Georgiev Et Al-2014-Engineering in Life Sciences-10-DR3

Eng. Life Sci.
2014, 14, 607621
Vasil Georgiev1
Anika Schumann2
Atanas Pavlov3,4
Thomas Bley5
1
Center for Viticulture and Small

Fruit Research, Florida A & M
University, Tallahassee, FL, USA
Vita 34 AG, Leipzig, Germany
Department of Analytical
Chemistry, University of Food
Technologies, Plovdiv, Bulgaria
Laboratory of Applied
Biotechnologies, The Stephan
Angeloff Institute of
Microbiology, Bulgarian
Academy of Sciences, Plovdiv,
Bulgaria
Institute of Food Technology

and Bioprocess Engineering,
Technische Universitt Dresden,
Dresden, Germany
www.els-journal.com
Review
Temporary immersion systems in plant

biotechnology
Plant tissue and organ cultures in vitro usually face technological challenges. When
submerged cultivation of plant cells in a controlled environment is desired, the
characteristic growth morphology and physiology of differentiated organ cultures
present a problem in process scale-up. Temporary immersion systems (TIS) were
developed several decades ago. These systems are providing the most natural environment 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 on
similar or divergent technological principles, have been developed and successfully
applied in the cultivation of various plant in vitro systems, including somatic embryos and transformed root cultures. In this article, the operational principle and
technological design of the most popular TIS are reviewed. In addition, recent examples of the application of temporary immersion technology for in vitro cultivation
of plant tissue and organ cultures at laboratory and pilot scales are discussed. Finally,
future prospects and challenges to the industrial realization of that fast-developing
technique 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
Introduction
Plant cells, tissue, and organ cultures have been recognized as

powerful tools for clonal propagation of commercially important
crops (micropropagation), production of valuable secondary
metabolites, expression of complex foreign proteins (molecular
farming), as well as for phytoremediation of waste waters (phytotransformation 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 liquid
media, under controlled environmental conditions in bioreactor 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 (vasgeorgiev@gmail.com),
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, temporary immersion systems

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2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
equipment, designed for intensive culture by regulating various

nutritional and/or physical factors [1]. Bioreactor systems usually 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 microenvironmental conditions, nutrients, and gaseous mass transfers.
The automated 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
overlay gaseous environment, and the level of the liquid 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 bioreactors (including mechanically agitated, pneumatically agitated,
hydraulically agitated, and membrane bioreactors) are currently
the best studied systems, revealing almost unlimited potential
for application in 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 organ cultures into the liquid medium often causes malformations
and loss of material due to asphyxia and hyperhydricity [3].
Asphyxia and hyperhydricity are undesirable physiological conditions, 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 existing difficulties, gas-phase bioreactors [6, 7], TIS, and hybrid
bioreactors [810] have been developed. TIS are simple automated systems, designed to provide optimal environment, improved 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 variations 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 secondary metabolite production, molecular farming, and even in
phytoremediation of toxic compounds [5]. The technical realization 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 are
pointed out as well.
Design and operation of TIS
The development of TIS is closely related with the commercialization of plant micropropagation. TIS are periodic semiautomated or fully automated cultivation systems, based on alternating 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
period is shorter (a few minutes), whereas the air exposure period
is prolonged (several hours). The precise adjustment of the durations of the immersion and exposure periods may significantly
reduce the hyperhydricity of the cultured plant tissue by creating
conditions for optimal humidity and nutrients supply with minimal liquid contact [14]. The direct exposure of the plant tissue to
the gaseous environment significantly simplifies the interphase
oxygen transport from the gas to the cultured cells, 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
with CO2 during the gas exposure period. The higher level of CO2
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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,
TIS do not utilize mechanical agitation devices, thus the dislocation of the cultured propagules, if any, is done only by the power
of hydrodynamic forces during immersion periods. Under these
conditions, the cultivated plant tissues undergo minimal shear
stress that preserves the culture integrity and additionally improves the morphology and physiology of the organs. TIS are
usually constructed with transparent 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
Temporal) is
The Twin-Flask system (Biorreactores de Inmersion
one of the earliest developed TIS [13,18,19]. Basically, the TwinFlask system consists of two containers (wide-mouth flasks, bottles, 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 container is used as a medium storage tank. The culture chamber
container may or may not be equipped with support 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 independent timer clocks, coupled with three-way solenoid valves.
The simple and reliable design makes Twin-Flask systems favorable for many laboratories. They are generally easy to operate
and the construction can maintain sterility for long periods of
cultivation [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 CO2 concentrations in the gaseous environment of the culture chamber [17,28].
Twin-Flask systems have been successfully applied in the propagation 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 ventilation
and CO2 enrichment;
Low headspace humidity in growth
chamber;
No nutrient medium renewal
Ebb-and-Flow
Pneumatic
and gravity
Glass
Autoclavable
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 CO2 enrichment
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 CO2
enrichment
Thermo-photobioreactor
Pneumatic
and gravity
Pyrex glass
Autoclavable
Complex construction;
enrichment
Hybrid
Ebb-and-Flow
with saturated
tubular
convective flow
Hydraulic
and
pneumatic
Glass, stainless
steel
Autoclavable
Bioreactor of
Immersion by
bubbles
Pneumatic
and gravity
Glass, stainless
steel
Autoclavable
Precise temperature control;

