Definition of hairy root culture ,multiple shoot culture ,Production of hairy root and multiple shoot , advantages an disadvantages of hairy root and multiple shoot culture, Sterilization and sterilizing agents wit concentration and exposure time

1. Hairy Root culture, Multiple shoot
culture , Micropropagation,
Sterilization Methods
Presented by : Shikha Bhardwaj
M.Pharma 2nd sem
Pharmacognosy

2. HAIRY ROOT CULTURE
DEFINITION: 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 or recombinant proteins,
often with plant genetic engineering.
• It is the culture produced after the infection of the
explant or culture by Agrobacterium rhizogenes is a
Gram negative soil-borne bacterium of the family
Rhizobiaceae

3. CONTD…
• A naturally occurring soil bacterium Rhizobium
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, opines, and
to grow abnormally.[2]
• The abnormal roots are particularly easy to culture in
artificial media because hormones are not needed in
contrast to adventitious roots and they are neoplastic,
with indefinite growth.

4. Explant(leave, stem,
or roots
Agrobacterium
tumefaciens
Infection with
Agrobacterium
rhizogenes
In vitro
organogenesis
Callus formation
Shoot teratoma lines Hairy root lines Untransformed shoot & root
Genetic
stability
Screening on
productivity
Production in bioreactor

5. PROPERTIES OF HAIRY ROOT
• Genotype and phenotype stability
• Autotrophy in plant hormones
• Fast growth
• High levels of secondary metabolite production
• High degree of lateral
• Do not require medium condition

6. Induction of hairy root cultures in
vitro
• Explant are wounded and then inoculated with A.
rhizogenes
• After three days, the explant can be transferred into
solid media with antibiotic such as cefatoxime or
ampicillin to kill or eliminate unwanted bacteria
• The hairy roots will be induced within a short period of
time which varies from one week to over a month
depending on different plant species

8. Production of hairy root in vivo
• Agrobacterium recognizes some signal molecules exuded
by wounded plant cells and becomes attached to it
• The bacteria contains the root inducing plasmid
• The bacteria genetically transfer part of the Ri-plasmid
called the transfer DNA to the plant genome, where it
gets expressed and make the plant cell to:
1.Proliferate by increasing the rate of cell division (cytokine
expression) and cell elongation (auxin expression) to produce
hairy root
2. Produce the opines which is a type unusual amino acids
(opctopine, agropine, nopaline, mannopine) which is
used by the bacterium as a carbon, nitrogen and energy
source

11. BIOREACTOR FOR HAIRY ROOT CULTURE
• Stirred tank bioreactor
• Air lift bioreactor
• Bubble column bioreactor
• Turbine blade bioreactor
• Mist trickle bed bioreactor
• Rotating drum bioreactor
• Spin filter bioreactor

12. Bubble column with hairy root of
B.vulgaris

13. Air lift bioreactor with hairy root

14. Applications of HRC
• Production of secondary metabolites
• Culture may produce the compounds which are
not found in untransformed roots
• Culture may change the composition of the
metabolites
• Culture can be used to regenerate a whole plant

15. MICROPROPAGATION

16. MICROPROPAGATION
Micropropagation is the production of whole plant
from small section of plant such as stem tip, node,
meristem, embryo or even a seed
It is a advanced vegetative propagation technology
for producing large number genetically superior
and pathogen free transplants in a limited time and
space
Multiplication of genetically identical copies of a
cultivar by asexual reproduction is called clonal
propagation.
 Use of tissue culture for microprogation was
initiated by G.Morel (1960) in orchid.

18. STEPS INVOLVED IN
MICROPROPAGATION
 Micropropagation procedure is divided in stages
 Murashige proposed three (I to III) stages, Debergh and
Maene added stage ‘0’. Currently we have accepted five
stages procedure (0 to IV)
 Stage 0- Selection and maintenance of stock plants for
culture initiation
 Stage I- Initiation and establishment of aseptic culture
 Stage II- Multiplication of shoots or somatic embryo
formation (rapid) using a defined culture medium
 Stage III- Rooting of regenerated shoots or germination of
somatic embryos in vitro
 Stage IV- Hardening

19. Selection and maintenance of stock plants for culture
initiation
• This stage was basically introduced to overcome the problem
of contamination. Stock plants are grown under more hygienic
conditions to reduce the risk of contamination.
Initiation and establishment of aseptic culture
• Explant isolation –any part of the plant can be used as explant like
vegetative parts (Shoot tip, meristem, leaves, stems, roots) or
reproductive parts (Anthers, pollen, ovules, embryo, seed, spores).
Shoot tip and auxiliary buds are most often used
• Surface sterilization – Explants are surface sterilized by treating it with
disinfectant solution for a specific period. Ethyl alcohol, bromine
water, mercuric chloride, silver nitrate, sodium hypochlorite, calcium
hypochlorite etc can be used as disinfectant.

20. • Washing – Washed with water.
• Establishment of explant on appropriate medium – There is no one
universal culture medium; however modifications of Murashige and
Skoog basal medium (Murashige and Skoog, 1962) are most frequently
used.
 Multiplication of shoots or somatic embryo formation (rapid) using a
defined culture medium.
• In this stage, rapid multiplication of the regenerative system is carried
out for obtaining large number of shoots
 Rooting of regenerated shoots or germination of somatic embryos in
vitro
– In this stage, shoots or shoot clusters from stage II are prepared to
transfer to soil. Shoots are separated manually from clusters and
transferred on a rooting medium containing an auxin.
 Hardening- Transfer of plantlets to sterilized soil for hardening under
greenhouse environment.

