Hairy Root Culture: History, Formulation and Application

Read this article to learn about Hairy Root Culture. After reading this article you will learn about: 1. History of Hairy Root Culture 2. Formation of Hairy Root Culture 3. Applications.

History of Hairy Root Culture:

The term “hairy root” was first coined by Steward et al. (1900). In 1930, Ricker et al., first named the hairy root causing organism Phytomonas rhizogenes, which was later renamed A. rhizo­genes. The first transformation of higher plants using A. rhizogenes was done by Ackermann in 1973.

More than 450 species of many different genera and families of higher plants are known to be susceptible to the infec­tion by A. rhizogenes. A. rhizogenes is a gram negative, non-sporing, motile, rod shaped bacterium, closely related to rhizobium which, produce nitrogen fixing nod­ules on leguminous plants.

The Genome Structure:

The ability of root induction by A.rhizogenes lies in its typical genome structure, the molecular bi­ology of which has not been properly understood till date. Virulent strains of A. rhizogenes, con­tains a large mega plasmid (more than 200 kb) which plays a key role in hairy root formation. Most of the genes involved in hairy root forma­tion are not borne on the chromosome, but on this root inducing (Ri) Plasmid.

This plasmid car­ries three genetic components, first is a mobile DNA element, t-DNA which is integrated into the nucleus of infected cells where it is subsequently stable and integrated into the host genome and transcribed causing the formation of proliferative multi-branched adventitious roots at the site of infection; called as hairy root diseases in dicoty­ledonous plants.

The transferred DNA (t-DNA) lo­cated on the Ri plasmids are approximately 10 to 30 kbp in size, generally represent less than 10% of the Ri plasmid. The second one is the virulence area (vir), which contains several vir genes which do not enter the plant cell but, together with the chro­mosomal DNA, cause the transfer of t-DNA.

The third one is, so-called border sequences (25 bp), resides in the Agrobacterium chromosome. The Ri-plasmids are grouped into two main classes according to the opines, which are produced as specific food for bacteria synthesized by hairy roots.

First, agropine-type strains induce roots to synthesise agropine, sec­ond, Mann opine-type strains induce roots to pro­duce Mann opine. Agropine-type strains are con­sidered to be the most virulent and therefore fre­quently used in the establishment of hairy root cultures.

Formation of Hairy Root Culture:

The t-DNA of the agropine-type Ri-plasmid con­sists of two separate t-DNA region tl-DNA and tr- DNA. The genes encoding auxin synthesis (tmsl and tms2) and agropine synthesis (ags) have been localized on the tr-DNA of the agropine type of Ri-plasmid.

While in Mann opine type Ri-plasmids contain only one t-DNA that shares considerable DNA sequence homology with tl of the agropine- type plasmids. ti-DNA regions of t-DNA consists of 4 genetic loci, rolA, rolB, rolC, and rolD, which affect hairy root induction.

The complete nucle­otide sequence of the tl-region revealed the pres­ence of 18 open-reading frames (ORFs), 4 of which, ORFs 10, 11, 12 and 15, respectively, cor­respond to the rolA, rolB, rolC, and rolD loci.

It was also shown that rolA, rolB, and rolC play the most pivotal role in hairy root induction, rolB seems to be the most crucial in the differentiation process of transformed cells, protruding stigmas and reduced length of stamens; while rolA is associated with internodes shortening and leaf wrin­kling; rolC causes internodes shortening and re­duced apical dominance and rolD provide the accessory functions.

Although the tr-DNA is not essential for hairy root formation it has been shown that the aux 1 gene harboured in this seg­ment provides to the transformed cells with an additional source of auxin. The plasmid vir genes are essential for the formation of the product that controls t-DNA movement. Vir A, B, C, D, E, G and Vir H are organized regions of 30 kbp vir regions.

The product of Vir A specifies and inner membrane protein that recognize plant phenolic compounds due to the wound of plant tissue and passes the information to the product of Vir G which then itself act as transcriptional activator which finally leads to t-DNA transfer. Vir H locus encodes a protein which is responsible for bacte­rial survival during infection process.