Integrated light source;
Simple automation;
chamber;
Sampling port
Designed for high-density hairy root
cultures;
Fully automated;
Uniform distribution of root
biomass;
Improved oxygen transfer
Better utilization of growth chamber
space;
Low share stress;
Better gas exchange
Rocker systems
Mechanical
Polycarbonate
Autoclavable

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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
Designed only for hairy root cultures;

Complex design;
Two stage operation;
Difficult for biomass harvesting
Requires the presence of detergent

into nutrient medium;
Uncontrolled time for drainage;
Expensive;
enrichment
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;
enrichment.
High energy costs
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Table 1. Continued
TIS
Power input
Construction
materials
Sterilization
Pros
Cons
BioMINT
Mechanical
Polycarbonate
Autoclavable
Requires tilting platforms;

chamber;
High energy costs
Rotating drum
Mechanical
Glass or plastic
Autoclavable
Simple design;
Cultivation boxes could be stack on
racks;
Full separation of explants from
liquid medium;
Easy to handle;
Option for forced ventilation and
CO2 enrichment.
Ready for large-scale process
Low investment costs;
Suitable for embryo, shoots, and
hairy root cultures;
Easy to handle
Bioreactor RALM
Pneumatic
Polycarbonate
and
polypropylene
Autoclavable
Easy to handle;
CO2 enrichment;
SETIS
Pneumatic
and gravity
Polypropylene
Autoclavable;
Gamma
irradiation
No internal elements;
Easy to handle;
Simplified automation;
Large illuminated area;
Improved drainage;
Low energy costs.
Optimal usage of growth room space

enrichment;
PLANTIMA
Pneumatic
and gravity
Polycarbonate
Autoclavable
Simple automation;
Reliable operation;
Easy to handle;
chamber;
Apparatus may be stacked one on the
other to save space. Low investment
costs
Construction with several internal

elements;
enrichment
PLANTFORM
bioreactor
Pneumatic
and gravity
Polycarbonate
Autoclavable
Simple automation;
Reliable operation;
Easy to handle;
chamber;
Improved access to light;
other to save space.
CO2 enrichment;

elements;
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Requires rotating platform;

liquid medium;
No control of times for immersion
and exposure;
No air exchange;
enrichment.
chamber
Complex automation;
elements;
Low headspace humidity in growth
chamber
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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;
other during transportation;
Disposable;
Two levels assembly requires more

space;
Time for drainage is increased with
the culture growth;
Low diameter of inoculation port;
enrichment
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;
Low labor costs
Requires specialized and expensive

control module and tilting platform;
Low diameter of inoculation port;
liquid medium;
Equal times for immersion and
exposure periods;
High investments costs
Figure 1. Technological design and operational principle of TwinFlask system: (A) period of exposure. The whole volume of liquid
medium is located into the medium storage tank. Air lines of both
containers are closed and the solenoid valves are opened to atmosphere; (B) dislocation of liquid medium from medium storage
tank to culture chamber. The air line of cultivation chamber is
closed, and the air line of medium storage tank is opened. The
overpressure moves the medium into the cultivation chamber;
(C) period of immersion. The propagules are immersed into the
liquid medium. The medium storage tank is empty. Air lines for
both containers are closed and the solenoid valves are opened
to atmosphere; (D) draining out the nutrient medium back to
the culture medium tank. The air line of cultivation chamber is
opened, whereas the air line of medium storage tank is closed.
The overpressure moves back the medium into the medium storage tank.

