In vitro plantlet production of Ilex paraguariensis adult plants using BIT bioreactors

Abstract

Ilex paraguariensis St. Hil. is cultivated in South America to prepare a tea-like infusion with pharmacological properties. Its recalcitrant character has hindered the development of a suitable method for propagating selected genotypes. This study aimed to produce in vitro plantlets from axillary shoots of adult plants using a temporary immersion bioreactor. During the elongation phase, the impact of ammonium and nitrate concentration on Murashige and Skoog quarter-strength (¼MS) formulation, macronutrient uptake, hormone supplementation, and explant density (5–30 explants) were analysed. In addition, during the induction and expression stages of root formation, the effect of indole-3-butyric acid (IBA), cadaverine, quercetin, and chlorogenic acid supplementation was studied. As a result, we propose a protocol for in vitro plantlet production of I. paraguariensis adult plants in bioreactors. Twenty-day-old stem segments established in semisolid ¼MS medium plus sucrose 30 g·L^− 1 and indole-3-acetic acid (IAA) 0.5 µM are transferred to the elongation phase during 40 days in 300 cc BIT bioreactor (20 explants per flask) containing 100 mL of ¼MS modified with double ammonium and nitrate content and N6-benzyladenine (BA) 20 µM. Then, shoots longer than one centimetre are removed from the explant and placed into a rooting medium consisting of ¼MS with sucrose 30 g·L^− 1 and supplemented with IBA 7.5 µM, cadaverine 20 µM, quercetin 20 µM, and chlorogenic acid 20 µM. Finally, the in vitro 30-day-old plantlets are acclimatised on 150 cc pots filled with non-sterile composted pine bark and controlled-release micro-fertiliser. An inter-simple sequence repeat assessment revealed no difference between in vitro plantlets and mother plants.