22. METHODS OF MICROPROPAGATION
 AXILLARY BUD PROLIFERATION APPROACH
 Meristem and shoot tip culture- This method is more successful
in herbaceous plant
Cymbidium orchid was the first plant to be propagated through
this technique by G. Morel
 Bud culture
 ORGANOGENESIS
 EMBRYOGENESIS- development of embryos or
embryo like structure through somatic cells,
involves callus intermediate stage

23. High rate of Plant Propagation- large number of plants
can be grown from a piece of plant tissue.
Micropropagation can be carried out throughout the
year.
Production of disease free plants- Meristem tip cultures
are generally employed to develop pathogen free
plants.
Cost effective process- Process requires minimum
growing space. Millions of species can be maintained
inside culture vials in a small room in nursery.
Propagation of seeds in some crops- Through axillary
bud profile for production of seeds in some plant (e.g.
cauliflower, onion).
APPLICATIONS OF
MICROPROPAGATION

24. It is the only viable method for regenerating
genetically modified cells after protoplast fusion.
Micropropagation produces more robust plant,
leading to accelerated growth compared to similar
plants produced by conventional methods. Such as
Narcissus and other bulbous crops.
In-vitro cultures can be stored for longer time
through cryptopreservation.
Breeding cycle can be shortened.

25.  Micropropagation of sugarcane (Saccharum spp.) was
studied using two procedures: (1) shoot tip culture; (2)
indirect somatic embryogenesis from callus.
 Shoot tip culture was considered a better method for
micropropagation, since it produced plants phenotypically
similar to the mother plant and gave a much more rapid
multiplication rate when compared to the other procedure.
MICROPROPAGATION BY SHOOT TIP CULTURE

26. DISADVANTAGES
 CONTAMINATION OF CULTURE- During course of micro
propagation, several slow growing microorganisms (e.g. Eswinia
sp, Bacillus sp) contaminate and grow in cultures. The microbial
infection can be controlled by addition of antibiotics and
fungicides.
 BREWING OF MEDIUM- Micro propagation of certain plants (e.g.
woody perennials) is often associated with accumulation of
growth inhibitory substance in the medium. Chemically these
substances are phenolic compounds are toxic and can inhibit the
growth of tissues. Brewing of the medium can be prevented by
the addition of ascorbic acid or citric acid or polyvinyl
pyrrolidone to the medium.

27.  GENETIC VARIABILITY- when micro propagation is carried
out through shoot tip cultures, genetic variability is very
low. However, the use of adventitious shoots is often
associated with pronounced genetic variability.
 VITRIFICATION- During the course of repeated in-vitro
shoot multiplication, the cultures exhibit water soaked or
almost translucent leaves. Such shoots cannot grow and
even may die. This phenomenon is referred to as
vitrification. This can be prevented by increasing the agar
concentration (from0.6 to 1%) in the medium. However
increased agar concentration reduces the growth rate of
tissues.
 Expensive equipment, sophisticated facilities and trained
manpower are needed. This limits its use.

28. • How does micro propagation work?
 Plant cell have the ability to reproduce the whole plant
from single cell. This is called TOTIPOTENCY.
 Totipotency is the ability of a single cell to express the
full genome in the cell to which it gives rise by cell
division.
• WHY DO WE DO IT?
 To regenerate plant from single cell or tissue
 To produce large quantities of identical plant
 To create new plant varieties

29. Shoot Tip Culture

32. Shoot tip culture for banana micropropagation: a. sword sucker and explant; b. shooting
after apical disabling; c. proliferation; d. multiple shooting; e. rooting; f. nursery
hardening

35. Stages Of Shoot Culture
Murashige reported that there are three stages of
culture:
Stage 1: is the culture establishment stage when explant
may develop into single shoot or multiple shoots. At this
stage explant are supplements with cytokinin like BA,
Kinetin and 2IP
Stage 2: in the objective is to multiple the propagules
and for this axillary shoot proliferation is followed as it
maintains higher genetic stability.
Stage 3:purpose is regeneration of adventitious roots
from the shoot obtain stage 2 numerous studies have
indicated that NAA is followed by IBA, IAA, 2,4-D and
other auxins are used for induction of root generation.

38. Sterilization
Sterilization refers to any process that eliminates,
removes, kills, or deactivate all form of life and
other biological agents(such as fungi, bacteria,
viruses, spore form etc.
Sterilization procedures may be enhanced by:
 With 70% ethyl alcohol
 Using wetting agent such as TWEEN 20 or 80 to
reduce surface tension and allow better surface
contact
 Conducting sterilization under vacuum.

39. Commonly used Sterilizing Agent For
Plant Tissue Culture
STERILIZING AGENT CONCENTRATION(%) EXPOSURE(min)
Calcium hypochlorite 9-10 5-30
Sodium hypochlorite 0.5-5 5-30
Hydrogen peroxide 3-12 5-15
Ethyl alcohol 70-95 0.1-5
Silver nitrate 1 5-30
Benzalkonium chloride 0.01-0.1 5-20

40. REFERENCE
 http://theagricos.com/tissue-culture/micropropagation/stages-of-
micropropagation/
 http://www.biologydiscussion.com/plant-breeding/micro-
propagation/micro-propagation-methods-and-stages-
biotechnology/61317
 https://www.sciencedirect.com/topics/agricultural-and-biological-
sciences/micropropagationhttps://www.slideshare.net/DrSureshSolleti/ha
iry-root-culture-78587463
 https://www.slideshare.net/anjaliparab_87/hairy-root-cell-culture
 https://en.wikipedia.org/wiki/Hairy_root_culture
 Article in advancing experimental medicine and biology 2010 (author
laura pistelli)
 www.sigmaaldrich.com/explant sterilization