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Large Scale Cultivation of Plant Cell and Tissue Culture in Bioreactors, 2010: 1- 54
ISBN: 978-81-7895-474-5 Authors: Ozlem Yesil-Celiktas, Aynur Gurel and Fazilet Vardar-Sukan
Large scale cultivation of
plant cell and tissue culture in bioreactors
Ozlem Yesil-Celiktas, Aynur Gurel and Fazilet Vardar-Sukan
Department of Bioengineering, Faculty of Engineering, Ege University
35100 Bornova-Izmir, Turkey
1. Introduction
Bioactive compounds extracted from plants are widely used. The natural
habitats for a large number of plants are rapidly destroyed leading to
extinction of many valuable and even endemic species. Studies on the
production of plant metabolites by callus and cell suspension cultures have
been carried out on an increasing scale since the end of the 1950's. The
prospect of using such culturing techniques is for obtaining secondary
metabolites, such as active compounds for pharmaceuticalss and cosmetics,
hormones, enzymes, proteins, antigens, food additives and natural pesticides
from the harvest of the cultured cells or tissues.
The large scale cultivation of tobacco and a variety of vegetable cells was
examined from the late 1950's to early 1960's initiating more recent studies
on the industrial application of plant cell culture techniquesin many countries.
The first patent for the cultivation of plant tissue was received in 1956.
Shortly afterwards, the National Aeronautics and Space Administration
(NASA) started to support research in the field of plant cell cultures for
regenerative life support systems (Sajc et al., 2000).
Correspondence/Reprint request: Dr. Ozlem Yesil-Celiktas, Department of Bioengineering, Faculty of
Engineering, Ege University, 35100 Bornova-Izmir, Turkey. E-mail: [email protected]
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Ozlem Yesil-Celiktas et al.
In paralel to these achievements, industrial companies in Japan have tried
to apply this technology for the commercial production of useful compounds
in collaboration with some university groups. For instance, The Japan
Tobacco Inc.'s interest involved around mass-production of tobacco cells as
raw materials of cigarettes. Meiji Seika in Japan also elucidated the
fundamentals of production of Panax ginseng cells in large volumes. The
work was followed by Nitto Denko Co. which started manufacturing cell
mass of ginseng commercially. Other firms such as Ajinomoto and Nippon
Shin-yaku also made efforts to increase the level of accumulation of
alkaloids, steroids and other secondary products in cultured cells (Misawa,
1994). Consequently, plant cell culture techniques have been used increasingly
in basic research to improve cognitive interrelations and finally for
exploitation in industry.
The field of plant cell culture covers diverse sub-fields providing
alternative approaches which may be attractive under certain circumstances;
if the source plant:
•
•
•
•
is difficult to cultivate,
has a long cultivation period,
produces a commercially significant compound that can not be
chemically synthesized in large scale,
has a low metabolite of the compound of interest.
Furthermore, in some cases mass cultivation of the source plant may not
be possible under natural conditions due to environmental, ecological or
climatic conditions and/or insufficient agricultural lands may limit the
cultivation processes. Secondary metabolites can be produced by plant cell
and tissue culture techniques under controlled and reproducible conditions,
independent of geographic and climatic conditions.
Biotechnological cultivation of plant cells and tissues involve two major
methodologies namely, cell culture studies and clonal propagation
techniques. Cell culture studies begin with callus initiation using in vitro
cultures for the purpose of determining the medium that best adapts for
cultivation. When calli are obtained, they can undergo somaclonal
variation, usually during several subcultures (Gurel, 1989). When genetic
stability is reached, callus lines need to be screened in order to evaluate the
productivity of each cell line so that the best performing lines can be taken
to cell suspensions. Various approaches can be used to increase the
production of secondary metabolites in cell suspensions but elicitation is
usually one of the most succesful. The final step is the bioreactor studies
leading to a possible commercial production of secondary metabolites. This
Large scale cultivation of plant cell and tissue culture in bioreactors
3
is a critical step as various problems can arise when scaling-up from shake
flasks to bioreactors.
However bioreactors act as a biological factory for the production of
high-quality products and provide many advantages listed as follows
(Tautorus et al. 1991; Fulzele, 2000; Su, 2006).
•
•
•
•
•
•
•
•
•
•
controlled supply independent of plant availability,
increased working volumes,
homogeneous culture due to mechanical or pneumatic stirring
mechanism,
better control of cultural and physical environment, therefore easy
optimization of growth parameters such as pH, nutrient media,
temperature, etc. for achieving metabolite production,
reproducible yields of end product under controlled growth conditions,
enhanced nutrient uptake stimulating multiplication rates and yielding a
higher concentration of yield of bioactive compounds,
simpler and faster harvest of cells,
the opportunity to perform biosynthetic and/or biotransformation
experiments related to metabolite production with enzyme availability.
easier separation of target compounds because of lower complexity of
extract,
better control for scale-up,
Successful operation of these bioreactors often requires the
implementation of aeration technologies which constantly need to be
improved. Glass and stainless steel bioreactors incorporating impellers are
often used for the production of cell and/or tissue culture at industrial scale.
However, it is of prime importance to design bioreactor configurations which
can provide adequate mixing and mass transfer while minimizing the
intensity of shear stress and hydrodynamic pressure. Thus, problems arise
when scaling up from pilot plant fermentors to large scale bioreactors.
As for the clonal propagation of a plant in large scale, regeneration from
cells belonging to an explant of plants is potentially very useful. Liquid
media have been used for plant cells, somatic embryos, and organ cultures in
both agitated flasks or various types of bioreactors. Scale up of plant
regeneration in a liquid medium is easier than on a solid medium (Okamoto
et al. 1996). Although the use of bioreactors has been directed mainly for cell
suspension cultures and secondary metabolite production, research directed at
improving bioreactors for somatic embryogenesis has been reported for
several plant species as well. Utilization of bioreactors for clonal propagation
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Ozlem Yesil-Celiktas et al.
has been attracting interest recently due to scale-up and automation
advantages (Gurel, 2009). Bioreactors have also been used for the cultivation
of hairy roots mainly as a system for secondary metabolite production (Ziv,
2000). Furthermore, innovative processes have been proposed for producing
secondary metabolites selectively by enzymatic reactions (Takemoto, 2009;
Abraham et al., 2005).
In spite of all these advantages, cell culture techniques have not been
effectively exploited in industrial scale bioreactors due to the slow growth
rates of plant cell cultures and the high capital costs involved and have been
limited to a handful of applications such as the production of shikonin from
Lithospermum erythrorhizon, berberine from Coptis japonica and
ginsenosides from Panax ginseng (Kieran et al., 1997). Phyton Biotech
located in Germany which is another good example of industrial exploitation,
owns and operates the world's largest cGMP plant cell culture facility with
bioreactors up to75000 L in size and specifically designed to meet the need of
plant cells in culture. The total production capacity of the Taxanes, one of its
products, runs up to 880.000 Litres/year (Phyton Biotech, 2006). On the other
hand, the large scale commercial propagation of plant material based on plant
tissue culture was pioneered in the USA. More recently, some companies
from Israel, USA and UK have shifted their production facilities to
developing countries.
The aim of this book is to critically outline the recent developments in
large scale cultivation of plant cell and tissue cultures by presenting scale-up
studies and state-of-the-art technologies, while focusing on different cell
culture applications and bioreactors. Moreover, a SWOT (Strengths,
Weaknesses, Opportunities, Threats) analysis has been carried out to evaluate
the methodologies intended for scale-up.
Large scale cultivation of plant cell and tissue culture in bioreactors
5
2. Bioreactor considerations
Plant cell suspension, organ cultures and hairy roots are potential sources
of secondary metabolites and recombinant proteins. As they are produced
under controlled conditions in bioreactors, variations in product yield and
quality are minimized and consequently product registration process is
simplified.
Different bioreactor designs are available providing the optimum
environment for effective cell growth and secondary metabolite production.
The bioreactors used for various plant cell cultures are classified as
mechanically agitated, pneumatically driven, disposable and non-agitated
(Fig. 1).
The most common bioreactor is the stirred tank bioreactor and sufficent
know-how exists about the design. In order to minimize the shear forces,
numerous modifications have been developed by employing a variety of
impeller designs and seals (Fig. 1 A1). Examples of varying capacities from
1–2 L lab scale to 20.000 L industrial scale systems exist in the literature.
For instance, biomass with a density of 6.3 g dry weight L-1 was produced
from Acer psedoplatanus using a 20 L stirred tank bioreactor with a
magnetic stirrer which is regarded as safer than the mechanical stirrers
against contamination risks (Asenjo and Merchuk, 1995). Despite their
popularity, stirred tank bioreactors have several limitations, such as high
power consumption, high shear, and problems with sealing and stability of
shafts in tall bioreactors. Horizontal vessels, or rotary drum reactors (Fig.
1 A2) have significantly higher surface area to volume ratio than other
reactor types. As a consequence, mass transfer is achieved with comparably
less power consumption. Rotary drum reactors used for the cultivation of
high-density plant suspensions have shown advantages in terms of
suspension homogeneity, low shear environment and reduced wall growth,
over either airlift or stirred tank reactors (Sajc et al., 2000). However, the
disadvantage is their comparatively high energy consumption in industrial
scale operations.