Gene Transfer Mechanism from Agro-bacterium Rhizogenes to Plant Genome:

The vir gene expression, generation of t-DNA copy, formation of T strand protein complex, movement of the T-complex through the bacteri­al membranes, targeting of the T complex into and within the plant cell, targeting of the T com­plex into the cell nucleus, it’s stabilization, and finally integration of T strand into cell DNA are seven successive steps of transfer of DNA from Agrobacterium to plant cell.

Step 1:

Bacterial colonization on the wounding site of plant tissue is prerequisite for transforma­tion. The production of phenolic compounds at the wounding site is sensed by one of the Vir A gene product which initiates induction of expression of remaining Vir loci.

Step 2:

The product of Vir C and Vir D play pivotal role in this step. Two Vir D specific product Vir D₁ and Vir D₂ are essentially required for synthesis of t-DNA strand. The Vir C locus decodes for two polypeptides Vir Q and Vir C₂ that are shown to enhance t-DNA border nicking.

Step 3:

The t-DNA strand is likely to exist as a DNA protein complex. The Vir E, specially Vir E₂ protein is the most abundant protein synthe­sized in Vir induced Agrobacterium cells. The Vir D₂ bounds to the leading end of the T- complex. Thus T-complex is compressed of the t-DNA strand, Vir D₂ and Vir E₂.

Step 4:

The product of Vir B locus produces trans membrane channel outside the bacterial cell wall because of its 11 open reading frame known as Vir B₁ to Vir B₁₁ the last one helps to pump the T complex out of the bacterial cell.

Step 5:

The uptake of T-complex into the plant cell though yet not understood clearly but assume this mechanism somewhat analogues to bac­terial conjugation.

Step 6:

The T-complex (T- DNA strand, Vir D₂ and Vir E₂) in this step enters within plant cell – nucleus. The N terminal of Vir D₂ has role to nick the T-DNA border while C terminus helps in the nuclear uptake of the T strand. The Vir E₂ help to Vir D₂ to target the T complex to the nuclear pore in a polar direction which facilitates it’s linear uptake.

Step 7:

Generally t-DNA insertions can occur in any chromosome of the plant genome or it may occur randomly.

Single strand of plant DNA participates in this process. At the 5′ end of the T strands invades a short nick the plant DNA’s short starch of homology, very few nucleotides which par­tial pairing between both ends followed by repairing of any overhangs of t-DNA end fol­lowed by it’s legation to the plant DNA ends.

As hairy root formation involves the transfer of DNA from the bacterium to the plant nucleus and the response of plant cell to the root inducing plant hormone-auxin. It was noticed that t| DNA of Ri plasmid appear to sensitize the transformed cell to auxin, which determined the root growth and typical char­acteristics of hairy roots.

Transformation Protocol:

Different scientists in different time proposed many protocols for successful transformation. Surface sterilization, overnight co culture and direct in­fection at the wound site of plant tissue with A. rhizogenes are commonly used procedure for transformation. However successful infection pro­cedure depends on particular plant species and specific plant tissue.

Hairy Root Induction and Establishment of Hairy Root Culture:

To succeed in establishing a hairy root culture system for a certain species, several essential con­ditions should be taken into consideration. These conditions include the bacterial strain of A. rhizo­genes, an appropriate explants, a proper antibi­otic to eliminate redundant bacteria after infec­tion, and a suitable culture medium.

Agropine strains are the most used strains owing to their strongest ability. Most plant materials such as hypocotyls, leaf, stem, stalk, petiole, shoot tip, cot­yledon, protoplast, storage root or tuber can be used to induce hairy root. The level of tissue differentiation determines the ability to give rise to transformed roots after A. rhizogenes inocula­tion.

In this case, successful infection of some species can be achieved by the addition of acetosyringone. Cefotaxime sodium, Carbencilin disodium, vancomycin, ampicilin sodium or strep­tomycin sulphate ranging in concentration from 100 to 500 µg/ml are used to kill or eliminate bac­teria.

However for different species, the proper explant material, antibiotic treatment, first emergence of hairy root after infection may vary and the age of the material is most critical, with juvenile material being optimal. The decon­taminated hairy root can be sub-cultured on phytohormones free medium.

