Phenylethanoids, phenylpropanoids, and phenolic acids quantification vis-à-vis gene expression profiling in leaf and root derived callus lines of Rhodiola imbricata (Edgew.)

Highlights

• First ever study on high frequency callus induction from R. imbricata root explant.

• UPLC-PDA based method developed and validated for metabolite quantification.

• Established callus morpho-variants for metabolite enriched biomass accumulation.

• RT-qPCR gene expression showed a positive correlation with metabolite content.

• Year-round callus biomass production to obtained desired metabolites.

Abstract

Salidroside and rosavins have been found as the most potent ingredients used in Rhodiola based herbal formulations. Rhodiola imbricata is a medicinal herb of the trans-Himalayan Ladakh region of India. Since, the natural supply of this herb is rapidly decreasing, due to its over-exploitation, high altitude region, and traditional usage in the Amchi system of medicine. In the present study, friable callus culture was developed for the first time from leaf and root explants of R. imbricata. Furthermore, callus cell lines were also evaluated for rapid growth rate and high metabolite accumulation. The results revealed that callus induction frequency observed in juvenile leaves (100 %) and roots (87.50 %) in MS medium enriched with 0.5 mg/L TDZ and 1 mg/L NAA. Scanning electron microscopy (SEM) analysis was done for histological observations of the callus surface. A selective and efficient ultra-performance liquid chromatography with PDA detector (UPLC-PDA) method was developed and validated for the quantification of phenylethanoids, phenylpropanoids, and phenolic acids. The maximum salidroside was detected in leaf derived friable green calli (3.59 mg/g DW) subsequently followed by leaf derived friable white calli (2.31 mg/g DW). While rosavin and rosarin were detected maximum in root derived compact green calli (0.15 mg/g DW) and root derived friable green calli (0.07 mg/g DW). Genes encoding enzymes involved in salidroside and rosavins biosynthesis were also investigated for transcript abundance in wild as well as in vitro cultures using the RT-qPCR approach. The present study explained the friable callus culture as a potential alternative approach to obtain higher metabolite yield in R. imbricata. Further, it can be used for sequential scale-up of metabolite at bioreactor level to meet the industrial demand.

Introduction

Rhodiola imbricata (Crassulaceae) is a perennial medicinal herb; found at rocky slopes, wet places, and high altitude passes of Ladakh region, India (Chaurasia et al., 2003). It is well recognized for its valuable medicinal properties like radioprotection, immunomodulatory, antioxidant, anticancerous, freeze tolerance, anti-aging, wound healing, anti-fatigue and anti-stress due to the presence of diverse secondary metabolites, mainly phenylethanoids and phenylpropanoids (Chaurasia et al., 2003; Gupta et al., 2007; Kumar et al., 2011; Bhardwaj et al., 2018; Kapoor et al., 2018; Tao et al., 2018). Salidroside and rosavins are the most important bioactive component of Rhodiola sp. due to their potential applications used in pharmaceutical, nutraceutical, aerospace, sports sector, and health care industries (Furmanowa et al., 1995; Arora et al., 2005; Chawla et al., 2010; Gupta et al., 2007; Mishra et al., 2009; Panossian et al., 2010; Galambosi et al., 2010; Tayade et al., 2013; Tao et al., 2018). In recent decades interest of researchers on applications of Rhodiola has increased tremendously due to their industrial and market demand (approximately 20–30 tons raw material/year) (Kotiranta et al., 1998; Grech-Baran et al., 2015; Tao et al., 2018). At present, the worldwide market price for Rhodiola is about 6800 Rs/Kg of raw material and around thirty companies are listed as ingredient suppliers for different herbal formulation preparations such as Medi herbs- Rhodiola and ginseng complex (60 tablets-Rs. 2640) and The vitamin shoppe (500 mg Rhodiola Rs. 1631) (Ampong-Nyarko, 2005; Tao et al., 2018). Top key players i.e. Nature’s way; Chansha Organic Herb Inc; MB-holding GmbH & Co.; Amax Nutra Source; iHerb; Swanson health products; Xian Yuensun Biological Technology Co. Ltd; PLT Health Solutions, Inc; Xian Greena Biotech Co. Ltd. produced, promoted and marketed high-value Rhodiola enriched products and formulations (Anonymous, 2019).

Over the years, the rising demand of R. imbricata facilitated the over-exploitation vis-à-vis decreasing its natural population from wild, leads to put under a rare category in the red data list of Indian flora (Kumar et al., 2011). Conventional cultivation of R. imbricata has been challenged due to low seed viability, embryo abortion, and the inability of the plant to grow besides its natural habitat (Chaurasia et al., 2003; Kumar et al., 2011; Bhardwaj et al., 2018). Although, the average time to accumulate an ample level of active ingredients in naturally grown Rhodiola plants is 5–7 years (Galambosi, 2006). This is mainly due to slow growing nature, poor seed germination, and pollinator specificity due to the high altitude environment (Kumar et al., 2011; Galambosi, 2006). Therefore, considering the above fact there is a need to establish more effective alternative production strategies for its bioactive constituents (Chaurasia et al., 2003; Kumar et al., 2011; Bhardwaj et al., 2018).

Plant cell culture offers an economically feasible and recognized as a potential alternative sustainable platform for year-round production of desired metabolites (Kapoor et al., 2018; Eibl et al., 2018; Nordlund et al., 2018; Efferth, 2019; Kumar et al., 2020). Therefore, the present study investigated for the first time to develop a friable callus cell lines derived from leaf and root explants of R. imbricata for the production of commercially important metabolites. Furthermore, UPLC-PDA based analytical method was developed and validated using qualitative quantification of phenylethanoids, phenylpropanoids, and phenolic acid marker compounds i.e. gallic acid (1), salidroside (2), tyrosol (3), rosavin (4), rosarin (5), p-coumaric acid (6) and cinnamic acid (7). Also, to correlate and validate the metabolite content analysis, transcript level gene expression (RT-qPCR) profiling was investigated using key biosynthetic pathway genes of Rhodiola species.