A bubble column bioreactor (Fig. 1 B1) is a reactor, in the shape of a
column, in which the reaction medium is kept mixed and aerated by the
introduction of air at the bottom (IUPAC, 1997). The main advantages of
bubble column bioreactors are the low capital costs, uncomplicated
mechanical configurations and not too high operational costs due to low
energy requirements. On the other hand, they are less suitable for the
processes where highly viscous liquids exist. An airlift bioreactor (Fig. 1 B1-5)
is defined as a bioreactor in which the reaction medium is agitated and
aerated by the introduction of air or another gas mixture and the circulation is
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Ozlem Yesil-Celiktas et al.
(A)
(1)
(2)
(B)
(1)
(2)
(3)
(4)
(5)
(C)
(1)
(2)
(D)
(1)
(2)
(3)
Figure 1. Depictions of bioreactor types for plant cell and tissue cultures. (A) Mechanically
agitated bioreactors; (1) stirred tank reactor equipped with various propellers (spin, helix,
bladed and paddle), (2) rotary drum tank reactor, (B) Pneumatically driven bioreactors; (1)
bubble column, (2) internal loop airlift, (3) external loop airlift, (4) propeller loop, (5) jet
loop, (C) Hydrodynamically driven disposable bioreactors; (1) Wave&Undertow
bioreactor, (2) Biowave reactor, (D) Non-agitated bioreactors; (1) packed bed, (2) fluidized
bed, (3) membrane reactor, (Adapted from Sajc et al., 2000 and Eibl and Eibl, 2008).
Large scale cultivation of plant cell and tissue culture in bioreactors
7
enhanced by internal draught tubes or external loops. Thus, the reactor
volume is seperated into gassed and ungassed regions generating a
vertically circulating flow (IUPAC, 1997). Airlift bioreactors can supply
the low O2 demands of plant cell cultures with low shear effects. Breuling
and coworkers (1985) reported the production of protoberberine from
Berberis wilsonae using 20 and 200 L airlift bioreactors. Maximum
biomass density was stated as 23 g dry weight L-1 in 200 L reactor. Airlift
bioreactors have several advantages such as combining high loading of
solid particles, providing good mass transfer, relatively low shear rate, low
energy requirements, and simple design. The major drawback is their
unsuitability for high density plant cultures. Stirred tank bioreactors are
therefore preferable for culturing plant cell suspensions at high densities
(Tanaka, 2000).
It is also worth mentioning the use of disposable bioreactors such as
the Wave&Undertow bioreactors (Fig. 1 C1) and the Biowave (Fig. 1 C2)
which are modern alternatives to traditional cultivation systems. These
bioreactors consist of a sterile plastic chamber that is partially filled with
media (10–50 %), inoculated with cells and discarded after harvest. Two
novel flexible plastic-based disposable bioreactors have been reported
lately. The first one, was based on the principle of a wave and undertow
mechanism that provided agitation while offering convenient mixing and
aeration to the plant cell culture contained within the bioreactor. The
second one was a high aspect ratio bubble column bioreactor, where
agitation and aeration were achieved through the intermittent generation
of large diameter bubbles, "Taylor-like" or "slug bubbles". It was possible
to increase the volume from a few liters to larger volumes up to several
hundred liters with the use of multiple units. The cultivation of tobacco
and soya cells producing isoflavones was described up to 70 and 100 L
working volumes for both types. Cleaning, sterilization, and maintenance
operations were reduced or eliminated with the disposable bioreactors
(Terrier et al., 2007).
Although packed bed (Fig. 1 D1) and membrane reactors (Fig. 1 D3) are
advantageous in terms of immobilizing large amounts of cells per unit
volume, diffusional limitations of mass transfer and difficulties in handling
gaseous components can limit the use of both configurations. (Sajc et al.,
2000).
Table 1 summarizes the major studies involving plant cell and tissue
cultures with different types of reactors. As can be seen from literature, plant
cell and tissue cultures have been cultivated in reactors of various designs
and configurations in scales ranging between 0.25 and 14 L.
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2.1. Production of secondary metabolites by plant cell suspension
cultures
The main advantages associated with in vitro plant systems include the
manipulation of environmental conditions, the rapidity of production, and the
use of simpler and cheaper downstream processing schemes for product
recovery from the culture medium (Doran, 2000). Plant cell culture is a
flexible system that is easily manipulated to increase product yields (Roberts
and Shuler, 1997). Cultures of plant cells are not limited by environmental,
ecological or climatic conditions (Zhong et al., 1995). Suspended cells with
high embryogenic potentials should be selected to maintain a stable
production of quality somatic embryos (Ibaraki and Kenji, 2001). Plant cell
cultures combine the merits of whole-plant systems with those of microbial
and animal cell cultures for the production of valuable therapeutic secondary
metabolites (Hellwing et al., 2004).
Recent progress in molecular biology, enzymology, physiology and
process technology of plant cell cultures suggest that these systems will
become a viable source of important natural products (Dicosmo and Misawa,
1995).
The capability to cultivate plant callus cells and organs in liquid media
has also made an important contribution to modern plant biotechnology with
respect to the production of commercially valuable compounds (Su and Lee,
2007). Callus cells obtained from the transgenic plants can be grown in
simple, chemically defined liquid media to establish transgenic cell
suspension cultures for recombinant protein production (Su, 2006).
Furthermore, the use of whole plants for the synthesis of recombinant
proteins has received a great deal of attention recently due to advantages in
economy, scalability and safety compared with traditional microbial and
mammalian production systems. However, plant cells generally have a longer
doubling time than bacterial or yeast cells. Genetic instability associated with
de-differentiated callus cells due to somaclonal variation is a potential
disadvantage in using cultured plant cells for recombinant protein production
(Su, 2006).
Oxygen transfer, fluid mixing and the magnitude of the acceptable shear
rates are the primary parameters in choosing a suitable bioreactor. For
successful cultivation of plant cell suspensions, the bioreactor has to provide
good mixing at low shear stress levels with a moderate oxygen supply.
Alternative designs to conventional stirred tank bioreactors have led to the
use of different impellers for the cultivation of plant cell suspensions. For
instance, helical impellers proved to be low shear and provided good mixing
with viscous non-Newtonian fluids (Cormier et al., 1996). Indeed, the
Large scale cultivation of plant cell and tissue culture in bioreactors
9
majority of plant cell culture studies in laboratory scale which have provided
basic information for large scale cultivation have been stirred tank bioreactors
equipped with different impellers. Usage of airlift bioreactors were reported
for thesuspension cultivation of Frangula alnus cells to produce
anthraquinones (Sajc et al., 1995) and for plantlet production by Oxalis
triangularis (Teng & Ngai, 1999). Cell suspensions of Lilium Oriental Hybrid
were cultivated in balloon type bubble bioreactors to produce bulblets (Lian
et al., 2003).
The adoption of plant cell cultures as an industrial process depends
greatly on the economics of such a process. The multicycle or draw-fill
culture technique offered by Lipsky (1992) is one method for improving the
productivity and, hence, cost of a process. Mathematical models have been
devised for the functional relationships between the nominal costs of
biomass and secondary metabolites and the plant cell growth characteristics
in a multicycle growth system. The models were used to evaluate the data
obtained with cultures of Dioscorea deltoidea (which produces diosgenin)
and Panax ginseng, grown in various types of bioreactors. The multicycle
system gave an increase of 1.5–2 in biomass productivity compared with
batch culture, but was probably only commercially viable if the cost of the
process in the bioreactor was at least 30 times that of the medium and if an
inoculum of about 30% of the culture of the previous cycle was left in the
bioreactor. In the multicycle system incompletely utilized nutrient or
metabolite accumulation can only reach 1.43 times or less that of the
initial values. With the P. ginseng culture, about 75% of the calculated
maximum cell packing density per fresh weight (≈ 530 g l−1) in this regime
was achieved. The possibility of growth in the standard bioreactor of a
shear sensitive type culture was shown with a marine impeller speed up to
330 cm s−1.
2.2. Production of secondary metabolites by organ cultures
Regeneration is provided by two morphogenic pathways: organogenesis
(the formation of unipolar organs) and somatic embryogenesis (the
production of bipolar structures, somatic embryos with a root and a shoot
meristem). When plant somatic cells are isolated and cultured in vitro
conditions, they are capable of expressing totipotency. The injured cells in
the outer layers of the isolated explant evolve ethylene that induces
dedifferentiation. Cell division can occur in an unorganized pattern with
callus formation, or in an organized pattern with the formation of
meristematic centers directly in the explant tissues. Meristematic centers
form directly on the explant in some plants and can develop into either
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Ozlem Yesil-Celiktas et al.
shoots, roots or somatic embryos. Organ cultures are used for secondary
metabolite production as well as for plant propagation.
2.2.1. Shoots
The development of spherical meristematic or bud clusters in liquid
cultures was shown to provide a highly proliferative and rapid growing
system amenable to automated inoculation, control of the medium
components, mechanical separation, and efficient delivery to the final stage
for plant growth and development (Ziv, 2000).
