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Eng. Life Sci.2014,14, 607621 www.els-journal.com
Vasil Georgiev1
Anika Schumann2
Atanas Pavlov3,4
Thomas Bley5
1Center for Viticulture and Small
Fruit Research, Florida A & M
University, Tallahassee, FL, USA
2Vita 34 AG, Leipzig, Germany
3Department of Analytical
Chemistry, University of Food
Technologies, Plovdiv, Bulgaria
4Laboratory of Applied
Biotechnologies, The Stephan
Angeloff Institute of
Microbiology, BulgarianAcademy of Sciences, Plovdiv,
Bulgaria
5Institute of Food Technology
and Bioprocess Engineering,
Technische Universitt Dresden,
Dresden, Germany
Review
Temporary immersion systems in plant
biotechnologyPlant tissue and organ cultures in vitro usually face technological challenges. Whensubmerged cultivation of plant cells in a controlled environment is desired, thecharacteristic growth morphology and physiology of differentiated organ culturespresent a problem in process scale-up. Temporary immersion systems (TIS) weredeveloped several decades ago. These systems are providing the most natural envi-ronment for in vitro culture of plant shoots and seedlings. Over the past few years,TIS have been recognized as a perspective technology for plant micropropagation,production of plant-derived secondary metabolites, expression of foreign proteins,and potential solutions in phytoremediation. Nowadays, several TIS, operating onsimilar or divergent technological principles, have been developed and successfullyapplied in the cultivation of various plant in vitro systems, including somatic em-bryos and transformed root cultures. In this article, the operational principle andtechnological design of the most popular TIS are reviewed. In addition, recent exam-ples of the application of temporary immersion technology for in vitro cultivationof plant tissue and organ cultures at laboratory and pilot scales are discussed. Finally,future prospects and challenges to the industrial realization of that fast-developingtechnique are outlined.
Keywords:Bioreactors / Micropropagation / Molecular farming / Secondary metabolites /Tissue and organ cultures
Received:April 9, 2014;revised:June 11, 2014;accepted:July 11, 2014
DOI:10.1002/elsc.201300166
1 Introduction
Plant cells, tissue, and organ cultures have been recognized as
powerfultools for clonal propagation of commerciallyimportant
crops (micropropagation), production of valuable secondary
metabolites, expression of complex foreign proteins (molecular
farming), as well as for phytoremediation of waste waters (phy-
totransformation and phytoextraction). Large-scale cultivation
of differentiated (embryos, shoots, seedlings, transformed or
adventitious roots) and dedifferentiated (suspended cells) plant
cultures could be realized by growing them in vitro in liquidmedia, under controlled environmental conditions in bioreac-
tor systems. The core concept of that approach is to achieve
economically feasible production of maximal amounts of plant
biomass, ready for direct application or for subsequent isolation
of valuable products. The bioreactor is specialized technological
Correspondence: Dr. Vasil Georgiev ([email protected]),
Center for Viticulture and Small Fruit Research, Florida A & M
University, 6505 Mahan Drive, Tallahassee, FL 32317, USA
Abbreviations: BIB, bioreactor of immersion by bubbles; DW, dry
weight;RITA, recipient for automated temporary immersion;TIS, tem-
porary immersion systems
equipment, designed for intensive culture by regulating various
nutritional and/or physical factors [1]. Bioreactor systems usu-
ally consist of a culture vessel and an automated control block.
The culture vessel is designed to accommodate the cultivated
cells in aseptic environment and to ensure their maximal growth
by providing opportunities for maintaining optimal microen-
vironmental conditions, nutrients, and gaseous mass transfers.
Theautomated control block is a computerized, fully automated
or semiautomated system, designed to monitor and control the
cultivation conditions in the culture vessel, such as the agitation
speed, temperature, dissolved oxygen and carbon dioxide (CO2)concentrations, illumination regime, pH, composition of the
overlaygaseous environment, and the levelof theliquid medium.
According to the nature of the environment surrounding the
cultured cells, existing bioreactors could be classified into four
main classes: liquid-phase bioreactors, gas-phase bioreactors,
temporary immersion systems (TIS), and hybrid bioreactors. In
liquid-phase bioreactors, the cultivated cultures are completely
immersed in a liquid nutrient medium. Liquid-phase bioreac-
tors (including mechanically agitated, pneumatically agitated,
hydraulically agitated, and membrane bioreactors) are currently
the best studied systems, revealing almost unlimited potential
for applicationin growing undifferentiated plant cell suspension
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cultures [2]. However, in most cases, liquid-phase bioreactor
systems fail to secure satisfactory growth of differentiated plant
in vitro systems. The complete immersion of plant tissue or or-
gan cultures into the liquid mediumoften causes malformations
and loss of material due to asphyxia and hyperhydricity [3].
Asphyxia and hyperhydricity are undesirable physiological con-ditions, caused exclusively by the low oxygen contents and water
potential of the culture media [3, 4]. The complex morphology
of differentiated plant tissue and organs requires development
of bioreactors with a sophisticated design, capable of providing
a specific microenvironment in order to secure the growth and
physiological integrity of the cultures [5]. To overcome the ex-
isting difficulties, gas-phase bioreactors [6, 7], TIS, and hybrid
bioreactors [810] have been developed. TIS are simple auto-
mated systems, designed to provide optimal environment, im-
proved nutrients and gas transfers, and lower mechanical stress
in order to reduce physiological disorders, and to preserve the
morphological integrity of the fast growing differentiated plant
in vitro cultures. TIS provide the most natural environment for
plant tissue and organ in vitro cultures, where the cultivated
propagules are periodically immersed into a liquid medium and
then exposed to a gaseous environment [5]. Different varia-
tions of TIS have been developed and are widely applied in
commercial micropropagation of economically important plant
species [1,1113]. Moreover, because of their simple design and
flexible operation, TIS have been adapted in the research of sec-
ondary metabolite production, molecular farming, and even in
phytoremediation of toxic compounds [5]. The technical real-
ization and principal operation of most popular TIS, including
some recently developed designs, are discussed in this review.
