1. HAIRY ROOT CULTURES AND THEIR ROLE IN SECONDARY METABOLITE PRODUCTION
Presented By;
Parul Chaudhari
Microbiology 3rd sem

2. CONTENTS :- Introduction Genetically transformed cultures
Marker to confirm transformation
Establishment and growth characteristics of hairy roots
Secondary metabolite production by hairy root cultures
Growth of hairy root cultures in various bioreactor for production of secondary metabolite

3. INTRODUCTION:-
Hairy root culture , also called transformed root culture , is a type of plant tissue culture that is used to study plant metabolic processes or to produce valuable secondary metabolites ,often with plant genetic engineering.
Hairy root cultures can be useful especially for the production of root-associated metabolites because of their high-growth rate and genetic stability.
Establishment of hairy root cultures by genetic transformation of plant tissue by the pathgenic bacterium Agrobactium rhizogenes.
A naturally occurring soil bacterium Agrobacterium rhizogenes that contains root inducing plasmids(also called Ri-plasmids) can infect plant roots and cause them to produce a food source for the bacterium.

4. Two most common species of Agrobacterium are A. tumefaciens and A
Two most common species of Agrobacterium are A. tumefaciens and A. rhizogenes which cause neoplastic disease in dicotyledonous plants , referred to as crown gall and hairy root disease respectively.
A. tumefaciens causes neoplastic growth of tissue to produce crown gall tumors , while A.rhizogenes causes production of roots at the site of infection.
The virulent plasmid of A. tumefaciens is called the Ti(tumor inducing) plasmid while that of A. rhizogenes is named Ri(root inducing)plasmid.

5. GENETICALLY TRANSFORMED CULTURES :-
The Ri plasmids can be engineered to also contain T-DNA used for genetic transformation of the plant cell.
The resulting genetically transformed root cultures can produced high levels of secondary metabolites , comparable or even higher than those of intact plants.
Three genetic elements are known to be essential for the control and regulation of successful transfer of the T-DNA from Ti or Ri plasmids to plant cells.
The right T-DNA border sequence
The plasmid vir genes and
Three bacterial chromosomal vir genes

6. The transfer of DNA from Agrobacterium to the plant cell is completed in seven successive steps:
Induction of vir gene expression
Generation of a 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 plant cell nucleus
Intergration of T-strand into plant cell DNA

7. MARKERS TO CONFIRM TRANSFORMATION :-
Confirmation that a plant cell is transformed on infection with Agrobacterium rhizogenes can be obtained in several different ways using markers:
Morphological marker :
Roots transformed with A.rhizogenes have an altered phenotype and these roots with their profusion of laterals and lack of geotropism grow readily in culture.
Also , the transformed regenerants of hairy roots inherit an aberrant phenotype in having wrinkled leaves , shortened inter node and several other morphologically distinct features compared to their normal counterparts.

8. Thus , Ri T-DNA of A. rhizogenes can be said to provide two kinds of markers that function in root organ culture.
The selectable , rapid growth
The morphological , increased branching and plagiotropism
2) Biochemical marker :
Since the opine synthesis A.rhizogenes infected plants is encoded by T-DNA of pRi plasmid.
Opines serve as an effective biochemical marker in elucidating transformed nature of the cultured root tissue.

9. 3) Genetic marker :
T-DNA localized in plant genome serves as a reliable genetic marker to confirm transformation.
There are number of techniques available to demonstrate and locate T-DNA incorporation in the host plant chromosomal DNA.
This include localization of T-DNA by southern Hybridization.
For confirmation of genetic transformation induced by A. rhizogenes , T-DNA localization in the transformed plant tissue genome appear to be the most reliable method.

10. Genetic studies have attributed the cause of such morphological differences to the randomness of the processing and integration of Ri plasmid T-DNA into plant genomic DNA during the infection process.
Although rapid proliferation of hairy roots with profuse branching and growth on medium without phytohormone are the readily observed characteristics of hairy roots induced in variety of plant species.
Agrobacterium rhizogenes can be a useful alternative , as genetically transformed roots , called hairy roots ,induced by Ri plasmid , compared to tumors induced by Ti plasmid , readily regenerate into whole plants , which transmit the Ri T-DNA to their progeny with differential utilization of T-DNA end borders.

11. ESTABLISHMENT AND GROWTH CHARACTERISTICS OF HAIRY ROOTS
A feature of hairy root system of paramount importance for their commercial exploitation is their stable , high-level production of secondary metabolites.
When establishing hairy root cultures , the isolation of plant showing desirable biosynthetic characteristics should be an important first step in obtaining root cultures that are suitable for commeracial exploitation.

