Agrobacterium-Mediated Transformation of Chrysanthemum with Artemisinin Biosynthesis Pathway Genes

Abstract

Artemisinin-based drugs are the most effective medicine for the malaria treatment. To date, the main method of artemisinin production is its extraction from wormwood plants Artemisia annua L. Due to the limitation of this source, considerable efforts are now directed to the development of methods for artemisinin production using heterologous expression systems. Artemisinin is a sesquiterpene lactone, synthesized through the cyclization of farnesyl diphosphate involved in other sesquiterpene biosynthetic systems. Chrysanthemum species as well as A. annua, belong to Asteraceae family, and had been characterized by containing highly content of sesquiterpenes and their precursors. This makes chrysanthemum a promising target for the production of artemisinin in heterologous host plants. Chrysanthemum (C. morifolium Ramat.) was transformed by Agrobacterium tumefaciens carrying with the binary vectors p1240 and p1250, bearing artemisinin biosynthesis genes coding: amorpha-4,11-diene synthase, artemisinic aldehyde Δ11(13) reductase, amorpha-4,11-diene monooxygenase (p1240 was targeted to the mitochondria and p1250 was targeted to the cytosol), cytochrome P450 reductase from A. annua, as well as yeast truncated 3-hydroxy-3-methylglutarylcoenzyme A reductase. This study obtained 8 kanamycin-resistant lines after transformation with the p1240 and 2 lines from p1250. All target genes were detected in 2 and 1 transgenic lines of the 2 vectors. The transformation frequency of all target genes were 0.33% and 0.17% for p1240 and p1250, relative to the total transformed explant numbers. RT-PCR analysis revealed the transcription of all transferred genes in two lines obtained after transformation with the p1240 vector, confirming the possibility of transferring genetic modules encoding entire biochemical pathways into the chrysanthemum genome. This holds promise for the development of a chrysanthemum-based expression system to produce non-protein substances, such as artemisinin.

Standardized Genetic Transformation Protocol for Chrysanthemum cv. ‘Jinba’ with TERMINAL FLOWER 1 Homolog CmTFL1a

“…Among them, Agrobacterium-mediated transformation has a unique significance and is being used for the insertion of specific genes into numerous ornamental plant species because of its authenticity and reliability. Several studies have been reported concerning the evaluation of Agrobacterium-mediated transformation in chrysanthemum [6][7][8]. The susceptibility of chrysanthemum to Agrobacterium-mediated transformation is well established and has been characterized by low transformation efficiency since 1975 [9,10].…”

Establishment of a Rapid and Effective Agrobacterium-Mediated Genetic Transformation System of Oxalis triangularis ‘Purpurea’

“…The genetic transformation procedure established in this study has the advantage of high transformation efficiency, ranging from 1.02% to 9.88% ( Table 2 ). The transformation efficiency is comparable to that of other ornamental flower plants, such as Chrysanthemum morifolium Ramat (the transformation efficiency is between 1% and 8%) [ 47 , 48 , 49 ], Broccoli oleracea var. italica (2.7–6.4%) [ 27 ], B. oleraceae var Botrytis (7–13.6%) [ 50 ], and L. pumilum DC Fisch (5.7–13.0%) [ 51 ].…”

Plant Metabolic Engineering by Multigene Stacking: Synthesis of Diverse Mogrosides

“…Therefore, the loss of exogenous genes can be reduced and can improve the transgenic efficiency. Due to the significant advantages of the multigene vector transformation, this method is more widely used in current synthetic biology, and a variety of multigene stacking strategies have been developed recently [ 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 ]. Researchers developed a TGS II system for multigene assembly, and astaxanthin, β-carotene, and anthocyanins have been produced in rice endosperm by the transformation of 4-10 structural genes, respectively [ 34 , 35 ].…”