There are few examples in the literature focusing on secondary
metabolite production from shoot cultures. Shoot cultures of Spathiphyllum
cannifolium (Dewir et al., 2007) and Stevia rebaudiana (Sreedhar et al., 2008)
were carried out in bubble column bioreactors in order to produce flowers and
biomass, respectively. Organ culture of Lavandula officinalis was cultivated
in 5 L bubble column bioreactor to obtain rosmarinic acid (Wilken et al.,
2005). Moreover, shoot culture of Ananas comosus was performed in a 10 L
airlift bioreactor (Firoozabady & Gutterson, 2003) and organ culture of
Hypericum perforatum in 2 L stirred tank bioreactor to produce hypericin
(Wilken et al., 2005) (Table 1). The necessity to expose shoot cultures to
light can be a problem with large stirred tank bioreactors made from steel
(Bourgaud et al. 2001).
2.2.2. Hairy roots
Hairy roots grow rapidly, show plagiotropic growth, and are highly
branched on phytohormone-free medium (Hu and Du, 2006). Agrobacterium
rhizogenes-derived hairy roots and plants have application for many different
areas. Hairy root cultures have been tested extensively in root nodule
research, for production of plant secondary metabolites (Christey, 2001).
Transformed roots have been widely studied for in vitro production of
secondary metabolites in many plant species and for artificial seed production.
Hairy root cultures produce secondary metabolites over successive generations
without losing genetic or biosynthetic stability (Giri ve Narasu, 2000). Many
aspects of plant secondary metabolite biosynthesis have been studied using
transformed root cultures (Kuzovkina and Schneider, 2006). The strong
corelation between secondary metabolite production and morphological
differentiation gives more impetus to the application of organized cell
cultures for large scale production of phytochemicals.
Intergeneric co-culture of genetically transformed hairy roots and shooty
teratomas is effective for improving tissue specific secondary metabolites.
Table 1. Laboratory scale plant cell and tissue culture studies useful for large scale cultivation in different types of bioreactors.
Large scale cultivation of plant cell and tissue culture in bioreactors
11
Table 1. Continued
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Ozlem Yesil-Celiktas et al.
Large scale cultivation of plant cell and tissue culture in bioreactors
13
Figure 2. Hairy root cultures actively growing in hormone-free MS medium in Rubia
tinctorum L. (Cetin et al. 2005).
This mimics the situation observed in the whole plant where a localized
metabolite synthesis is translocated throughout the organs for further
bioconversion (Giri and Narasu, 2000). Also, production of two different
secondary metabolites is possible simultaneously by adventitious root cocultures (Wu et al., 2008).
This vast potential of hairy root cultures (Fig. 2) (Cetin et al. 2005) as a
stable source of biologically active chemicals has provided the exploitation of
in vitro system through up scaling in novel bioreactors (Mehrotra et al., 2008).
Hairy root cultures of Lithospermum erythrorhizon (Sim &Chang, 1993),
Harpagophytum procumbens (Ludwig-Muller etal., 2008) and adventitious
roots of Panax ginseng (Yu et al., 2002; Jeong et al., 2008) and Scopolia
parviflora (Min et al., 2007) were studied in various volumes of bubble
column bioreactors to obtain shikonin, harpagide, ginsenosides and alkoloids
respectively. Ginsenoside was also produced in 5 L stirred tank bioreactor
using adventitious root culture (Jeong et al., 2006). Hairy root culture of
Stizolobium hassjo to yield 3,4-dihydroxyphenylalanine was reported
using 9L mist bioreactor (Table 1). Present scale-up technology dictates
the use of stainless steel tanks for growth of plant cells on an industrial scale.
The usage of bioreactors equipped with special hangers inside the vessel is
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Ozlem Yesil-Celiktas et al.
reported (Rhodes et al., 1989). Hairy root cultures continue to attract interest
as a potential resource for large-scale production of commercially valuable
compounds.
2.2.3. Somatic embryogenesis
Somatic embryogenesis has been considered to be a distinct
developmental pathway, different from either shoot or root organogenesis, in
which a single cell gives rise to a structure containing bipolar meristems and
with no direct vascular connections to the maternal tissue (Brown et al.,
1995). Somatic embryogenesis is also an alternative method for the
production of certain metabolites (Tejavathi et al. 2007) and has been
regarded as an especially suitable system for the investigation of various
morphological and physiological phenomena (Hirai et al. 1997).
In most cases the somatic embryos or the embryogenic cultures can be
cryopreserved, which makes it possible to establish gene banks. Embryogenic
cultures are also an attractive target for genetic modification (Von Arnold
et al. 2002). Plant regeneration from cell suspension cultures offers a suitable
in vitro system for the mass propagation of economically important plants. If
this could be scaled up to fit bioreactor design, it could become an excellent
tool for the large-scale production of somatic embryos that would greatly aid
the commercial production of artificial seeds (Patnaik et al. 1997).
The ability to perform all the stages of somatic embryogenesis, from the
growth of embryogenic cells to embryo maturation, in liquid media in
bioreactors is essential for the succesful scale-up of the process (Mavituna
and Buyukalaca, 1996).
2.3. In vitro propagation
Propagation of some commercial plants which are difficult to reproduce
conventionally by seed or vegetative propagules conventionally is realized by
in vitro tissue culture techniques. Plant regeneration can follow the
organogenic or embryogenic pathways and is dependent on the manipulation
of the inorganic and organic constituents in the medium, the type of explant
and the species. In most plants, subcultures in various media are very
important for successful regenerations from the callus or directly from the
explants (Ziv, 2000).
Four basic methods are used to propagate plants in vitro. Depending on
the species and cultural conditions, in vitro propagation can be achieved by:
•
•
enhanced axillary shoot proliferation,
node culture,
Large scale cultivation of plant cell and tissue culture in bioreactors
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15
de novo formation of adventitious shoots through shoot organogenesis,
nonzygotic embryogenesis (Trigiano and Gray, 2000).
Micropropagation offers the advantages in quality, quantity and
economics over conventional vegetative propagation for many species
providing an efficient method for production of a large number of uniform
plantlets in a short time (Altman and Colwell, 1997). Furthermore, they
enable the rapid propagation of valuable genotypes, production of diseasefree plants, non-seasonal production throughout the year and germplasm
conservation (Gurel, 2009). The parameters affecting vegetative regeneration
such as nutrient and growth regulator levels, light and temperature are
adjustable and flexible. Plant propagation and stock plant maintenance require
less energy and space (George et al., 2007). Exploring micropropagation to
solve special problems connected with selective breeding is expected to prove
to be beneficial in the future (Jha and Ghosha, 2005). The other advantages of
micropropagation are production of large stocks of true-to-type propagation
material as well as a large number of plants from elite or difficult-to-grow and
slow-to-grow plants, bringing new plants to the market rapidly, easier
transportation of plant material and conservation of plant genetic resources
(Trigiano and Gray, 2000; Scragg, 2007).
Clonally propagated plants are suitable materials to improve cultural
practices for commercial productions (Jain and Ishii, 2003). Techniques used
to propagate commercially important plants in vitro are based on axillary
branching, adventitious shoot formation and somatic embryogenesis.
Micropropagation by conventional techniques is typically a labourintensive means of clonal propagation. The work by Paek et al. (2005)
describe lower cost and less labour-intensive clonal propagation through
the use of modified air-lift, bubble column, bioreactors (a balloon-type
bubble bioreactor), together with temporary immersion systems for the
propagation of shoots, bud-clusters and somatic embryos. Propagations
of Anoectochilus, apple, Chrysanthemum, garlic, ginseng, grape, Lilium,
Phalaenopsis and potato were described. Studies are being carried out
along these lines to improve the productivity and cost efficiecy of in vitro
propagation techniques.
2.3.1. Propagation by shoot cultures
Propagation from axillary shoots has proved to be a reliable method for
the micropropagation of a large number of species, although not the most
efficient procedure. Most commercially micropropagated plants are derived
from axillary shoot formations. (Gurel, 2009) and the major application of
16
Ozlem Yesil-Celiktas et al.
shoot cultures is in plant propagation. Compared to other micropropagation
methods, shoot cultures:
•
•
•
provide reliable rates and consistency of multiplication following culture
stabilization,
are less susceptible to genetic variation,
may provide for clonal propagation of periclinical chimeras (Trigiano
and Gray, 2000).
Depending on the species, two methods, shoot and node culture, are
used. Both methods rely on the stimulation of axillary shoot growth from
lateral buds following disruption of apical dominance of the shoot apex. The
axillary shoots produced are either subdivided into shoot tips and nodal
segments that serve as secondary explants for further proliferation or are
treated as microcuttings for rooting. Shoot culture (shoot tip culture) refers to
the in vitro propagation by repeated enhanced formation of axillary shoots
from shoot tips or lateral buds cultured on medium supplemented with
growth regulators, usually a cytokinin. Node culture, a simplified form of
shoot culture, is another method for production from preexisting meristems.
Although node culture is the simplest method, it is associated with the least
genetic variation (Trigiano and Gray, 2000).