Some recent examples of the application of TIS in plant-derived
secondary metabolites production, foreign proteins expression,
phytoremediation, micropropagation, and clonal selection arepointed out as well.
2 Design and operation of TIS
The development of TIS is closely related with the commercial-
ization of plant micropropagation. TIS are periodic semiauto-
mated or fully automated cultivation systems, based on alternat-
ing cycles of temporary immersion of the cultured plant tissue
into the liquid medium followed by draining and exposing the
plant tissue to a gaseous environment. Usually, the immersion
periodis shorter (afew minutes),whereastheair exposureperiod
is prolonged (several hours). The precise adjustment of the du-
rationsof theimmersion and exposure periods may significantly
reduce the hyperhydricity of theculturedplant tissue by creating
conditions for optimal humidity and nutrients supply with min-
imal liquid contact [14]. Thedirect exposureof theplanttissue to
the gaseous environment significantly simplifies the interphase
oxygen transport from the gas to theculturedcells, in contrast to
the submerged culture, where the interphase oxygen transport
faces resistance in a few boundary layers (gasliquid and liquid
solid interfaces) [15]. Improved oxygen transport contributes to
the better gas exchange, reduced oxygen limitations, and thus,
a lower occurrence of physiological disorders such as asphyxia.
Some TIS have the additional option of enriching the headspace
withCO2duringthe gasexposureperiod. The higherlevel of CO2
may have positive effects on the multiplication of the cultivated
plant tissue, photosynthetic activity, organs morphology, and on
the accumulation of secondary metabolites [16, 17]. Moreover,
TISdo not utilize mechanical agitation devices, thus the disloca-
tion of the cultured propagules, if any, is done only by the power
of hydrodynamic forces during immersion periods. Under theseconditions, the cultivated plant tissues undergo minimal shear
stress that preserves the culture integrity and additionally im-
proves the morphology and physiology of the organs. TIS are
usually constructed withtransparent glass or plastic vessels, thus
the light from external sources may be used to illuminate the
cultivated plant materials. The technological design of some of
the most common TIS is discussed in more details below. Their
basic characteristics are shortly summarized in Table 1.
2.1 Twin-Flask system
TheTwin-Flask system (Biorreactoresde Inmersion Temporal) is
one of the earliest developed TIS [13,18,19]. Basically, the Twin-
Flask system consists of two containers (wide-mouth flasks, bot-
tles, or jars), connected together by a U pipe (glass or plastic)
or a silicone tube (Fig. 1) [16,17,2024]. One of the containers
has the function of a culture chamber, whereas the other con-
tainer is used as a medium storage tank. The culture chamber
container may or may not be equipped withsupport material for
explants (glass beads, polyurethane foam, metal or nylon sieves
may be used) at its bottom [21, 22, 2430]. Each container is
connected to its own pressurized-air line, controlled by two in-
dependent timer clocks, coupled with three-way solenoid valves.
The simple and reliable design makes Twin-Flask systems favor-
able for many laboratories. They are generally easy to operate
and the construction can maintain sterility for long periods ofcultivation [28]. Some of the major disadvantages of Twin-Flask
systems are the comprehensive automation (the need of two
timer clocks and two three-way solenoid valves) and the lack
of options for nutrient medium renewal and forced ventilation.
Twin-Flask systems are also not equipped with a specialized port
for external CO2 supply during the exposure period. However,
CO2-enriched air may be used to ensure higher CO2concentra-
tions in thegaseous environmentof theculturechamber[17,28].
Twin-Flask systems have been successfully applied in the prop-
agation of plant seedlings, shoots, nodule cluster, and embryo
cultures [2022, 27, 30]. Recently, Twin-Flask systems have been
used in research work on secondary metabolites accumulation
by differentiated in vitro cultures as well [17, 23,24,29].
2.2 Ebb-and-Flow
Ebb-and-Flow systems could be described as a simplified
modification of the Twin-Flask systems. The system consists
of two vessels one large wide-mouth vessel functioning as
a culture chamber, and one smaller vessel functioning as a
medium storage tank (Fig. 2). Both vessels are interconnected
by external ports, mounted on the bottoms. The bigger vessel
is the culture chamber, where the plant explants are placed
on polyurethane foam support. The polyurethane support
maintains sufficient humidity (8590%) during the exposure
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Table 1.General features, advantages, and disadvantages of most common TIS
TIS Power input Construction
materials
Sterilization Pros Cons
Twin-Flask Pneumatic Glass Autoclavable Widely accessible;
Simple construction;Easy to operate;
Maintains sterility for long period;
Low investment costs
Complex automation;
Not suitable for forced ventilationand CO2enrichment;
Low headspace humidity in g rowth
chamber;
No nutrient medium renewal
Ebb-and-Flow Pneumatic
and gravity
Glass Autoclavable Simple construction;
Can be realized in large volumes (up
to 50 L);
Easy to handle;
Simplified automation;
Option for nutrient medium renewal;
Low energy costs
Two levels assembly requires more
space;
Time for drainage is increased with
the culture growth;
Nonuniform light distribution inside
the growth chamber;
Not suitable for forced ventilation
and CO2enrichment
RITA Pneumatic
and gravity
Polypropylene Autoclavable Simple automation;
Reliable operation;Easy to handle;
Unified organization of internal
elements;
High headspace humidity in growth
chamber;
Compact space for apparatus
accommodation
No nutrient medium renewal;
No forced ventilation and CO2enrichment
Thermo-photo-
bioreactor
Pneumatic
and gravity
Pyrex glass Autoclavable Precise temperature control;
Integrated light source;
Simple automation;
High headspace humidity in growth
chamber;
Sampling port
Complex construction;
No nutrient medium renewal;
No forced ventilation and CO2enrichment
Hybrid
Ebb-and-Flowwith saturated
tubular
convective flow
Hydraulic
andpneumatic
Glass, stainless
steel
Autoclavable Designed for high-density hairy root
cultures;Fully automated;
Uniform distribution of root
biomass;
Improved oxygen transfer
Designed only for hairy root cultures;
Complex design;Two stage operation;
Difficult for biomass harvesting
Bioreactor of
Immersion by
bubbles
Pneumatic
and gravity
Glass, stainless
steel
Autoclavable Simple construction;
Better utilization of growth chamber
space;
Low share stress;
Better gas exchange
Requires the presence of detergent
into nutrient medium;
Uncontrolled time for drainage;
Expensive;
No nutrient medium renewal;
No forced ventilation and CO2enrichment
Rocker systems Mechanical Polycarbonate Autoclavable Simple design;
Cultivation boxes could be stack on
racks;Availability of external air source is
not mandatory;
Easy to handle;
Maintains high headspace humidity;
Improved access to light;
Ready for large scale process
Requires tilting platforms;
Occupies more space in growth
chamber;No full separation of explants from
liquid medium;
Equal times for immersion and
exposure periods;
Difficult to air exchange;
No nutrient medium renewal;
No forced ventilation and CO2enrichment.
