Role of bioreactor in

micropropagation

Soobedar
Yadav
Roll. No. 5004
Discipline
of
Horticulture
IARI, New Delhi

Bioreactor micro propagationAutomation in micropropogation is

best achieved by adopting
bioreactor propagation

BIOREACTOR

Bioreactor is a vessel, made up of glass or steel, in which plant

cells are cultivated under controlled environment is suspension to
obtain a propagules in large numbers.

Ammirato et al., 1983

Brief History..
Microbiology fermentation industry
Secondary metabolites

The first patent for the cultivation of

plant tissue was received in 1956(
NASA)
In micropropagation first reported
in 1981 for begonia propagation
(Takayama and Misawa, 1981)

SCHEMATIC DIAGRAM OF
BIOREACTOR

Why bioreactor for micro-propagation?

Better control of the culture conditions
Automated and mechanized
Low energy requirement
Avoid intensive manual handling
Reduces the cost
Reduce the time.
Less chance of contamination

Applicatio
ns
Pharmaceutical industries
Biomedical applications
Micropropogation
A. Somatic embryogenesis
B. Organogenesis
C. Bud or Meristem Clusters

Plant Cell Culture

Plant Part
(Leaf, Shoot, Root, Embryo)
Callus culture
(Solid/Semi solid media)
Suspension culture
(Liquid media)
Bioreactor

Sharp et al.,

BIOREACTOR PROCESS
STEPS
1

10
CIP Post-Use
CIP
2

Set Up

Recovery
8

Pressure Hold

Growth
7

Inoculation
SIP
6

5
Media
Equilibration
Fill
4

The set up of the bioreactors and the process steps

are performed in a specific order

CLASSIFICATION OF
BIOREACTOR

A. MECHANICALLY AGITATED
.Stirred tank bioreactor
. Rotating drum bioreactor
.Spin filter bioreactor

B.PNEUMATICALLY AGITATED AND NONAGITATED BIOREACTORS

. Simple aeration bioreactor
. Bubble column bioreactor
. Air-lift bioreactor
. Balloon type bubble bioreactor-BTBB)

C.TEMPORARY IMMERSION BIOREACTORS

STIRRED TANK BIOREACTOR

The use even flow of the medium in different directions
Proper oxygenation of the cultured tissue
MERITS
Uniform flow pattern
Low operation cost
DEMERITS
High shear force
Complicated configuration
High power required

BUBBLE-COLUMN REACTOR
Advantage
Enhance
mixing

dispersion

Low-Shear
Low power required
Disadvantage

Foaming

and

BALLOON TYPE BUBBLE

BIOREACTOR(BTBB)
By using a concentric tube on top of the vessel for reduced
the foam

TEMPORARY IMMERSION BIOREACTORS

(TIB)
The principal equipment in an TIB is the same
as that in the BTBB
Avoid the complete submersion of explants in
the liquid medium
Hyperhydricity is completely reduced

Plant
perform
acclimatization

better

during

the

Sherrington et al., 1999

TEMPORARY IMMERSION BIOREACTORS (TIB)

Impact of(TIB) culture on production

costs
Reduced costs by 46% of coffee embryogenesis to
standard procedure compare with semi-solid medium
( Lorenzo et al., 1998)
Develop protocol reduced production costs of pineapple
plant 42% when compared to the conventional method
with liquid medium (Ziv et al., 1998)
Based on that harvesting rate, this system is around 7
times cheaper than the normal method for sub culturing
Pinus radiata shoots on semi-solid medium (Smith et al.,
1996)
Pineapple shoots resulted in a 100-fold increase in the
number of shoots during culture establishment (Escalona

Research1-5 L
Product development5-10L

Industrial scale 31-1000 L

Introduction to large scale11-30 L

Culture parameters affecting the efficacy of

temporary immersion systems

Immersion time
Aeration and forced ventilation
Volume of liquid medium
Culture container volume

Commercial application in
plant propogation
Shoot cultures of Spathiphyllum cannifolium (Dewir et al., 2007)
Organ culture Stevia rebaudiana ( Sreedhar et al., 2008)

Shoot culture of Ananas comosus (Firoozabady & Gutterson, 2003)

Micropropagation of Boston fern, banana and gladiolus were
carried out at 2 L bioreactors ( Ziv et al., 1998)
Coffee embryogenesis (Ziv et al.,1999)

