In Vitro Culture of Rosmarinus officinalis L. in a Temporary Immersion System: Influence of Two Phytohormones on Plant Growth and Carnosol Production

Abstract

Emerging infectious diseases have become a major global problem with public health and economic consequences. It is an urgent need to develop new anti-infective therapies. The natural diterpene carnosol exhibit a wide variety of interesting antibacterial and antiviral properties, and it is considered a theoretical inhibitor of COVID-19 Mpro. However, this compound is present in the family Lamiaceae in low quantities. To obtain carnosol in concentrations high enough to develop pharmacological studies, we evaluated the efficiency of a micropropagation protocol of Rosmarinus officinalis using a solid medium and a temporary immersion system (TIS), as well as the effect of 6-benzylaminopurine (6-BAP) and α-naphthaleneacetic acid (NAA) on the growth of shoots. Moreover, we developed and validated an analytical method to quantify carnosol using the H-point standard additions method in the high-performance liquid chromatography diode array detector (HPLC-DAD). After 30 days of culture, TIS produced the maximum number of shoots per explant (24.33 ± 1.15) on a liquid medium supplemented with 6-BAP at 5.0 mg L−1. Next, we also evaluated the effect of immersion time and frequency for TIS. After 72 days of culture, the best results were obtained with an immersion cycle of 1 min every 12 h, yielding 170.33 ± 29.40 shoots. The quantification of carnosol on the samples was performed at a flow rate of 1.2 mL min−1 using binary isocratic mobile phase system 60:40 (v/v) 10 mM formic acid (pH 3.0) (A) and acetonitrile (B) on a reverse-phase column. The content of carnosol in the in vitro cultures was around 8-fold higher than in the wild plant. The present study represents an efficient alternative method to obtain carnosol for its pre-clinical and clinical development.

Evaluation of the yield, chemical composition and biological properties of essential oil from bioreactor-grown cultures of Salvia apiana microshoots

“…5 B, C). So far, bioreactor cultivation of sage in vitro shoots has been scarcely studied 51 , 52 .

Figure 6 Growth parameters (dry weight – DW and growth index—Gi) of S. apiana microshoots, maintained in different bioreactors, during 7–42 days of cultivation.

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“…However, research demonstrated that a temporary immersion system is effective for cultivating in vitro shoots of S. rosmarinus 52 . In the current work, the concentrations of the major constituents of S. apiana essential oil varied depending on the bioreactor type.…”

An Efficient Method of Pennisetum × advena ‘Rubrum’ Plantlets Production Using the Temporary Immersion Bioreactor Systems and Agar Cultures

“…In our research, we used a 1min immersion time with a 1h interval to achieve short but frequent contact of the explant with the medium. Perez et al [ 27 ] found that immersion for 1 min every 4 h induced 114 cotyledonary embryos in Quercus suber , which was significantly more than the 48 embryos obtained with immersion for 1 min every 6 h and the 14 embryos obtained with immersion for 1 min every 12 h. Similarly, Villegas-Sanchez et al [ 47 ] demonstrated that more frequent immersion of Rosmarinus officinalis explants leads to excellent results. Immersion for 1 min every 12 h resulted in 170 new shoots, while immersion for 1 min every 24 h produced only three new shoots.…”

A Temporary Immersion System to Improve Cannabis sativa Micropropagation

“…Recently, several studies have shown that TIS have numerous advantages as regards the semi-solid methods (Vidal and Sánchez, 2019 ). These systems have been successfully used for micropropagation of Stevia rebaudiana (Melviana et al, 2021 ), Colocasia esculenta L. Schott (Mancilla-Álvarez et al, 2021 ), Agave angustifolia (Monja-Mio et al, 2021 ), Rosmarinus officinalis L. (Villegas-Sánchez et al, 2021 ), Dracocephalum forrestii (Weremczuk-Jezyna et al, 2020 ), Guarianthe skinneri (Leyva-Ovalle et al, 2020 ), and the number is increasing steadily. In our laboratory, we have developed protocols using bioreactor propagation systems with liquid medium for axillary shoots of chestnut, alder and willow (Vidal et al, 2015 ; Cuenca et al, 2017 ; Regueira et al, 2018 ; San José et al, 2020 ; Gago et al, 2021 ), and somatic embryos of Quercus robur (Mallón et al, 2012 , 2013 ).…”