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Figure 2. Technological design and operational principle of Ebband-Flow system: (A) period of exposure; (B) dislocation of liquid medium. Air pressure is applied to the medium storage
tank 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 the
medium storage tank due to gravity.
period and has the function of an air sparger during the immersion 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 RITA

system: (A) period of exposure; (B) Dislocation of liquid medium.
Air pressure is applied to the bottom compartment through the
central pipe. The liquid medium is moving to the upper compartment; (C) period of immersion; (D) draining out the nutrient
medium. The air flow is stopped and the medium flows back to
the bottom compartment due to gravity.
vessel and the lack of options for forced ventilation and CO2
enrichment are among the main disadvantages of the system.
2.3
Figure 4. Technological design and operational principle of

thermo-photo-bioreactor TIS: (A) period of exposure; (B) dislocation of liquid medium. Air is supplied to medium storage tank
and medium is moving to the culture chamber; (C) period of immersion; (D) draining out the nutrient medium. The air supply
was stop and the medium was drained out back by the gravity.
RITA system
2.4
The RITA (recipient a` immersion temporaire automatique) TIS

(CIRAD, France, distributed by VITROPIC, France) have been
developed for intensive in vitro plant culture. The system consists 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 the vessel is the culture chamber, whereas
the bottom compartment is the medium storage tank. The advantages of the RITA TIS are the simple and reliable operation,
the compact space for apparatus accommodation, and the support 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 disadvantages of the systems are the inability for nutrient medium
renewal and the lack of options for forced ventilation and CO2
enrichment.
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Thermo-photo-bioreactor TIS
This TIS bioreactor has been developed exclusively for micropropagation and secondary metabolite production of Antarctic
hair grass (Deschampsia antarctica E. 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 temperature 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, none of them can compete with

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Figure 5. Technological design and operational principle of hybrid Ebb-and-Flow with saturated tubular convective flow: (A) operation in
bubble column mode until uniform distribution and immobilization of the transformed hairy roots is achieved; (BE) operation in temporary
immersion mode similar to that of Twin-Flask system (see text for details).
the precise temperature control of thermo-photo-bioreactor

TIS.
2.5
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.
Hybrid Ebb-and-Flow with saturated tubular

convective flow
2.6
The Ebb-and-Flow with saturated tubular convective flow was

exclusively developed for cultivation of high-density hairy root
cultures [10]. This system is hybrid bioreactor, acting as a bubble
column for the first days after inoculation, and then switches over
to operation as a Twin-Flask system (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, the reactor starts to operate as a Twin-Flask TIS (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
(2 min each) and the pump speed is set up to ensure full medium
dislocation from one vessel to the other for 1 min, so that a tubular 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
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Bioreactor of immersion by bubbles
The bioreactor of immersion by bubbles (BIBBiorreator de