Currently the most frequently used micropropagation method for
commercial production utilizes enhanced axillary shoot proliferation from
cultured meristems. This method provides genetic stability due to the highly
organized structure and is easily attainable for many plant species.
Besides propagation, shoot meristems are cultured in vitro for two other
purposes: production of pathogen eradicated plants and preservation of
pathogen eradicated germplasm. Shoot growth in mature plants is restricted
to specialized regions which exhibit little differentiation and in which the
cells retain the embryonic capacity for unlimited division. These regions,
called apical meristems, are located in the apices of the main and lateral buds
of the plant. Cells derived from these apical meristems subsequently undergo
differentiation to form the mature tissues of the plant body. Rejeneration of
shoots from meristem tips are often used as initial explants for large-scale
micropropagation (Pérez-Tornero et al. 1999; Trigiano and Gray, 2000; Vila
et al. 2002; Goleniowski, et al. 2003; Prehn et al. 2003; Wang et al. 2003;
Hosokawa et al. 2004; Verma et al. 2004).
Scaling-up in bioreactors requires the use of liquid instead of agar-gelled
media during the proliferation and biomass production stages. This
introduces some problems. The culture of plants in liquid medium is known
Large scale cultivation of plant cell and tissue culture in bioreactors
17
to cause anomalous morphogenesis, resulting in plant hyperhydricity. The
plants that develop in liquid media are fragile, have a glassy appearance, with
succulent leaves or shoots and a poor root system. The leaves are the organs
that are affected most severely in liquid cultures. The two major processes
carried out by the leaves, photosynthesis and transpiration, are not fully
functional in hyperhydric leaves and thus cause the poor performance of the
transplanted plants ex vitro (Ziv, 2000). Another study dealing with the
commercial micropropagation was smooth Cayenne pineapple. In vitro
shoots were used as starting materials, and either longitudinal sections of the
shoots or leaf bases were used as the explants to regenerate shoots. When
these explants were used, the axillary meristems, which usually remain
quiescent during shoot multiplication, were able to form new shoots.
Subsequent to the regeneration step, additional multiplication was achieved
inside a 10 L periodic immersion bioreactor with shoots immersed in liquid
medium for 5-10 min/h (Firoozabady et al., 2003). Currently studies are
conducted to overcome these difficulties in shoot culture systems.
Although a number of possible scale up techniques for shoot culture in
bioreactors have been described and the mass propagation of shoots of Stevia
rebaudiana was reported using a bioreactor of 500 L volume (Akita et al.
1994), efficient techniques for the mass propagation of shoots are needed for
the production of disease free seed plants.
2.3.2. Propagation by hairy root cultures
Hairy root explants can also be propagated clonally and such hairy root
lines facilitate studies of plant responses during early stages of
mycorrhization. In addition to the speed and ease with which Agrobacterium
rhizogenes transformed hairy roots can be generated, a valuable characteristic
of such roots is that they can be propagated clonally as independent organs,
with a vigorous growth that does not require the addition of plant hormones.
Furthermore, these root explants have the potential to be colonized by
endomycorrhizal fungi in vitro (Boisson-Dernier et al. 2001).
Another alternative strategy for evaluating of transformed roots is the
production of composite plants. These are generated by inducing transformed
roots on non-transformed shoots. These have been widely used in legumes as
they represent an ideal system for analyses of gene expression involving
infection of rhizobia and nitrogen fixation (Christey, 2001).
A. rhizogenes can transfer T-DNA from binary vectors and enable the
production of transgenic plants containing foreign genes carried on a
second plasmid. This character has been used to produce transgenic plants
18
Ozlem Yesil-Celiktas et al.
(Hu and Du, 2006). A. rhizogenes-mediated transformation has also the
potential to introduce foreign genes specifically into the root system, such
as resistance to root pathogens, pests, resistance to heavy metals. Hairy root
cultures represent an ideal system to study root function, the effects of root
pathogens and to test genes reletad with pathogen resistance (Christey, 2001).
Transformed hairy roots are very important materials for remediation of soil,
groundwater, and biowastes (Kuzovkina and Scheider, 2006).
2.3.3. Propagation by somatic embryogenesis
The embryogenic pathway, as opposed to the organogenic pathway, can
be a more efficient and productive system for large-scale clonal propagation
(Fulzele and Satdive, 2003). Plant regeneration via somatic embryogenesis is
the in vitro process used to reduce multiplication time, and potentially offers
an efficient system for mass clonal propagation and for regeneration of plants
from genetic transformation and somatic hybridisation experiments (Siragusa
et al. 2007, Fulzele and Satdive, 2003). The method has the potential to
produce a large number of plantlets in a relatively short period of time (Wu
et al. 2007).
Somatic embryogenesis may result in less variation through chimerism.
However, somaclonal variation will be less likely to take place in direct
regeneration, but might be more pronounced in somatic embryos developing
Figure 3. In vitro propagation of olive (Olea europaea L.) plants (Gurel et al. 2006).
Large scale cultivation of plant cell and tissue culture in bioreactors
19
from continuously cultured callus tissue. Since the embryo contains both a
root and an apical shoot meristem, the rooting stage required in conventional
in vitro bud or shoot propagation technology is obviated. Somatic embryos
are small and can be adequately handled in scaled-up procedures. They are
amenable to sorting and separation by image analysis, dispensing by
automated systems, and can be encapsulated and either stored or planted
directly, with the aid of mechanized systems (Ziv, 2000).
Furthermore, manipulation of somatic embryos to function as synthetic
seeds would allow clonal germplasm to be stored efficiently in seed
repositories if viability can be maintained for adequate lengths of time
(Jayasankar et al. 2005).
Bacopa australis
Cryptocoryne wendtiti
Figure 4. In vitro micropropagation of some aquarium plants (Gurel et al. 2007).
20
Ozlem Yesil-Celiktas et al.
Figure 5. Clonal propagation of Rubia tinctorum L. (Cetin et al. 2005).
The technology is currently applied to a large number of agricultural,
ornamental, vegetable crop and forestry plant species. In vitro propagation
of different plants carried out in our laboratories are presented in Fig. 3, 4
and 5.
Conventional in vitro plant propagation methods on semi-solid media
involve a considerable amount of handling such as transfers to fresh media
and plantlet separation (Etienne et al. 1997, Gurel et al. 2006; Gurel et al. 2007;
2007; Cetin et al. 2005). Various liquid medium culture techniques have
therefore been developed to reduce labor costs, stimulate growth and improve
multiplication rates, and to increase culture uniformity by the elimination of
nutritional gradients (Etienne et al. 1997).
Efficient commercial micropropagation depends on rapid and extensive
proliferation in conjunction with the use of large-scale cultures for the
multiplication phase. Mechanization and automation of the micropropagation
process can greatly overcome the limitation imposed by existing conventional
labor-intensive methods. Considerable attention has been directed toward
buds, shoots, or plantlets during the multiplication and transplanting phases
(Ziv, 2000).
However, the establishment of efficient automated micropropagation
techniques to overcome some of the present labor input limitations requires,
among other technologies, the development of scaled-up liquid cultures in
bioreactors. Bioreactors are used in micropropagation for both the
embryogenic and organogenic pathways. Successful micropropagation in
shake liquid cultures or bioreactors has been reported previously for
Large scale cultivation of plant cell and tissue culture in bioreactors
21
ornamental and vegetable crop species, as well as for woody plants. The
airlift and immersion bioreactors used in laboratory scale plant cell culture
studies provide a basis for large scale cultivation. Micropropagation of
potato, Boston fern, banana and gladiolus were carried out at 2 L disposable,
presterilized bioreactors to obtain meristem clusters (Ziv et al., 1998). The
micropropagation of plantlets from protocorm-like bodies was also realized
in 3 L airlift bioreactor (Young et al., 2000). Usage of immersion bioreactors
were reported for micropropagation of shoots and buds from Ananas comosus
L. (Escalona et al., 1999) and plantlets from Potato Atlantic (Piao et al.,
2003) (Table 1).
Nevertheless, two major problems still limit the use of bioreactors for
plant culture and micropropagation:
•
•
the phenomenon of hyperhydricity (vitrification), which results from
abnormal plant morphogenesis in liquid media
the high initial capital and operartional costs of bioreactors.
While growth retardants added to liquid media were found to reduce
shoots into meristematic or bud clusters and thus limit hyperhydricity, simple
inexpensive bioreactors are as yet not prevalent. Disposable presterilized
plastic film bioreactors may reduce costs and simplify plant processing in
scaled-up liquid cultures. The use of clusters can provide an improved system
for normal plant development and further growth and hardening after
transplanting as the problem of hyperhydricity in liquid cultures is obviated
(Ziv et al. 1998).
22
Ozlem Yesil-Celiktas et al.
3. Scale-up
Scale-up generally involves taking a lab-scale bioprocess and replicating it
as closely as possible to produce larger amounts of product. A typical scale-up
sequence in plant cell and tissue culture studies start with jars, moves to 1 L
shake flasks, after that to 1 - 10 L glass bioreactors, then scales up through to
stainless steel vessels of varying sizes from 30 – 150 L to 1000 L (Figure 6).
