Germline transformation of Artemisia annuaL. plant via in planta transformation technology “Floral dip”

Abstract

The therapeutic efficacy of Artemisia annua L. is governed by artemisinin (ART), prevalently produced by A. annua extraction. Due to the modest amount of ART (0.01-1 %dw) in this plant, commercialization of ACTs is difficult. In this study, the floral-dip based transformation protocol for A. annua was developed to enhance expression of artemisinin biosynthesis genes and ART content. For dipping, the effective infiltration media components were optimized, and to obtain high transformation (26.9%) partially open bud stage capitulum of floral development was used. Hygromycin phospho-transferase (hptII) selection marker was used to validate the transformed T1 progenies. The copy numbers of the transgene (hptII) in T1 progenies were determined using a sensitive, high-throughput SYBR Green based quantitative RT-PCR. The results of the hptII transgene were compared with those of the low copy number, internal standard (hmgr). Using optimised PCR conditions, one, two and three transgene copies in T1 transformants were achieved.

Keywords

• Artemisinin
• Copy number
• hptII
• hmgr
• qRT-PCR