Role of bioreactor in micropropagation of horticultural crop

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

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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 propagation Bioreactor micro propagation- BIOREACTOR Ammirato et al., 1983

Brief History….. : 

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)

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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 : 

Plant Cell Culture Sharp et al., 1980 Plant Part (Leaf, Shoot, Root, Embryo) Callus culture (Solid/Semi solid media) Suspension culture (Liquid media) Bioreactor

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

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MECHANICALLY 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

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


BUBBLE-COLUMN REACTOR Advantage Enhance dispersion and mixing Low-Shear Low power required Disadvantage Foaming

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

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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

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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 et al., 1999)

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Research1-5 L Product development5-10L Introduction to large scale11-30 L Industrial scale 31-1000 L

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Immersion time Aeration and forced ventilation Volume of liquid medium Culture container volume Culture parameters affecting the efficacy of temporary immersion systems

Commercial application in plant propogation : 

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)

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Banana somatic embryogenesis bioreactor micropropagation Sondhal et al.,2007

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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 Explants from each plate were transferred to a temporary immersion bioreactor vessel [RITA† bioreactors Shoot clumps developed After 4 week After 8 week Transferred to glass vessels, containing gelled zeatin for rooting

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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 Samir et al.,2008 TIB RITA @ bioreactors

Adventitious shoot regeneration in a bioreactor system and EST-PCR based clonal fidelity in lowbush blueberry (Vaccinium angustifolium Ait.) : 

Adventitious 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.,2011

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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

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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

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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

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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 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

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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

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Growth of Chrysanthemum plantlets in gelled and liquid culture Joo Hahn et al.2005

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Table – Effects of growth in liquid medium, of the number of single nodes inoculated. Shoots from liquid culture grew vigorously without hyperhydricity, showing 100% ex vitro survival Joo Hahn et al.2005

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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)

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These results indicate that shoot growth did not totally depend on the photosynthesis process Plantlets showed large increases in sugar and nitrogen Escalona et al .,2003 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

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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

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(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.

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Types of bioreactors for PLB proliferation A temporary immersion culture with charcoal filter attached was t suitable for PLB culture Young et al., 2005

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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 Effect of MS, Hyponex, VW, KC and LM media on plantlet regeneration from PLB sections, Young et al., 2005

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This is the first report of multiplication of PLBs in orchid species using bioreactor system Young et al., 2005 This 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

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Efficiency of liquid culture systems over conventional micropropagation Mehrotra et al. (2007)

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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 ADVANTAGES

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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 Hyperhydricity

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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 Hyperhydricty (or vitrification) Contamination Major Problems in Bioreactor micropropagation Ziv et al., 2001

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Hyperhydricity management Use of temporary immersion bioreactor (TIB) B .Use of growth retardant ( paclobutrazol)

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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 effective CONCLUSION

Future Thrusts : 

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.

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Questions?? & suggestion