Transformation and characterization of transgenic Bidens pilosa L.

Bidens pilosa L. is an erect perennial herb that is used as a folk medicine for the treatment of a variety of illnesses including diabetes. Genetic engineering is an effective means of producing desired phytocompounds in certain medicinal plants; however, plant regeneration and genetic transformation have not yet been reported for B. pilosa. Here, we determined the optimal conditions for plant regeneration from cotyledon explants of in vitro-grown B. pilosa L. var. radiata, one of the three common variants of this species found in Taiwan. An Agrobacterium-mediated method was developed to transform the vector pCHS, which is carrying the Petunia chalcone synthase (chs) and neomycin phosphotransferase II (nptII) genes into B. pilosa var. radiata. From a total of 1,373 cotyledon explants, 21 putative transgenic lines were regenerated on selection medium, and 15 lines carrying both Petunia chs and nptII transgenes were obtained. Out of these, one line, BpCHS21, showed an anomalous flower phenotype. The presence of Petunia chs transgene in randomly-selected transgenic plants was confirmed by Southern blot analysis. Reverse transcription-polymerase chain reaction analysis revealed that the transgenic chs was differentially expressed in the leaf tissue of all the transformants, but not in wild type. T-1 progeny assay of outcross seeds (i.e., wild-type pollens crossed with transgenic flowers) from selected transformants showed that the ratio of kanamycin-resistant and kanamycin-sensitive seedlings was close to 1 in kanamycin-containing medium, verifying again the single integration of foreign DNA into the nuclear chromosome of transformants and the inheritance of the nptII transgene. The present protocol will be useful for the introduction of genes of interest into B. pilosa paving the way for metabolic engineering in this medicinal plant.