Generally hairy roots are considered to be stable and easy to subcul­ture. Nonetheless, hairy root culture also possess a certain amount of heterogeneity, even if derived from a single root tip, and the repeated selection seems to be important to obtain high scopolamine producing hairy root lines, so selection of appro­priate hairy root line are prerequisite for estab­lishing hairy root culture.

The confirmatory test for hairy root culture:

(a) Morphological Characteristics:

Pal-geotropism is common phenomenon in the roots transformed with A. rhizogenes, have an alter phenotype such as profuse lateral branching, as a result due to increase bio- mass and consequent increase in the number of elongating tips.

(b) Biochemical Markers:

The opines are effective biochemical marker for identification of transformed roots has been done through paper electrophoresis. Due to instability of opine genes within transformed roots this proc­ess is not popularly used.

(c) Genetic Marker:

t-DNA identification of the host plant genome acts as a reliable genetic marker to confirm trans­formation. The most widely used procedure is Southern blot hybridization. Other procedures include DNA “dot blotting“, localization of t-DNA in plant chromosome by “in situ hybridization” and Polymerase chain re­action.

Applications of Hairy Root Culture:

Production of Secondary Metabolite For­mation:

For 25 years, hairy roots have been investigated as a biological system for the production of valu­able phytochemicals that are useful as pharma­ceuticals, cosmetics and food additives. These roots can also synthesize more than a single me­tabolite and therefore prove economical for com­mercial production purposes.

Many medicinal plants have been transformed successfully by rhizogenes and induced a relatively high produc­tivity of secondary metabolites which are poten­tially important pharmaceutical products.

Sevon et al., (2002) has summarized the most important alkaloid produced by hairy root culture includ­ing Atropa belladonna L., Catharanthus tricophyllus L., and Datura Candida L. A substantial re­search effort has already been put in for the pro­duction of anti-tumour alkaloids such as campothecin, vinblstine and taxol with this technique.

Hairy root culture of Chinese medicinal plant tri-chosanthes kirilowii have been seen producing trichosanthin, a ribosome inactivating proteins, can inhibit multiplication of the Human Immuno Deficiency (HIV) THE AIDS virus. In recent year, hairy root culture has emerged as useful to study the biology and engineering of plant chemical production.

Understanding and Manipulating of Root Specific Metabolism:

There are several ways in which we can visual­ize roots are chemical factories, the underground growth habit of roots poses major technical diffi­culties for their study and has hindered biochem­ical research in particular.

A recent reincarnation of a classic plant organ culture system has ex­tremely useful in reinvigorating research on root metabolism. Hairy root culture provided many insights into root specific metabolism and its reg­ulation.

This system has been used to follow the production of bioactive proteins. Hairy root cul­ture provided many insights into root specific metabolism and its regulation. This system has been used to follow the production of bioactive proteins. Hairy roots will have a continuing role as an experimental model in plant metabolic en­gineering.

Plants also have considerable poten­tial for the production of biopharmaceutical pro­teins and peptides. Applications vary from the production of natural products and foreign pro­teins to a model for phyto-remediation of organic and metal contaminants.

As the demand for biopharmaceuticals is expected to increase, it would be wise to ensure that they will be available in significantly larger amounts on a cost-effective basis.

Production Compounds not Found in Untransformed Roots:

Transformation may affect the metabolic pathway and produce new compounds that cannot be produced normally in untransformed roots. In Scutellaria baicalensis Ceorgi, accumulated glucoside conjugates of flavonoids instead of the glu­cose conjugates accumulated in untransformed roots.

Regeneration of Whole Plants:

Most importantly can transfer T-DNA from binary vectors and enables the production of trans­genic plants containing the foreign genes carried on a second plasmid. This property has been used to produce transgenic plants.

Regeneration of transformed plants through hairy roots have been reported in medicinal plant species is limited. Evaluation of secondary metabolite from regenerated plants from hairy roots has been started quite recently Oung and Tepfer.

Why Hairy Root Culture prefers over other Plant Tissue Culture?