12. TRANSFORMATION PROTOCOLS :
In 1987 , Hamill et al. and Rhodes et al,described to protocols for successful establishment of axenic hairy root cultures.
The technique for a routine establishment of axenic hairy root cultures in a laboratory involves three major steps ,
Root induction
Establishment of roots on solid agar medium for decontamination
Establishment of liquid root culture

13. Transformation protocol I : (In 1985, Ooms et al.)
Different transformation protocols are available some of the widely used are as follows :
Transformation protocol I : (In 1985, Ooms et al.)
Aseptic seedling are inoculated by using a sterile scarpel which has been touched with an overnight culture of A. rhizogenes.
This method works best with actively growing organs like the hypocotyl.

14. Transformation protocol II :(1988 ,Rech et al.)
Aseptic parts of the plant like stem , leaf , hypocotyl segment and cotyledons.
The organs are punctured with hypodermic needle attached to a syringe containing an overnight culture of A. rhizogenes.

15. Transformation protocol III : ( In 1988 , Horsh et al.)
Leaf disc from presterilised leaf explants are infected with A.rhizigenes in the following manner :
Inoculation and preincubation.
The leaf disc are immersed in an overnight bacterial suspension.
After shaking for 4-5 minutes leaf discs are removed and directly placed on sterilized filter paper to remove excess bacterial suspension.
The inoculated leaf disc are placed upside down on 1% agar plate or sterilized moist bated paper and t incubated 24 ̊C under white fluorescent light.

16. Post incubation :- After 3 days of preincubation , the inoculated leaf discs are transferred on to MS modified solid medium containing ampicillin at a concentration of 250 mg/liter.

17. Transformation protocol IV : (In 1987, Jung and Tepfer)
Stems from mature plants grown in green house are surface sterilized with A. rhizogenes and introduced into defined media.

18. Growth characteristics of the established root clones :
Doubling time :
In transformed root cultures , an exponential increase in biomass occurs as a result of lateral root formation and a consequent exponential increase in the number of elongating tips.
In 1988 , Flores et al. have reported an average doubling time for different hairy root cultures of family Asteraceae and shown it to be rarely more than 24 hours , explaining higher growth rate associated with hairy roots tissues in culture.
An average doubling time for cultured hairy roots of Hyoscyamus species is reported to be between hours.

19. plagiotropism of roots in culture :
Plagiotropsim of hairy roots is a phenomenon not uncommon with hairy roots and is infect reported as one of the readily observed characteristic during A. rhizogenes mediated transformation.
This characteristic of hairy roots , in particular , is considered advantageous as it increases the aeration in liquid medium since roots grow in air resulting in an elevated accumulation of the biomass.

20. Product release into the growth medium:
Release of metabolites into the growth medium by cultured hairy root tissue though may not be a phenomenon of wide occurrence , reports to this effect are available.
Reports are available on product release from normal and hairy roots in culture , with reference to the metabolites of interest like alkaloids , terpenes , anthraquinones , vitamins , nucleotides , sugars , enzymes , amino acids and auxins.

21. Auxin sensitivity in hairy roots :
The symptoms of hairy root disease like abundant root formation at the site of infection and high incidence of lateral root formation , are suggestive of auxin effect , culture s of hairy roots are sensitive to exogenously added growth hormones.
Shen et al. have shown transformed roots of Lotus corniculatus to be 100 to 1000 fold more sensitive to exogenous auxin than untrasformed roots.
In 1987 , Flores et al. have demostrated that transformed root cultures which are cycle back and forth between growth in auxin and auxin deficient medium, callus versus root is induced.
Mode of auxin addition and the culture age are the two most important factors in determining the response of given root tissue to exogenously added auxin.

22. SECONDARY METABOLITE PRODUCTION BY HAIRY ROOT CULTURES :-
IN 1941 ,Dawson first reported that roots may contribute significantly to secondary metabolism of the whole plant.
In classical experiment involving reciprocal grafts between tobacco and tomato, he presented evidence that the roots are the major , if not the only , site of synthesis of the alkaloid nicotine.
A tomato grafted onto a tobacco rootstock accumulated nicotine in the leaves .

23. Reciprocal grafting experiments later suggested that the tropane alkaloids found in several solanaceae species are also synthesized in the roots.
Isolated tobacco root cultures showed conclusively that roots have the make nicotine.
It observed that the increase in nicotine levels in tobacco root cultures closely paralleled the increase in root tissue as measured by root length , number of branches and dry weight accumulation.
Root cultures of Hyoscyamus niger have also been shown to synthesize a secondary metabolite hyoscyamine.

