Secondary metabolite production through hairy root culture, obtained via Agrobacterium rhizogenes mediated gene transfer techniques, has drawn much attention in last few decades. Hairy root system is a convenient and viable approach to get the target metabolites.

1. HAIRY ROOT CULTURE
Submitted to,
Dr. Elsam Joseph
Associate
Professor
Dept. of Botany
St. Teresa’s college
Submitted by,
Silpa Selvaraj
Roll no: 13
Dept. of Botany
St. Teresa’s
college

2.  As medicinal plants are the exclusive sources of these beneficial bioactive
compounds, they are disappearing at a high speed.
 More than 95% of the medicinal plants are being collected from the wild to meet
the requirement of secondary metabolites.
 Leaves, bark, roots, fruits, seeds or even the whole plant are indiscriminately
being collected from the wild habitats, without taking care of the mother plants.
 Due to this over-exploitation and habitat destruction, many of these important
useful species are on the verge of extinction.
 The content of secondary metabolites in wild plants is highly dependent on
seasonal and environmental factors and not sufficient to meet the industrial
demand.
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INTRODUCTIO
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3.  So, there is a clear need to encourage multiplication of these plants in an
alternative way.
 For these reasons, different in vitro systems like plant cell and organ
cultures, immobilized plant cultures, transformed cultures, bioreactor cultures
are now being utilized for the synthesis of these compounds.
 Secondary metabolite production through hairy root culture, obtained via
Agrobacterium rhizogenes mediated gene transfer techniques, has drawn
much attention in last few decades.
 Hairy root system is a convenient and viable approach to get the target
metabolites.
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4.  A.rhizogenes contain root inducing plasmid
called Ri plasmid.
 The Ri plasmid integrates its T-DNA(25 bp long)
region into the plant genome.
 As a result rapidly growing and intensely branched
(hairy) adventitious roots are developed.
 When roots induced by the bacteria in aseptic
conditions are maintained in vitro on a nutrient
media, the transformed root tissue can grow for
years.
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5. Agrobacterium rhizogenes
 Also called as Rhizobium rhizogenes.
 Gram negative, aerobic soil bacterium.
 Family – Rhizobiaceae.
 Naturally causes hairy root disease in higher plants.
 R. rhizogenes induces the formation of proliferative multi
branched adventitious roots at the site of infection,
called hairy roots.
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6. HAIRY ROOT DISEASE
 Hairy root and the crown gall tumor are two plant diseases caused by
two Gram-negative soil bacteria, Agrobacterium rhizogenes and A.
tumefaciens, respectively.
 The transferred T-DNA derived from the Ti-plasmid causes the plant cells
to proliferate, to form crown gall tumors, and
 In the case of the Ri-plasmid, the extensive formation of adventitious
roots at or near the site of infection.
 The transformed plant tissues are also directed by T-DNA genes to
produce unusual metabolites called opines, that serve as specific nutrients
for the bacteria.
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7.  The physiological basis of the hairy root disease is not totally understood.
 Alteration of auxin metabolism in transformed cells has been supposed to play
an important role in expression of the hairy root phenotype.
 TL-DNA plays the major role in hairy root induction, and the genes encoding
auxin synthesis have a somewhat accessory role.
 Spanó and co-workers have suggested that the genes responsible for increased
sensitivity of hairy root cells to auxin are located on the TL-DNA.
 The hairy roots of Hyoscyamus muticus L. have been demonstrated to tolerate
high auxin levels.
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8. CHARACTERIZATION OF AGROBACTERIUM
PLASMID
 The plasmids are large (200 to greater than 800 kb).
 The Ri-plasmids are grouped into two main classes according to the opines
synthesised by hairy roots.
 Agropine-type strains (e. g., A4, 15834, LBA9402, 1855) induce roots to
synthesise agropine, mannopine and the related acids.
 Mannopine-type strains (e.g., 8196, TR7, TR101) induce roots to produce
mannopine and the corresponding acids.
 Other types of opines (e.g., cucumopine, mikimopine) have also been
described.
 The most studied Ri-plasmids are agropine-type strains (most virulent).
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9. GENES RESPONSIBLE FOR HAIRY ROOT
FORMATION
 The T-DNA of the agropine-type Ri-plasmid consists of two separate T-DNA
regions; TL-DNA and TR-DNA.
