Biolistic-mediated Transformation of Lilium longiflorum cv. Nellie White

Abstract

Slow-growing compact calluses were initiated from bulb scales of Lilium longiflorum cv. Nellie White that had been cultured for at least 6 months on Murashige and Skoog (MS) medium with 9 μm dicamba. To develop a reliable selection system, the sensitivity of nontransformed calluses and in vitro plants to different selective agents such as phosphinothricin, kanamycin, geneticin, paromomycin, and hygromycin was tested when grown on MS medium. Nontransformed calluses showed high sensitivity to 0.5 mg·L−1 phosphinothricin, 25 mg·L−1 geneticin, and 5 mg·L−1 hygromycin. Nontransformed plants grown in vitro died on either 2 mg·L−1 phosphinothricin or 75 mg·L−1 hygromycin. Plants did not die when grown on either 200 mg·L−1 kanamycin or 100 mg·L−1 geneticin, and 100 mg·L−1 paromomycin stimulated plant growth. Transformation was achieved using biolistics on callus bombarded with either the bar-uidA fusion gene under control of the CaMV 35S promoter or npt II and uidA under control of the CaMV 35S promoter. One week after biolistic bombardment, callus bombarded with the bar-uidA fusion gene was cultured for 1 month on MS medium supplemented with 9 μm dicamba and 0.1 mg·L−1 phosphinothricin and then transferred to 0.2 mg·L−1 phosphinothricin for 1 month followed by 1.0 mg·L−1 for the next 4 months. Regenerating shoots and well-established plants were cultured on MS medium lacking hormones and with either 0.2 mg·L−1 or 2.0 mg·L−1 phosphinothricin, respectively. Callus bombarded with the npt II gene was cultured on MS medium with 50 mg·L−1 geneticin until shoots regenerated. Regenerated shoots were cultured on MS medium lacking hormones. Under optimal conditions, 10 transgenic plants were selected from seven plates of callus bombarded with the bar-uidA fusion gene using phosphinothricin for selection. Both Southern hybridization of genomic DNA and polymerase chain reaction analysis verified the presence of the transgene in transformed ‘Nellie White’ plants. Transgenic plants were phenotypically normal, and they were crossed with nontransformed plants of L. longiflorum cvs. Sakai, Yin tung, Sakai, and Flavo. The presence of the bar gene in 41% of the T1 progeny plants confirmed stable integration of the transgene into the genomic DNA of transgenic lily plants. β-glucuronidase expression resulting from the uidA gene was demonstrated in leaves and roots of some of the transgenic lily plants by histochemical staining, determination of the specific activity of the β-glucuronidase enzyme, and Northern hybridization.