Isolated microspore culture for embryoid production in Artemisia annua L.

The haploidy technique is a useful tool for quickly producing pure, fully homozygous lines. Artemisia annua L. is a medicinal plant that produces artemisinin, a widely used antimalarial drug. Because of its extremely small flowers (≤ 3.0 mm), this study conducted microscopic observations to determine the types of flowers of A. annua suitable for microspore embryogenesis, as well as their corresponding microspore development stages, and obtained embryoids (non-zygotic embryos) from isolated microspore cultures. The media for inducing embryoid production were based on Nitsch and Nitsch medium containing 13% or 17% sucrose and the following plant growth regulators: (1) a combination of naphthaleneacetic acid and 6-benzyladenine (MCA medium) and (2) a combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (MCAD medium). The results indicated that based on the proportions of uninucleate and binucleate microspores, flowers at the prebloom and early bloom stages contained sufficient late uninucleate to early binucleate microspores suitable for inducing embryogenesis. The production of microspore-derived embryogenic (MDE) structures was faster in MCA13 and MCA17 media than in MCAD13 and MCAD17 media. MCA13 and MCAD13 media induced the production of more callus-like structures than MCA17 and MCAD17 media. Thus, the addition of 2, 4-D to MCAD medium inhibited the growth of MDE structures. Globular embryoids emerged from the multicellular cluster.