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Soobedar YadavRoll. No. 5004Discipline of Horticulture IARI, New Delhi Role of bioreactor in micropropagation

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. Automation in micropropogation is best achieved by adopting bioreactor propagationBioreactor micro propagation-BIOREACTOR

Ammirato et al., 1983

Brief History..Microbiology fermentation industry

Secondary metabolites In micropropagation first reported in 1981 for begonia propagation (Takayama and Misawa, 1981) The first patent for the cultivation of plant tissue was received in 1956( NASA)

SCHEMATIC DIAGRAM OF BIOREACTOR

Why bioreactor for micro-propagation? Better control of the culture conditions Avoid intensive manual handling

Reduces the cost

Reduce the time.

Less chance of contamination Automated and mechanized

Low energy requirement

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Pharmaceutical industries

Biomedical applications

Micropropogation

Somatic embryogenesis

Organogenesis

Bud or Meristem Clusters

Applications Plant Cell Culture Sharp et al., 1980Plant Part(Leaf, Shoot, Root, Embryo)

Callus culture(Solid/Semi solid media)

Suspension culture(Liquid media)

Bioreactor

The set up of the bioreactors and the process steps are performed in a specific order

GrowthEquilibrationMedia FillInoculationPressure HoldSIPSet UpCIP12456789Post-Use CIP103RecoveryBIOREACTOR PROCESS STEPS8MECHANICALLY AGITATED

Stirred tank bioreactor

Rotating drum bioreactor

Spin filter bioreactor

B.PNEUMATICALLY AGITATED AND NON-AGITATED BIOREACTORS

Simple aeration bioreactor

Bubble column bioreactor

Air-lift bioreactor

Balloon type bubble bioreactor-BTBB)CLASSIFICATION OF BIOREACTOR C.TEMPORARY IMMERSION BIOREACTORS STIRRED TANK BIOREACTOR DEMERITS High shear force

Complicated configuration

High power requiredThe use even flow of the medium in different directions

Proper oxygenation of the cultured tissue

MERITS

Uniform flow pattern

Low operation costBUBBLE-COLUMN REACTORAdvantageEnhance dispersion and mixing

Low-Shear

Low power required

Disadvantage

Foaming

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 better during the acclimatization

Sherrington et al., 1999

TEMPORARY IMMERSION BIOREACTORS (TIB)Impact of(TIB) culture on production costsReduced 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 et al., 1999)

Research1-5 LProduct development5-10LIntroduction to large scale11-30 LIndustrial scale 31-1000 L16Immersion time

Aeration and forced ventilation

Volume of liquid medium

Culture container volumeCulture parameters affecting the efficacy of temporary immersion systems

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 bioreactorLeaf, sepal and petiole explants24 mM thidiazuron (TDZ)-containing mediumExplants from each plate were transferred to a temporary immersion bioreactor vessel [RITA bioreactorsShoot clumps developedAfter 4 weekAfter 8 weekTransferred to glass vessels, containing gelled zeatin for rooting

20This 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 mediumSamir et al.,2008

TIB RITA @ bioreactorsAdventitious shoot regeneration in a bioreactor system and EST-PCR based clonal fidelity in lowbush blueberry (Vaccinium angustifolium Ait.)

Axillary shoots explants on gelled basal medium(BM)

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

Debnath et al.,201122Expressed 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 23 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 bioreactorsThis 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., 2009Multiplication of Chrysanthemum shoots in bioreactors as affected by culture method and inoculation density of single node stemsSingle node cuttings (1 cm in length) of Chrysanthemum were cultured on gelled and liquid media to compare shoot multiplication efficiency

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 cuttingsJoo Hahn et al.2005

Effects of culture system on fresh weight, stem length, leaf number, leaf area of Chrysanthemum plantletsResults 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 cultureJoo Hahn et al.2005Plantlet parameter GelledcultureLiquidcultureFresh weight(mg per plantlet)844 211986 226Dry weight(mg per plantlet)66 4160 14Shoot length (cm)4.8 0.18.3 0.4No. leaves/plantlet11.2 0.28.3 0.4Leaf area(cm2 per plantlet)20.4 1.549.9 1.3Growth of Chrysanthemum plantlets in gelled and liquid culture