 TL-DNA has four 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 (OFRs), 4 of which ORFs 10, 11, 12 and 15 respectively,
correspond to the rolA, rolB, rolC, and rolD loci.
 rolA, rolB, and rolC play the most important role in hairy root induction.
 rolB is most crucial in the differentiation process of transformed cells.
 rolA and rolC provide with accessory functions.
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10.  Although the TR-DNA is not essential for hairy root formation, aux1
gene harboured in this segment provides the cells with an additional source
of auxin.
 Recently, Moyano and co-workers found that auxin genes play a significant
role in the morphology and alkaloid production of transformed roots of
Datura metel and Duboisia hybrid.
 The studies with Panax ginseng c. v. Meyer hairy roots also support this
finding.
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11. MECHANISM OF AGROBACTERIUM PLANT CELL
INTERACTION
 One of the earliest stages in the interaction between Agrobacterium and a
plant is the attachment of the bacterium to the surface of the plant cell.
 A plant cell becomes susceptible to Agrobacterium when it is wounded.
 The wounded cells release phenolic compounds, such as
acetosyringone, that activate the vir-region of the bacterial plasmid.
 The transformation is induced on aseptic, wounded plants or plant parts by
inoculating them with a thick, viable A. rhizogenes suspension.
 After 1-4 weeks, when roots emerge at the site of inoculation, they are
individually cut off and transferred into a hormone-free growth medium e.g.,
MS or Gamborg B5 containing antibiotics to kill the bacteria.
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12.  The susceptibility of plant species to Agrobacterium strains varies greatly.
 Plant species, which were shown to be insusceptible to A. rhizogenes, e. g.,
strain A4, have been successfully transformed with other strains.
 The age and differentiation status of plant tissue can also affect the chances
of successful transformation.
 The level of tissue differentiation also determines the ability to give rise to
transformed roots after A. rhizogenes inoculation.
 Successful infection of some species can be achieved by the addition of
acetosyringone .
 The genetic transformation can be confirmed by assaying the opines.
 For this reason, detection of T-DNA by Southern blot hybridization is often
necessary to confirm the genetic transformation.
 Gamborg's B5 medium is the most widely used medium for the hairy roots
of many species .
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13. • Mechanical agitation is seldom suitable for hairy roots because they are
susceptible to shear stress that causes disorganisation and callus formation,
which lowers productivity.
• Conventional stirred-tank reactors have been successfully applied to hairy roots
even though the mixing system of such bioreactors has been reported to cause
shear damage.
• It seems to be clear that standard reactors are not suitable for hairy root
cultures.
• However, the best growth characteristics were obtained with bio reactors without
mechanical stirring.
• The use of airlift reactors makes it possible to avoid shear stress completely.
BIOREACTORS IN HRC
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14. • The most promising bioreactors for the cultivation of hairy roots seem to be
so-called wave reactors.
• This reactor system has three components: a rocker unit, the disposable
bioreactor chamber, and the measuring and control units.
• This reactor has been demonstrated to increase the growth of hairy root
cultures producing tropane alkaloids and ginsenosides significantly more
than optimised stirred reactors, rotating drum reactors and droplet phase
reactors.
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15. PROPERTIES OF HAIRY ROOTS
• Unlimited Growth Potential: Hairy roots exhibit rapid and continuous
growth, providing a sustainable and scalable source of biomass for various
applications.
• Genetic Stability: Hairy roots tend to maintain genetic stability over
multiple sub-culturing cycles, ensuring consistent production of desired
compounds.
• High Metabolic Activity: Hairy root cultures often display enhanced
metabolic activity, leading to increased production of secondary metabolites
such as alkaloids, flavonoids, and terpenoids.
• Autotrophic Nature: Hairy roots can be grown in vitro without the need for
exogenous hormones, making them relatively independent in terms of
growth regulators.
• Root Hair-Like Morphology: Hairy roots resemble natural root hairs,
providing a unique morphology that facilitates nutrient absorption and
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16. • Ability to Synthesize Phytohormones: Hairy roots can produce
phytohormones, contributing to their own growth regulation and creating a
dynamic microenvironment.
• Adaptability to Suspension Culture: Hairy roots can be adapted to
suspension culture systems, enabling efficient nutrient uptake and metabolite
production in bioreactors.