Large scale cultivation of plant cell and tissue culture is an alternative to
the traditional methods of plantation. However, plant cell suspensions, shoot
and root cultures pose very different problems in bioreactors when compared
to microbial cultures during scale-up. Briefly, plant cells grow slowly, the
cells are large and generally form clumps which make them more sensitive to
shear associated with agitation and exhibit long processing times. Organ
cultures are far more sensitive to shear. These characteristics have lead to the
necessity to design alternative bioreactor configurations, particularly those
that avoid or reduce shear within the large scale bioreactor (Scragg, 2007).
Therefore, a lot of effort is currently being put into novel bioreactor designs.
In order to control plant morphogenesis and biomass growth in
bioreactors, various culture conditions must be monitored and/or controlled,
such as the morphology, oxygen supply and CO2 exchange, mixing,
consequently pH and temperature. Most of these parameters are interrelated,
therefore a holistic approach is required for a successful scale-up.
3.1. Culture conditions and requirements
A thorough understanding of substrate requirements as well as culture
conditions is essential for the optimization of the scale-up of plant cell and
tissue cultures. Carbon and oxygen are two major substrates affecting
biomass production therefore the utilization rates of these substrates should
be closely monitored. The physical conditions within the reactor are
interrelated with the chemical conditions which in turn affect both productivity
and cost effectiveness.
There are a number of findings related to substrates types and
concentrations in order to optimize process parameters. Adding a saccharide
mixture to the culture medium as a carbon source to increase the productivity
of the secondary metabolites, increasing the productivity of secondary
metabolites and shortening the culture time by using a mixture of glucose and
fructose or growing plant cells in a medium treated with alkanoic acid or salt
are typical examples (Kim et al., 2005; 2006). Choi et al., (1999) suggested
increasing the paclitaxel production by inoculating the cells at a high initial
concentration and by increasing the saccharide concentration in the medium.
Large scale cultivation of plant cell and tissue culture in bioreactors
23
Figure 6. Scale-up studies starting with shake flasks at laboratory scale to industrial
scale.
Another innovation focused on modifications of culture conditions such
as media composition and operating modes which were found to enhance the
yield of various taxanes and taxol-like compounds from cell culture of all
Taxus species. Particularly preferred enhancement agents included silver ion
or complex, jasmonic acid (especially the methyl ester), auxin-related growth
regulators, and inhibitors of the phenylpropanoid pathway, such as 3,4methylenedioxy-6-nitrocinnamic acid. These enhancement agents were
suggested to be used alone or in combination with one another or other yieldenhancing conditions (Bringi et al., 1997).
These innovations could lead to the economic production of valuable
secondary metabolites on an industrial scale using plant cell culture.
3.1.1. Cell morphology
Plant cells in suspension cultures display a range of shapes, with the
largely spherical and the rod shapes being the most common. Suspension
24
Ozlem Yesil-Celiktas et al.
cultures normally exhibit various degrees of cell aggregation with the
aggregate sizes varying dependent on the plant species, growth stage and
culture conditions. While some plant cells such as Nicotiana tabacum and
Anchusa officinalis, form fine suspensions with few large aggregates smaller
than 1 mm others form aggregates as large as 2 cm in diameter, as in the case
of Panax ginseng suspension culture (Su, 2006). Unlike cell suspension
cultures, clusters that are compact, consist mainly of small meristematic cells
and are apparently less shear sensitive (Ziv, 2000).
The morphological character of the plant material, eg.: cell aggregates,
calli or hairy roots becomes a more important factor for the design and
optimization of the culture conditions as the structure changes from cell
aggregates to tissue cultures (Choi, et al., 2006).
3.1.2. Aeration and agitation
The requirements for O2 may vary from one species to another. Oxygen
must be supplied continuously to provide adequate aeration, since it affects
metabolic activity and energy supply. The level of O2 in liquid cultures in
bioreactors can be regulated by agitation or stirring methodologies and
intensities as well as aeration techniques and gas flow rates, which affect
bubble sizes, mixing and circulation times, gas hold-up values and mass
transfer coefficients (Vardar-Sukan, 1985; Vardar-Sukan, 1986; Takayama
and Akita 1998). Air-flow supply to the reactor determines the degree of
aeration and agitation as well as prevents settling of the plant biomass, thus
affecting growth and proliferation of the biomass.
The effect of the O2 partial pressure on proliferation and differentiation
of embryogenic suspension cultures has been reported for different crops:
Daucus carota, Medicago sativa, Euphorbia pulcherrima, Triticum aestivum
and Picea abies (Feria et al., 2003). In these studies high aeration rates
appear to reduce the biomass growth (Ziv, 2000). This is attributed is to the
stripping of CO2 and essential volatiles from the system (Kato et al., 1975;
Smart and Fowler, 1981, Kieran et al., 1997).
In many plants cultivated in bioreactors, intensive aeration, mixing, and
circulation cause shearing damage, cell wall breakdown, and thus
accumulation of cell debris and release of polysaccharides. Cell death and
debris accumulation results in foaming, in turn causing adhesion of cells and
aggregates onto the culture vessel walls, and the development of a layer at the
upper part of the vessel, thus disrupting the homogeneous culture conditions,
causing additional cell debris formation. As the biomass increases and the
cultures become viscous, higher rates of aeration are required to allow for
oxygen supply and circulation and demand for higher aeration rates that
Large scale cultivation of plant cell and tissue culture in bioreactors
25
intensify foaming and the clogging problem (Scragg 1992). Similarly cell
aggregates exhibit intercellular mass transfer limitations leading to more
vigorous aeration and agitation requirements.
Although plant cells growing in liquid cultures are better exposed to the
medium components and the uptake and consumption are faster (Ziv, 2000),
individual cells in an organized tissue or cell aggregate can be oxygen
deprived within the tissue due to the intra-particle resistances, even if the
surface is exposed to oxygen saturated medium. Cultured plants and plant
tissue present very large structures which must have considerable oxygen
transport rates within the tissue to maintain aerobic respiration. As the
oxygen moves into the tissues, it is consumed by respiration. The transport
rate through the outermost tissues must be sufficient to supply the oxygen to
all tissues that are deeper within (Curtis and Tuerk, 2006). In the case of
hairy root cultures, the growth of hairy roots in liquid medium results in the
packed root mass playing an inhibitory role in fluid flow and limiting oxygen
availability. In addition, the roots hairs play a detrimental role for liquid
circulation environment because they inhibit fluid flow, induce the stagnation
thus create micro-environmental deficiencies and limit the availability of
oxygen.
On the other hand, Shuler (1993) suggested that metabolite productivity
might be influenced by the degree of cellular association and be affected by
variations in aggregation patterns arising on scale-up. Therefore, the aeration
and agitation rates have to be optimized for large-scale production
(Ramachandra and Ravishankar, 2002). Oxygen transfer in large scale
cultures have to be investigated in-depth since plant cells have a tendency to
aggregate. Understanding the oxygen requirements is essential in order to
design high performance and more efficient bioreactors for large scale
applications.
3.2. Mixing
Fluid mixing is an important factor in the process optimization and scaleup of plant cell and tissue culture cultivation and for efficient production of
valuable metabolites. However, in most of the studies cited in the literature,
mixing time was not controlled at a constant level during cultivation. There is
a need to investigate the effect of a constant mixing time on plant cell
physiology and metabolism in long-term agitated cultures (Zhong et al.,
2002). Nevertheless, environmental factors such as the composition of the
gas phase, pH and temperature cannot be controlled precisely while handling
large volumes in bioreactors. Thus, the effect of these factors on cell
proliferation and differentiation can be investigated in order to improve
culture performance (Hohe et al., 1999).
26
Ozlem Yesil-Celiktas et al.
3.2.1. Shear sensitivity
Hydrodynamic shear sensitivity is considered one of the obstacles for
large-scale plant cell culture and has attracted considerable research attention.
Studies in this field can be expressed in two categories:
•
•
The cells are exposed to shear forces under growth conditions for the
entire duration of the cultivation.
The cells are exposed to well-defined laminar or turbulent flow
conditions for short periods of time, generally under non-growth
conditions (Kieran et al., 1997; Sajc et al., 2000).
Although, varying levels of shear stresses were identified for different
species (i.e., 80-200 Nm-2 for M. citrifolia, 50 Nm-2 for Daucus carota), no
single mechanism for cell damage has been conclusively identified.
Moreover, conducted studies revealed the difficulty of comparing the shear
sensitivity of different species and drive conclusions (Sajc et al., 2000). But
in general terms, the dependence of the hydrodynamic stress sensitivity of a
cell line is predicted to be governed by a combination of effects:
•
•
•
•
•
•
•
•
the species investigated,
medium components,
the subcultivation regime,
age of cells,
cell morphology,
types of cells in case of differentiation,
liquid viscosity,
degree of aeration.