Joo Hahn et al.2005Table Effects of growth in liquid medium, of the number of single nodes inoculated.Number of singlenodes inoculatedStem length (cm)No. branchesper plantletEx vitrosurvival (%)2023.4 2.38.33 1.51004026.7 1.56.61 0.81006026.9 2.46.58 1.01008028.3 2.04.52 0.9100Shoots from liquid culture grew vigorously without hyperhydricity, showing 100% ex vitro survival

Joo Hahn et al.2005PHYSIOLOGY OF EFFECTS OF TEMPORARY IMMERSION BIOREACTORSON MICROPROPAGATED PINEAPPLE PLANTLETSTwo levels of photosynthetic photon flux (PPF)

80 mmolm-2 s-1 (low) 225 mmolm-2 s-1(high)Growth conditionCO2 (mmol )Dry massper shoot (g)Sugar(mg g21 FW)Nitrate(mg g21 FW)Chlorophyll(mg g21 FW)Conventional micro propagationLow PPF2851 1.2777 16.80.040.17High PPF2113 1.2550 25.80.600.20Temporary immersion bioreactor.Low PPF

3372 1.5392 189.80.600.23High PPF

14171 1.7858 275.50.720.25

These results indicate that shoot growth did not totally depend on the photosynthesis processPlantlets showed large increases in sugar and nitrogen

Escalona et al .,2003Photosynthetic rate as well as the maximum quantum yield of photosystem II were low for plantlets cultivated in the temporary immersion bioreactor at high PPF

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 bioreactorsHyponex 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 bioreactorsBiomass of PLBs(fresh weight g 1)Air lift balloon type94.6Air lift column type93.9Temporary immersion type87.9Temporary immersiontype with charcoal filterattached138.9Types of bioreactors for PLB proliferation A temporary immersion culture with charcoal filter attached was t suitable for PLB culture

Young et al., 2005Medium% of regenerationFresh weight of plantlets(mg/plantletMS22.673.1Hyponex83207.4Vacin andWent45.198.3Knudson C25.0104.3Lindemann23.9127.7Hyponex 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 weeksEffect of MS, Hyponex, VW, KC and LM media on plantlet regeneration from PLB sections,

Young et al., 200535This is the first report of multiplication of PLBs in orchid species using bioreactor systemYoung et al., 2005This procedure can be conveniently applied for mass multiplication

This system/methodology will be-

Reducethe labor and space

Cost of micropropagation,

Also overcomes the problems of hyperhydricity

Efficiency of liquid culture systems over conventional micropropagationLiquid (bioreactor)Conventional(semi solid)Less time needMore time Better phytohormone & nutrient uptakeLess nutrient uptakeBetter oxygen and co2 availability to rootlessAutomated and mechanizedMechanized but not automatedLess cost of per unit plantmoreMore initial establishment costlessLess but highly skilled man power need More but less skilled man power need More plant produce per unit time Less Hyperhydricty problemlessMehrotra et al. (2007)

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 environmentADVANTAGES38

LIMITATIONS High initial input and running costs of bioreactors Leakage of endogenous growth factors Foam development Airlift type bioreactors is the evaporation of culture medium HyperhydricityAbnormal 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 &transpirationHyperhydricty (or vitrification)Contamination Major Problems in Bioreactor micropropagation

Ziv et al., 2001Hyperhydricity management Use of temporary immersionbioreactor (TIB)

B .Use of growth retardant ( paclobutrazol)

It is more efficient alternative system for plant propagation 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 Temperature, dissolved oxygen and pH are important to cell growth

Hardware and materials are cleanable and sterilizable Plus they are cost effectiveCONCLUSION

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

Questions??

& suggestion45