Banana somatic embryogenesis bioreactor

micropropagation

Sondhal et al.,2007

Developing a scale-up system for the in vitro multiplication of

thidiazuron-induced strawberry shoots using a bioreactor

Leaf, sepal and petiole explants

24 mM thidiazuron (TDZ)-containing
medium
After 4 week
Explants from each plate were transferred to a
temporary immersion bioreactor vessel [RITA
bioreactors
After 8 week
Shoot clumps developed

Transferred to glass vessels, containing gelled zeatin for

rooting

This study presents, for the first time, a protocol for adventitious
shoot regeneration, proliferation and rooting of strawberry using
a bioreactor system in a liquid medium combined with in vitro
culture on semisolid gelled medium

TIB RITA @ bioreactors

Samir et al.,2008

Adventitious shoot regeneration in

EST-PCR based clonal fidelity
(Vaccinium angustifolium Ait.)

a bioreactor system and

in lowbush blueberry

(A) Axillary shoots explants on gelled basal medium(BM)

(B) Morphogenesis on gelled BM
(C) Shoot proliferation of bioreactor containing liquid BM
(D) Hardened-off plant in greenhouse

Debnath et al.,2011

1
2

Expressed sequence tag-polymerase chain reaction (EST-PCR)

banding pattern of donor plants .
primer NA1068. 1: Standard molecular size (1 kb ladder).

EST-PCR analysis showed 100% similarity among 12 random

tissue culture plants and the donor plant with monomorphic
bands

This is the first report of use of molecular markers to

monitor true-to-type of bioreactor micropropagated in
Vaccinium species

Plant is true-to-type low bush blueberry micro propagation

using a bioreactor system combined with gelled medium

Debnath et al.,2011

Mass propagation of blue berry in bioreactors

This is the first report on the use of
permanent immersion bioreactors for
the micro propagation of Blue berry

Plant quality (size, hardiness,

survival ex vitro), for the evaluated
was better than for plants grown in
semi-solid media

Multiplication of six-fold in eight

weeks, without transfer of explants to
fresh medium.
Ross et al., 2009

Multiplication of Chrysanthemum shoots in bioreactors

as affected by culture method and inoculation density of
single node stems
Single node cuttings (1 cm in length) of Chrysanthemum were cultured on
gelled and liquid media to compare shoot multiplication efficiency

(A)
(B)
(C)
(D)
(E)

Shoots induced from meristem culture

Single node stems (1.5 cm in length)
Bioreactor culture of single node stems
Shoot multiplication in a bioreactor
Ex vitro rooting of single node cuttings

Joo Hahn et al.2005

Effects of culture system on fresh weight, stem length,

leaf number, leaf area of Chrysanthemum plantlets

Results indicated that the deep flow technique (DFT ) culture led
to the greatest fresh weight, shoot length and leaf area, followed by
the ebb and flood culture

Joo Hahn et al.2005

Growth of Chrysanthemum plantlets in gelled and

liquid culture

Plantlet
parameter

Gelled
culture

Liquid
culture

Fresh weight
(mg per
plantlet)

844 21

1986 226

Dry weight
(mg per
plantlet)

66 4

160 14

Shoot length
(cm)

4.8 0.1

8.3 0.4

No.
leaves/plantlet

11.2 0.2

8.3 0.4

Leaf area
(cm2 per
plantlet)

20.4 1.5

49.9 1.3

Joo Hahn et al.2005

Table Effects of growth in liquid medium, of the number of

single nodes inoculated.

Number
of single
nodes
inoculate
d

Stem
length
(cm)

No.
branches
per
plantlet

Ex vitro
survival
(%)

20

23.4 2.3

8.33 1.5

100

40

26.7 1.5

6.61 0.8

100

60

26.9 2.4

6.58 1.0

100

80

28.3 2.0

4.52 0.9

100

Shoots from liquid culture grew vigorously without hyperhydricity,

showing 100% ex vitro survival

Joo Hahn et al.2005

PHYSIOLOGY OF EFFECTS OF TEMPORARY IMMERSION

BIOREACTORSON
MICROPROPAGATED
PINEAPPLE
PLANTLETS
Two levels of photosynthetic photon flux (PPF)
80 mmolm-2 s-1 (low)
225 mmolm-2 s-1(high)
Growth
condition

CO2
Dry
Sugar
(mmo mass
(mg g21
l)
per
FW)
shoot (g)

Nitrate
(mg g21
FW)

Chloro
phyll
(mg
g21
FW)