High energy costs
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Table 1.Continued
TIS Power input Construction
materials
Sterilization Pros Cons
BioMINT Mechanical Polycarbonate Autoclavable Simple design;
Cultivation boxes could be stack onracks;
Full separation of explants from
liquid medium;
Easy to handle;
Maintains high headspace humidity;
Option for forced ventilation and
CO2enrichment.
Option for nutrient medium renewal;
Ready for large-scale process
Requires tilting platforms;
Occupies more space in growthchamber;
High energy costs
Rotating drum Mechanical Glass or plastic Autoclavable Simple construction;
Low investment costs;
Suitable for embryo, shoots, and
hairy root cultures;
Maintains high headspace humidity;
Easy to handle
Requires rotating platform;
No full separation of explants from
liquid medium;
No control of times for immersion
and exposure;
No air exchange;
No nutrient medium renewal;
No forced ventilation and CO2enrichment.
Occupies more space in growth
chamber
Bioreactor RALM Pneumatic Polycarbonate
and
polypropylene
Autoclavable Easy to handle;
Option for forced ventilation and
CO2enrichment;
Option for nutrient medium renewal;
Low investment costs
Complex automation;
Construction with several internal
elements;
Low headspace humidity in growth
chamber
SETIS Pneumatic
and gravity
Polypropylene Autoclavable;
Gamma
irradiation
Simple construction;
No internal elements;
Easy to handle;
Simplified automation;Large illuminated area;
Improved drainage;
Low energy costs.
Optimal usage of growth room space
Low investment costs
No forced ventilation and CO2enrichment;
No nutrient medium renewal
PLANTIMA Pneumatic
and gravity
Polycarbonate Autoclavable Simple automation;
Reliable operation;
Easy to handle;
High headspace humidity in growth
chamber;
Apparatus may be stacked one on the
other to save space. Low investment
costs
Construction with several internal
elements;
No nutrient medium renewal;
No forced ventilation and CO2enrichment
PLANTFORM
bioreactor
Pneumatic
and gravity
Polycarbonate Autoclavable Simple automation;
Reliable operation;
Easy to handle;
High headspace humidity in growth
chamber;
Improved access to light;
Apparatus may be stacked one on the
other to save space.
Option for forced ventilation and
CO2enrichment;
Low investment costs
Construction with several internal
elements;
No nutrient medium renewal
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Table 1.Continued
TIS Power input Construction
materials
Sterilization Pros Cons
Box-in-Bag Pneumatic
and gravity
Polycarbonate,
polyethylene,and nylon
Gamma
irradiation,single use
Reliable operation;
Large surface-to-volume ratio;Improved light access;
Option for nutrient medium renewal;
Apparatus may be stacked one on the
other during transportation;
Disposable;
Low investment costs
Two levels assembly requires more
space;Time for drainage is increased with
the culture growth;
Low diameter of inoculation port;
No forced ventilation and CO2enrichment
WAVE bioreactor Mechanical Biocompatible
transparent
plastics
Presterilized
single use
Simple design;
Scalable disposable technology;
Fully automated precise monitoring
and control of pH, dissolved oxygen,
carbon dioxide, and temperature;
Easy to handle;
Maintains high headspace humidity;
Low labor costs
Requires specialized and expensive
control module and tilting platform;
Low diameter of inoculation port;
No full separation of explants from
liquid medium;
Equal times for immersion and
exposure periods;
No nutrient medium renewal;
High investments costs
Figure 1. Technological design and operational principle of Twin-Flask system: (A) period of exposure. The whole volume of liquidmedium is located into the medium storage tank. Air lines of bothcontainers areclosed and thesolenoid valvesare openedto atmo-sphere; (B) dislocation of liquid medium from medium storagetank to culture chamber. The air line of cultivation chamber isclosed, and the air line of medium storage tank is opened. Theoverpressure moves the medium into the cultivation chamber;(C) period of immersion. The propagules are immersed into theliquid medium. The medium storage tank is empty. Air lines forboth containers are closed and the solenoid valves are openedto atmosphere; (D) draining out the nutrient medium back tothe culture medium tank. The air line of cultivation chamber isopened, whereas the air line of medium storage tank is closed.The overpressure moves back the medium into the medium stor-age tank.
Figure 2. Technological design and operational principle of Ebb-and-Flow system: (A) period of exposure; (B) dislocation of liq-
uid medium. Air pressure is applied to the medium storagetank and the liquid medium is moving to the culture chamber;(C) period of immersion; (D) draining out the nutrient medium.The air pressure is switched off and the medium flows back to themedium storage tank due to gravity.
period and has the function of an air sparger during the immer-
sion phase [31, 32]. The smaller vessel is the nutrient medium
storage tank and is placed below the culture chamber vessel.
The advantages of Ebb-and-Flow systems are the simple and
reliable construction, simplified automation, and lower energy
input. The nonuniform light distribution inside the cultivation
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Figure 3. Technological design and operational principle of RITAsystem: (A) period of exposure; (B) Dislocation of liquid medium.Air pressure is applied to the bottom compartment through thecentral pipe. The liquid medium is moving to the upper com-partment; (C) period of immersion; (D) draining out the nutrientmedium. The air flow is stopped and the medium flows back tothe bottom compartment due to gravity.
vessel and the lack of options for forced ventilation and CO 2enrichment are among the main disadvantages of the system.