Traditional agricultural methods often require months to years to obtain a crop. Furthermore the levels of secondary me­tabolite production are affected by eco-geographical factors.

The transformed roots are highly dif­ferentiated and can cause stable and extensive production of secondary metabolites, whereas other plant cell cultures have a strong tendency to be genetically and biochemically unstable and often synthesize very low levels of secondary me­tabolites.

Normally, root cultures need an exogenous phytohormone supply and grow very slowly, result­ing in the very poor or negligible synthesis of sec­ondary metabolites.

These hairy roots can be maintained as or­gan cultures for a long time and subsequent shoot regeneration can be obtained without any cytological abnormality. Rapid growth of hairy roots on hormone-free medium and high plantlet re­generation frequency allows clonal propagation of elite plants. This property can be utilized by genetic manipulations to increase biosynthetic capacity.

The secondary metabolite production of hairy roots is highly linked to cell differentia­tion. Alkaloid production decreased clearly when roots were induced to form callus, and reappeared when the roots were allowed to re-differentiate. For this reason undifferentiated callus culture sometime loosed biosynthetic ability to accumu­late secondary metabolite.

Production Enhancement in Hairy Root Culture:

To obtain a high density root culture, the culture conditions should be maintained at the optimum level. The composition of the culture medium; sucrose percentage; supply of exogenous growth hormone, specially auxin sensitivity, nature of nitrogen source; light; temperature and the pres­ence of biotic and abiotic elicitors; even use of heavy metals enhanced secondary metabolite production.

Media manipulations have been re­ported to aid in the release of metabolites.

Use of Bioreactors for Commercial Pro­duction of Secondary Metabolite from Hairy Root Culture:

In many industrialized countries, more and more individuals are turning to herbal medicines and plant-derived products to offset the high costs of personal healthcare. Many efforts have been made to commercialize production of the medical plant metabolites from plant-cell culture. Specific yields for several natural products are higher in plant cell and hairy root culture.

Currently, the cost of biopharmaceuticals limits their availability. Plant-derived biopharmaceuticals are cheap to produce and store, easy to scale up for mass production, and safer than those derived from animals and microorganisms.

Bioreactor is a vessel construct­ed of glass, polycarbonate or steel in which cell or organ is cultured. Bioreactors must have suffi­cient mixing and mass transfer ability to provide adequate nutrient supply under operating _pH, temperature, dissolved oxygen etc.) conditions.

Scaling up of hairy roots in novel bioreactors can provide the best conditions for optimum growth and secondary metabolite production than in native root culture. Hairy root morphology is quite plastic so the bio­reactor design for root culture is a balancing act between plant tissues and surrounding control­led environment.

Thus exploitation of hairy root culture in commercial scale depends on the development of suitable bi­oreactor system. Several bioreactor designs have been reported for hairy root culture roughly di­vided into two types namely Liquid phase; Gas phase and Hybrid reactors that are a combina­tion of both.

Among liquid phase reactors stirred tank; air lift; bubble col­umns and turbine blade reactor are commonly used. Here roots are submerged in the liquid medium. The major disadvantage of this reactor is the requirement of high energy for large scale operation.

Reactors in which liquid is the depressed phase and gas in the continuous phase appear to offer continuous conditions and roots are exposed to air and liquid nutrients are delivered as mist. One of the major advan­tages of this type is the complete control of the gases in the culture environment.

Different types of gas phase reactors have been used in research activities among them trickle bed; radial flow and nutrient mist reactors are important. The disad­vantage of this reactor is that there is no way to uniformly distribute the roots in the chamber with­out manual loading.

A hybrid reactor system made up of bubble column and nutrient mist bioreac­tor was used to study the transient growth char­acteristics and nutrient utilization rates of Artem­isia annua hairy roots.

Potential Problems Encountered In Hairy Root Culture:

Several decades have passed since the hairy root culture was established, however this system has not been utilized globally in commercial scale.

To resolve the bottleneck of this application fu­ture research should focus on the establishment of effective and economical scaled up culture systems that can reduce the consumption but obtain the biggest benefits. If such breakthrough is achieved, the application is more likely.