Bio Reactor Technology for Plant Micro Propagation

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.



� 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.



� 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.



PLANT DEVELOPMENTAL

PATHWAYS IN BIOREACTORS� Bud or meristem clusters

� Organogenic pathways

� Somatic embryogenesis

� Anomalous plant morphogenesis



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.



� 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



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.



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.



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.



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.



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



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



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



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



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.



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.



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.



� 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.



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



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



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



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



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



� 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.



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



� 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.

Documents

Bio Reactor Technology for Plant Micro Propagation