24. Hairy cultures have the same metabolic features as normal root cultures , yet are as rapid growing as suspension cultures.
These roots grow much faster than normal roots.
Hairy roots can be cultivated in a simple defined medium without addition of growth hormones and can grow from low inocula to high final biomass densities with only minimal lag phase.

25. PRODUCT ENHANCEMENT : Media parameter :-
The biosynthesis of secondary metabolites in transformed roots is genetically controlled ,is strongly influenced by nutritional and environmental factors.
Composition of the culture medium affects both the growth and production of metabolite.
The variables examined include the effects of different basal media an growth and production , sucrose level , sucrose/nitrate ratios , ammonium/nitrate ratio , exogenous growth hormones like auxins and gibberellic acid etc.

26. Some of the physical factors examined for their influence on growth and regulation of secondary metabolite production in transformed roots include , light , temperature , presence of chemicals inducing physical stress and applied magnetic field.
In plant tissue culture techniques sucrose is the principal carbon source , although the use of alternative carbon sources have also been investigated from time to time for both cell suspension and hairy root cultures.
The effect of various concentrations of sucrose on biomass and thiophene production respectively by hairy root cultures of Tagetes patula.

27. The plant cells show ability to use a wide range of nitrogen sources including urea , glutamate , caesin hydrolysate and amino acid mixtures, but yet, nitrates and ammonia are the two most preferred sources of nitrogen in the growth medium.
The relative amounts of nitrate and ammonia in the growth medium too affect both growth and secondary product formation.
In hairy root cultures of Lithspermum erythrorhizon , shikonin production is inhibited when ratio of nitrate to ammonia is low in the growth medium.

28. 1n 1994, Kino-oka et al. has communicated that in case of hairy root cultures of Rubia tinctorius , presence of either only ammonia nitrogen or nitrate nitrogen as a nitrogen source in the growth medium results in poor growth of the cultured root tissue.
Light as a physical factor of growth has been found to have an important role in controlling growth and stimulating secondary product formation in plant tissue cultures.
For root culture , several morphological changes such as change in biomass yield as a direct effect of light , greening of root clones on exposure to the light or an increased ability for shoot production on phytohormone free medium in response to light have been reported.

29. Elicitation :
One of the most successful strategies used to increase the productivity of plant cell cultures has been to induce the de novo biosynthesis of secondary metabolites by exposing the cells to various elicitors.
The term elicitor is used to describe compounds ranging from heavy metals to fungal cell wall fragments.
The strategy has been successfully employed and used in hairy root cultures leading to enhancement in secondary metabolite production by the culture.

30. In 1992 , Flores and Curtis have shown elicitation of the accumulation of specific metabolites in hairy root cultures during their study with hairy root cultures of Solanaceae and Asteraceae.
In 1989 , Signs and Flores have shown that if hairy root cultures of Solanaceae were treated with cell wall fragments from Rhizoctonia solani ,there is burst in the production of sesquiterpene phytoalexin within 24h.

31. Biotransformation :
In addition to producing constitutive and inducible metabolites , hairy roots have been recently shown to have the ability to biotransformation xenobiotics.
In 1992 , Flores and Curtis have shown that root cultures are capable of converting xenobiotics to bioactive compounds.
This may have interesting implications for the root-microbe interactions that take place in the rhizosphere.
It may also be possible to use this biotransformation aproach to screen for production of novel compounds with agrichemical and pharmaceutical potential.

32. Production of Macromolecules :
In addition to producing a diversity of low molecular compounds ,plant roots also specifically synthesize and accumulate storage proteins and defense related proteins.
Examples of defense related proteins produced in roots include proteinase inhibitors.
In 1994 ,Savary and Flores have described production of several extracellular and intracellular proteins in hairy root cultures of Trichosanthes kirilowii var japonica , demonstrating that class III chitinases are major root proteins and that TCN biosynthesis is associated developmentally with root secondary growth.

33. Hairy roots of horse radish have been studies as a production system for production of an important enzyme like peroxidase which has wide applications in clinical diagnosis and because of its wide range of substrate it finds newer applications like removal of phenolics from waste water.

34. GROWTH OF HAIRY ROOT CULTURES IN VARIOUS BIOREACTOR FOR PRODUCTION OF SECONDARY METABOLITE:-
Agrobacterium rhizogenes- mediated hairy-root cultures provide a promising approach to the biotechnological exploitation of plant cell cultures for fine chemical production.
Hairy root cultures are characterized by stable metabolite production over successive generations, inherent genetic stability reflected in stable productivity and the possibility of genetic manipulation to increase biosynthetic capacity.