Imersao por Bolhas) utilizes a completely new cultivation strategy, 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 (170220 m pores) plate (Fig. 6). The upper compartment is the growth chamber, in which a few stainless steel internal racks are stacked one upon another to support the 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 BIB provides better growth and higher shoot number
per explant than RITA TIS in propagation of tea-tree (Melaleuca
alternifolia) and orchid (Oncidium leucochilum) [37, 38]. However, the presence of detergent in the nutrient medium, as well
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cylindrical autoclavable polycarbonate vessels that are joined together 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 propagules in place when the bioreactors change position. The adaptor
also has two external ports that allow the application of forced
ventilation or CO2 enrichment [42]. Because of its flexible construction and easy handling, BioMINT bioreactors are popular
in shoots propagation [43, 44].
2.8
Figure 6. Technological design and operational principle of bioreactor 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 bubbles. When aeration stops, the foam density decreases with time
due to liquid drainage and the explants are exposed to gaseous
environment.
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 or
hairy 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
as the prolonged time for liquid drainage may restrict the application 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 lateral 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 accommodated on one rack and no additional connection to an
airline is necessary. The disadvantages of rocker systems are related with the necessity of an electromechanically driven tilting
platform that increases the investment and energy costs. Tilting 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 replacement exist. Some of these problems could be overcome by using
the BioMINT bioreactors as culture vessels on a rocker platform.
The BioMINT is a mid-sized (1.2 L) bioreactor, consisting of two
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Rotating drum system
Low-cost and disposable TIS
In an attempt to decrease the initial investment costs for equipment and to save space and labor, several TIS have been developed
and 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 Industria e Comercio ltda., Brazil) is a TIS, operating on the TwinFlask 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 Bioscientific Co., Ltd., Taiwan; Fig. 9C) is a
small volume TIS, operated on the RITA principle and has been
used for plantlet propagation [47, 48]. Another TIS, using the
principle of operation of the RITA system, are the PLANTFORM
bioreactor (Plant Form AB, Sweden & TC propagation Ltd.,
Ireland; Fig. 9D). Box-in-Bag (Fig. 9E) is a disposable TIS, operating 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].
TIS in micropropagation
Micropropagation on semisolid nutrient medium is a costly process, since the technology is based on manual handling of a

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Figure 7. Technological design

and operational principle of
(A, B) rocker TIS and (C, D)
BioMINT bioreactor.
Figure 8. Technological design

and operational principle of rotating drum bioreactor system.
large number of single containers [1, 52]. Labor accounts for

4060% of the final cost of propagated plants, and the technology is not subject to automation [1, 12]. To overcome those
negatives, automated TIS have been developed in order to reduce 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 canephora var.
robusta) by cultivating somatic embryos in Ebb-and-Flow TIS
has been reported [32]. The authors reported an annual production 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 improving the embryo growth ex 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
than those 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
higher when TIS (RITA system) are used. The authors have found
that the addition of the cytokinins metatopolin and benzylade
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nine has a beneficial effect on plant regeneration [53]. A similar

positive effect of metatopolin on plant regeneration has also been
reported in the micropropagation of plantain (Musa spp.) plants
in TIS (Twin-Flasks) [54]. More detailed information concerning the practical application of TIS in plant micropropagation
could be found elsewhere [1, 12, 13, 19].
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 compartmentally 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 cultures in liquid medium is closely associated with the availability
of specially designed bioreactor systems [5, 49]. Because of their
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Eng. Life Sci. 2014, 14, 607621
Figure 9. Technological design of (A) RALM bioreactor, (B) SETIS

temporary immersion bioreactor system, (C) PLANTIMA system,
(D) PLANTFORM bioreactor, (E) Box-in-Bag, and (F) WAVE bioreactor.
reasonable price, excellent performance, flexible operation,

and easier handling, TIS could be considered as very suitable
platforms for production of secondary metabolites by differentiated 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 of Beta vulgaris cv. 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 stramonium L. 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]. The option allowing the manipulation of the secretion level
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of secondary metabolites only by changing the durations of the

immersion or exposure periods may be a useful opportunity for
the development of a milking process, based on the continuous
recovery of metabolites released in the culture medium. Recently,
RITA TIS have been used in the study of secoiridoid glycosides
accumulation by Centaurium maritimum L. Fritch transformed
hairy 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 min exposure periods. The achieved yield was
10 times higher than the one registered for a culture fully submerged 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 by Panax
ginseng C.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 transgenic Nicotiana
tabacum L. cv. Petit Havana SR1 hairy roots, harboring the
VoGES gene (a geraniol synthase gene from Valeriana 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 additions
of 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 production of Amaryllidaceae alkaloids by sea daffodil (Pancratium
maritimum L.) shoot culture [67]. In optimal cultivation conditions (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. maritimum L. shoot culture.

