Laiq ur Rahman

Biotransformation studies using hairy root cultures - A review.

Agrobacterium rhizogenes induced hairy root cultures are entering into a new juncture of functional research in generating pharmaceutical lead compounds by bringing about chemical transformations aided through its inherent enzyme resources. Rational utilization of hairy root cultures as highly effective biotransformation systems has come into existence in the last twenty years involving a wide range of plant systems as well as exogenous substrates and diverse chemical reactions. To date, hairy root cultures are preferred over plant cell/callus and suspension cultures as biocatalyst due to their genetic/biochemical stability, hormone-autotrophy, multi-enzyme biosynthetic potential mimicking that of the parent plants and relatively low-cost cultural requirements. The resultant biotransformed molecules, that are difficult to make by synthetic organic chemistry, can unearth notable practical efficacies by acquiring improved physico-chemical properties, bioavailability, lower toxicity and broader therapeutic properties. The present review summarizes the overall reported advances made in the area of hairy root mediated biotransformation of exogenous substrates with regard to their reaction types, plant systems associated, bacterial strains/molecules involved and final product recovery.

Enhanced production of valepotriates by Agrobacterium rhizogenes induced hairy root cultures of Valeriana wallichii DC

Abstract Hairy roots of Valeriana wallichii DC were obtained following co-cultivation of detached leaf explants with Agrobacterium rhizogenes strains A 4 and LBA 9402. For V. wallichii the A 4 strain appeared to be better than the LBA 9402 strain in terms of both relative rate of hairy root formation and growth of the respective hairy root line. The optimal growth of both the hairy root lines occurred on half strength MS [1] salt medium with 40 gm/l sucrose. Valepotriate contents from the roots of 24 week old wild type field grown plants were compared with those of 12, 16 and 20 week old hairy root lines induced by the A 4 and LBA 9402 strains of A. rhizogenes . The homodidrovaltrate, didrovaltrate, IVHD and acevaltrate contents were quantified individually by HPLC. The valepotriates were localized only in the root tissues and were not detected in the culture medium. In terms of the production of total as well as individual valepotriates, the LBA 9402 induced hairy root line appeared to be a better performer than the A 4 induced one. The total valepotriate content was highest in the 20 week old LBA 9402 induced hairy root line (8.52% dw) followed by that of the A 4 induced line (5.10% dw) of the same age, which were 3.3 and 2 times higher, respectively than that of the control roots (2.58% dw). Rapid growth of the hairy roots of V. wallichii with in vitro valepotriates production potential may offer an attractive alternative to the exploitation of this endangered plant species.

In vitro -studies in Plumbago zeylanica : rapid micropropagation and establishment of higher plumbagin yielding hairy root cultures

Summary A protocol for rapid in vitro -multiplication of Plumbago zeylanica L. through axillary bud proliferation was developed as an essential prerequisite to conduct genetic transformation studies. A maximum of 3.5 ± 0.5 shoots were produced from a single nodal segment of a four year old field grown plant after 4 weeks of transfer to Murashige and Skoogs (MS) basal medium supplemented with 8.87 mmol/L BAP + 0.49 mmol/L IBA. Optimum root induction response was achieved upon transferring the individual regenerant to half strength MS medium containing 0.49 mmol/L IBA. Hairy roots initiated at 0.9 ± 0.05 relative transformation frequency with the A4 strain of Agrobacte riumrhizogenes exhibited optimum growth in half strength MS medium containing 4 % sucrose. Growth kinetic studies demonstrated a maximum 21 fold increase in biomass yield after 6 weeks of culture. The fresh hairy roots produced 2.5 times higher amounts of plumbagin than the fresh, untransformed control roots or the dry hairy roots of the same age. The present research findings revealed for the first time the potentialities of the hairy root cultures of P. zeylanica for the production of the important secondary metabolite plumbagin.

Agrobacterium rhizogenes -mediated transformation of Picrorhiza kurroa Royle ex Benth.: establishment and selection of superior hairy root clone

A protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Picrorhiza kurroa was developed through optimization of the explant type and the most suitable bacterial strain. The infection of leaf explants with the LBA9402 strain resulted in the emergence of hairy roots at 66.7% relative transformation frequency. Nine independent, opine and TL-positive hairy root clones were studied for their growth and specific glycoside (i.e., kutkoside and picroside I) productivities at different growth phases. Biosynthetic potentials for the commercially desirable active constituents have been expressed by all the tested hairy root clones, although distinct inter-clonal variations could be noted in terms of their quantity. The yield potentials of the 14-P clone, both in terms of biomass as well as individual glycoside contents (i.e., kutkoside and picroside I), superseded that of all other hairy root clones along with the non-transformed, in vitro-grown control roots of P. kurroa. The present communication reports the first successful establishment, maintenance, growth and selection of superior hairy root clone of Picrorhiza kurroa with desired phyto-molecule production potential, which can serve as an effective substitute to its roots and thereby prevent the indiscriminate up-rooting and exploitation of this commercially important, endangered medicinal plant species.

Bacoside production by suspension cultures of Bacopa monnieri (L.) Pennell

Cell suspension cultures of Bacopa monnieri (L.) Pennell, grown in modified MS medium, grew some 5–6 fold over 40 days. Selected cell lines produced the important saponin, bacoside A, up to 1 g/100 g dry wt after this time.

An extensive case study of hairy-root cultures for enhanced secondary-metabolite production through metabolic-pathway engineering.

