Generation of transgenic maize with enhanced provitamin A content

Abstract

Vitamin A deficiency (VAD) affects over 250 million people worldwide and is one of the most prevalent nutritional deficiencies in developing countries, resulting in significant socio-economic losses. Provitamin A carotenoids such as beta-carotene, are derived from plant foods and are a major source of vitamin A for the majority of the world's population. Several years of intense research has resulted in the production of 'Golden Rice 2' which contains sufficiently high levels of provitamin A carotenoids to combat VAD. In this report, the focus is on the generation of transgenic maize with enhanced provitamin A content in their kernels. Overexpression of the bacterial genes crtB (for phytoene synthase) and crtI (for the four desaturation steps of the carotenoid pathway catalysed by phytoene desaturase and zeta-carotene desaturase in plants), under the control of a 'super gamma-zein promoter' for endosperm-specific expression, resulted in an increase of total carotenoids of up to 34-fold with a preferential accumulation of beta-carotene in the maize endosperm. The levels attained approach those estimated to have a significant impact on the nutritional status of target populations in developing countries. The high beta-carotene trait was found to be reproducible over at least four generations. Gene expression analyses suggest that increased accumulation of beta-carotene is due to an up-regulation of the endogenous lycopene beta-cylase. These experiments set the stage for the design of transgenic approaches to generate provitamin A-rich maize that will help alleviate VAD.

Fig. 1.

Carotenoid biosynthetic pathway in maize. PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; CRTISO, carotenoid isomerase; βLCY, β-cyclase; εLCY, ε-cyclase; HYD, carotene hydroxylases; CRTB, bacterial homologue of PSY; CRTI, bacterial homologue of PDS and ZDS.

Fig. 2.

Constructs used in the maize transformations. Four constructs were generated: pRB, pRI, pRBS, and pRIS. The core plasmid was pRC4 (see Materials and methods). Pzein, 27 kDa γ-zein promoter; SPzein, ‘super 27 kDa γ-zein promoter’ obtained by repeating the -444/-174 region; TEV, tobacco etch virus 5′ untranslated region; TP, transit peptide from pea Rubisco small subunit (rbcS); crtB, Erwinia herbicola phytoene synthase; crtI, E. herbicola phytoene desaturase; Tvsp, soybean vegetative storage protein terminator.

Fig. 3.

Analysis of T₁ kernels. (A) Representative ears from transformed plants with enhanced provitamin A content. The crtB/crtI cotransformants were regenerated from callus and crossed with Hi-II (white maize). There is segregation of white and yellow in the T₁ kernels. The lines are 48 (A) and 49 (B). (B) Total number of T₁ seeds on ears from six individual transgenic maize lines.The number of yellow/orange and white seeds on each ear are shown. (C) Total carotenoid and provitamin A content of T₁ seeds from transformed plants. HPLC analysis was performed on bulked samples of 20 T₁ seeds from each T₀ ear. The parental plants were the pRBS/pRIS regenerants from the transformed callus lines crossed with Hi-II. The amount of total carotenoid (in μg g⁻¹ dry weight of the seed) is equal to the sum of all carotenoids, and the provitamin A carotenoids were calculated as the sum of α-carotene, β-cryptoxanthin, 13-cis-β-carotene, 15-cis-β-carotene, trans β-carotene, 9-cis-β-carotene contents present in the extracted sample.

Fig. 4.

Analysis of T₂ kernels. (A) Total carotenoid and provitamin A contents of transgenic T₂ maize lines. The transgenic T₁ plants in Fig. 3B were self-pollinated, and HPLC analysis was conducted on pooled samples of 20 T₂ seeds from the resulting ears. The amount of total carotenoid is equal to the sum of all carotenoids and the provitamin A carotenoids were calculated as the sum of α-carotene, β-cryptoxanthin, 13-cis-β-carotene, 15-cis-β-carotene, trans β-carotene, 9-cis-β-carotene contents present in the extracted sample. (B) Carotenoid composition of T₂ seeds from transgenic lines derived from lines 24 and 25. Data represent the average carotenoid content (±SD) of seeds from five individual ears per line.

Fig. 5.

Provitamin A content of T₂, T₃, and T₄ seeds from transgenic maize lines 24 and 25. HPLC analyses were conducted on pooled samples of 20 T₂, T₃, and T₄ seeds of individual maize lines, respectively. Data shown represent the average provitamin A content (±SD) of seeds from 3–8 ears for each line.

Fig. 6.

Maize carotenoid gene expression. Total RNA was isolated from T₄ kernels of lines 24 and 25. Transcript levels were measured by RT-PCR using gene-specific primers and the data were normalized using 18S rRNA accumulation as a control. Histogram represents average expression levels of determinations from two separate experiments conducted with pooled 3–4 seeds for each line.