2015

NPC Natural Product Communications Vol. 10

No. 5
Plant Cell, Tissue and Organ Culture: the Most Flexible Foundations 815 - 820
for Plant Metabolic Engineering Applications
Shinjiro Ogitaa, b*
a
Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Imizu,
Toyama, 939-0398, Japan
b
Faculty of Life and Environmental Sciences, Department of Life Sciences, Prefectural University of Hiroshima,
727-0023 Shoubara, Japan

ogita@pu-hiroshima.ac.jp

Received: March 17th, 2015; Accepted: March 19th, 2015

Significant advances in plant cell, tissue and organ culture (PCTOC) have been made in the last five decades. PCTOC is now thought to be the underlying
technique for understanding general or specific biological functions of the plant kingdom, and it is one of the most flexible foundations for morphological,
physiological and molecular biological applications of plants. Furthermore, the recent advances in the field of information technology (IT) have enabled access
to a large amount of information regarding all aspects of plant biology. For example, sequencing information is stored in mega repositories such as the
National Center for Biotechnology Information (NCBI), which can be easily accessed by researchers worldwide. To date, the PCTOC and IT combination
strategy for regulation of target plant metabolism and the utilization of bioactive plant metabolites for commercial purposes is essential. In this review, the
advantages and the limitations of these methodologies, especially regarding the production of bioactive plant secondary metabolites and metabolic engineering
in target plants are discussed mainly from the phenotypic view point.

Keywords: Bioactive compound, Biosynthesis, IT, Metabolic engineering, Plant cell biology, PCTOC, PDCA-cycle.

