bio reactor technology for plant micro propagation
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8/6/2019 Bio Reactor Technology for Plant Micro Propagation
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� In vitro micropropagation is based on enhanced axillary
bud proliferation and on the capability of differentiated to
redifferentiate and develop new meristematic centres
that are capable of regenerating fully normal plants.� Micropropagation ( in vitro propagation of axillary and or
adventitious buds as well as somatic embryos) is
presently used as an advanced biotechnological system
for the production of identical pathogen free, true to type
plants for agriculture and forestry.
� Efficient commercial micropropagation depends on rapid
and extensive proliferation along with the use of large
scale cultures for multiplication phase.
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� Normal development during the acclimatization and hardening stageis mandatory to ensure a high percent of survival after transplanting
to greenhouse.
� Mechanization and automation of the micropropagation process cangreatly contribute to overcoming the limitations imposed by existing
conventional labor intensive methods
Progress in tissue culture automation will depend on the use of liquid cultures in bioreactors.
� These techniques have been reported for some plant species andwere shown to reduce hand manipulation and thus reduce in vitro
plant production costs.
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� Various propagation aspects of several plant species in bioreactors
and some of the problems associated with the operation of
bioreactors have been reviewed. One major problem addressed was
microbial contamination as affected by both introduced plant
material and the operation procedures of large scale bioreactors.� Liquid media has been used for plant cells, somatic embryos and
organ cultures in both agitated flasks or various types of bioreactors.
� However liquid cultures confer several problems associated with
abnormal plant development, a phenomenon described as
hyperhydricity (vitrification) which causes poor development in vitroand later ex vitro.
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PLANT DEVELOPMENTAL
PATHWAYS IN BIOREACTORS� Bud or meristem clusters
� Organogenic pathways
� Somatic embryogenesis
� Anomalous plant morphogenesis
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BUD OR MERISTEM CULTURE
� The development of spherical meristematic or bud
clusters in liquid cultures provide a highly proliferative
and rapid growing system amenable to automated
inoculation, control of the medium components,mechanical separation and efficient delivery to the final
stage for plant growth and development.
� Cluster formation appears to be associated with the
continuous submergence , circulation and agitation of
the plant biomass in the medium as well as with abalanced ratio of growth promoting and growth retarding
regulators.
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� Clusters can form axillary buds and or adventitious buds
as well as from meristemoids in pro-embryogenic callus
that later differentiate to somatic embryos.
� The clusters are made up of densely packed cells,actively dividing and forming new meristematic centers
on the outer surface.
� Banana, carrot, gladiolus, potato, poplar, radiata pine,
several ornamental species, Philodendron
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ORGANOGENIC PATHWAY
� The Propagation of most plants is presently carried out commercially
through the organogenic pathway in agar gelled cultures even
though protocols are long and costly.
� The process has to be scaled up using liquid cultures in bioreactors
to amend it to automation to reduce hand manipulation and cost of plant production.
� First micropropagation procedure using liquid cultures- orchids
protocorms (1974)
� Control of shoot growth and providing culture conditions that reduce
abnormal leaf growth and enhanced the formation of bud or meristematic clusters a high proliferative rate was achieved.
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SOMATIC EMBRYOGENESIS
� The use of liquid cultures for cultivation of somatic cells in recapulatingembryogeny was reported as early as 1958.
� The underlying concept for cell totipotency and the ability to renew the
growth and express morphogenesis ±isolation and bathing of cells in a
specific medium will stimualte the zygotic embryo conditions in the ovary.
� Somatic embryogenesis was achieved by the initial use of 2,4-D and
coconut water-once the proembryogenic clusters formed removal or loweingof the auxin level initiate sequence of events similar to zygotic embryogeny
� Somatic embryogenesis in family Umbelliferae in liquid media
� Since the embryo contain both root and shoot meristem the rooting stage
required in conventional in vitro shoot or bud propagation technology is
obviated.
� Being small in size can be adequately handeled for scale up procedures,
are amenable to sorting and separation by image analysis,dispensing in
automated systems and can be encapsulated,stored or planted directly.
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ANOMALOUS PLANT MORPHOGENESIS
� The use of liquid medium is known to cause anomalous
morphogenesi resulting in plant hyperhydricity. Such plants are
fragile have a glossy appearance with succulent leaves or shoots
and poor root system.
� Unorganized mesophyll tissue made up of spongy parenchyma withlarge intercellular spaces, a deformed vascular tissue and abnormal
epidermis.
� Leaves lack a well developed cuticle and possessed malfunctioning
guard cells which can not respond to closure signals.
