Optimization of conditions for the induction of Tagetes patula L. hairy roots

L P Khlebova,O V Bychkova,N V Pavlova,E S Brovko

doi:10.15421/2019_119

Abstract

The hairy root cultures are promising sources of secondary metabolites of plants, including rare and endangered species. They possess genetic and biochemical stability, unlimited growth rate in free-hormone medium, short doubling times, high biosynthetic activity and ecological purity of plant raw materials. The hairy root cultures of Tagetes patula L. can be used to produce biologically active substances with biocidal activity. The study aimed to determine the virulent strain of Agrobacterium rhizogenes and the most effective period of co-cultivation of T. patula leaf explants with an agrobacterium to induce actively growing hairy root cultures. We used 3 strains (A-4b, 8196RT and 15834). The time of infection ranged from 3 to 33 hours in increments of 3 hours. We found that 24 h is the best time of infection to induce hairy roots with the highest transformation efficiency (92%). The wild strain A. rhizogenes 15834 turned out to be the most virulent when infected leaf explants of spreading marigold. This strain provided the maximum transformation effect, reaching 85.4%. We have identified 5 actively growing clones of hairy roots with intensive branching, the growth indices of which were 64-75. In the future, they will be transferred to a liquid medium for biomass accumulation and scaling.

Highlights

One of the most promising areas of modern phytobiotechnology is the use of cell cultures of higher plants to obtain biologically active substances for medicine, veterinary medicine, perfumery, agriculture, food and chemical industries (Dicosmo and Misawa, 1995; Ramachandra Rao and Ravishankar, 2002; Mulabagal and Tsay, 2004; Malik et al, 2011; Orlova et al, 2014; Isah et al, 2018; Kochkin et al, 2019; Watcharatanon et al, 2019)
The root culture obtained in this way has genetic and biochemical stability, unlimited growth rate in growth regulator‐free media, short doubling times, high biosynthetic activity and responsiveness to elicitor treatments as well as ecological purity of the plant raw materials (Vdovichenko et al, 2007; Kuzovkina and Vdovichenko, 2011; Khan et al, 2018; Halder et al, 2019)
The growth activity of the cultures was evaluated by the growth index (GI), which was calculated by the difference in the length of the roots at the end and beginning of the passage using the formula: GI = l1 – l0/l0, where l0 is the initial root length; l1 is the final length of the roots

Summary

Introduction

One of the most promising areas of modern phytobiotechnology is the use of cell cultures of higher plants to obtain biologically active substances for medicine, veterinary medicine, perfumery, agriculture, food and chemical industries (Dicosmo and Misawa, 1995; Ramachandra Rao and Ravishankar, 2002; Mulabagal and Tsay, 2004; Malik et al, 2011; Orlova et al, 2014; Isah et al, 2018; Kochkin et al, 2019; Watcharatanon et al, 2019). The chemicals that make up the plants determine the usefulness of representatives of this genus (marigolds) as sources of various classes of secondary metabolites that are used in the pharmaceutical and food industries (Giri et al, 2011; Chkhikvishvili et al, 2016) These substances can be used as the main component of agents with biocidal activity (Mares et al, 2004; Mulabagal and Tsay, 2004; Faizi et al, 2011; Politi et al, 2016; Ayub et al, 2017; Krzyzaniak et al, 2017; Mir et al, 2019). The aim of this work was the induction in in vitro of rapidly growing lines of hairy root cultures of T. patula for their further use in the production of biologically active substances with biocidal activity

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