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Another study demonstrated that the duration of the immersion

period had not a significant effect on the intracellular and
extracellular production of galanthamine (another important
Amaryllidaceae alkaloid) by a shoot culture of summer snowflake
(L. aestivum L.) cultivated in RITA TIS [68]. It was found that
the duration of the immersion period had more dramatic effect
on the biomass accumulation by in vitro cultivated L. aestivum
shoots [68]. Further research demonstrated that the alkaloid
pattern of L. aestivum shoots cultivated in RITA TIS could be
significantly affected by the temperature at which the cultivation
was performed [68, 69]. Moreover, the levels of Amaryllidacea
alkaloids produced by L. aestivum shoots could be additionally
increased by using an appropriate elicitation strategy [65, 70].
Twin-Flask TIS have also been adapted for in vitro cultivation
of L. aestivum, and its potential to stimulate galanthamine production 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 purpurea L. 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 DW per 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 of Digitalis lanata Ehrh. 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
by D. lanata shoots. 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 by D. lanata shoots [72].
TIS in molecular farming
Plants have been considered as excellent platforms for production of valuable recombinant proteins. The interest in the
industrial process of foreign protein expression using wholeplants 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

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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 different plant cells, tissue, and organ cultures [74]. Among them,
TIS have been considered as the most potential cultivation systems 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. tabacum cv. Petit Havana) transplastomic shoots [75]. The
authors calculated that 60 Ebb-and-Flow TIS of 10 L, accommodated 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 transgenic tobacco whole-plants, a floor area of 1800 m2 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 burgdorferi by transformed tobacco (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 ensure absolute containment of transgenic material and could be
used for large-scale propagation of transplastomic plant material
expressing proteins toxic to the host plant [76].
Other applications of TIS in plant

biotechnology
TIS are automated platforms for controlled short-time contact

of the plant explants with a liquid medium in an aseptic environment, which could be a particularly interesting option in terms
of Agrobacterium-mediated genetic transformation techniques.
The technological advantages of RITA TIS have been used for obtaining a transgenic strawberry (Fragaria ananassa) by genetic
transformation with Agrobacterium tumefaciens LBA4404 harboring the pCAMBIA1391Z vector with hygromycin selectable
marker [77]. It has been found that strawberries are sensitive
to cefotaxime (antibiotic used to kill Agrobacterium after transformation) 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 overcome that problem [77]. After the complete bacterial removal,
the culture medium was replaced with selective medium supplied with 10 mg/L hygromycin, the explants were cultivated for
10 days at the same immersion regime and then the concentration of hygromycin was increased to 15 mg/L for selecting only
the transformed explants. After the selection phase, the transformed explants were left in the RITAs culture chamber and the
medium was replaced with medium for regeneration. The cultivation 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 of Quercus robur L. after transformation with
A. tumefaciens EHA105:p35SGUSINT (containing the 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

explants in RITA TIS operated at short immersion (1 min at every
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 the
conventional 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 system has also been used to prevent the explants viability and
to protect young hairy roots formation after Agrobacterium
rhizogenes ATCC 15834 genetic transformation of balsamic sage
(Salvia tomentosa Mill.) [79]. It was found that the phenolics
released by the wounded S. tomentosa explants had lethal effects
on the explants themselves, as well as on the newly formed hairy
roots. To overcome that negative action, after the genetic transformation 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 the polymeric resin Amberlite XAD-4, which
plays the role of a second phase, and adsorb the released phenolic 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 metals from the soil [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 annuus L.)
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 efficiency
of hairy roots depends on the plant species [84]. The authors
have shown that rapeseed (Brasicca napus L.) hairy roots have
better potential for phenol remediation than tomato (Solanum
lycopersicon cv. Pera) hairy roots [84]. Moreover, the addition 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].
Scale-up and automation of TIS
The temporary immersion technology is generally based on utilization of small-to-medium size inexpensive cultivation vessels.
618
Although the volume of culture vessels could be increased up