Introduction
Totipotency is the inherent capacity of a plant cell or tissue to
develop into a whole plant. Theoretically, all plant cells contain all
the information necessary for the growth and reproduction of the
organism [1]. Numerous studies have been carried out to
characterize the totipotency of plant cells in terms of morphology,
physiology and molecular biology [2]. In this context, one of the
most important findings was the identification of plant growth
regulators (PGRs). For example, in 1928, auxin-type PGRs were
discovered via the Avena geo-curvature test [3]. The establishment
of the in vitro cell differentiation model in carrot [4] was the key
study that demonstrated totipotency for the first time. Another
important step in the study of plant cell totipotency was the
establishment of a common medium component for plant cell and
tissue cultures, i.e., Murashige and Skoog (MS) medium [5]. In the
last five decades, significant advances regarding plant cell, tissue
and organ culture (PCTOC) have been made [6-7]. At present,
PCTOC protocols (Figure 1) are used in all research and
development areas of plant biotechnology.
Another relevant point is the recent advances in information
technology (IT), known as bioinformatics in life sciences. The
regulatory dynamics of plant metabolism involved in general
responses during growth, differentiation, senescence, and in specific
responses to environmental stresses, pest damage, plant-plant and
plant-microbe interactions have been studied since the early 20th
century via morphological, physiological, and molecular
methodologies. Therefore, most of the basic metabolic pathways of
model plant species, such as Arabidopsis (Arabidopsis thaliana L.)
and rice (Oryza sativa L.), have already been elucidated [8-9]. At
present, owing to the use of the omics technologies, genomic
information, gene transcription, protein expression, and
metabolomics profiling (mainly of primary metabolites and
specifically of target secondary metabolites) can be obtained from
a model plant. Furthermore, plant secondary metabolites, which
Figure 1: Methodologies involved in Plant Cell, Tissue Organ Cultures (PCTOC). Bio-
reactor system (*1) is the ultimate bioprocess technology for bioactive plant secondary
metabolites production and metabolic engineering. Cryopreservation technique (*2) is
the non-lethal storage of plant tissues or cells at ultra-low temperatures. This is a useful
technique in metabolic engineering for maintaining superior mother plants or cell
strains that accumulate high amounts of target metabolite. For more information, see
references [21-22].
comprise more than 200,000 defined structures [10], including a
large number of natural bioactive compounds, have been published
and a large amount of this information is now available in mega-
data banks, such as the National Center for Biotechnology
Information (NCBI). For example, DNA sequences annotated
worldwide are deposited on the GenBank at the NCBI site. The
GenBank is part of the International Nucleotide Sequence Database
Collaboration, which comprises three organizations that exchange
data on a daily basis: the DNA Data Bank of Japan (DDBJ), the
European Molecular Biology Laboratory (EMBL), and the
GenBank itself. All types of data such as genomes, genes, proteins,
chemicals, and references are now deposited in the NCBI web-site
via the world-wide network (see http://www.ncbi.nlm.nih.gov/).
Many researchers studying fundamental or applied aspects of
the regulation of the target plant metabolisms and the utilization of
816 Natural Product Communications Vol. 10 (5) 2015
Figure 2: Representation of biological events, environmental stresses, plant-plant and plant-microbe interactions related to plant primary and secondary metabolism.
bioactive plant metabolites for commercial purposes have focused
on PCTOC and IT issues separately. In order to merge these two
areas for scientific research or commercial production of plant
secondary metabolites, researchers should understand phenotypic
and logical issues of PCTOC and bioinformatics research. In this
review, the advantages and the limitations of these methodologies,
especially regarding the production of bioactive plant secondary
metabolites and metabolic engineering in target plants are discussed
mainly from the phenotypic view point.
Biological events related to primary and secondary metabolic
activities in plants
As shown in Figure 2, primary and secondary metabolic activities
from photosynthesis to leaf senescence are regulated by the whole
plant under natural conditions. They are also regulated during
certain morphological and physiological changes such as fruit
ripening and flowering under specific conditions. Furthermore,
plants respond to stress-triggered phenomena such as pest damage,
wounding, and plant-microbe, or plant-plant interactions known to
trigger allelopathy [11-12]. It is widely known that phytohormones
are major endogenous regulatory factors. In natural environments,
primary and secondary metabolic activities are up- or down-
regulated under the control of phytohormone metabolisms. The
resulting primary and secondary metabolites are regulated at various
stages of the annual life-cycle of the whole plant. Thus, this
regulation is a dynamic and long-spanned reaction that is difficult to
