves. NT, non-transgenic plants; EV, empty vector transgenic plants. The 7, 13, 16 and 22 <i>β-PAC</i> transgenic lines and 5 and 6 <i>35S-PAC</i> transgenic lines were analyzed at the T₂ generation. The <i>Actin11</i> gene was used as the normalization control for both the seed and leaf RNA levels.</p

<i>Agrobacterium</i>-mediated transformation of half-seed explants and regeneration.

<p>(a) Half seed explants immediately after infection (left) and at five days after inoculation (right). (b) Shoot induction medium without DL-phosphinothricin. (c) Shoot induction medium containing DL-phosphinothricin (10 mg/L) for Basta® selection. (d) Shoot elongation medium with DL-phosphinothricin (5 mg/L). (e) Rooting medium. (f) Acclimated putative transgenic plant in a small pot. (g) Transgenic plant growing in a large pot. (h) Leaf painting assay showing wild type plant sensitivity (left) and transgenic plant resistance (right) to Basta® at five days after treatment with DL-phosphinothricin (100 mg/L).</p

Determination of insertion events of <i>β-PAC</i> and <i>35S-PAC</i> transgenes.

<p>(a) Genomic Southern blot analysis was performed with genomic DNAs from each leaf tissues and a <i>PAC</i> probe. The DNA molecular size markers are indicated on the left. (b) Quantitative real-time PCR analysis was carried out with the same genomic DNAs and a <i>Bar</i> primer set. Con, transgenic plant that its single <i>Bar</i> gene insertion was already confirmed NT, non-transgenic plants; EV, empty vector-transgenic plants.</p

Total carotenoid content and β/α ratio analysis of <i>β-PAC</i> and <i>35S-PAC</i> transgenic soybean plants.

<p>(a) Seeds. (b) Leaves. Total carotenoid levels were calculated as the sum of eight carotenoid subtype levels i.e. violaxanthin, antheraxanthin, lutein, zeaxanthin, α-cryptoxanthin, β-cryptoxanthin, α-carotene and β-carotene. The β-carotenoids include violaxanthin, antheraxanthin, zeaxanthin, β-cryptoxanthin and β-carotene and α-carotenoids include lutein and α-carotene in the determination of the β- to α- carotenoid ratios. Values (µg/g dry weight) are the means of three replicates. Error bars represent the standard deviations.</p

Schematic representation of the binary vectors used in the soybean transformations.

<p>Two vectors contained the same recombinant <i>PAC</i> gene as a closed box downstream of a soybean seed specific β-conglycinin promoter and a cauliflower mosaic virus 35S promoter, respectively. The remaining construct components had the same configuration, consisting of the <i>Bar</i> cassette to express the DL-phosphinothricin resistance gene driven by the 35S promoter and the cauliflower mosaic virus 35S terminator. LB and RB, left and right borders for <i>Agrobacterium</i>-mediated transformation, respectively; <i>5′-β</i>, a soybean seed specific β-conglycinin promoter; <i>5′-35S</i>, a cauliflower mosaic virus 35S promoter; <i>PAC</i>, a recombinant <i>Psy-2A-Tp-CrtI</i> gene; T35S, a cauliflower mosaic virus 35S terminator.</p