A novel in vitro whole plant system for analysis of polyphenolics and their antioxidant potential in cultivars of Ocimum basilicum

Plants are an important source for medicinal compounds. Chemical screening and selection is critical for identification of compounds of interest. Ocimum basilicum (Basil) is a rich source of polyphenolics and exhibits high diversity, therefore bioprospecting of a suitable cultivar is a necessity. This study reports on the development of a true to type novel "in vitro system" and its comparison with a conventional system for screening and selection of cultivars for high total phenolics, individual polyphenolics, and antioxidant content. We have shown for the first time using online acidic potassium permanganate chemiluminescence that extracts from Ocimum basilicum showed antioxidant potential. The current study identified the cultivar specific composition of polyphenolics and their antioxidant properties. Further, a distinct relationship between plant morphotype and polyphenolic content was also found. Of the 15 cultivars examined, "Holy Green", "Red Rubin", and "Basil Genovese" were identified as high polyphenolic producing cultivars while "Subja" was determined to be a low producer. The "in vitro system" enabled differentiation of the cultivars in their morphology, polyphenolic content, and antioxidant activity and is a cheap and efficient method for bioprospecting studies.

Acidic potassium permanganate chemiluminescence for the determination of antioxidant potential in three cultivars of Ocimum basilicum

Ocimum basilicum, a member of the family Lamiaceae, is a rich source of polyphenolics that have antioxidant properties. The present study describes the development and application of an online HPLC-coupled acidic potassium permanganate chemiluminescence assay for the qualitative and quantitative assessment of antioxidants in three cultivars of O. basilicum grown under greenhouse conditions. The chemiluminescence based assay was found to be a sensitive and efficient method for assessment of total and individual compound antioxidant potential. Leaves, flowers and roots were found to be rich reserves of the antioxidant compounds which showed intense chemiluminescence signals. The polyphenolics such as rosmarinic, chicoric, caffeic, p-coumaric, m-coumaric and ferulic acids showed antioxidant activity. Further, rosmarinic acid was found to be the major antioxidant component in water-ethanol extracts. The highest levels of rosmarinic acid was found in the leaves and roots of cultivars "holy green" (14.37; 11.52 mM/100 g DW respectively) followed by "red rubin" (10.02; 10.75 mM/100 g DW respectively) and "subja" (6.59; 4.97 mM/100 g DW respectively). The sensitivity, efficiency and ease of use of the chemiluminescence based assay should now be considered for its use as a primary method for the identification and quantification of antioxidants in plant extracts.

Mycorrhizal elicitation of secondary metabolites in hairy roots of Ocimum basilicum

 Mycorrhiza, a symbiotic soil fungus was identified as a biotic elicitor of antioxidant compounds found in the plant roots. In vitro developed technique and bioresources carry potential towards formation of biological and biochemical factories for application in the agricultural and pharmaceutical industries.

Elite hairy roots of Ocimum basilicum as a new source of rosmarinic acid and antioxidants

This study reports Agrobacterium rhizogenes-mediated transformation of three cultivars of Ocimum basilicum for hairy root establishment, screening and selection for the production of rosmarinic acid and antioxidants. Hairy root development was found to be explant-specific and virulence-dependent. Distinct inter-cultivar morphological variability was found between the seven axenically developed hairy root lines and morphological traits were found to be correlated with the presence of aux2 genes, their expression and endogenous IAA content. Further inter-cultivar variability in the content of total phenolics, rosmarinic acid and caffeic acid was also found. Production of rosmarinic acid was found to be age-dependent and cultivar-specific. Chemiluminescence analysis showed the hairy roots to be rich in antioxidants and that rosmarinic acid was the major antioxidant molecule. The concentration of rosmarinic acid was found to be positively correlated with the total antioxidant potential of the hairy root extracts. On the basis of origin, morphology and metabolite content, three elite hairy root lines were selected that had significantly higher rosmarinic acid production, biomass and antioxidant potential than non-transformed roots. These new lines are rich reserves of both antioxidants and rosmarinic acid.

Rhizophagus irregularis as an elicitor of rosmarinic acid and antioxidant production by transformed roots of Ocimum basilicum in an in vitro co-culture system

Arbuscular mycorrhiza is a symbiotic association formed between plant roots and soil borne fungi that alter and at times improve the production of secondary metabolites. Detailed information is available on mycorrhizal development and its influence on plants grown under various edapho-climatic conditions, however, very little is known about their influence on transformed roots that are rich reserves of secondary metabolites. This raises the question of how mycorrhizal colonization progresses in transformed roots grown in vitro and whether the mycorrhizal fungus presence influences the production of secondary metabolites. To fully understand mycorrhizal ontogenesis and its effect on root morphology, root biomass, total phenolics, rosmarinic acid, caffeic acid and antioxidant production under in vitro conditions, a co-culture was developed between three Agrobacterium rhizogenes-derived, elite-transformed root lines of Ocimum basilicum and Rhizophagus irregularis. We found that mycorrhizal ontogenesis in transformed roots was similar to mycorrhizal roots obtained from an in planta system. Mycorrhizal establishment was also found to be transformed root line-specific. Colonization of transformed roots increased the concentration of rosmarinic acid, caffeic acid and antioxidant production while no effect was observed on root morphological traits and biomass. Enhancement of total phenolics and rosmarinic acid in the three mycorrhizal transformed root lines was found to be transformed root line-specific and age dependent. We reveal the potential of R. irregularis as a biotic elicitor in vitro and propose its incorporation into commercial in vitro secondary metabolite production via transformed roots.