2.3 RITA system
The RITA (recipienta immersion temporaire automatique) TIS
(CIRAD, France, distributed by VITROPIC, France) have been
developed for intensive in vitro plant culture. The system con-
sists of a single autoclavable polypropylene vessel (500 mL) with
two compartments, separated by an installed tray with a mesh
support and a plastic pipe, mounted to its center (Fig. 3). The
vessel is closed by a wide screw lid, equipped with central and
lateral external ports on the top. Both ports are secured with
membrane filters, and the central port is connected to an airline
controlled by a timer clock and a three-way solenoid valve. The
upper compartment of thevessel is theculture chamber, whereas
the bottom compartment is the medium storage tank. The ad-
vantages of the RITA TIS are the simple and reliable operation,
the compact space for apparatus accommodation, and the sup-
port of sufficient relative humidity level with full separation of
the propagules and liquid medium. All of the internal elements
are connected to each other and can be manipulated as a single
piece that facilitates the handling of the biomass. The main dis-
advantages of the systems are the inability for nutrient medium
renewal and the lack of options for forced ventilation and CO 2enrichment.
Figure 4. Technological design and operational principle ofthermo-photo-bioreactor TIS: (A) period of exposure; (B) dislo-cation of liquid medium. Air is supplied to medium storage tankand medium is moving to the culture chamber; (C) period of im-
mersion; (D) draining out the nutrient medium. The air supplywas stop and the medium was drained out back by the gravity.
2.4 Thermo-photo-bioreactor TIS
This TIS bioreactor has been developed exclusively for micro-
propagation and secondary metabolite production of Antarctic
hair grass (Deschampsia antarcticaE. Desv.) [33]. The bioreactor
consists of two Pyrex glass vessels connected by stainless steel
joints and pipes (Fig. 4). The upper vessel is the culture chamber.
It is equipped with a water jacket for precise control of the tem-
perature and an integrated source of UV light, mounted on the
top lid. The plant material is supported by a stainless steel screen
installed inside the culture chamber. The lower vessel is the
nutrient medium storage tank. It is designed with two external
ports one on the upper end, used for air supply, and one on the
bottom used for loading the medium and sampling. The main
advantages of the thermo-photo-bioreactor TIS are the options
for precise control of temperature and UV irradiation, which is
very important for the cultivation of extremophile plants [34].
However, the complex and expensive construction is the main
argument against this design. Several low-cost TIS, operating
on the same principle, such as thermo-photo-bioreactors,
have been developed by using glass bottles [35] or NALGENE
filtration systems [36]. However, noneof them cancompetewith
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Figure 5. Technological design and operational principle of hybrid Ebb-and-Flow with saturated tubular convective flow: (A) operation inbubblecolumn mode until uniform distribution and immobilization of the transformed hairy roots is achieved; (BE) operation in temporaryimmersion mode similar to that of Twin-Flask system (see text for details).
the precise temperature control of thermo-photo-bioreactor
TIS.
2.5 Hybrid Ebb-and-Flow with saturated tubularconvective flow
The Ebb-and-Flow with saturated tubular convective flow was
exclusively developed for cultivation of high-density hairy root
cultures [10]. This systemis hybridbioreactor, acting as a bubble
column forthe first days afterinoculation,and then switches over
tooperationas a Twin-Flasksystem (Fig. 5).The initial operation
as a bubble column is necessary to secure uniform distribution
and immobilization of the transformed hairy roots (Fig. 5A).
Once the roots are attached to the internal immobilization mesh
support, thereactor starts to operate as a Twin-FlaskTIS (Fig. 5B
to E). Peristaltic pump is used to dislocate the nutrient medium
from the storage tank to the culture chamber and vice versa. The
system operates with short periods of immersion and exposure
(2min each) and the pumpspeed is set up toensurefullmedium
dislocation fromone vessel to the other for 1 min, so that a tubu-
lar convective flow is achieved [10]. The used nutrient medium
is presaturated with air, so it can supply oxygen to the very inner
zones of the compact growing roots. The main advantage of this
hybrid bioreactor is the improved oxygen transfer during cul-
tivation of high-density root biomass. The main disadvantages
are the complicated design, complex operation, limited use only
to root cultures, and difficult harvest of the immobilized root
biomass.
2.6 Bioreactor of immersion by bubbles
The bioreactor of immersion by bubbles (BIBBiorreator de
Imersao por Bolhas) utilizes a completely new cultivation strat-
egy, based on temporary immersion of propagated explants in
foam instead of liquid medium. The system consists of a single
glass cylinder, transversely divided into two compartments by a
microporous (170220m pores)plate(Fig. 6).The upper com-
partment is thegrowth chamber, in whicha fewstainlesssteel in-
ternalracksarestacked oneupon another to supportthe cultured
explants. The liquid nutrient medium with added detergent
(Tween 20) is filled at the bottom of the culture chamber as well.
The lower chamber is for uniform air distribution by the porous
plate. BIB is commercially available in Brazil at 1.5 L scale (Tec-
nal Equipamentos para Laboratorio, Brazil). Recent research has
shown that BIBprovidesbetter growthand highershootnumber
per explant than RITA TIS in propagation of tea-tree (Melaleuca
alternifolia) and orchid (Oncidium leucochilum) [37, 38]. How-
ever, the presence of detergent in the nutrient medium, as well
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Figure 6. Technological design and operational principle of biore-actor of immersion by bubbles system: (A) period of exposure;(B) period of immersion. Air is supplied and foam is formed.The explants are immersed by culture medium in a form of bub-bles. When aeration stops, the foam density decreases with timedue to liquid drainage and the explants are exposed to gaseousenvironment.
as the prolonged time for liquid drainage may restrict the appli-
cation of BIB for propagation of some sensitive plant species.