The study carried out by Meijer and coworkers (1994) supports this
general conclusion where plant cell suspensions of different species were
subjected to various levels of hydrodynamic stress generated by a Rushton
impeller in a fermenter. The cell lines Catharanthus roseus and Nicotiana
tabacum were capable of growth under conditions of high hydrodynamic
stress which were similar to those in a 25 m3 stirred-tank production
fermenter. Cinchona robusta and Tabernaemontana divaricata were found to
be more sensitive to hydrodynamic stress. The variation in the hydrodynamic
stress sensitivity of the cells depended strongly on the cell line used.
In another study by Gong and coworkers (2006), the responses of
suspension cultured Taxus cuspidata in different culture phases were
investigated under shear stress using a Couette reactor. It was found that the
Large scale cultivation of plant cell and tissue culture in bioreactors
27
pH in medium and peroxide hydrogen (H2O2) in cells increased more rapidly
in the exponential phase than that in lag phase under shear stress. The pH and
H2O2 concentration in the exponential phase were also higher than those in
the lag phase. Inhibition studies showed that there existed a 30–45 min delay
in the action time of G-protein, Ca2+ channel and phospholipase C of
T. cuspidata cells in the lag phase than that in the exponential phase. Agerelated different membrane fluidity and H+-ATPase activity may partially
contribute to the observed responses. These early responses might be
indicators for selecting shear-resistant cell lines and for cell damage caused
by shear stress.
As scaling up based on constant mixing rate is troublesome because of
the complex mechanism of shear sensitivity in bioreactors, research efforts
can be devoted to identification of shear resistant cell lines in order to
overcome shear related obstacles in industrial scale applications.
3.2.2. Effects of temperature and pH
The effects of temperature and pH are important parameters in plant
cell and tissue cultures and are dependent upon substrate utilization as well
as mixing and circulation within the reactor. Insufficient mixing leads to
spatial variations within the reactor and the resulting heterogeneity creates
gradients of temperature and pH due to variations in physiological
performance.
Cultures are maintained at mean temperatures to enhance growth and
morphogenesis in vitro, which are higher than those for the same plants in
vivo. The control of the temperature in the liquid medium inside the
bioreactor can be easily adjusted by a heating element or by circulating water
in a jacket outside the vessel. There is, however, limited information on the
effects of temperature in bioreactor cultures, which is usually kept between
17- 25° C for induction of callus tissues and growth of cultured cells. But,
each plant species may favor a different temperature.
Toivonen and coworkers (1992) found that lowering the cultivation
temperature increased the total fatty acid content per cell in dry weight. Akita
and Takayama (1994) investigated the temperature effects on potato tuber
formation in an airlift bioreactor and reported that a higher number and
larger-size tubers developed at 25°C than at 17°C; the higher temperature
caused an increase in tuber size. Ziv and Shemesh (1996), working with
potato internode explants in liquid cultures, found that tuber formation was
best at a 16 h photoperiod and 18/15°C day and night temperature. In another
study, the authors investigated the impact of temperature and light quality on
biomass accumulation and ginsenoside production by hairy roots cultivated in
28
Ozlem Yesil-Celiktas et al.
5 L bubble bioreactors. Biomass accumulation and ginsenoside production
was optimal under 20°C/13°C day (12 h)/night (8 h) cycle. Biomass of hairy
roots was highest in the cultures grown under dark or red light while
ginsenoside accumulation was optimum in the cultures grown under
fluorescent light (Yu et al., 2005). Furthermore, Choi et al. (1999)
investigated the effect of the temperature to increase the production level of
paclitaxel during the plant cell culture.
The medium pH is usually adjusted to between 5 and 5.9 before
autoclaving and extremes of pH are avoided. The optimum pH is determined
and controlled using a small scale bioreactor or a jar fermentor with pH
control equipment. Each plant species has different optimized conditions both
for growth of the cells and for production of useful products, so it is
necessary to optimize the conditions in each case. Moreover, temperature and
pH gradients due to inhomogeneous mixing are still issues to be tackled.
Knowledge produced from immobilized culture studies can be exploited to
elucidate the complex interactions between chemical and physical parameters
of a plant bioreactor.
Large scale cultivation of plant cell and tissue culture in bioreactors
29
4. SWOT analysis for scale-up
SWOT analysis is a general tool intended to be used in the preliminary
stages of decision-making and as a precursor to strategic planning in various
kinds of applications. It is an examination of internal strengths and
weaknesses of a specific topic as well as the opportunities and threats it is
facing due to outside factors. In this section, a structured set of information
compiled in the form of a SWOT analysis (Table 2) regarding the scale up of
plant cell culture, organ culture and propagation has been presented to
demonstrate the state-of-the-art and point out areas for further research.
4.1. Plant cell suspension culture
Cell suspension culture has more immediate potential for industrial
application than plant tissue or organ cultures, due to the extensive body of
expertise acquired from submerged microbial culture studies (Kieran et al.,
1997). Production can be more reliable, simpler, and more predictable and
isolation of the phytochemical can be rapid and efficient, as compared to
extraction from complex whole plants. Cell cultures cannot only yield
defined standard phytochemicals in large volumes but also eliminate the
presence of interfering compounds that occur in the field-grown plants (Lila,
2005). Therefore the metabolites can be produced under controlled and
reproducible conditions, independent of geographical and climatic factors.
Moreover, selection of high-growth cell lines to produce the desired
metabolites is possible.
Plant cell culture is still lacking long-term commercial success as
discussed in many reviews concerning the technology and strategies for its
optimization (Kieran et al., 1997). Problems associated to slow growth rates
and low products yields (Taticek et al., 1991) affecting intracellular
production of valuable metabolites have made plant cell culture-based
processes, with a few exceptions, economically unrealistic. Moreover,
contamination which is a result of slow growth is very risky and has to be
tackled. Plant cell suspensions are shear-sensitive. The large size and rigid
cell wall indicate that the cells and in particular the larger organized
structures could be sensitive to shear (Altman, 1997).
Oxygen transfer in large scale cultures have to be investigated in-depth
and the aeration and agitation rates have to be optimized for large-scale
production since plant cells have a tendency to aggregate. Information
obtained through immobilized cell cultures can be a starting point shedding
light on mass transfer limitations within aggregates.
Table 2. SWOT analysis of scale-up in plant cell and tissue cultures.
30
Ozlem Yesil-Celiktas et al.
Table 2. Continued
Large scale cultivation of plant cell and tissue culture in bioreactors
31
32
Table 2. Continued
Ozlem Yesil-Celiktas et al.
Large scale cultivation of plant cell and tissue culture in bioreactors
33
Finally, lack of genetic stability (Kieran et al., 1997) is also regarded as
a weakness. In terms of economical issues, the production cost of
metabolites is still high in spite of remarkable advances in plant cell culture
technology. Zenk (1978) stated that industrial plant cell culture techniques
would be introduced only if the plant product under consideration is
produced at a price equal to or preferably lower than the field-produced
product. On the other hand, Drapeau and coworkers (1987) suggested that a
quick way to assess the attractiveness of a cell culture method vs.
conventional agriculture is to calculate a specific biosynthesis rate "based
on the final dry weight and the total time of bioreactor process or land
occupation". Shikonin is a good example, in that the long growing period of
3-5 years and strict climatic requirements mean that the cost of the plant
raw material is high. With shikonin there is an 830-fold increase in plant
cell culture, making it feasible for industrial application. But the technology
is still too expensive to produce food stuffs, food additives and pharmaceuticals
since these can be more easily produced by alternative ways such as
chemical synthesis, extraction from plants or by microbial fermentation
(Misawa, 1994). A major aspect which has to be addressed is the problem
of contamination in large-scale liquid cultures, which can cause severe
losses (Leifert and Waites 1992). Attempts to control contamination in
liquid cultures of foliage plants were reported by Levin et al. (1997a), who
used continuous filtration systems to control bacterial growth in the
medium (Ziv, 2000).
Recent developments in bioreactor design and control, elicitor
technology, metabolic engineering and biotransformation chemistry are some
of the opportunities identified. Production of podopyllotoxin which is the
precursor of a semi-synthetic anticancer drug via biotransformation induced
by introduction of coniferin in the suspension culture of Podophyllum
hexandrum is a good example. Plant cell culture as a production strategy is
gaining even more importance when combined with the consumer demand
towards natural products and additives (Taticek et al., 1991). Moreover,
increased pharmaceutical applications such as phytotheraphy can boost the
industrial application of plant cell culture. Since genetic manipulation of cell
cultures has a great potential for altering the metabolic profile of plants,
profitability on industrial scale is expected to increase in the future (Razdan,
2003).
Reductions in the cost of conventional production due to the
development of more efficient agricultural techniques and cultivation of
plants with high secondary metabolite content, using genetically modified
organisms (GMOs) are regarded as threats for the large scale application of
plant cell cultures.
34
Ozlem Yesil-Celiktas et al.
4.2. Organ cultures
Organ culture enables a close control of secondary compound production
and repeated and regular harvests from the same biomass (Bourgaud et al.,
2001). Shoot and root cultures are relatively more stable. Compared to cell
suspension cultures, organ cultures generally display a lower sensitivity to
shear stress (Bourgaud et al., 2001) and are fairly well stable in metabolite
yields due to their genetic stability (Ramachandra and Ravishankar, 2002).
