The demand for decaffeinated coffee is increasing because the stimulatory effects of caffeine can adversely affect sensitive individuals by triggering palpitations, increased blood pressure and insomnia1. Three N-methyltransferase enzymes are involved in caffeine biosynthesis in coffee plants — CaXMT1, CaMXMT1 (theobromine synthase) and CaDXMT1 (caffeine synthase), which successively add methyl groups to xanthosine in converting it into caffeine2,3,4. Here we describe the construction of transgenic coffee plants in which expression of the gene encoding theobromine synthase (CaMXMT1) is repressed by RNA interference (RNAi). The caffeine content of these plants is reduced by up to 70%, indicating that it should be feasible to produce coffee beans that are intrinsically deficient in caffeine.
Specific sequences in the 3′ untranslated region (UTR) of CaMXMT1 messenger RNA were selected for construction of RNAi short and long fragments (Fig. 1). We transformed Agrobacterium tumefaciens EHA101 cells with these constructs and then used them to transform Coffea canephora5. After 2–4 months of culture, most infected tissues turned brown and necrotic; however, it was possible to regenerate hygromycin-resistant cells from these tissues. Seedlings were then cultured as described5.
More than 35 transgenic somatic seedlings were obtained from each transformant, each containing short or long RNAi fragments or a control gene encoding green fluorescent protein (GFP). The phenotypes were apparently normal when compared with the wild-type plant (Fig. 1a).
Young leaves of one-year-old seedlings were collected 2–3 weeks after flushing and their purine alkaloid content was measured. The wild-type and transgenic lines that expressed GFP contained similar amounts of endogenous theobromine and caffeine (about 1 and 8 mg per g of fresh plant tissue, respectively; Fig. 1b, c). By contrast, young leaves of transgenic lines expressing RNAi showed a 30–80% reduction in theobromine content (Fig. 1b) and a 50–70% reduction in caffeine content (Fig. 1c) in comparison with the controls.
At present, coffee is decaffeinated industrially, but the process is expensive and the flavour of the product is poor6 — problems that could potentially be overcome by the genetic engineering of coffee plants3,6. As CaMXMT1 is expressed in young leaves, buds and immature fruits4, the transgenic plants described here should yield coffee beans that are essentially normal apart from their low caffeine content at maturity.
We are now applying this RNAi-based technique to C. arabica, which produces high-quality Arabica coffee and accounts for roughly 70% of the world market. Our method not only shortens the breeding period, which is more than 25 years for conventional crossing, but also opens the way to develop new species of coffee plant.
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The authors declare no competing financial interests.
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Ogita, S., Uefuji, H., Yamaguchi, Y. et al. Producing decaffeinated coffee plants. Nature 423, 823 (2003). https://doi.org/10.1038/423823a
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