Withania somnifera is a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues. In vitro propagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential of in vitro cell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years. In vitro culture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field.

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Addressing Challenges to Enhance the Bioactives of Withania somnifera through Organ, Tissue, and Cell Culture Based Approaches

Agrobacterium rhizogenes and A. tumefaciens transfer DNA and effector proteins into plant cells, where transferred DNA (T-DNA) is inherited and expressed. Most transgenic plants are created using A. tumefaciens, but transformation by A. rhizogenes yields desirable single-copy transgenes more frequently than A. tumefaciens does. DNA transfer from both species resembles plasmid conjugation, but later events differ between species. Efficient transformation by A. tumefaciens requires single-strand DNA-binding protein VirE2, which A. rhizogenes lacks, so substrates for T-DNA integration differ greatly. In A. rhizogenes, the GALLS proteins substitute for (but do not resemble) VirE2. GALLS proteins occur in two forms: full-length (FL) and a more abundant C-terminal domain (CT). Both have protein-binding domains and type IV secretion signals. GALLS-FL has ATPase/ strand transferase and nuclear localization (NLS) domains, allowing it to enter the nucleus and bind VirD2, a pilot protein attached to single-stranded T-DNA (T-strands). GALLS-FL ATPase may pull T-strands into the nucleus. GALLS-CT stimulates an early step in gene transfer to plants; this effector protein alters host gene expression and stimulates T-DNA transfer, apparently by suppressing host defenses. These observations challenge the assumption that A. rhizogenes and A. tumefaciens transform plants and mitigate host defenses by the same mechanisms.

Production of Plant Derived Natural Compounds through Hairy Root Culture

Withania somnifera (L.) Dunal is one of the most important medicinal plant belonging to the family Solanaceae. Its root have been used as a drug since ancient times, and various pharmacological effects have been attributed to the occurrence of secondary compounds like withaferin-A and withanolide-A. Recently, huge interests are generated for production of these bioactive compounds through Agrobacterium rhizogenes-mediated hairy root culture techniques. The present review explores the culture conditions for efficient Agrobacterium-mediated hairy root culture system of W. somnifera for withanolide production. The hairy root induction is influenced by several factors like bacterial stain, type of explant, and cocultivation methods. The transformation efficiency could be enhanced by the addition of acetosyringone and SAAT treatments during cocultivation. Recent studies have also shown positive correlations of elicitors and biosynthetic pathway genes on withanolide production in hairy root culture of W. somnifera.

An Efficient Hairy Root System for Withanolide Production in Withania somnifera (L.) Dunal

Withania somnifera is a high value medicinal plant of the family Solanaceae. It is known for its anti-tumour, anti-stress, anti-aging, cardio-protective, neuro-protective and anti-inflammatory properties. Its pharmaceutical properties are attributed to a wide range of secondary metabolites, such as steroidal lactones, alkaloids, glycowithanolides, flavanol glycosides, phenolics and sterols. The traditional cultivation of W. somnifera is limited mainly by poor seed viability and germination, low yield and inconsistency in production of secondary metabolites. The infestation with various pests and pathogens also throw a major challenge in its cultivation. Biotechnological approaches involving organ, tissue and cell culture offer potential solution to the existing problems. In vitro propagation helps in rapid multiplication of elite cultivars and facilitate in raising quality planting materials. Genetic manipulation and secondary metabolite engineering hold great promise for enhancement of secondary metabolites and for overall crop improvement. The present chapter briefly discuss the challenges in W. somnifera and present a quick overview of biotechnological advances to address these challenges. It also highlights the futuristic approaches that would lay a foundation in the conglomeration of W. somnifera as an ideal model medicinal plant.

Biotechnological Approaches in Propagation and Improvement of Withania somnifera (L.) Dunal

Withania somnifera (L.) Dunal is one of the most reputed medicinal plants belonging to the family Solanaceae. The plant contains various steroidal lactones, alkaloids, polyphenols, flavanol glycosides, and sterols, which are responsible for its medicinal properties such as antitumor, neuroprotective, cardioprotective, antiaging, and anti-inflammatory. The main focus of research in W. somnifera is to increase the production of withanolides, the pharmaceutically important constituents. Various in vitro strategies like organ and cell culture and genetic manipulation and molecular biology tools such as metabolic engineering have been adapted to enhance secondary metabolite production. The present chapter provides a brief overview of challenges in W. somnifera and discusses some of the latest developments to address these challenges.

Biotechnological Strategies for Improvement of Withania somnifera (L.) Dunal

An efficient protocol was devised for rapid in vitro propagation of Withania somnifera through shoot bud culture. The proliferating auxiliary shoot was isolated from nodal explants of field grown Withania somnifera. established on Murashige and Skoogs medium (MS) and Gamborg B5 medium supplemented with six concentrations of Benzyl amino purine (BAP), Naphthalene acetic acid (NAA), Kinetin(Kn) and 2,4Dichlorophenoxy acetic acid (2, 4-D). After 30 days of culture of Withania somnifera, the maximum numbers of shoots was produced on MS medium supplemented with 1.0mg/L BAP. Each of the explants had developed more than two shoots per nodes, while in six concentrations each of kinetin, NAA and 2, 4-D developed either two or less than two shoots/explants. The study revealed highest frequency of shoot formation and maximum number of shoots per explants in MS medium supplemented with 1mg/L BAP than the other growth hormone.

Raghvendra Rao

Papers

Micropropagation of a medicinal plant withania somnifera l. dunal by shoot bud culture