Golden bananas in the field: elevated fruit pro-vitamin A from the expression of a single banana transgene

Abstract

Vitamin A deficiency remains one of the world's major public health problems despite food fortification and supplements strategies. Biofortification of staple crops with enhanced levels of pro-vitamin A (PVA) offers a sustainable alternative strategy to both food fortification and supplementation. As a proof of concept, PVA-biofortified transgenic Cavendish bananas were generated and field trialed in Australia with the aim of achieving a target level of 20 μg/g of dry weight (dw) β-carotene equivalent (β-CE) in the fruit. Expression of a Fe'i banana-derived phytoene synthase 2a (MtPsy2a) gene resulted in the generation of lines with PVA levels exceeding the target level with one line reaching 55 μg/g dw β-CE. Expression of the maize phytoene synthase 1 (ZmPsy1) gene, used to develop 'Golden Rice 2', also resulted in increased fruit PVA levels although many lines displayed undesirable phenotypes. Constitutive expression of either transgene with the maize polyubiquitin promoter increased PVA accumulation from the earliest stage of fruit development. In contrast, PVA accumulation was restricted to the late stages of fruit development when either the banana 1-aminocyclopropane-1-carboxylate oxidase or the expansin 1 promoters were used to drive the same transgenes. Wild-type plants with the longest fruit development time had also the highest fruit PVA concentrations. The results from this study suggest that early activation of the rate-limiting enzyme in the carotenoid biosynthetic pathway and extended fruit maturation time are essential factors to achieve optimal PVA concentrations in banana fruit.

Keywords: Uganda; Vitamin A deficiency; banana; biofortification; genetic modification; pro-vitamin A; staple food crop.

Figure 1

Analysis of promoter activity in wild‐type and transgenic Cavendish banana lines. GUS activity was measured in leaf (a, b, c), and peel (g, h, i), while GUS protein concentration was measured in pulp tissue (d, e, f). Ubi promoter (a, d, g); Exp1 promoter (b, e, h); and ACO promoter (c, f, i). WT, wild‐type FT432. S3, S6 and S9 represent 3, 6 and 9 weeks post‐bunch emergence, respectively. FG, full green and FR, full ripe. Error bars: ±SD.

Figure 2

Analysis of mRNA expression levels in the FG fruit pulp of selected transgenic Cavendish banana lines by qRT‐PCR. (a) MtPsy2a lines and (b) ZmPsy1 lines. FG, full green. WT, wild‐type with WT1 = FT167 and WT2 = FT430. Values are normalized expression levels ± SEM.

Figure 3

PVA carotenoid accumulation in the pulp of wild‐type and selected transgenic Cavendish banana lines during fruit development. S3, S6 and S9 represent 3, 6 and 9 weeks post‐bunch emergence, respectively. FG, full green and FR, full ripe. Error bars = ±SD.

Figure 4

Characteristic phenotypes observed in wild‐type and transgenic Cavendish banana lines. (a) Bunch from wild‐type line FT448; (b) fruit from wild‐type line FT448; (c) longitudinal section of fruit from wild‐type line FT448; (d) immature bunch from ACO‐ZmPsy1 line FT477; (e) fruit from ACO‐ZmPsy1 line FT477; (f) longitudinal section of fruit from ACO‐ZmPsy1 line FT477; (g) Exp1‐ZmPsy1 line FT192; (h) Ubi‐MtApsy2a line FT324; and (i) Ubi‐MtApsy2a line FT294. WT, wild‐type.

Figure 5

Percentage accumulation of individual carotenoids in the fruit pulp of wild‐type and transgenic bananas. Percentage (%) carotenoid content calculated based on total carotenoid content measured in the pulp of full green fruit collected from the sucker crop. Biological replicates: wild‐type (n = 5), ACO‐MtPsy2a (n = 5), ACO‐ZmPsy1 (n = 4), ACO‐ZmPsy1 + Exp1‐PaCrtI (n = 3), Exp1‐MtPsy2a (n = 4), Exp1‐ZmPsy1 (n = 5), Ubi‐MtPsy2a (n = 4) and Ubi‐ZmPsy1 (n = 2). All samples were analyzed in three technical replicates.

Figure 6

Influence of temperature and time to fruit maturity on the concentration of PVA carotenoids in the FG pulp of wild‐type Cavendish banana. For each month, β‐CE levels and time from bunch emergence to harvest were averaged from all samples collected. FG, full green.