2.7 Rocker systems
Rocker systems use a mechanical platform to tilt the cultured
boxes at a given angle, so that the medium can be dislocated
from one end of the cultured box to the other, and vice versa
(Fig. 7A and B). The cultivation boxes are made of autoclavable
transparent polycarbonate and are rectangle-shaped with a lat-
eral wide-mouth opening, closed by a wide screw cap with filter
membrane inside. After inoculation, the boxes are placed on
racks with mechanically tilted shelves. The tilts of the shelves
create small wave fronts and alternately immerse and aerate the
cultured propagules [3941]. The main advantage of the rocker
system is that large numbers of cultivation boxes could be ac-
commodated on one rack and no additional connection to an
airline is necessary. The disadvantages of rocker systems are re-
lated with the necessity of an electromechanically driven tilting
platform that increases the investment and energy costs. Tilt-
ing platforms require more space to operate properly and this
may reflect on the production cost per unit space in the growth
chamber. The cultured boxes have no good air renewal, and
no options for forced ventilation or nutrient medium replace-
ment exist. Some of these problems could be overcome by using
theBioMINT bioreactors as culture vessels on a rocker platform.
TheBioMINT is a mid-sized(1.2 L) bioreactor, consisting of two
cylindrical autoclavable polycarbonate vessels that are joined to-
gether by a perforated adaptor with two female screw threads
(Fig. 7C and D). One vessel is for the plant tissues and the other
for the liquid culture medium. The perforated adaptor permits
the free flow of the liquid medium while keeping the propag-
ules in place when the bioreactors change position. The adaptoralso has two external ports that allow the application of forced
ventilation or CO2enrichment [42]. Because of its flexible con-
struction and easy handling, BioMINT bioreactors are popular
in shoots propagation [43,44].
2.8 Rotating drum system
The system consists of a roller apparatus and an autoclavable
plastic or glass bottle lying on it (Fig. 8). A stainless steel net
or a mat of polyurethane foam is placed inside the bottle to
support the explants [45]. When the roller apparatus is rotating
at low speed, the immobilized plants are periodically immersed
and exposed to air environment. In the case of adventitious orhairy roots cultivation, the installation of internal support is not
necessary, since the roots are adsorbed onto the bottle walls by
adhesion [46]. The advantage of the rotating drum system is the
simple construction. The main disadvantages are the inability
to set up independent and prolonged times for immersion and
exposure periods, higher shear stress due to mechanical mixing,
and the lack of options for ventilation and exchange of internal
atmosphere.
2.9 Low-cost and disposable TIS
In an attempt to decrease the initial investment costs for equip-
mentandtosavespaceandlabor,severalTIShavebeendevelopedand distributed on the market in the last few years (Fig. 9). A
common feature of all of them is the simple design, inexpensive
utilization, and interchangeable plastic elements. The systems
are easy to handle, compact to store, autoclavable, and ready
for multiple use. A few disposable variants are also available at
present.
The bioreactor RALM (Biorreatores RALM, Ralm Indus-
tria e Comercio ltda., Brazil) is a TIS, operating on the Twin-
Flask principle (Fig. 9A). The SETIS system (Vervit, Belgium,
distributed by Duchefa Biochemie, The Netherlands; Fig. 9B)
operates in a similar way as the Ebb-and-Flow TIS system.
PLANTIMA (A-Tech BioscientificCo., Ltd., Taiwan; Fig.9C) is a
small volume TIS, operated on the RITA principle and has beenused for plantlet propagation [47, 48]. Another TIS, using the
principle of operation of theRITA system, arethe PLANTFORM
bioreactor (Plant Form AB, Sweden & TC propagation Ltd.,
Ireland; Fig. 9D). Box-in-Bag (Fig. 9E) is a disposable TIS, op-
erating on the principle of the Ebb-and-Flow TIS. The WAVE
bioreactor (Fig. 9F) is a mechanical rocking platform that uses
disposable presterilized cultivation bags [4951].
3 TIS in micropropagation
Micropropagation on semisolid nutrient medium is a costlypro-
cess, since the technology is based on manual handling of a
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Figure 7. Technological designand operational principle of(A, B) rocker TIS and (C, D)BioMINT bioreactor.
Figure 8. Technological designand operational principle of ro-tating drum bioreactor system.
large number of single containers [1, 52]. Labor accounts for
4060% of the final cost of propagated plants, and the tech-
nology is not subject to automation [1, 12]. To overcome those
negatives, automated TIS have been developed in order to re-
duce the labor component and to intensify the culture by using
a liquid nutrient medium in plant propagation. Recently, a pilot
scale process for propagation of Robusta (Coffea canephoravar.robusta) by cultivating somatic embryos in Ebb-and-Flow TIS
has been reported [32]. The authors reported an annual pro-
duction of 2.5 million pregerminated embryos by using 100
Ebb-and-Flow TIS with 10 L culture vessels, installed in a 40 m2
growth room [32]. Moreover, the authors succeeded in improv-
ingthe embryogrowthex vitro germination phase by developing
and using the new disposable Box-in-Bag TIS and reported the
production of 600 000 somatic Robusta seedlings [11, 31]. It
has been shown that blueberry plants (Vaccinium corymbosum
L.), multiplied in TIS (Twin-Flasks), have higher adaptability
thanthose cultured by the conventional approach [28]. Recently,
Ptak et al. [53] have reported that the number of regenerated
Leucojum aestivum L. plants from somatic embryos is twice
higherwhenTIS(RITAsystem)areused.Theauthorshavefound
that the addition of the cytokinins metatopolin and benzylade-
nine has a beneficial effect on plant regeneration [53]. A similar
positiveeffect of metatopolinon plant regenerationhas also been
reported in the micropropagationof plantain (Musaspp.) plants
in TIS (Twin-Flasks) [54]. More detailed information concern-
ing the practical application of TIS in plant micropropagation
could be found elsewhere [1, 12,13, 19].
4 TIS in secondary metabolite production
The commercialization of the plant in vitro technology could
be considered as the most perspective alternative for sustainable
supply of valuable phytochemicals in the near future [2,5558].
Since the biosynthesis of some secondary metabolites in plants
may involve the active participation of several, often compart-
mentally separated biosynthetic pathways, a certain level of cell
and/or organ differentiation is required for their production.