replicate in the laboratory. This is disadvantageous for modern
metabolic engineering approaches. Therefore, in the following
section, the advantages of PCTOC methodologies are described,
especially considering bioactive plant secondary metabolite
production and metabolic engineering in target plants.
PCTOC methodologies
The PCTOC theory, techniques, and case studies developed during
the last five decades [7] can be obtained from the following sources:
research and review articles, protocol books, databases accessible
via the internet, and practical knowledge in traditional laboratories
under the guidance of senior staff members. Junior researchers, who
Ogita
are particularly interested in the latest plant biology research, tend
to access the databases as their first source of information due to
convenience and easy accessibility. Although IT development has
contributed to the development of all the fields of plant biology, the
development of mega databases has also complicated the
identification of the relevant information. Thus, without an
extensive knowledge of all the fields of plant biology, it might be
difficult to choose the appropriate answer.
There are vital issues that need answering to design a well-
organized PCTOC strategy for the target plant, especially in the
area of plant metabolic engineering. These issues include how the
PCTOC technology works in the focused area, which protocols are
available to us, and whether there is any particular step (trick) in the
protocol. However, research and review articles, and the databases
rarely provide clear answers to these questions. In some cases,
asking senior staff members might be the ideal solution to find out
about particularly difficult steps in the protocol, though it is
sometimes not convenient either. In order to solve these problems,
combinations of the glossary of PCTOC [13] and some critical
points of PCTOC technologies are described below. For basic
information of PCTOC, a plant biotechnology textbook [14] is
recommended.
Micropropagation: This is a general term referring to vegetative
plant propagation under aseptic conditions in a synthetic medium in
vitro. It sometimes refers specifically to whole clonal plant
multiplication via shoot/axillary bud cultures. In general, the
following culture methods are classified under micropropagation.
Organ culture: Culture propagation by using plant organs
(explants) such as leaf, node, internode, shoot, axillary bud, root,
and seedling. This is a basic micropropagation technique for target
plants without somaclonal variation. It is a useful technique in
metabolic engineering for maintaining superior mother plant
varieties that accumulate high amounts of target metabolites.
Solidified medium conditions are usually preferred for organ
culture. In some cases, organ culture is used as a synonym for tissue
culture.
Plant cell, tissue and organ culture for plant metabolic engineering
Meristem tip culture: This is an advantageous culture type derived
from meristem tip explants (meristem tip consists of the
meristematic dome and usually a pair of leaf primordia). This
technique is used for virus elimination or axillary shoot
multiplication purposes. It is a useful technique to maintain superior
mother plant varieties that accumulate high amount of target
metabolite. Liquid medium conditions are used in this technique,
occasionally.
Organogenesis: This is the term used to mean organ initiation and
growth (usually shoots and roots) from cells or tissues. It also refers
to the culture process. Organs may form on the surface of the
explants (direct organogenesis) or upon an intervening callus phase
(indirect organogenesis; see callus culture). Since cultured shoots
can sometimes show heterogeneous differentiation levels, culture
conditions should be carefully controlled to select a suitable culture.
After culture condition optimization, the handling procedure for
organogenesis is easy and can be used for metabolic engineering
purposes [15].
Somatic embryogenesis: This is the term used to mean production
of embryos from explant somatic cells. It also refers to the culture
process. Somatic embryos may form on the surface of explants
(direct embryogenesis) or upon an intervening embryogenic callus
phase (indirect embryogenesis). Usually, suitable explants for
somatic embryogenesis are immature zygotic embryos and
seedlings. The embryogenic capacity of mature organs such as
leaves and roots from adult plants is low. After culture condition
optimization, somatic embryogenesis can be used for metabolic
engineering purposes [16].
Cell and tissue culture: This is a general term referring to the
cultivation of plant parts (cells, tissues or organs) under aseptic
conditions in synthetic medium in vitro. It also refers to the culture
of single or groups of cells on solidified or liquid media. In some
cases, tissue culture is a synonym for organ culture. In general, the
following culture methods are classified as cell and tissue culture.
Adventitious root/ hairy root culture (rhizogenesis): This is a
useful technique to produce target metabolites. Isolated root tips of
apical or lateral origin may produce in vitro root systems with
indeterminate growth habits. The process of in vitro root formation
is also called rhizogenesis. The root inducing (Ri) plasmid of
