Root cultures

In vitro production of alkaloids is possible also with the use of another in vitro technique: organ culture. Root cultures are the most common concerning alkaloids, as this part of plants is of great importance for alkaloid synthesis. Root cultures that produce alkaloids have been studied as far back as the 1950s658, but this subject did not come under wide scrutiny until the 1970s-1990s659'660'661'662'663'664'665'666'667'668'669'670. Root cultures with the ability to produce alkaloids have importance nowadays in biotechnology and genetic engineering671'672,673,674,675. Modifications of roots by hybridization, gene transfer and the transformation of root cultures led to establishing organs with high alkaloid production potential. This research area is growing all the time. Richter et al.673 reported an increase of total tropane alkaloids in the transformed root cultures of A. belladonna. Moreover, the transformation with cDNA of tropinone reductases successfully altered the ratio of tropine-derived alkaloids versus pseu-dotropine alkaloids. Similar results have also been reported by Dechaux and Boitel-Conti671. The optimization of the medium led to an increase of scopolamine production in hairy root cultures of Datura innoxia by increasing the produced biomass. Genetic engineering is the best way to increase the accumulation of scopolamine. Hong et al.674 have studied Catharantus roseus hairy root cultures transgenic for the rol ABC genes from T-L-DNA of the agropine-type Agrobacterium rhizogenes strain A4. They concluded that the three genes rol ABC are sufficient to induce high-quality hairy roots in Catharantus roseus. A recent study by Hu and Du675 proved that hairy root cultures have proved to be an efficient means of producing secondary metabolites that are normally biosynthesized in the roots of differentiated plants. Moreover, a transgenic root system offers tremendous potential for introducing additional genes along with the Ri plasmid, especially with the modified genes. Root cultures can be used to elucidate the intermediates and key enzymes involved in the biosynthesis of secondary metabolites. A prototype basket-bubble bioreactor has been designed and built to upgrade the scale of Genista tinctoria hairy root cultures672. The industrial mass scale production of alkaloids from bioreactors with transformed hairy root cultures is possible.

Many alkaloids can be produced by root cultures. The most known applications are with anabasine, nicotine, harmine, harmaline, hyoscyamine, calystegine, scopolamine, senecionine263'670'673'676'677'678'679'680'681'682'683'684. Enzymes such as putrescine N-methyltransferase545, N-methylputrescine oxidase685, tropinione reductase I and ii548 683, pseudotropine acyltransferase, hyoscyamine 6ft-hydroxylase553, homospermidine synthase686 and tropine reductase673 can be purified from root cultures producing alkaloids. The root cultures are useable in the production of enzymes active in alkaloid biosynthesis pathways.

CHAPTER 5

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