An intrinsic improvement is taking place in the methodologies for the development of culture systems with first‐rate production of plant‐based molecules. The blending of HR (hairy root) cultures with ME (metabolic engineering) approaches offers new insights into, and possibilities for, improving the system productivity for known and/or novel high‐value plant‐derived active compounds. The introduction and expression of foreign genes in plants results in improvement of cellular activities by manipulating enzymatic, regulatory and transport function of the cell. The rational amendments in the rate‐limiting steps of a biosynthetic pathway as well as inactivating the inefficient pathway(s) for by‐product formation can be accomplished either through single‐step engineering or through the multi‐step engineering. The hierarchical control of any metabolic process can lead the engineer to apply the ME ideas and principles to any of the strata, including transcriptional, moving on to translational and enzymatic activity. The HR culture systems offer a remarkable potential for commercial production of a number of low‐volume, but high‐value, secondary metabolites. Taking HR as a model system, in the present review, we discuss engineering principles and perceptions to exploit secondary‐metabolite pathways for the production of important bioactive compounds. We also talk about requisites and possible challenges that occur during ME, with emphasis on examples of various HR systems. Furthermore, it also highlights the utilization of global information obtained from ‘‐omic’ platforms in order to explore pathway architecture, structural and functional aspects of important enzymes and genes that can support the design of sets of engineering, resulting in the generation of wide‐ranging views of DNA sequence‐to‐metabolite passageway networking and their control to obtain desired results.

Root tip-dependent, active riboflavin secretion by Hyoscyamus albus hairy roots under iron deficiency

Hyoscyamus albus hairy roots with/without an exogenous gene (11 clones) were established by inoculation of Agrobacterium rhizogenes. All clones cultured under iron-deficient condition secreted riboflavin from the root tips into the culture medium and the productivity depended on the number and size of root tips among the clones. A decline of pH was observed before riboflavin production and root development. By studying effects of proton-pump inhibitors, medium acidification with external organic acid, and riboflavin addition upon pH change and riboflavin productivity, we indicate that riboflavin efflux is not directly connected to active pH reduction, and more significantly active riboflavin secretion occurs as a response to an internal requirement in H. albus hairy roots under iron deficiency.

Hairy root biotechnology of Rauwolfia serpentina: a potent approach for the production of pharmaceutically important terpenoid indole alkaloids

Hairy root cultures of Rauwolfia serpentina induced by Agrobacterium rhizogenes have been investigated extensively for the production of terpenoid indole alkaloids. Various biotechnological developments, such as scaling up in bioreactors, pathway engineering etc., have been explored to improve their metabolite production potential. These hairy roots are competent for regenerating into complete plants and show survival and unaltered biosynthetic potential during storage at low temperature. This review provides a comprehensive account of the hairy root cultures of R. serpentina, their biosynthetic potential and various biotechnological methods used to explore the production of pharmaceutically important terpenoid indole alkaloids. The review also indicates how biotechnological endeavors might improve the future progress of research for production of alkaloids using Rauwolfia hairy roots.

HCHL expression in hairy roots of Beta vulgaris yields a high accumulation of p-hydroxybenzoic acid (pHBA) glucose ester, and linkage of pHBA into cell walls.

As part of a study to explore the potential for new or modified bio-product formation, Beta vulgaris (sugar beet) has been genetically modified to express in root-organ culture a bacterial gene of phenylpropanoid catabolism. The HCHL gene, encoding p-hydroxycinnamoyl-CoA hydratase/lyase, was introduced into B. vulgaris under the control of a CaMV 35S promoter, using Agrobacterium rhizogenes LBA 9402. Hairy root clones expressing the HCHL gene, together with non-expressing clones, were analysed and revealed that one expression-positive clone accumulated the glucose ester of p-hydroxybenzoic acid (pHBA) at about 14% on a dry weight basis. This is the best yield achieved in plant systems so far. Determination of cell-wall components liberated by alkaline hydrolysis confirmed that the ratio of pHBA to ferulic acid was considerably higher in the HCHL-expressing clones, whereas only ferulic acid was detected in a non-expressing clone. The change in cell-wall components also resulted in a decrease in tensile strength in the HCHL-expressing clones.

Essential oil composition of Pelargonium graveolens L’Her ex Ait. cultivars harvested in different seasons

To determine the seasonal influence on essential oil yield and composition of three rose-scented geranium cultivars, namely Bourbon type, CIM-Pawan and Kelkar, an experiment was carried out in western Himalayan region, India. The essential oil yield varied from 0.05% to 0.20%, from 0.10% to 0.25% and from 0.03% to 0.12% in fresh biomass of the cv. Bourbon type, CIM-Pawan and Kelkar, respectively. Gas chromatography/flame ionization detector (GC/FID) and /mass spectrometry (GC/MS) analyses revealed significant variations in the essential oil composition of rose-scented geranium due to cultivar and season of harvesting. The major components in the essential oil of cv. Bourbon type were geraniol (14.1–34.6%), citronellol (15.2–31.3%), linalool (2.9–9.2%), citronellyl formate (4.4–9.2%), isomenthone (4.5–6.6%), 10-epi-γ-eudesmol (4.7–6.7%) and geranyl formate (3.8–6.2%). The dominant constituents of the cv. CIM-Pawan essential oil were geraniol (11.9–31.9%), citronellol (16.1–30.2%), citronellyl formate (5.2–8.9%), linalool (3.7–6.4%), isomenthone (4.0–6.3%), 10-epi-γ-eudesmol (4.4–5.2%) and geranyl formate (4.3–5.0%). However, the chemical composition and odor of cv. Kelkar was quite different from the other two cultivars and the major components found in this oil were citronellol (51.0–63.4%) and isomenthone (9.8–17.8%).