Establishment of efficient callus genetic transformation system for Hemerocallis fulva ‘Kanai’

Abstract

Abstract Hemerocallis is regarded as a model plant for future breeding because of its high ornamental value and strong resistance. However, there are few reports on regeneration and genetic transformation of this plant. In this study, mature seeds of Hemerocallis fulva ‘Kanai’ were used as explants for regeneration and Agrobacterium infection. The results showed that the germination rate of explants with inner and outer seed coats peeled was 98% and the contamination rate was only 11%. The callus induction efficiency of MS medium supplemented with 3.5 mg∙L− 1 6-Benzylaminopurine (6-BA) and 0.1 mg∙L− 1 1-naphthylcetic acid (NAA) was 95.2%. Based on this, transformation was successfully achieved using the following protocol: callus were soaked in Agrobacterium tumefaciens EHA105 (OD600 = 0.6) containing pCambia1300-35S-FT and pCambia1300-35S-GUS plasmid for 15 min. After 3 days co-culture with 100 uM Acetosyringone (AS) in MS medium, it was transferred to MS medium containing 300 mg∙mL− 1 Timentin for 5 days. The Transgenic plants were obtained by hygromycin (9 mg∙mL− 1) screening. The presence of transgenic plants was confirmed by histochemical GUS detection and PCR (Polymerase chain reaction). Overall, the establishment of this efficient regeneration and genetic transformation will contribute to the functional gene research and genetic improvement of Hemerocallis fulva ‘Kanai’.