• Expression of Foreign Genes: Genetic modification of hairy roots allows the
expression of foreign genes, making them a versatile platform for producing
specific proteins or bioactive compounds.
• Inducible Response to Elicitors: Hairy roots can respond to elicitors
(substances that induce a specific response) by triggering the synthesis of
secondary metabolites, enhancing their utility in pharmaceutical and
biotechnological applications.
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17. Why hairy root cultures
do not require
exogenous hormone
supply??
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18. • The Ri plasmid carries genes responsible for synthesizing plant hormones,
such as auxins and cytokinins, which are essential for root development.
• Auxin Production: The Ri plasmid in hairy roots carries genes for auxin
biosynthesis. This leads to the continuous production of auxins within the hairy
root tissue, promoting uncontrolled and vigorous growth.
• Cytokinin Synthesis: Similarly, the Ri plasmid also carries genes involved in
cytokinin synthesis. This results in the production of cytokinins, which balance
the auxin effects and contribute to the overall autonomous growth of the hairy
roots.
• Autoregulation:The continuous production and presence of auxins and
cytokinins within the culture sustain the growth and development of the hairy
roots without the need for additional hormone supplementation.
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19. HAIRY ROOTS V/S NORMAL ROOTS
1. Induction Method:
a)Hairy Roots: Initiated through infection with Agrobacterium rhizogenes,
which transfers the Ri plasmid and induces uncontrolled growth of transformed
roots.
b)Normal Roots: Developed through traditional methods such as seed
germination, root cutting, or tissue culture without the involvement of
Agrobacterium.
2. Growth Characteristics:
a)Hairy Roots: Exhibit rapid and continuous growth, often surpassing the
growth rates of normal roots.
b)Normal Roots: Typically grow at a slower rate compared to hairy roots.
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20. 3) Morphology:
a)Hairy Roots: Possess a dense covering of fine root hairs, creating a unique
morphology that enhances nutrient absorption.
b)Normal Roots: Have a more typical root structure without the dense covering
of fine root hairs.
4) Genetic Stability:
a)Hairy Roots: Tend to maintain genetic stability over prolonged sub-culturing,
ensuring consistent production of desired compounds.
b)Normal Roots: May undergo genetic changes over time, leading to variability
in characteristics.
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21. 5) Secondary Metabolite Production:
a)Hairy Roots: Known for enhanced production of secondary metabolites, making
them valuable for the synthesis of bioactive compounds.
b)Normal Roots: Produce secondary metabolites at a natural rate without the
pronounced increase seen in hairy roots.
6) Autotrophic Nature:
a)Hairy Roots: Can be maintained in vitro without the need for exogenous
hormones once established, exhibiting a degree of autonomy in growth regulation.
b)Normal Roots: Often require the presence of external hormones for proper
growth and development in culture.
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22. 7) Research Applications:
a)Hairy Roots: Commonly used in molecular biology and genetic engineering
studies due to their amenability to genetic modifications.
b)Normal Roots: Utilized in basic plant biology and physiology research.
8) Phytoremediation Potential:
a)Hairy Roots: Engineered hairy roots can be used in phytoremediation efforts,
expressing enzymes to detoxify environmental pollutants.
b)Normal Roots: May contribute to phytoremediation but are not typically
engineered for specific pollutant degradation.
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23. ADVANTAGES
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24. • Availability Throughout the Year: Unlike intact plants that may have specific
growth seasons, hairy root cultures can be maintained and utilized throughout
the year.
• Ease of Manipulation: Genetic engineering and manipulation are relatively
easier in hairy root cultures, enabling the development of customized plant lines
for specific purposes.
• Conservation of Endangered Species: Hairy root culture can contribute to the
conservation of endangered plant species by providing an alternative method
for obtaining valuable compounds without harvesting plants from their natural
habitats.
• Potential for Large-Scale Production: Hairy root cultures can be scaled up for
large-scale production, making them suitable for industrial applications.
These advantages make hairy root culture a valuable tool in plant biotechnology
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25. • Dependency on Agrobacterium: Hairy root culture initiation relies on the
transformation by Agrobacterium rhizogenes, which may introduce genetic
changes or alter the physiology of the plant cells.