� Photosynthesis and transpiration processes are not fully functional .� Hyperhydricity affects plant survival after transplanting and plants
wilt or die.
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PLANT CELL AND TISSUE GROWTH IN BIOREACTOR
� Advantages provided by aerated liquid cultures inbioreactors- better contact between the plant biomassand the medium; no restrictions of gas exchange, thecontrol of the composition of both the medium andgaseous atmosphere, ability to manipulate the plantbiomass in relation to medium volume.
� Efficient circulation and mixing of plant biomassespecially for cluster and embryogenic tissue is essentialto prevent sedimentation and allow optimal growth.
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TYPES OF BIOREACTORS
Mechanical stirred bioreactors
Mechanically stirred bioreactors-impellers, magnetic
stirrers,vibrating perforated plates
Gas sparged mixingBubble coloumn or air lift bioreactors-air is provided from
sideor bottom placed air sparger
Low shearing stress in bubble coloumn or air lift reactors ,
simple construction,the lack og regions of highshear,reasonably high mass and heat transfer, high
yields at low input rates
Mist bioreactor-hairy root cultures
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MAJOR PROBLEMS
� Contamination-fungi, bacteria, yeast and insects are the
major source of contaminations
� Contamination due to manipulation of the bioreactor
apparatus-various stages of preparing and maintainingthe equipment
� Keep operation area clean by positive pressure air flow
� Continuous screening of the plant tissue for
contaminants and continuous indexing
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PHYSICAL AND CHEMICAL FACTORS IN
LIQUID CULTURES
� The gaseous atmosphere- the oxygen level, Carbon
dioxide effects, Ethylene
� Mineral nutrient consumption
� Carbohydrate supply and utilization� pH effects
� Growth regulator effects
� Temperature effects
� Cell and aggregate density,
� Foaming
� Medium rheology
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The gaseous atmosphere
� Atmosphere in the culture vessel of bioreactor-nitrogen(78%),
oxygen(21%) and carbon dioxide(0.036%)
� Volume of the culture vessel and the extent of ventilation affect
composition of gas in vessel
� Gas flow manipulate gaseous phase and can be easily manipulatedto the required levels of all three gases
� Importance of aeration and gaseous phase- potato cultured in air lift
bioreactors
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OXYGEN LEVEL
� Oxygen Level- presence of oxygen in the gas phase above and and
in the air bubbles in the medium as well as in the dissolved oxygen
in the medium
� Air is released in bioreactor through a sparger at the base of the
bioreactor � The available oxygen for the plant cells in the liquid cultures
determined by oxygen transfer coefficient (kLa) is the part that
dissolves in water
� Plant cells have lower metabolic rate than microbial cells and a slow
doubling time and therefore have low oxygen requirement.� High flow rate reduce the biomass growth.
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Carbon-di-oxide
effects
� Reported mainly for agar gelled cultures or cell suspension cultures
for secondary metabolite production
� The contribution during proliferation and multiplication stage in
media supplied with sucrose is debatable.
� High levels -Beneficial promotion of plant growth during plantacclimatization and transplanting in ex vivo
� If photoautotrophic conditions donot prevail the gas enrichment
beyond 0.036% in the air supply is not fruitful.
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MINERAL NUTRIENTS CONSUMPTION
� Many different mineral nutrient formulations have been used.
� The availability of minerals nutrients depends upon the type of
culture (liquid/agar gelled) the type and size of the plant biomass
and the physical properties of cultures.
� Factors such as pH temperature, light, aeration, the concentrationsof the minerals, the medium volume, and the viscosity of the
medium will determine the rate of absorption of the nutritional
constituents.
� Plants cells growing in the liquid culture medium are better exposed
to the medium components and the uptake and consumption arefaster.
� A drop in pH to 4.5 and lower values and the susbsequent increase
in pH to 5.5 was attributed to the initial utilization of ammonium and
to the uptake at a later stage of nitrate.
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� Depletion of ammonium ions is the first major limiting factor of
biomass and somatic embryos development.
� In Eschcholtzia californica embryonic cultures in helical ribbon
impeller bioreactor after a lag phase of about 140 hours first
ammonium and then nitrate, phosphate potassium and sulphate ionsuptake was observed to coincide with biomass and somatic embryo
development.
� In somatic embryos of the spruce cultured in bioreactors 80% of the
ammonium was consumed by the growing biomass.
� In general biomass growth is limited by the availability of phosphate,nitrogen and carbohydrates and to a lesser extent by the calcium,
magnesium and other ions.