The strong correlation between secondary metabolite production and
morphological differentiation gives more impetus to the application of
organized cell cultures for large scale production of phytochemicals. The vast
potential of hairy root cultures as a stable source of biologically active
chemicals has focused the attention of researchers towards the exploitation of
this excellent in vitro system through up scaling in novel bioreactors which
would provide the best conditions for maximum growth. The main
constraints for the commercial exploitation and large scale cultures of hairy
roots in bioreactor relate to the operational phases in which several
parameters are required to be optimized (Mehrotra et al., 2008).
Shoot regeneration from shoot apex is direct, relatively simple and is not
prone to somaclonal variation and chromosomal abnormalities. Shoot and
root development from meristematic shoot tips is also quite simple and needs
less time and labor to regenerate large numbers of plants (Saeed et al., 1997).
Hence the storage of shoot tip cultures may be an alternative method for the
maintenance of material in gene banks for medium and long term storage
(Grout and Henshaw, 1978).
One of the major weaknesses encountered with organ cultures in
bioreactors is due to the inherent heterogeneous nature of the biomass
compared to cell suspensions and thus the high degree of spatial
heterogeneity in the reactor. This heterogeneity can be enhanced by
consequent mass transfer limitations of oxygen and nutrients.
Root systems of higher plants generally exhibit slower growth and are
difficult to harvest. In comparison to a cell suspension culture, the growth of
organs in liquid medium results in the packed root mass playing an inhibitory
role in fluid flow and limiting oxygen availability. Experience and expertise
from packed and expanded bed cultures could be used or adapted to develop
more efficient techniques for organ culture cultivation.
The major opportunity identified for this technique is that a wide variety
of chemical compounds including aromatics, steroids, alkaloids, coumarins
and terpenoids can undergo biotransformations using plant cells, organ
cultures and enzymes (Giri et al., 2001). The metabolite pattern found in
hairy roots is similar, if not always identical to that of plant roots. Moreover,
Large scale cultivation of plant cell and tissue culture in bioreactors
35
the ability to engineer the genomic DNA through tailor-made Ri plasmid is
predicted to find a tremendous use in producing novel compounds
(Ramachandra and Ravishankar, 2002). The fact that the medium is less
amenable to contamination in open conditions (Bourgaud et al., 2001) makes
it even more interesting.
Cheaper conventional production and cultivation of genetically
engineered plants yielding higher amounts of secondary metabolites are also
regarded as threats for large scale application of organ cultures.
4.3. Propagation
Clonal propagation has created new challenges in global trading for
producers, farmers, nursery owners, and for rural employment in terms of
producing high quality and healthy planting material of ornamentals,
forest and fruit trees, propagated from vegetative parts (Anonymous,
2002). The possibility of propagating the plants under a controlled
environment, anywhere, irrespective of the season and weather, on a yearround basis (Anonymous, 2002) and cultivation of true-to-type plants
have made this technique appealing. Production of very large number of
clonal propagules within a short time span and obtaining disease-free
plant material by eliminating viral, bacterial and fungal contamination are
the major strengths. Moreover, the possibility of air-shipping large
numbers of plantlets efficiently and bringing newly bred plants and
selections to market quickly and in large quantities (Altman, 1997) are
regarded as strengths of propagation. Somatic embryogenesis as a
propagation technique provides an ideal system for the investigation of
the whole process of differentiation of plants and presents a potential of
delivering high-quality products with predictable and reproducible
characteristics. Useful systems can be developed to produce transgenic
clonal plants as well as to obtain materials for artificial seeds
(Zimmerman, 1993) by using this technique. Synthetic seed application is
a promising field. Genetically heterozygous plants or plants with unique
gene combinations which can not be maintained by conventional seed
production techniques due to increases in genetic recombinations at each
generation, can be manufactured as synthetic seeds. This technology
might provide an alternative to fields on green house nurseries for storing
germplasm. Implementation of recycling procedures involving immature
somatic embryos can be a challenge for large scale applications. The
major emphasis during the production scale-up period should be to
overlay the established fundamental principles of manufacturing planning
and control (Grossnickle et al., 1996).
36
Ozlem Yesil-Celiktas et al.
The organization of the specialized organs and the nature of the various
tissues of cultured material, determine the survival potential of plants ex
vitro. Vitrification which is the phenomenon of hyperhydricity in organs and
plants in vitro, and in plants after they have been transplanted ex vitro is
identified as a weakness. Vitrified organs appear to be water soaked and are
brittle with an abnormal structure. Hyperhydric shoots are easily damaged by
desiccation and survive very poorly when subcultured or transferred to an
external environment (Ziv and Chen, 2007). In economical terms, clonal
propagation is again more expensive than the conventional methods of
planting and requires several types of skills and a constant monitoring of the
input (Anonymous, 2002). The technique is costly due to intensive hand
manipulation of the various culture phases. The need for multiple subcultures
on different media makes shoot and node culture extremely labor intensive.
Presently, total labor costs, typically ranging from 50 to 70% of production
costs, limit the expansion of micropropagation industry. Thus the current
application of the technology is restricted to high value horticultural crops
such as ornamental plants. Expansion of the industry to include production of
vegetable, plantation and forest crops depends on the development of more
efficient micropropagation systems. Cost-reduction strategies including
elimination of production steps and development of reliable automated
micropropagation systems will facilitate this expansion (Trigiano et al.,
1999). Abnormal plant development is sometimes observed and thus the
method can not be used commercially for all plant species (Ziv, 2000).
Genetic and epigenetic variations may occur during vegetative propagation
(Podwyszynska, 2005) and can seriously impair the quality of the product.
Microbial contamination as affected by both the introduced plant material
and operation procedures (Ziv, 2000) can be a drawback in large scale
bioreactors. Additionally, the existence of recalcitrant species for bioreactor
application is regarded as a weakness as such species are difficult to be
cultured in liquid medium even if they could be propagated on agar medium
(Gray et al., 1995). Moreover, synchronization of development remains one
of the major difficulties for moving somatic embryogenesis technique on to
an industrial scale (Molle and Freyssinet, 1992). Such synchronization needs
to be controlled at all stages of the process. Studies are now under way to
understand and control factors leading to morphological heterogeneity of
somatic embryos in the bioreactor, in order to achieve more uniform plantlet
development in the nursery. Overcoming this obstacle is essential before
industrial production can even be envisaged (Barry-Etienne et al., 2002).
Despite the difficulties, somatic embryogenesis continues to be a highly
attractive biological strategy for large scale production research. The
financing and funding of various projects and companies have been erratic,
Large scale cultivation of plant cell and tissue culture in bioreactors
37
often resulting in a lack of continuity and instability, as evidenced by
ventures such as Plant Genetics, of the United States based example of the
scale-up of somatic embryogenesis (Sluis, 2008).
Market demands for newly bred and ornamental plants and industrial
demands for standardized raw materials with high valuable compounds
contents are considered as opportunities. Hvosleif-Eide and coworkers (2003)
proposed three different approaches to scale-up micropropagation which then
are referred to as opportunities:
•
•
•
Standardization of plant tissue
Automation through use of liquid cultures and bioreactors and
Robotics
National and global regulatory and legal restrictions are identified as
threats. For instance, research on genetically engineered plants is developing
at a rapid pace and these GMO’s will be commercially produced by clonal
propagation (Sedjo, 2006), but this is very likely to face a barrier because of
the regulations.
38
Ozlem Yesil-Celiktas et al.
5. State of the art for large scale cultivation
Shikonin, ginsenosides, taxol and berberine are presently produced on
large scale. But, how do we define large scale? The rule of thumb is that any
production equal or above 20 L can be considered as large scale. The
production of secondary metabolites from C. roseus such as alkaloids,
ajmalicine and serpentine have been studied intensively and form ideal model
systems for testing new methods to stimulate secondary metabolite formation
(Asada and Shuler, 1989). Scale-up applications in plant cell and tissue
cultures is presented in Table 3. Air driven bioreactors are generally
preferred over the agitated tanks, and the stirred tank bioreactors equipped
with specially designed impellers are preferred when mechanical agitation is
indispensible. The volumes are in the range of 20 to 1000 L yielding
secondary metabolites such as alkaloids, ajmalicine, taxol and ginsenosides
from undifferentiated biomass and/or roots, shoots.
Only few studies have been carried out for the production of
adventitious roots in large scale at the industrial level. The first successful
attempt for scale-up was by Choi et al (2000) who have successfully
achieved 150-fold growth increases when ginseng adventitious roots were
grown in 500 L balloon type bubble bioreactors (air-lift bioreactors) for
7 weeks. The adventitious roots grown in bioreactors contained 1% of dry
root weight, which corresponds half of the content for the field grown
plants. Another successful example of scale-up process was by Wu et al
(2007) who have cultivated adventitious roots of Echinacea purpurea in
1000 L air lift bioreactors. They were able to achieve 5.1 kg dry biomass of
adventitious roots and these roots were possessing higher amounts of
cichoric acid (22 mg/g dry mass), chlorogenic acid (5 mg/g dry mass)
and caftaric acid (4 mg/g dry mass).
