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Title: Selection and clonal propagation of high artemisinin genotypes of Artemisia annua

Author
item WETZSTEIN, HAZEL - Purdue University
item PORTER, JUSTIN - Purdue University
item JANICK, JULES - Purdue University
item Ferreira, Jorge
item MUTUI, THEOPHILUS - University Of Eldoret

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2018
Publication Date: 3/27/2018
Citation: Wetzstein, H.Y., Porter, J.A., Janick, J., Ferreira, J.F., Mutui, T.M. 2018. Selection and clonal propagation of high artemisinin genotypes of Artemisia annua. Frontiers in Plant Science. 9:358. https://doi.org/10.3389/fpls.2018.00358.
DOI: https://doi.org/10.3389/fpls.2018.00358

Interpretive Summary: Malaria affects approximately 2.5 million people a year worldwide and artemisinin (ART) combination therapy (ACT) is the only medicine that cures quinine-resistant malaria. Prices for ACT continue to be higher than people in malaria-afflicted countries can afford, and are tightly linked to the fluctuation in ART prices and availability worldwide. While, annual wormwood is the only commercial ART source, commercial yield is hampered by low and variable levels of ART in shoots. Artemisinin yields by farmers rely on seed-generated plants produced by cross-pollinated parents. However, seed-generated plants have highly variable ART concentration that leads to low ART yields per hectare. Breeding efforts to produce improved F1 hybrids (as used for agricultural crops, such as corn) have been hampered by both plant self-incompatibility and by the lack of parents that are homozygous for artemisinin. Thus, we have combined conventional hybridization and selection with clonal propagation of selected high-artemisinin genotypes from three distinct geographic regions (Brazil, China, and Switzerland) as a means to enhance crop artemisinin production. Typical seed-propagated annual wormwood plants produce less than 1% (dry weight) artemisinin with crude ART yields below 25 kg/ha. Our selected genotypes produced high artemisinin levels of over 2% (w/w) and some possessed improved agronomic characteristics such as high leaf area and shoot biomass production. Selected plants were vegetatively propagated through tissue culture and cuttings for field and greenhouse studies, and confirmed enhanced plant uniformity with the potential to produce up to 70 kg/ha of crude ART. Vegetative propagation by tissue culture and cuttings for the mass clonal propagation of annual wormwood were developed for shoot regeneration, rooting, acclimatization, and field cultivation. Proof of concept studies showed that rooted tissue culture-regenerated plants performed better than plants derived from seed in terms of uniformity, yield, and consistently high ART concentration. Use of this technology will lead to plants with homogeneously-high ART that can double ART worldwide yield without need to expand the cultivated area, benefiting farmers and communities that rely on this crop as the base for their economy. Farming plants with high ART will also lead to decreased prices of the antimalarial ACT.

Technical Abstract: Artemisinin, produced in the glandular trichomes of Artemisia annua L. is a vital antimalarial drug effective against Plasmodium falciparum resistant to quinine-derived medicines. Although work has progressed on the semi-synthetic production of artemisinin, field production of A. annua remains the principal commercial source of the compound. Crop production of artemisia must be increased to meet the growing worldwide demand for artemisinin combination therapies (ACTs) to treat malaria. Grower artemisinin yields rely on plants generated from seeds from open-pollinated parents. Although selection has considerably increased plant artemisinin concentration in the past 15 years, seed-generated plants have highly variable artemisinin content that lowers artemisinin yield per hectare. Breeding efforts to produce improved F1 hybrids have been hampered by the inability to produce inbred lines due to selfincompatibility. An approach combining conventional hybridization and selection with clonal propagation of superior genotypes is proposed as a means to enhance crop yield and artemisinin production. Typical seed-propagated artemisia plants produce less than 1% (dry weight) artemisinin with yields below 25 kg/ha. Genotypes were identified producing high artemisinin levels of over 2% and possessing improved agronomic characteristics such as high leaf area and shoot biomass production. Field studies of clonally-propagated high-artemisinin plants verified enhanced plant uniformity and an estimated gross primary productivity of up to 70 kg/ha artemisinin, with a crop density of one plant ^(-2). Tissue culture and cutting protocols for the mass clonal propagation of A. annua were developed for shoot regeneration, rooting, acclimatization, and field cultivation. Proof of concept studies showed that both tissue culture-regenerated plants and rooted cutting performed better than plants derived from seed in terms of uniformity, yield, and consistently high artemisinin content. Use of this technology to produce plants with homogeneously-high artemisinin can help farmers markedly increase the artemisinin yield per cultivated area. This would lead to increased profit to farmers and decreased prices of ACT.