Agrobacterium rhizogenes is used for hairy root culture induction
[17]. Since cultured roots can sometimes show heterogeneous
differentiation levels, culture conditions should be carefully
controlled to select a suitable culture. Dark condition is preferred to
maintain rhizogenesis. A liquid culture system is applicable to
target metabolite production. The bioreactor system can also be
used to scale-up target metabolite production.
Callus culture (Callogenesis): The term callus refers to
disorganized tumor-like masses of plant cells. Calli proliferate in
irregular tissue masses, and vary widely in texture, appearance, and
growth rate. The process of callus formation is also known as
callogenesis. The histological and physiological characteristics of
calli vary depending on the explant source and culture medium
composition. The callus culture is usually initiated on solidified
medium by inoculation of small explants or sections from
established organ cultures. After culture condition optimization, the
handling procedure of callus culture is easy and can be used for
metabolic engineering purposes [18].
Suspension culture: This is an advantageous technique to maintain
cells and small cell aggregates suspended in a liquid medium.
Natural Product Communications Vol. 10 (5) 2015 817
Explants, or calli derived from these, are transferred to liquid
medium and the cultures are then agitated on a mechanical shaker.
The cells and small cell aggregates that show homogenous growth
in liquid medium are useful for metabolic engineering. A liquid
culture system is applicable to target metabolite production. The
bioreactor system can also be used to scale-up target metabolite
production [19].
Protoplast culture and fusion: A protoplast is a cell without its
cellulosic wall. Protoplasts are isolated by either mechanical
disruption or by enzymatic digestion of plant tissue or cultured
cells. Protoplasts are utilized for selection or hybridization at the
cellular level and for a variety of other purposes [20]. Protoplast
fusion (sometimes called cell fusion) can be mediated by either
chemical or electrical techniques. Polyethylene glycol (PEG) is the
most commonly employed fusogen. The equipment necessary for
electrical induction of fused cells consists of a function generator to
produce the AC field, a DC source, a switching unit to apply the AC
field or DC pulses, and a suitable fusion chamber.
Bio-reactor systems: This is the ultimate bioprocess technology for
bioactive plant secondary metabolites production and metabolic
engineering. The bioreactor design/operation optimization,
development of an advanced strategy for bio-reactor functioning, or
the adoption of disposable bioreactors need to be investigated. The
industrial exploitation of bioprocessing using plant cells also
requires systematic scale-up studies [21].
Cryopreservation: Cryopreservation is the non-lethal storage of
plant tissues or cells at ultra-low temperatures. This technique is
applicable to a wide-range of plant biological research purposes
[22]. It is a useful technique in metabolic engineering for
maintaining superior mother plants or cell strains that accumulate
high amounts of target metabolite.
PCTOC approaches for metabolic analysis and engineering
The establishment of a strategy for suitable PCTOC protocol
implementation for metabolic analysis and engineering in a target
plant requires several systematic studies. The Plan-Do-Check-Act
(PDCA) cycle generally used in the business administration field
can be adopted in PCTOC (Figure 3). Researchers can logically
consider the requirements for solving the issues in the P and D
cycles, such as targeting, explant selection, medium preparation,
sterilization, and PCTOC and environment setting. IT can be of
great use for these logical considerations. On the other hand, cycle
C requires the knowledge acquired through experience. The
important trick is whether the researcher is able to observe
phenomena such as browning, growth habits, and phenotype of the
plant cells and make appropriate theoretical judgments.
PCTOC methodologies
An example of a PDCA-cycle applied to PCTOC is shown in Figure
4. This is a case study involving the establishment of an efficient
cell culture protocol in bamboo plant and its application in our
laboratory. Bamboos are fast-growing plants and they have recently
been considered a prime renewable resource for biomass
production. Furthermore, the importance of bamboo forests as a
potential global environment modulator has also been proposed [23-
24]. In cycle P, we systematically maintained in vitro node (organ)
culture stocks of several bamboo species. Node portions that had
apical and intercalary meristems were selected explants for callus
culture in Bambusa multiplex. In cycle D, in vitro node culture
stocks could directly be used as explant source without sterilization.
When node portions of B. multiplex were cultured on an optimized
818 Natural Product Communications Vol. 10 (5) 2015
Figure 3: The Plan-Do-Check-Act (PDCA) cycle for PCTOC approaches.
proliferation medium of bamboo [25], active callus induction and
organ differentiation could be seen. The important factors to
consider with macroscopic observation of a culture are coloration,
browning (both medium and cultured tissue), and growth habits
(proliferation, hardness, and water content). The typical variations
