Tissue culture and transformation of the antisense DFR gene into lotus (Nelumbo nucifera Gaertn.) through particle bombardment

The effects of explant and plant growth regulators on callus induction and plant regeneration and particle bombardment-induced transformation were examined in lotus (Nelumbo nucifera Gaertn.) cv. Buntharik. The apical buds from embryos (∼3 mm) formed an embryogenic callus when cultured on MS medium supplemented with 40 μM 1-napthaleneactic acid (NAA) and 0.5 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ) for 8 weeks. All of the embryogenic calluses were transferred to MS medium supplemented with 0, 40, 50 and 60 μM 6-benzyladenine (BA) and the highest number of shoots was achieved in the medium supplemented with 50 μM BA after 8 weeks of culture. The transformation of lotus using a particle bombardment device was examined with the shoot clusters from embryogenic apices. The pCAMBIA2301anti-DFR plasmid contained β-glucuronidase (GUS) as a reporter gene, neomycin phosphotransferase (NPTII) as a selectable marker, and antisense dihydroflavonol 4-reductase (anti-DFR) as the gene of interest. The particle bombardment procedure with a helium gas pressure of 1100 psi and a target distance of 6 cm yielded the highest number of blue spots (4.8 spots/shoot cluster). However, stable transformations, as confirmed by polymerase chain reaction and reverse transcription polymerase chain reaction analysis, were only achieved using a helium gas pressure of 1100 psi and a target distance of 9 cm. Our work illustrates that the stable transformation of lotus is possible, and we suggest that this methodology can be used to target and modify specific charactics of this plant.