Establishment, Culture, and Scale-up of Brugmansia candida Hairy Roots for the Production of Tropane Alkaloids

Abstract

Brugmansia candida (syn. Datura candida) is a South American native plant that produces tropane alkaloids. Hyoscyamine, 6β-hydroxyhyoscyamine (anisodamine), and scopolamine are the most important ones due to their anticholinergic activity. These bioactive compounds have been historically and widely applied in medicine and their demand is continuous. Their chemical synthesis is costly and complex, and thereby, these alkaloids are industrially produced from natural producer plants. The production of these secondary metabolites by plant in vitro cultures such as hairy roots presents certain advantages over the natural source and chemical synthesis. It is well known that hairy roots produced by Agrobacterium rhizogenes infection are fast-growing cultures, genetically stable and able to grow in hormone-free media. Additionally, recent progress achieved in the scaling up of hairy root cultures makes this technology an attractive tool for industrial processes. This chapter is focused on the methods for the induction and establishment of B. candida hairy roots. In addition, the scaling up of hairy root cultures in bioreactors and tropane alkaloid analysis is discussed.

Establishment of hairy roots culture of Perilla frutescens L. and production of phenolic acids

“…For instance, studies have shown that S. bulleyana hairy roots can be used to produce polyphenolic compounds (Wojciechowska et al, 2020), Trachyspermum ammi hairy roots can produce thymol (Vamenani et al, 2020), Gymnema sylvestre hairy roots can produce gymnemic acid (Rajashekar et al, 2016), Artemisia hairy roots can produce phenol, avonoid, sterol, and essential oil (Mottaki et al, 2019). Furthermore, Salvia viridis hairy roots can be utilized to produce polyphenolic compounds (Grzegorczyk-Karolak et al, 2018), Brugmansia candidas hairy roots can produce tropane alkaloids (Cardillo et al, 2016), Rehmannia elata hairy roots can produce iridoid and phenylethanoid glycosides (Piątczak et al, 2019), and Corylus avellana hairy roots can produce saponins (Jalalipour . These examples demonstrate the potential of using hairy root cultures to produce a variety of bioactive compounds for multiple applications.…”

Enhancement of anthraquinone production and release by combination of culture medium selection and methyl jasmonate elicitation in hairy root cultures of Rubia tinctorum

“…Transformation with A. rhizogenes was confirmed by PCR amplification of rolC, aux1 and ags genes in genomic DNA extracted from hairy roots. Genomic DNA was isolated according to Cardillo et al (Cardillo et al, 2016). Persistence of A. rhizogenes was evaluated by PCR detection of virD gene.…”

“…Persistence of A. rhizogenes was evaluated by PCR detection of virD gene. The sequences of the primers were obtained from (Cardillo et al, 2016) and are given in Table 1.…”

“…The FW biomass quantification was performed according to Cardillo et al (Cardillo et al, 2016). Growth index (GI) was calculated according to the following formula:…”

Production of tropane alkaloids by biotransformation using recombinant Escherichia coli whole cells

“…In order to extract the alkaloids from the reaction mixture described above, samples were made alkaline at pH 9 by the addition of 1 vol of carbonate buffer as it was described above [26]. Samples were extracted twice with 5 ml chloroform by vortexing and the organic phase was evaporated under gaseous N 2 [27,28].…”