The profitability of a product is determined by the effectiveness of the
scale-up and the resulting cost of the transition into manufacturing. Capital
and operating costs associated with most glass and stainless steel bioreactors
are high, due to low yields coupled with high sterility criteria subsequent to
each culturing cycle. Consequently, the products extracted from cells or
tissues grown in such bioreactors are expensive, and cannot at present
compete commercially with comparable products produced by alternative
techniques.
Industrial scale bioreactor devices are traditionally permanent or semipermanent structures. Lately a patent has been filed proposing a concept of a
disposable bioreactor device for solving the aforementioned problems
regarding large scale cell/tissue culture production. The recent literature
reveals the predominance of disposable or in other words single use bioreactors.
Table 3. Large scale applications of plant cell and tissue culture.
Large scale cultivation of plant cell and tissue culture in bioreactors
39
40
Ozlem Yesil-Celiktas et al.
The disposable device for axenically culturing and harvesting cells and/or
tissue in at least one cycle, comprises of a sterilisable disposable container
having a top and a bottom end, which can be filled partially with a suitable
sterile biological cell and/or tissue culture medium (Shaaltiel, 2005).
In many cases, maintaining sterile conditions outside the disposable
bioreactor devices are not possible. Once opened to extract the harvestable
yield, these devices have no further use after one culturing cycle. But in spite
of this, disposable bioreactors are relatively inexpensive for the quantities and
production volumes which are typically required by non-industrial-scale
users, and are relatively easy to use by non-professional personnel. In fact
this aspect of simplicity of use and low economic cost is a major attraction of
disposable bioreactor devices. But on the other hand, the disposable
bioreactor devices have very little in common with industrial scale
bioreactors structurally, operationally or in the economies of scale (Shaaltiel,
2005) and the performance of electrochemical sensors for pH and oxygen is
often compromised.
Moreover, the control systems lack both performance and sophistication
when compared with their stainless steel counterparts at large scale
applications. Therefore, the usage of disposable bioreactors at industrial scale
is still in its infancy.
Shiau (2009) developed a bioreactor for growing fungus, plant cell,
tissue, organ, hairy roots and plantlet. The bioreactor comprised a nutrient
reservoir, a rotatable culture bed, culture medium, a mist generator and a mist
delivery pipe. The nutrient reservoir is used to store a nutrient solution
required for the explants and the culture medium is supported on the rotatable
culture bed which is rotated at a specific speed. Mists of the nutrient solution
is produced by the mist generator and delivered by the mist delivery pipe
which has a mist-exit port adjusted to a desired level above the culture
medium so as to discharge and spray the mists over the explants.
Although, the commercial production of anticancer drug paclitaxel using
plant cell culture was achieved by the Samyang Genex (Taejon, Korea) and
Phyton Catalytic (NY, USA) (Zhong et al., 2002), many research studies
have been concentrated on increasing the production of taxol-like compounds
from various Taxus species.
Apart from these, the majority of the innovations are related to
developing methods and optimization of process parameters such as the
innovation related to conducting the plant cell culture by adding a saccharide
mixture to the culture medium as a carbon source, to increase the productivity
of the secondary metabolites, increasing the productivity of secondary
metabolites and shortening the culture time by the use of the mixture of
glucose and fructose. This could contribute in producing useful secondary
Large scale cultivation of plant cell and tissue culture in bioreactors
41
metabolites on an industrial scale using plant cell culture (Kim et al., 2006).
Kim and coworkers (2005) developed a method for production of secondary
metabolites comprising the step of culturing plant cells in a medium treated
with alkanoic acid or salt which may contribute to the commercial production
of some secondary metabolites. In another patent, a method of reproducing
plants by somatic embryogenesis is described where the embryogenic tissue
is placed onto a semi-permeable membrane located in a culture medium and
incubating it to produce a normal somatic embryo (Bausher et al., 2004). A
method for clonal propagation of scented Pandanus amaryllifolius was
patented involving the establishment of aseptic shoot cultures of
P. amaryllifolius using both erminal and lateral shoot buds as explants
(Neelwarne et al., 2005).
In addition to the well-known applications of plant cell and tissue
cultures, another novel approach has been introduced in recent literature,
where the enzymatic content of the plant material is used to carry out
enzymatic modifications to produce value-added products. Takemoto (2009)
proposed an innovative process for producing theaflavin selectively, in large
quantity, in high yield, simply, and at low cost. The process was
characterized by mixing a processed plant extract containing epicatechin,
epigallocatechin, epicatechin-3-O-gallate and epigallocatechin-3-O-gallate
with a plant cell culture material having a peroxidase activity to thereby
produce theaflavin selectively.
Another innovation focused on a process development for the production
of peroxidase which comprised of a plant cell culture producing cells from
neem (Azardiracta indica) and nirgundi (Vitex negundo) wherein the
peroxidase obtained had higher enzymatic activity not reported earlier.
Compact callus aggregates showing tremendous enzyme activity were
induced so that it could be recovered and reused. In most of the commercial
preparations, the general plant body is used for the extraction of the enzyme
thereby leading to the destruction of the plants as well as accumulation of
waste materials. Apart from that, the enzyme purification from the general
plant body is very difficult and usually nearly 70 % o the purified enzyme
price may be akin to purification cost. A method was developed by changing
the concentrations of hormone combinations and the supplement (coconut
water) in order to optimize the biomass growth and enzyme production
(Abraham et al., 2008).
42
Ozlem Yesil-Celiktas et al.
6. Concluding remarks
In this study, the large-scale cultivation of plant cell and tissue cultures
for the purpose of commercially valuable secondary metabolite production as
well as in vitro plant propagation are discussed. With respect to secondary
metabolite production by plant cell and tissue cultures, the characteristics and
the configuration of the industrial plant, consisting of reactors and separation
equipment, determines the economic feasibility of the process. In general, the
industrial plant must be capable of for the production of more than one
product so that an economic utilization is ensured. Even when the plant is
used for a single product, a flexible design allowing for different flows of
material, new strains of higher productivity, different heat evolution and
aeration requirements should be used.
Technological, economical and scale-up aspects show that air driven
bioreactors such as airlift and bubble column reactors are more commonly
adapted to large scale applications. The successful and commercial exploitation
of plant cell and tissue cultures will depend on multidisciplinary studies of
plant morphogenesis in liquid media and on the understanding of the control
mechanisms of organ and embryo development from meristematic or bud
clusters. The chemical and physical environment, in relation to biomass
growth and controlled regeneration, should be further investigated. The
chemical composition of the growth media such as the level of carbohydrates
and specifically the levels and ratios of growth-promoting and -retarding
regulators will need to be further studied in more detail as well as the
accompanying physical conditions. The wide spread exploitation of plant cell
and tissue cultures for secondary metabolite production will be dependent on
the development of low cost, high productivity processes specifically tailormade for low volume high cost products.
The high efficiency of plant propagation using bioreactors has been
revealed by the work of many researchers. Bioreactor technology seems to be
applicable to commercial propagation for the several plant species discussed
herein. However, many problems still exist in scale-up and in the adaption of
this know-how to other plant species. The main cause for the problems comes
from the difficulty in the preparation and handling of the bioreactors, in the
preparation of ‘seed cultures’ and in determining the optimum culture
conditions, which depends mainly on the types of culture, different genera or
species of plants, etc. The advantages of bioreactor technology exist in the
high efficiency and ease of operation. However, the very special requirements
of mass propagation provide significant challenges to process engineers. For
Large scale cultivation of plant cell and tissue culture in bioreactors
43
example, the mass propagation of shoots in large scale bioreactors is realized
and the propagated shoots can be used as seed plants after acclimatization. In
the future, it will be necessary to develop a system of illumination to
uniformly propagate shoots. The problems discussed as the weaknesses of the
technique have to be overcome before the wide-spread use of the technique
for commercial propagation.
In parallel to the future developments in process automation and robotics,
bioreactors will be indispensible for industrial scale in vitro plant
propagation. Implementations of scale-up and mechanization techniques are
mandatory for the expansion of commercial micropropagation. New
technologies in robotics and machine vision systems for cutting, sorting, and
dispensing have been reported. However, these technologies have actually
increased the cost of plant micropropagation.
Automation is potentially effective for the development of a practical
embryo production system. It could offer labor cost reductions for the
established culture protocol, as well as contribute to the study somatic
embryogenesis and to improvement of embryo production efficiency.
Automation techniques are expected to contribute to the acquisition of basic
knowledge of the biological processes. Methods for monitoring and control
of culture parameters will improve the reliability and/or reproducibility of
culture processes, replacing expert judgements. Many improvements are still
needed for economical automated somatic embryo production systems and
this can only be achieved through progress in mechanization techniques
coupled with studies in the response and mathematical modelling of
biological processes.
Further studies should also be carried out on developing techniques to
efficiently remove plants from the bioreactor for transplanting because the
cultures often form large clumps.
In conclusion it can be stated that the future success of large scale
cultivation of plant cell and tissue culture will depend on the collaborative
efforts of researchers from different disciplines as well as the evolving
consumer demands and innovative spirit of entrepreneurs.
44
Ozlem Yesil-Celiktas et al.
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