Establishment of an efficient Agrobacterium tumefaciens-mediated leaf disc transformation of Thellungiella halophila.
Thellungiella halophila is a salt-tolerant close relative of Arabidopsis, which is adopted as a halophytic model for stress tolerance research. We established an Agrobacterium tumefaciens-mediated transformation procedure for T. halophila. Leaf explants of T. halophila were incubated with A. tumefaciens strain EHA105 containing a binary vector pCAMBIA1301 with the hpt gene as a selectable marker for hygromycin resistance and an intron-containing beta-glucuronidase gene as a reporter gene. Following co-cultivation, leaf explants were cultured on selective medium containing 10 mg l(-1) hygromycin and 500 mg l(-1) cefotaxime. Hygromycin-resistant calluses were induced from the leaf explants after 3 weeks. Shoot regeneration was achieved after transferring the calluses onto fresh medium of the same composition. Finally, the shoots were rooted on half strength MS basal medium supplemented with 10 mg l(-1) hygromycin. Incorporation and expression of the transgenes were confirmed by PCR, Southern blot analysis and GUS histochemical assay. Using this protocol, transgenic T. halophila plants can be obtained in approximately 2 months with a high transformation frequency of 26%.
Li
Hong-Qing
HQ
Guangdong Provincial Key Lab of Biotechnology for Plant Development, South China Normal University, Guangzhou 510631, People's Republic of China.
Xu
Jie
J
Chen
Lei
L
Li
Mei-Ru
MR
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D019607:Brassicaceae / Q000235:genetics*; D018515:Plant Leaves; D030821:Plants, Genetically Modified; D014170:Transformation, Genetic