ESTABLISHMENT OF HAIRY ROOT CULTURE WITH GENETIC

TRANSFORMATION IN MEDICINAL PLANTS

PROTEGES-Kartikey Vikram SinghAnd

Vineet DubeyMENTOR-Santosh Kr. Mishra

INTRODUCTION

Recently there has been a perceptible revival of interest in the use of medicinal plants throughout the world culminating into a manifold increase of medicinal plant based products.

At present there are about 130 clinically useful prescription drugs of known chemical structures, solely derived from higher plants and used in modern system of medicines.

In view of the spectacular increase in the demand of plant based compounds, the concerned medicinal plants have been indiscriminately exploited leading to scarcity or extinction of many valuable plant species.

plant species product Cost($/kg)

Cath. roseus Ajmalicine 37,000

P. somniferum Codeine 17,000

Cath. roseus Vincristine 20,00,000

Cath. roseus Vinblastine 10,00,000

Colchium autumnale colchicine 35,000

Panax ginseng Ginsenosides 50,000

Project Idea

This project deals with the production of high-value secondary metabolites including

pharmaceuticals and food additives through root cultures and transgenic roots obtained through biotechnological means.

Recent developments in transgenic research have opened up the possibility of the metabolic engineering of biosynthetic pathways to produce high-value secondary metabolites.

Hairy root cultures:

Definition It is the culture produced after the

infection of explants or cultures by the gram negative soil bacterium Agrobacterium rhizogenes.

This processes take advantage of the naturally occurring hairy root disease in Dicotyledons.

Production of hairy roots in plant cells

Agrobacterium recognizes some signal molecules exuded by wounded plant cells and becomes attached to it.

The bacteria contain the Root inducing plasmid (Ri-plasmid) The bacteria genetically transfer part of the Ri-plasmid called the transfer DNA (T-DNA) to the plant genome.

Production of hairy roots in plant cells

where it gets expressed and make the plant cell to proliferate by increasing the rate of cell division (cytokine expression) and cell elongation ( auxin expression) to produce the hairy roots.

 Produce the opines which is a type of unusual amino acids (octopine, agropine, nopaline, mannopine, and cucumopine) which is used by the bacterium as a carbon, nitrogen and energy source.

The Process

Ri-Plasmid

Advantages of hairy root cultures

The hairy root system is genetically and biosynthetically stable.

High production of secondary metabolites.

The culture can grow under phyto-hormone-free conditions.

The culture shows fast growth which reduces the culture time and easy the handling

Aim and Objective

The main aim of this project is to synthesize secondary metabolites from hairy root culture of medicinal plants this process will comprise of following stages:

1. Selecting a plant species appropriate for the desired product.

2. Through Micropropagation obtaining an infection free sample.

3. By means of genetic transformation induce the hairy root culture.

4. Obtaining the product and analysing the final product for quality.

5. Observing effect of different chemicals on the culture.

Plan of Work

Time Objective

two month Literature and Review

two month Micro propagation of the Selected Plant

two month Biotransformation

two month Optimisation of different parameters of culture

two month effect of different types of chemical factor on hairy root culture

two month Analysis of secondary metabolites produced by hairy roots

Progress till date

To grow the culture of A. rhizogenes we can prepare media either LB or YEB.

To grow the hairy root we are preparing MS media and enzyme supplement stock solutions.

Establishment of culture room is in progress.

Selection of the plant species for micro propagation.

References:-

S. Ramachandra Rao and G.A. Ravishankar; Biotechnology advances 20 (2002) 101 – 153; Plant cell cultures: Chemical factories of secondary metabolites.

Suchitra Banerjee, Sailendra Singh, Laiq Ur Rahman; Biotechnology advances 30 (2012) 461–468; Biotransformation studies using hairy root cultures.

Flores HE, Dai YR, Freyer AJ, Michaels PJ. Biotransformation of menthol and geraniol by hairy root cultures. Plant Physiol Biochem 1994;32:511–9.

Jung G, Tepfer D. Use of genetic transformation by the T-DNA of Agrobacterium rhizogenesto stimulate biomass and tropane alkaloid production in Atropa belladonna and Calystegiasepium roots grown in vitro. Plant Sci1987;50:145 – 51.

Ambros PF, Matzke AJM, Matzyke MA. Localization of Agrobacterium rhizogenes T-DNA in plant chromosomes by in situ hybridization. EMBO J 1986;5:2073 – 7

Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years.J Nat Prod 2007;70:461–77

Subroto MA, Kwok K, Hamill JD, Doran PM. Coculture of genetically transformed roots and shoots for synthesis, translocation and biotransformation of secondary metabolites. Biotechnol Bioeng 1996;49:481 – 94.

Banerjee S, Shang TQ, Wilson AM, Moore AL, Strand SE, Gordon MP, et al. Expression of functional mammalian P450 2E1 in hairy root cultures. Biotechnol Bioeng 2002;77:462–6.