in callus/organ originated from the same explant are shown color-
coded in Figure 4. The whitish callus, soft to friable in texture,
indicated an active proliferation capacity (24-fold), greenish callus,
friable to compact in texture, showed a moderate growth (9-fold),
and adventitious roots, organ bunches, proliferated by
elongating/branching (5-fold). Although calli usually proliferate in
irregular tissue masses and vary widely in texture, it is possible to
generate different cell lines under the same medium conditions by
careful separation of these cultures from the explant. The growth
habits of each culture were recorded during subcultures for 3-6
months, and the phenotypes were homogenized during the C and A
cycles.
Phenotypic information from these bamboo cell and organ cultures
recorded via macroscopic and microscopic observations
(morphological and histochemical characteristics), and their
metabolic profiling obtained e.g. via HPLC analysis were merged
with the available IT data (Figure 5). The biosynthesis of secondary
metabolites shown in the background of Figure 5 represents the
information for Oryza sativa (japonica rice) from the Kyoto
Encyclopedia of Genes and Genomes (KEGG:
http://www.genome.jp/kegg/). This schematic representation
captures well the important relation between phenotypes and the
metabolic profiling of the active biosynthesis in the cultures
characterized in this study.
Perspectives
Model plant cultured cell lines: Plant cultured cell line collections,
especially suspension cultured cells, are of great use in many
aspects of basic and applied metabolic engineering. For example, 52
optimized unique cell lines are supplied by the Bioresource Center,
RIKEN, Japan (see
http://www.brc.riken.jp/lab/epd/Eng/catalog/pcellc.shtml). The BY-
2 cell line (rpc00001) of tobacco (Nicotiana tabacum cv. ‘Bright
Yellow 2’), which is supplied by the center mentioned above, is the
most widely used cell line for the study of the different aspects of
plant cell biology [26-28]. Furthermore, suspension cell lines such
as the T87 line (rpc00008) of Arabidopsis (Arabidopsis thaliana L.
Ogita
Heynh., ecotype Columbia) and the Oc line (rpc00031) of rice
(Oryza sativa L. C5924) have also been established and used as
model plant cell lines [29-30].
We also proposed two prominent bamboo suspension cell lines, Pn
(rpc00047) and Pb (rpc00048) as bio-resource models [31-32, 25].
A series of morphological and histochemical characterizations, and
the metabolic profiling of the Pn cell line were carried out under
several culture conditions, and the profiling data were characterized
[33-34]. At present, our efforts focus on metabolic engineering via
functional gene introduction or transcriptional regulation.
Future impact of omics methodologies on PCTOC applications:
Large-scale omics methodologies such as DNA sequencing,
transcription profiling, proteomics, and metabolite profiling have
become important tools, mainly in the field of inverse metabolic
engineering. These techniques allow the identification of the diverse
genetic players involved in the biosynthesis of target metabolites
and provide an insight into their cellular effects or interactions [35].
The future impacts of omics methodologies on PCTOC applications
for metabolic engineering might include the well standardized
phenotyping of target plant cells. This process has a great demand
because the phenotypes of cultured cells are still evaluated
manually at present, and the process is subjective and not precise
enough for high-throughput profiling. As previously mentioned, the
important trick is whether the researcher is able to observe
phenomena such as browning, growth habits, and the phenotype of
plant cells or organs, and make appropriate theoretical judgments.
Therefore, a so-called phenomics approach [36] in PCTOC should
be developed, which focuses not on a series of data but on the better
integration of the knowledge acquired through experience.
Acknowledgements – The author (SO) would like to express his
deep appreciation to all the members of the Plant and Cell
Engineering Laboratory of Toyama Prefectural University. The
author also thanks Ms Mishizu Hayashi for her technical assistance.
This study was supported in part by Grants-in-Aid for Scientific
Research C (no. 22580387, 25450519, and 25450134 to SO), from
the Japan Society for the Promotion of Science. This study was also
partially supported by a plant research grant to SO from the New
Technology Development Foundation (2013-2014).
Plant cell, tissue and organ culture for plant metabolic engineering Natural Product Communications Vol. 10 (5) 2015 819

Figure 4: An example of PDCA-cycle in callus and organ cultures of Bambusa multiplex. Scale bars indicate 1 cm.

Figure 5: Schematic representation of phenotyping and profiling of active biosynthesis in callus and organ cultures of Bambusa multiplex. Lugol and Wisner staining methods
[33,37] were used for the detection of starch accumulation and lignin deposition under a white light, respectively. DPBA and NileRed staining methods [38,39] were also used for the
evaluation of flavonoid and lipid biosynthetic activities, respectively. Scale bars indicate 1 cm.
820 Natural Product Communications Vol. 10 (5) 2015 Ogita

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