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

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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;


chamber;

Sampling port

Complex construction;



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;



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 forced ventilation and CO2enrichment.

High energy costs



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Table 1.Continued


materials


BioMINT Mechanical Polycarbonate Autoclavable Simple design;

Cultivation boxes could be stack onracks;

Full separation of explants from

liquid medium;

Easy to handle;


Option for forced ventilation and

CO2enrichment.


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;


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 forced ventilation and CO2enrichment.

Occupies more space in growth

chamber

Bioreactor RALM Pneumatic Polycarbonate

and

polypropylene

Autoclavable Easy to handle;


CO2enrichment;



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


No forced ventilation and CO2enrichment;


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


elements;



PLANTFORM

bioreactor

Pneumatic

and gravity

Polycarbonate Autoclavable Simple automation;

Reliable operation;

Easy to handle;


chamber;

Improved access to light;


other to save space.


CO2enrichment;



elements;




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Table 1.Continued


materials


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;


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;

No full separation of explants from

liquid medium;

Equal times for immersion and

exposure periods;


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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