Agrobacterium-mediated genetic transformation of the most widely cultivated superior clone Eucalyptus urophylla × Eucalyptus grandis DH32-29 in Southern China

Abstract

Eucalyptus, as an economically important species for wood and paper industries, still remains a challenge to genetic improvement by transgenic technology owing to the deficiency of a highly efficient and stable genetic transformation system, especially in cultivated superior clones.Eucalyptus urophylla×Eucalyptus grandisclone DH32-29 is most widely planted in southern China, but it is relatively recalcitrant to adventitious bud regeneration, which blocks the establishment of a genetic transformation system. Here, an efficient adventitious bud regeneration and transformation system ofEucalyptuswas established usingE. urophylla×E. grandisDH32-29 as material. Thein vitroleaves from microshoots that were subcultured for 20–25 days were immersed into liquid Woody Plant Medium supplemented with 0.02 mg·L−1of α-naphthaleneacetic acid (NAA) and 0.24 mg·L−1of forchlorfenuron (callus-inducing medium (CIM)). After 15 days, explants were transferred to a medium containing 0.10 mg·L−1of NAA and 0.50 mg·L−1of 6-benzyladenine (shoot-inducing medium (SIM)) for adventitious bud induction. The highest regeneration efficiency of adventitious buds was 76.5%. Therefore, anAgrobacterium tumefaciens-mediated genetic transformation system was optimized. The leaves were precultured for 7 days and infected for 30 min withA. tumefaciensstrain EHA105 grown to a bacterial density of 0.3 (OD600). After 72 h of cocultivation in the dark, leaves were transferred to CIM supplemented with 100 mg·L−1of cefotaxime (Cef), 100 mg·L−1of timentin, and 15 mg·L−1of kanamycin (Kan) for 15 days to induce calluses. Then, the explants were transferred to SIM supplemented with the same concentration of antibiotics, and the fresh medium was replaced every 15 days until resistant adventitious buds appeared. After induction of roots in root-inducing medium supplemented with 200 mg·L−1of Cef and 75 mg·L−1of Kan, completely transgenic plants were obtained. With the use of the aforementioned method, the transformation frequency can reach 1.9%. This provides a powerful approach for genetic improvement ofE. urophylla×E. grandisDH32-29 and gene function analysis inEucalyptus.