35. These characteristics of hairy root cultures have initiated considerable interest both as fundamental research tool and as a source of valuable products.
One of the important limitations for commercial exploitation of “hairy root” cultures is the development of technologies for large-scale culture.
Though these roots have been grown in various bioreactors- stirred tank , air-lift column , trickling film , mist , bubble column.

36. The growth and productivity of hairy root cultures are reviewed with an emphasis on successful bioreactors and important culture considerations.
Bioreactors used to culture hairy toots can be roughly divided into three types: liquid-phase , gas-phase , or hybrid reactors that are a combination of both.
The growth phase , media composition , the gas regime , use of elicitors , the role of light , are together with genetic engineering and process optimization , proper reactor design plays a key role in the development of successful large scale production of secondary metabolites from plat cultures.

37. On a laboratory scale ,transformed root cultures grow in a liquid medium containing sucrose and mineral salt , including nitrogen source in 150 ml Erlenmeyer flasks on a rotary shaker at 100rpm and incubated at 25+2 ̊C temperature.
Bioreactors have two advantages over flasks.
That better control can be exerted on the system
That since most bioreactors are scaleable , they are better able to reproduce on a large scale those conditions which were observed on a smaller scale to be most desirable for culture performance.

38. That bioreactors design and applications.
Stirred tank :-
This bioreactor is a classical representative of bioreactor types where air is dispersed by mechanical agitation.
Various types of stirrers , e.g spin , helix , bladed , paddle , etc.
Temperature ,pH , amount of dissolved oxygen and nutrient concentration can be controlled better in this type of reactor.

39. Various cell cultures and hairy root cultures have been cultivated successfully in this type of reactor.
It is useful in high density of culture.
The stirred tank is the simplest configuration for scale up since it is a proven technology for the ‘fermentation’ industry.
Growth of Beta vulgaris in a stirred reactor and found that growth rate was much poorer than controls in Erlenmeyer flasks.

40. 2) Air-lift column :
It is gas liquid bioreactor where compressed air is used for aeration and agitation is based on the draught tube principle.
Air lift reactors upto 100 liter capacity have been used extensively for the cultivation of cathranthus roseus.
Pneumatically agitated fermenters are more suitable for the production of secondary metabolites than stirred tank reactor.
Oxygen transfer at low shear , less contamination due to non-movable parts , low operation cost because of simple design are a few merits of air-lift type reactors.

41. 3) Trickling film bioreactor:-
In this type of bioreactor the medium trickles over glass beads.
The media is recirculated from a reservoir and sprayed over the fixed bed of roots and permitted to flow down through the root bed.
Roots are inoculated on top of the glass beads.
These grow over the surface of bed and down between beads in the film of circulating medium.

42. 4) Mist bioreactor :-
In this type of reactor medium is pumped through mist head using peristaltic pump fitted with timer
Glass fiber is inserted at the base of the bioreactor vessel to filter out cells sloughed from the root cap and any other debris which could clog the jet the mist head.
A fine stainless steel mesh filter is also incorporated into the mist head itself to further guard against blocking of jet by cell debris.

43. This type of bioreactors have the advantage that the medium can be drained off and the wet weight can be determined directly.

44. 5) Bubble column :- Bubble columns are simple to construct and operate.
They consist of vessel usually cylindrical in which gas is sparged into liquid.
In bubble column reactors , the energy needed for agitation , as well as oxygen required for the culture is provided by the sparged air.
The most important advantage of bubble columns , results from their simplicity.

45. Many commercially important bioprocesses involve shear sensitive cultures.
Hairy root cultures are also shear sensitive , have been grown successfully in bubble column reactors.
Experimental observations showed that the growth of plant cell cultures in bubbles column reactors ,stirred tank reactors and shake flasks was similar.
1n 1987,Tanaka reported that bubble column reactors gave better results than stirred tank reactors and aeration at high energy input rate is much simpler than stirring.

46. Therefore , the use of bubble column reactors for shear sensitive cultures may be practical alternative to other bioreactor configurations.
Oxygen transfer at low shear , low operation cost and low hydrodynamic stress makes bubble column reactor an attractive choice for scale up studies.
Hairy root cultures present a tremendous potential for commercial production of secondary metabolites.
As a result , the scale up of commercial production must be quite large and systematic approaches must be developed.

47. References : Hairy root culture and secondary metabolites production
Usha Mukundan
Himanshu G. Dawda
Seema Ratnaparkhi

48. THANK YOU