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CARBOHYDRATE SUPPLY AND
UTILIZATION
A constant supply of carbohydrates as source of energy is required.
Sucrose, glucose, fructose,or sorbitol are the most commonly used
carbohydrates in vitro.
30gms/l sucrose deplet to 5-10 gms/l within 10-15 days and glucose
and fructose increase in levels and increase 5-10 gms/lIn C atharanthus roseus in a column airlift bioreactor -total hydrolysis of
sucrose to glucoase and fructose during first five days.
Most of the sugar uptake occurs after 5 days and glucose is taken up
preferentially over fructose.
Increase in sucrose level from 30 to 60gm/l in bioreactor decreasedbiomass in gladiolus by 50% whereas in ferns meristematic clusters
in a bubble column bioreactor increase in sucrose concentration
from 7.5 to 30 gm/l increased
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pH effects
� Initial pH in most cultures ranges between 5.5-5.9. Since media are
not buffered changes during autoclaving and during biomass growth
in cultures occurs.
� Initial ammonium uptake and acidification due to cell lysis results in
initial drop in pH upto 4.0-4.5 within 24-48 hours. However pHincreased to 5.5 after few days related to uptake of nitrates
� Carrot somatic embryo- maximum production acidic, low pH 4.3
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GROWTH REGULATOR EFFECTS
� The use of growth regulators in liquid cultures can be more effective
in controlling the proliferation and regeneration potential than in agar
gelled medium due to direct contact of plant cells and aggregates
with the medium.
� A balance of auxin and cytokinin is essential to promote anymorphogenetic response.
� Somatic embryogenesis and growth of developed embryos may
need different growth regulator in the medium.
� High levels of cytokinins and growth retardents which inhibit
giberellic acid biosynthesis reduce shoot and leaf growth andenhance meristematic cluster formation
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TEMPERATURE EFFECTS
� The control of the temperature in the liquid medium inside the
bioreactor can be easily manipulated by a heating element in vessel
or by circulating water in an enveloping jacket outside the vessel
� Little information of effect of temperature in bioreactors- constant 25
degree celcius. In lower temperature potato tuber size in bioreactorsdecreased
� Tuber formation was best at a 16 h photoperiod and 18/15 degree
celsius day and night temperature in potato internodes explants
cultured in bioreactor
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CELL AND AGGREGATE DENSITY,FOAMING
AND MEDIUM RHEOLOGY
� Airflow in the medium regulates growth and proliferation of the biomass inbioreactors, helps in aeration and prevents plant biomass sedimentation.
� Continuous aeration, mixing and circulation cause shearing damage, cellwall breakdown and accumulation of cell debris which is made up of polysaccharides.
� Increase in mixing speed from 60 to 100 rpm results in poor embryogenic
devlopment in helical ribbon impeller bioreactor � Cell debris results in foaming, adhesion of cells and aggregates to the
culture vessel walls and develop a crust on the upper part of bioreactor vessel. This layer prevents adequate circulation causing additional celldebris formation and demand for higher rates of aeration that intensify theclogging problem.
� High biomass increases viscocity higher rates of aeration are required for
oxygen supply.� Medium viscosity and foaming are reduced by using half strength MS
nutrients and lowering calcium levels
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� PEG 6000 changed rheology of the medium in alfalfa liquid cultures
and improved somatic embryo development beyond the globular
stage while it was arrested in a less viscous medium.
� The problems of cell damage, foaming and culture density can be
better controlled by developing bioreactors with an optimal shapesuitable for micropropagation through meristematic or bud clusters.
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CONCLUSIONS
� The Application of liquid cultures for micropropagation in bioreactorsusing the organogenic or the embryogenic pathway is becoming amore efficient alternative system for scale-up and automation invitro.
� Immplementation of scale up and mechanization are mandatory for
the expansion of commercial micropropagation.
� The successful exploitation of bioreactors as a commercialmicropropagation system will depend upon carefull studies of plantmorphogenesis in liquid media and understanding of controlmechanisms of organ and embryo development from meristematicor bud clusters.
� The physical and chemical environment in relation to the biomassgrowth and controlled regeneration should be further investigated.
of bioreactor
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� The levels of carbohydrates and specifically the levels and ratios of
growth promoting and retarding regulators will need to be further
studied in more detail.
� A major aspect which will have to be addressed is the problem of
contamination in large scale liquid cultures which can cause severedamage.
� The understanding of the effects of aeration,mixing,consumption
and depletion of various components present in medium will provide
information foe establishing semi-continuous or continuous culture
systemsand thus provide optimal conditions for biomass growth,
differentiation and eventual production of quality plants.