Thus, the utilization of differentiated plant tissue or organ
cultures is the most natural way to produce such substances in
vitro. However, in vitro cultivation of plant tissue or organ cul-
tures in liquid medium is closely associated with the availability
of specially designed bioreactor systems [5,49]. Because of their
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Figure 9. Technologicaldesign of (A) RALM bioreactor, (B) SETIStemporary immersion bioreactor system, (C) PLANTIMA system,(D) PLANTFORM bioreactor, (E) Box-in-Bag, and (F) WAVE biore-actor.
reasonable price, excellent performance, flexible operation,
and easier handling, TIS could be considered as very suitable
platforms for production of secondary metabolites by differ-entiated plant in vitro cultures in laboratory and pilot scales.
TIS (RITA system and hybrid bioreactors) have been used in
the study of pigment and alkaloid production by transformed
hairy root systems from different species [5, 810, 59]. It has
been reported that hairy root culture ofBeta vulgariscv. Detroit
Dark Red show rapid growth and stable accumulation of
betalaines when cultivated in RITA TIS [60]. By using 15 min
of immersion and 60 min of exposure periods, the authors
achieved a concentration of accumulated betalaines of 18.8 mg/g
dry weight (DW), which is comparable with the concentration
registered in a culture fully submerged in shaking flasks [60].
A temporary immersion RITA system has also been used for
studying hyoscyamine production by diploid and tetraploid
Datura stramoniumL. hairy roots [61]. It has been found that
the durations of 15 min of immersion and 105 min of exposure
are optimal for both diploid and tetraploid hairy root cultures.
In these conditions, the diploid and tetrasploid hairy roots
produced 3.11- and 2.82-fold higher amounts of hyoscyamine,
compared to the same cultures, cultivated in shaking flasks [61].
Moreover, it was demonstrated that the duration of exposure
periods may affect the secretion of alkaloids into the liquid
medium. Thus, by increasing the duration of the exposure
period (from 65 to 165 min), the amount of extracellular
hyoscyamine secreted by the diploid D. stramonium hairy
root culture increased from 0.6 mg/200 mL to 2.1 mg/200 mL
[61]. Theoption allowing themanipulation of thesecretionlevel
of secondary metabolites only by changing the durations of the
immersion or exposure periods may be a useful opportunity for
thedevelopment of a milking process, based on thecontinuous
recoveryof metabolites released in the culture medium. Recently,
RITA TIS have been used in the study of secoiridoid glycosides
accumulation byCentaurium maritimumL. Fritch transformedhairy root cultures [62]. The selected line (HR3) showed the
best productivity of secoiridoid glycosides (4 mg/L/day), when
cultivated in a RITA system for 28 days with cycles of 15 min
immersion and 45 minexposureperiods. Theachieved yield was
10 times higher than the one registered for a culture fully sub-
merged in shaking flasks (about 0.4 mg/L/day), which supports
the authors conclusion that the RITA TIS are the most efficient
system for the production of secoiridoid glycosides [62]. RITA
TIS have also been used to study the effects of phytohormones
on the growth and ginsenosides saponins production byPanax
ginsengC.A. Meyer adventitious root culture [63]. It has been
found that the combination of 3-benzo(b)selenienyl acetic
acid (3.0 mg/L) and kinetin (0.02 mg/L) leads to the best
growth rate (5.62) and the maximal ginsenosides accumulation
(15.94 mg/g DW) after 8 wk of cultivation at immersion periods
of 5 min and exposure periods of 60 min [63]. Recently, a
mechanically mixed WAVE bioreactor TIS has been used for
cultivation and geraniol accumulation by transgenicNicotiana
tabacum L. cv. Petit Havana SR1 hairy roots, harboring the
VoGESgene (a geraniol synthase gene fromValeriana officinalis
L.) [64]. Experiments with a 2 L disposable culture chamber
(CultiBag RM 2L basic screw cap) showed that the fed-batch
mode of cultivation (initial medium volume of 200 mL and
three additions of 40 mL each) resulted in 56% more dry
biomass than the batch mode [64]. The cultivation process was
successfully scaledup to 20 L scale (CultiBag RM 20L basic screw
cap) by using 1 L of initial nutrient medium and three additionsof fresh medium of 200 mL each [64]. The rocking platform was
operated for 28 days at a rocking rate of 8 rpm and a rocking
angle of 6, and a final amount of 10.7 g dry biomass with a
concentration of geraniol of 204.3 g/g DW was produced.
The achieved geraniol yield was 2325% higher than the yields
recorded in 2 L bags operated in batch or fed-batch modes
[64].
During the last few years, greater attention has been focused
on the cultivation of differentiated shoot in vitro cultures for
production of valuable plant-derived secondary metabolites [5].
Several new modifications of liquid-phase bioreactors have
been developed and applied for submerged cultivation of shoot
cultures, but more attempts for successful scale up are yet to be
made [24,65,66]. TIS offer a flexible and perspective cultivation
technology that could be adopted for the needs of large-scale
production of secondary metabolites by plant in vitro shoot
cultures. Recently, RITA TIS have been used to study the pro-
duction of Amaryllidaceae alkaloids by sea daffodil (Pancratium
maritimum L.) shoot culture [67]. In optimal cultivation con-
ditions (immersion periods of 15 min and exposure periods of
12 h), the shoots produced 900.1 g/g DW hemanthamine and
799.9 g/g DW lycorine [67]. Moreover, it was demonstrated
that the duration of the exposure periods had a significant
effect on both the alkaloids pattern and the levels of alkaloids
released into the culture medium [67]. However, the observed
effect seems to be specific for the P. maritimumL. shoot culture.
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Another study demonstrated that the duration of theimmersion
period had not a significant effect on the intracellular and
extracellular production of galanthamine (another important
Amaryllidaceaealkaloid)by a shootcultureof summer snowflake
(L. aestivumL.) cultivated in RITA TIS [68]. It was found that
the duration of the immersion period had more dramatic effecton the biomass accumulation by in vitro cultivatedL. aestivum
shoots [68]. Further research demonstrated that the alkaloid
pattern ofL. aestivum shoots cultivated in RITA TIS could be
significantly affected by thetemperatureat whichthe cultivation
was performed [68, 69]. Moreover, the levels of Amaryllidacea
alkaloids produced byL. aestivumshoots could be additionally
increased by using an appropriate elicitation strategy [65, 70].
