<i>Agrobacterium</i> -mediated transformation of <i>Eucalyptus camaldulensis</i> to suppress hydrolyzable tannin biosynthesis

Eucalyptus camaldulensis is a Myrtaceae tree species accumulating high concentrations of hydrolyzable tannins. This study was conducted to generate transgenic E. camaldulensis lines with decreased hydrolyzable tannin levels suitable for investigations of their physiological functions. Transformation was based on co-cultivation of Agrobacterium tumefaciens strain EHA105 cells harboring binary vectors with E. camaldulensis explants. Because genetic transformation of E. camaldulensis remains technically challenging, we initially improved the protocol by optimizing explant preparation and selection conditions. Hypocotyl explants were prepared from seeds (lot 19708) that are readily available from the Australian Tree Seed Centre. A kanamycin sensitivity test using wild-type hypocotyls indicated that 25 mg L−1 kanamycin was effective for selecting transformants on Murashige and Skoog agar medium. Although hypocotyls without shoot apices showed a slightly lower transformation frequency (2.5%) than hypocotyls with shoot apices (4.0%), the former approach was more efficient overall because it required less effort for explant preparation. The overexpression of a bacterial β-glucuronidase gene demonstrated that the cauliflower mosaic virus 35S promoter was functional throughout transgenic E. camaldulensis plants. We used the optimized transformation method to downregulate hydrolyzable tannin biosynthesis. Antisense approaches targeting UDP glycosyltransferase genes (UGT84A25 and UGT84A26) significantly reduced the concentration of the hydrolyzable tannin oenothein B in the roots of a transgenic line, AS251, which may serve as a valuable resource for elucidating the physiological functions of hydrolyzable tannins in plants.