to 10 or 20 L, it has been found that this approach cannot improve the overall system performance. Pilot scale cultivation of
somatic embryos of Robusta (C. canephora var. robusta) in 10 L
Ebb-and-Flow TIS jars has shown that most of the embryos
do not have normal development because of the nonuniform
light distribution inside of the vessel [32]. With the increase of
the seedlings biomass, light becomes a rate-limiting factor during 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
appear that may generate significant resistance to the mass transfer 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 operation 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 integrated 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 accommodate as many TIS as 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 system 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 simple 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 enrichment, then a fully automated computerized system operated
with appropriate software should be used [87].
Concluding remarks
The efficiency of TIS for micropropagation of commercially important 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
combination with the features of TIS may be combined for development of local biological installations for treatment of phenolcontaminated waste waters. However, the full potential of the
temporary immersion technology for phytoremediation of industrial wastes is still to be revealed. The operational principle
and the option for full control of the contact between the cultured explants and the liquid medium make TIS an attractive
technique for improving existing protocols for genetic transformation of plants. Because of the absolute containment of cultivated explants from the surrounding environment, TIS could be
considered also as excellent eco-friendly platforms for large-scale
production of valuable recombinant proteins by transgenic plant
tissue. However, the scale-up of temporary immersion technology is closely related with the occupation of considerable area
in the air-conditioned growth rooms, which may raise the production 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 application of temporary immersion technology for laboratory
and large-scale cultivation of plant tissue and organ cultures. Principle operation and technological implementation of various temporary immersion systems are described
in detail. Recent examples of the application of temporary immersion technology in micropropagation, production of secondary metabolites, molecular farming, genetic
transformation, clonal selection, and phytoremediation are
discussed. This review could be useful for scientists, researchers, and students focusing their work on in vitro
manipulation and cultivation of plant tissue and organ cultures.
The authors have declared no conflict of interest.
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Eng. Life Sci.

2014, 14, 607621

Center for Viticulture and Small

Vita 34 AG, Leipzig, Germany

Institute of Food Technology

Temporary immersion systems in plant

Plant cells, tissue, and organ cultures have been recognized as

2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

equipment, designed for intensive culture by regulating various

Eng. Life Sci. 2014, 14, 607621

cultures [2]. However, in most cases, liquid-phase bioreactor

Design and operation of TIS

may have positive effects on the multiplication of the cultivated

Ebb-and-Flow systems could be described as a simplified

2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Eng. Life Sci. 2014, 14, 607621

Table 1. General features, advantages, and disadvantages of most common TIS

Two levels assembly requires more

No nutrient medium renewal;

Precise temperature control;

2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Designed only for hairy root cultures;

Requires the presence of detergent

Eng. Life Sci. 2014, 14, 607621

Requires tilting platforms;

No forced ventilation and CO2

Construction with several internal

Construction with several internal

Requires rotating platform;

2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Eng. Life Sci. 2014, 14, 607621

Two levels assembly requires more

Requires specialized and expensive

2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Eng. Life Sci. 2014, 14, 607621

Figure 3. Technological design and operational principle of RITA

Figure 4. Technological design and operational principle of

The RITA (recipient a` immersion temporaire automatique) TIS

2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Eng. Life Sci. 2014, 14, 607621

the precise temperature control of thermo-photo-bioreactor

tivation of high-density root biomass. The main disadvantages

Hybrid Ebb-and-Flow with saturated tubular

The Ebb-and-Flow with saturated tubular convective flow was

2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Bioreactor of immersion by bubbles

The bioreactor of immersion by bubbles (BIBBiorreator de

Eng. Life Sci. 2014, 14, 607621

The system consists of a roller apparatus and an autoclavable

Rocker systems use a mechanical platform to tilt the cultured

Rotating drum system

Low-cost and disposable TIS

2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Eng. Life Sci. 2014, 14, 607621

Figure 7. Technological design

Figure 8. Technological design

large number of single containers [1, 52]. Labor accounts for

2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

nine has a beneficial effect on plant regeneration [53]. A similar

TIS in secondary metabolite production

The commercialization of the plant in vitro technology could

Eng. Life Sci. 2014, 14, 607621

Figure 9. Technological design of (A) RALM bioreactor, (B) SETIS

reasonable price, excellent performance, flexible operation,