Twin-Flask TIS have also been adapted for in vitro cultivation
ofL. aestivum, and its potential to stimulate galanthamine pro-
duction in shoots has been compared with that of several air-lift
bioreactors [24]. However, although the accumulated biomass
in Twin-Flask TIS was higher than the one observed in other
investigated systems, the galanthamine concentration remained
very low (0.06 mg/g DW), which significantly reduced its overall
productivity compared to the air-lift systems [24]. Further
research on elicitation showed that the addition of methyl
jasmonate stimulated the accumulated dry biomass and slightly
improved galanthamine accumulation (from 47.5 to 48.7%) in
the leaves of cultured L. aestivum shoots [65]. Twin-Flask TIS
have been used to study cardiotonic glycosides production by
Digitalis purpureaL. shoot culture as well [71]. It was found that
the duration of the exposure period did not alter the content of
digitoxin in shoots,but rather had a strong effect on the biomass
accumulation [71]. When cultivated at 2 min immersion and
4 h exposure periods, the D. purpurea L. shoots accumulated
5.82 g DWper 250 mL medium with concentrations of digitoxin
and digoxin of 28.8 and 20.68 g/g DW, respectively [71].The same Twin-Flask system has been further adapted for
cultivation ofDigitalis lanataEhrh. shoot culture in order to
study the biosynthesis of cardiotonic glycosides lanatoside C
and digoxin [72]. The authors investigated the effects of three
elicitors (Chitoplant, Silioplant, and methyl jasmonate) on the
biomass accumulation and cardiotonic glycosides production
byD. lanatashoots. It was found that the addition of Chitoplant
or Silioplant could lead to a 2.2-fold increase in the lanatoside
C content, when compared to the nonelicited shoots [72]. The
authors concluded that the combination of Twin-Flask TIS
and elicitation could be a useful strategy for enhancing the
production of cardiotonic glycosides byD. lanatashoots [72].
5 TIS in molecular farming
Plants have been considered as excellent platforms for pro-
duction of valuable recombinant proteins. The interest in the
industrial process of foreign protein expression using whole-
plants or their in vitro cultured cells, tissue, or organs, known
also as molecular farming has grown rapidly in the past two
decades [73]. However, product yields in field-grown transgenic
plants can be highly variable due to environmental impacts,
and the harvested material has a limited shelf life and must be
processed immediately after harvest, which may have ecological
implications for disposal of transgenic biomass waste [73]. Most
of the listed disadvantages could be overcome by using plant in
vitro technique [73, 74]. Several bioreactor configurations have
been adapted for in vitro production of foreign proteins by dif-
ferent plant cells, tissue, and organ cultures [74]. Among them,
TIS have been considered as the most potential cultivation sys-
tems for differentiated shoots and hairy roots cultures [5, 74].Recently, 2 L Ebb-and-Flow TIS have been used as platforms for
expression of a modified form of the green fluorescent protein
and, a vaccine antigen, fragment C of tetanus toxin by tobacco
(N. tabacumcv. Petit Havana) transplastomic shoots [75]. The
authors calculated that 60 Ebb-and-Flow TIS of 10 L, accom-
modated in 30 m2 growth room, could produce about 3.5 kg of
green fluorescent protein and 0.5 kg of fragment C of tetanus
toxin for 1 year, whereas to produce the same amounts in trans-
genic tobacco whole-plants, a floor area of 1800 m 2 of a level
II biosafety greenhouse would be required [75]. Recently, RITA
TIS have been used to study the expression of a bacterial outer
surface protein A from Borrelia burgdorferiby transformed to-
bacco (N. tabacum cv. Petit Havana) chloroplasts [76]. After
40 days of cultivation at 4 min immersion and 8 h exposure
periods, a maximum yield of outer surface protein A of about
108 mg/L was achieved [76]. The authors pointed that TIS en-
sure absolute containment of transgenic material and could be
used for large-scale propagationof transplastomic plant material
expressing proteins toxic to the host plant [76].
6 Other applications of TIS in plantbiotechnology
TIS are automated platforms for controlled short-time contact
of theplant explants witha liquid mediumin an aseptic environ-
ment, which could be a particularly interesting option in termsofAgrobacterium-mediated genetic transformation techniques.
Thetechnological advantages of RITA TIShave been used forob-
taining a transgenic strawberry (Fragaria ananassa) by genetic
transformation with Agrobacterium tumefaciensLBA4404 har-
boring the pCAMBIA1391Z vector with hygromycin selectable
marker [77]. It has been found that strawberries are sensitive
to cefotaxime (antibiotic used to kill Agrobacteriumafter trans-
formation) and the explants rarely survive after the treatment.
Cultivation in RITA TIS with short immersion (10 s for every
4 h) with nutrient medium supplied with high concentration of
cefotaxime (200 mg/L) was applied after cocultivation to over-
come that problem [77]. After the complete bacterial removal,
the culture medium was replaced with selective medium sup-
plied with 10 mg/L hygromycin, the explants were cultivated for
10 days at the same immersion regime and then the concentra-
tion of hygromycin was increased to 15 mg/L for selecting only
the transformed explants. After the selection phase, the trans-
formed explants were left in the RITAs culture chamber and the
medium was replaced with medium for regeneration. The culti-
vation continued at the same immersion regime until vigorous
shoot formation and visible roots were observed [77]. Recently,
RITA TIS have been applied in the selection of transformed
somatic embryos ofQuercus roburL. after transformation with
A. tumefaciensEHA105:p35SGUSINT (containingthe neomycin
phosphotransferase II [nptII] and the intron-containing uidA
reporter (GUS) genes) [78]. After transformation and bacterial
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removal, a two-step selection procedure involving cultivation of
explantsinRITATISoperatedatshortimmersion(1minatevery
12 h) in liquid medium supplemented with 25 mg/L kanamycin,
and then with 75 mg/L kanamycin, was applied [78]. It was
found that the transformation frequency by the new selection
procedure was five times higher than that achieved with theconventional protocol (a selection on semisolid medium) [78].