Low PPF

2851

1.2777

16.8

0.04

0.17

High PPF

2113

1.2550

25.8

0.60

0.20

Conventional
micro propagation

Temporary
immersion
bioreactor
Low PPF

3372

1.5392

189.8

0.60

0.23

Plantlets showed large increases in

sugar and nitrogen

Photosynthetic rate as well as the maximum

quantum yield of photosystem II were low
for plantlets cultivated in the temporary
immersion bioreactor at high PPF

These results indicate that shoot growth did not totally

depend on the photosynthesis process

Escalona et al .,2003

Mass multiplication of protocorm-like bodies using bioreactor

system and subsequent plant regeneration in Phalaenopsis
(Orchid)
Nodal buds (2 cm long)
They were cultured on Murashige and Skoog
(1962)
The leaves & shoot emerging from nodes were used for
PLB induction.
Inoculated into bioreactors
Hyponex media

Plantlet regeneration

(A) Multiplication of PLBs charcoal filter attached to temporary

immersion bioreactor system
(B) Biomass of PLBs harvested from temporary immersion
bioreactor system
(C) Plantlet regeneration from PLBs on Hyponex medium.
(D) Acclimatized plantlets.

Types of bioreactors for PLB proliferation

Types of bioreactors

Biomass of PLBs
(fresh weight g 1)

Air lift balloon type

94.6

Air lift column type

93.9

Temporary immersion
type

87.9

Temporary immersion
type with charcoal
filter
attached

138.9

A temporary immersion culture with charcoal filter

attached was t suitable for PLB culture

Young et al., 2005

Effect of MS, Hyponex, VW, KC and LM media on plantlet

regeneration from PLB sections,
Medium

% of
regeneration

Fresh weight
of plantlets
(mg/plantlet

MS

22.6

73.1

Hyponex

83

207.4

Vacin and
Went

45.1

98.3

Knudson C

25.0

104.3

Lindemann

23.9

127.7

Hyponex medium is suitable for plantlet regeneration from PLBs

and on this medium 83% and fresh weight 207.5 mg of PLBs
regenerated into plantlets in 8 weeks

Young et al., 2005

This is the first report of multiplication of PLBs in orchid

species using bioreactor system

This procedure can be conveniently applied for mass

multiplication
This system/methodology will beReducethe labor and space
Cost of micropropagation,
Also overcomes the problems of hyperhydricity

Young et al., 2005

Efficiency of liquid culture systems over conventional

micropropagation
Liquid (bioreactor)
Conventional(semi solid)
Less time need

More time

Better phytohormone & nutrient

uptake

Less nutrient uptake

Better oxygen and co2 availability to

root

less

Automated and mechanized

Mechanized but not automated

Less cost of per unit plant

more

More initial establishment cost

less

Less but highly skilled man power

need

More but less skilled man power need

More plant produce per unit time

Less

Hyperhydricty problem

less

Mehrotra et al. (2007)

ADVANTAGES
Short time large quantity plant production
Provision of adequate oxygen transfer
Less cost per unit plant production
Less labour requirement
Aautomated and mechanized
Reducing contamination
Automated control of environment

LIMITATIONS
High initial input and running costs of bioreactors
Hyperhydricity
Leakage of endogenous growth factors
Foam development
Airlift type bioreactors is the evaporation of
culture medium

Major Problems in Bioreactor micropropagation

Hyperhydricty (or
vitrification)
Contamination
Abnormal development of plant
grown in liquid culture brittle, glossy
succulent leave ,shoot &poor growth
of root
Poor plant development continuous
ex
vitro
as
leave
unable
to
photosynthesis &transpiration

Ziv et al., 2001

Hyperhydricity
management
A. Use of temporary immersion
bioreactor (TIB)

B .Use of growth retardant

( paclobutrazol)

CONCLUSION

It is more efficient alternative system for plant propagation

Temperature, dissolved oxygen and pH are important to cell growth
Hardware and materials are cleanable and sterilizable Plus they are
cost effective
Best method for increase cost: benefit ratio
Use rapid multiplication of endangered plant species
Possible of rapid multiplication less known plant species
(Specialty Flower) e.g.- ( Heliconia , Bird of Paradise, Red ginger
flower

Future Thrusts
More efficient designs with easy operation and change of
media and propagule harvest facilities
Problems of hyperhydricity is still a problem in several
plant species
Plantlet conversion ratio in some woody species is still a
challenge
Genetic stability or clonal fidelity of propagaules using
molecular marker techniques.
Cytogenetic abnormalities in long term culture - a concern
Synthetic seed production for in vitro genetic
conservation rare palms, orchids, etc.

t
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