Suchitra Banerjee

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.

An efficient in vitro procedure for micropropagation and generation of somaclones of rose scented Pelargonium.

Efficient protocols have been established for both direct and indirect regeneration of plants in Pelargonium graveolens Indian cultivar Hemanti (Algerian type). Murashige and Skoogs (MS) medium [T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15 (1962) 473-497] supplemented with 5.0 mg/l kinetin and 1.0 mg/l NAA was optimal for direct regeneration of plants from leaf explants while 8.0 mg/l kinetin and 1.0 mg/l NAA proved optimum for nodal explants for maximum number of shoots per explant. Callus induction was observed from nodal explants on MS medium supplemented with 10 mg/l kinetin and 1.0 mg/l NAA. Callus on further transfer to MS medium with 0.5 mg/l BAP and 0.1 mg/l NAA exhibited regeneration of maximum number of shoots. In vitro grown shoots of both direct and indirect origin rooted within 7-10 days following transfer to half strength MS medium with 1.0 mg/l IBA. Plantlets were acclimatized under glass house conditions with 90% survival. Randomly selected 85 individual Calliclones were subjected to field trial with 85-95% survival for two successive years along with control in randomized block design with three replicates. Screening of these calliclones revealed two distinct morphotypes, one with parental type highly dentated leaves (HDL) and the other with less dentated, round leaves (LDL). Only HDL calliclones flowered under field conditions. The LDL clones differed in several herb related agronomic characteristics such as plant height, herb yield, canopy size and number of branches per plant from the parental type as well as from the parent, which seems advantageous for commercial exploitation of such clones. The HDL clones closely resemble the parent in having higher content of citronellol than geraniol while the LDL clones contain almost equal contents of citronellol and geraniol in their essential oils as revealed by gas chromatography analysis. It is noticeable that the variability both in terms of agronomic characters and essential oil profiles among the clones were stable over 2 years of field trials.

A bioactive labdane diterpenoid from Curcuma amada and its semisynthetic analogues as antitubercular agents

A labdane diterpene dialdehyde was first time isolated from the chloroform extract of rhizomes of Curcuma amada. This compound exhibited antitubercular activity (MIC=500 microg/mL) against Mycobacterium tuberculosis H(37)Rv strain in BACTEC-460 assay. Two of its semisynthetic analogues also exhibited antitubercular activity at 250-500 microg/mL. It is the first report on isolation and antimycobacterial activity of this dialdehyde from C. amada.

Analysis of hairy root culture of Rauvolfia serpentina using direct analysis in real time mass spectrometric technique.

The applicability of a new mass spectrometric technique, DART (direct analysis in real time) has been studied in the analysis of the hairy root culture of Rauvolfia serpentina. The intact hairy roots were analyzed by holding them in the gap between the DART source and the mass spectrometer for measurements. Two nitrogen-containing compounds, vomilenine and reserpine, were characterized from the analysis of the hairy roots almost instantaneously. The confirmation of the structures of the identified compounds was made through their accurate molecular formula determinations. This is the first report of the application of DART technique for the characterization of compounds that are expressed in the hairy root cultures of Rauvolfia serpentina. Moreover, this also constitutes the first report of expression of reserpine in the hairy root culture of Rauvolfia serpentina.

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.

Effect of Altitude on the Essential Oil Constituents of Artemisia roxburghiana Besser var. purpurascens (Jacq.) Hook.

Abstract Mature vegetative plants of Artemisia roxburghiana Besser var. purpurascens (Jacq.) Hook were collected from Mussoorie (2205 m), Bhatwari (1218 m) and Bhaldana (850 m) from Garhwal Himalayas and their essential oils were isolated and subjected to GC and GC/MS analysis. The oil yield was lowest (0.2%) in the plants collected from the relatively higher altitude of Mussoorie; it was rich in borneol (21.2%) followed by linalyl acetate (7.4%) and α- humulene (6.7%). The oils from plants collected from the lower altitudes of Bhatwari and Bhaldana yielded higher percentage of oils (0.8–0.85%) which were dominated by β-caryophyllene (16.3%, 18.4%) followed by α-thujone (12.0%) in the former and eugenol (16.2%) in the later.

Variation in the growth and alkaloid production capability of the hairy roots of Hyoscyamus albus, H. muticus and their somatic hybrid

Hairy root cultures have been established by inoculating the somatic hybrid (Hyoscyamus muticus + H. albus) as well as parent leaf explants with two strains of Agrobacterium rhizogenes (A4 and LBA 9402). The transformation frequency obtained with the A4 strain was found to be better than that of the LBA 9402 strain in all three test systems. Six lines each of H. albus and H. muticus, and four lines of the hybrid were studied for their growth and secondary metabolite production after 30 days of culture. Great variation was observed among the various lines induced by the two strains of bacteria. The maximum biomass had been noted in the somatic hybrid line 2D of A4 origin which is 1.26 and 1.55 times higher than that of the maximum biomass yielding lines 3M and 43A of H. muticus and H. albus respectively of the same bacterial origin. The somatic hybrid hairy root line 1D induced by A4 strain revealed maximum yield of atropine (9.73mg/culture) which was 9.18 times and 1.45 times higher than that of the highest atropine yielding hairy root line of H. muticus (35M) and H. albus (28A) respectively, of A4 origin. The scopolamine content of the same somatic hybrid line was found to be 1.13 times higher than that of the maximum scopolamine yielding H. albus line (28A).