Moreover, by using TIS in selection, the transgenic lines were
established 1216 wk earlier than in the selection performed on
a semisolid medium [78]. The temporary immersion RITA sys-
tem has also been used to prevent the explants viability and
to protect young hairy roots formation after Agrobacterium
rhizogenesATCC 15834 genetic transformation of balsamic sage
(Salvia tomentosaMill.) [79]. It was found that the phenolics
released by the woundedS. tomentosaexplants had lethal effects
on the explants themselves, as well as on the newly formed hairy
roots. To overcome that negative action, after the genetic trans-
formation the explants were cultivated in RITA TIS (operated
at 15 min immersion and 12 h exposure periods). The nutrient
medium storage compartment was loaded with liquid medium
and a package with thepolymeric resin Amberlite XAD-4, which
plays the role of a second phase, and adsorb the released pheno-
lic compounds [79]. Cultivated under these conditions, 100% of
the explants gave rise to fast growing transformed hairy roots at
the end of the second week [79].
Phytoremediation is another perspective field for application
of TIS. It is well known that plants can extract and accumulate
heavy metalsfrom thesoil [80,81]. Plant roots play an important
role in that process [82]. Recently, it has been shown that the
phenol removal efficiency of sunflower (Helianthus annuusL.)
hairy roots ranges from 99 to 63% when exposed to varying
concentrations of phenol (100400 mg/L)[83]. In another study,
it has been demonstrated that the phenol removal efficiencyof hairy roots depends on the plant species [84]. The authors
have shown that rapeseed (Brasicca napusL.) hairy roots have
better potential for phenol remediation than tomato (Solanum
lycopersicon cv. Pera) hairy roots [84]. Moreover, the addi-
tion of PEG may significantly enhance the phenol removal
efficiency of rapeseed hairy roots (up to 9888%) [84]. More
detailed information about the potential of transformed
hairy root cultures to remove toxic compounds could be
found elsewhere [82, 85]. Since TIS are considered as one
of the best bioreactor systems for scale-up the cultivation
of transformed hairy root cultures, their adaptation for the
needs of industrial phytoremediation remains to be done.
Moreover, recently successful application of TIS (RITA system)
for decoloration of the recalcitrant anthraquinonic textile
dye C.I. Reactive Blue 19 by using solid-state fermentation
with white rot fungus (Trametes pubescens MB89) grown on
sunflower seed shells [86] has been demonstrated. The author
has pointed the RITA system as very promising for scaling
up the process of detoxification of reactive industrial dyes
[86].
7 Scale-up and automation of TIS
The temporary immersion technology is generally based on uti-
lization of small-to-medium size inexpensive cultivation vessels.
Although the volume of culture vessels could be increased up
to 10 or 20 L, it has been found that this approach cannot im-
prove the overall system performance. Pilot scale cultivation of
somatic embryos of Robusta (C. canephoravar.robusta) in 10 L
Ebb-and-Flow TIS jars has shown that most of the embryos
do not have normal development because of the nonuniformlight distribution inside of the vessel [32]. With the increase of
the seedlings biomass, light becomes a rate-limiting factor dur-
ing the culture, since it cannot penetrate into the center of the
culture vessel [31]. Moreover, with the increase of the biomass
density in the larger culture vessels, more thickened zones could
appearthat may generate significantresistance to themass trans-
fer of oxygen and nutrients [8,10]. It is obvious that the increase
in the size and volume of the cultivation vessel is not the most
effective way to achieve a reasonable scale-up in a case of TIS.
For that reason a different strategy, based on simultaneous op-
eration of huge numbers of small-to-medium size single TIS
apparatus, is applied to achieve large-scale production. The TIS
are usually accommodated in racks with several shelves and in-
tegrated light sources hosted in air-conditioned growth rooms.
The racks should be arranged in an appropriate way to secure
easy access for operation and handling of the single vessels, and
to accommodateas many TISas possible, which will improve the
production cost per unit space in the growth room. The single
TIS are usually connected to a common central automated sys-
tem to control their simultaneous work. If the operation of all
TIS involves a cycle with identical durations of immersion and
exposure periods, a semiautomated system, controlled by a sim-
ple timer clock, could be used. However, if the different TIS have
to be operated at different immersion or exposure periods, or
the cultivation requires forced ventilation or carbon dioxide en-
richment, then a fully automated computerized system operated
with appropriate software should be used [87].
8 Concluding remarks
The efficiency of TIS for micropropagation of commercially im-
portant crops is unquestionable. TIS have potential application
for the production of plant-derived secondary metabolites with
high added value. The potential of transformed hairy roots in
combinationwith thefeatures of TISmaybe combined fordevel-
opment of local biological installations for treatment of phenol-
contaminated waste waters. However, the full potential of the
temporary immersion technology for phytoremediation of in-
dustrial wastes is still to be revealed. The operational principle
and the option for full control of the contact between the cul-
tured explants and the liquid medium make TIS an attractive
technique for improving existing protocols for genetic transfor-
mation of plants. Because of the absolute containment of culti-
vated explants from the surrounding environment, TIS could be
considered also as excellenteco-friendly platforms for large-scale
productionof valuable recombinant proteins by transgenic plant
tissue. However, the scale-up of temporary immersion technol-
ogy is closely related with the occupation of considerable area
in the air-conditioned growth rooms, which may raise the pro-
duction cost per unit space. Many of the newly developed TIS,
especially the low-cost and disposable variants, could contribute
to the effective solution of that problem.
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Practical application
This review summarizes the recent progress in applica-tion of temporary immersion technology for laboratory
and large-scale cultivation of plant tissue and organ cul-tures. Principle operation and technological implementa-tion of various temporaryimmersionsystems aredescribedin detail. Recent examples of the application of tempo-rary immersion technology in micropropagation, produc-tion of secondary metabolites, molecular farming, genetictransformation, clonal selection, and phytoremediation arediscussed. This review could be useful for scientists, re-searchers, and students focusing their work on in vitromanipulation and cultivation of plant tissue and organ cul-tures.
The authors have declared no conflict of interest.
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