• Risk of Contamination: Maintaining sterile conditions is crucial, as
contamination with other microorganisms can affect the culture and lead to
undesirable outcomes.
• Potential Genetic Variation: Despite efforts to maintain genetic stability,
some hairy root cultures may undergo genetic changes over prolonged sub-
culturing, affecting the consistency of metabolite production.
• Complex Medium Requirements: Hairy roots often require complex nutrient
media, making the cultivation process more intricate and expensive compared
to other cell culture systems.
LIMITATIONS
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26. • Difficulty in Scale-Up: Scaling up hairy root cultures for large scale production
can be challenging due to factors like mass transfer limitations, oxygen supply,
and the need for specialized bioreactor systems.
• Limited Commercial Success: Despite promising features, the commercial
success of products derived from hairy root cultures is not as widespread as
anticipated, partly due to economic considerations and competition from
alternative production methods.
• Ethical and Regulatory Concerns: Genetically modified organisms, including
those generated through hairy root culture, may face regulatory challenges and
ethical considerations, impacting their widespread adoption.
• Lack of Standardization: Standardizing hairy root culture protocols for different
plant species can be challenging, leading to variations in outcomes across
different laboratories.
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27. APPLICATIONS
1.Secondary Metabolite Production:
Pharmaceuticals: Hairy root cultures are employed for the production of medicinal
compounds, including alkaloids and anti-cancer agents, providing a controlled and
sustainable source for drug development.
Flavours and Fragrances: Hairy root cultures produce aromatic compounds used
in the food and fragrance industries, offering a consistent supply of natural
flavours.
2.Phytoremediation: Hairy root cultures can be engineered to express genes
involved in pollutant detoxification, making them useful for phytoremediation, a
process that utilizes plants to remove or degrade environmental contaminants.
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28. 3. Molecular Biology and Genetic Engineering:
Hairy root cultures serve as a model system for studying gene expression and
regulation in plants, facilitating research in plant molecular biology.
Genetic engineering allows the modification of hairy roots to enhance traits, such
as increased metabolite production or stress resistance.
4. Biopharmaceutical Production:
Hairy root cultures can be genetically modified to produce therapeutic proteins,
vaccines, and antibodies, offering an alternative to traditional biopharmaceutical
production methods.
5. Vaccine Production:
Modified hairy roots expressing antigens can be used for plant-based vaccine
production, offering a cost-effective and scalable method for vaccine development
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29. 6. Research in Plant-Microbe Interactions:
Hairy root cultures aid in understanding the molecular mechanisms underlying plant-
microbe interactions, particularly the interactions with Agrobacterium rhizogenes.
7. Conservation of Endangered Species:
Hairy root cultures provide a non-destructive way to obtain bioactive compounds
from endangered plants, contributing to the conservation of rare and threatened
species.
8. Bioactive Compound Screening:
Hairy root cultures are utilized in high-throughput screening for bioactive
compounds, identifying potential candidates for pharmaceutical, agricultural, or
industrial applications.
9. Educational and Training Purposes:
Hairy root cultures are valuable tools in educational settings, allowing students to
learn and practice plant tissue culture techniques, genetic engineering, and
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30. REFERENCES
1. Hussain, M. J., Abbas, Y., Nazli, N., Fatima, S., Drouet, S., Hano, C., &
Abbasi, B. H. (2022). Root Cultures, a Boon for the Production of Valuable
Compounds: A Comparative Review. Plants (Basel, Switzerland), 11(3), 439.
https://doi.org/10.3390/plants11030439
2. Morey, K. J., & Peebles, C. A. M. (2022). Hairy roots: An untapped potential
for production of plant products. Frontiers in plant science, 13, 937095.
https://doi.org/10.3389/fpls.2022.93
3. Sandra Irene Pitta-Alvarez, & Giulietti, A. M. (1995). Advantages and
Limitations in the Use of Hairy Root Cultures for the Production of Tropane
Alkaloids: Use of Anti-Auxins in the Maintenance of Normal Root Morphology.
In Vitro Cellular & Developmental Biology. Plant, 31(4), 215–220.
http://www.jstor.org/stable/4293096
4. Thi Thuy Tien, L. (2021). Root Cultures for Secondary Products. IntechOpen.
doi: 10.5772/intechopen.94419
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31. THANK YOU