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In Vitro Cell. Dev. Biol.—Plant 40:342–345, July/August 2004
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DOI: 10.1079/IVP2004555
INVITED REVIEW:
IN VITRO MORPHOGENESIS IN PLANTS – RECENT ADVANCES
GREGORY C. PHILLIPS*
Arkansas State University, College of Agriculture, P.O. Box 1080, State University, AR 72467-1080
(Received 1 January 2004; accepted 16 March 2004; editor P. Lakshmanan)
Summary
The capacity of cultured plant tissues and cells to undergo morphogenesis, resulting in the formation of discrete organs
or whole plants, has provided opportunities for numerous applications of in vitro plant biology in studies of basic botany,
biochemistry, propagation, breeding, and development of transgenic crops. While the fundamental techniques to achieve
in vitro plant morphogenesis have been well established for a number of years, innovations in particular aspects of the
technology continue to be made. Tremendous progress has been made in recent years regarding the genetic bases
underlying both in vitro and in situ plant morphogenesis, stimulated by progress in functional genomics research.
Advances in the identification of specific genes that are involved in plant morphogenesis in vitro, as well as some selected
technical innovations, will be discussed.
Key words: somatic embryogenesis; shoot organogenesis; root organogenesis; floral organogenesis; gene expression;
morphogenesis; in vitro.
pH; humidity; light quality and quantity or absence of light;
temperature; gaseous environment), and osmotic potential. Many of
these factors have to be adjusted (e.g., carbohydrates, nitrogen
sources) or completely changed (e.g., withdrawal or reduction in
auxin signal; perhaps an increase in other plant growth regulators
such as abscisic acid; osmotic potential change to encourage
desiccation) during maturation of somatic embryos, during which
time they become competent for conversion into plantlets (Thorpe,
2000).
Many of the same culture factors described above for somatic
embryogenesis are also manipulated to induce and optimize
organogenesis, but often these factors are manipulated in different
ways (Joy and Thorpe, 1999). For example, a high auxin signal (often
specifically using 2,4-dichlorophenoxyacetic acid) is usually
important to induce somatic embryogenesis, whereas a high
cytokinin to auxin ratio (or high cytokinin with no auxin) is typically
required to induce shoot organogenesis. Root initiation also typically
requires a moderate to high auxin signal – but rarely with the use of
2,4-dichlorophenoxyacetic acid, rather with the use of a more
‘natural’ source of auxin (Gamborg and Phillips, 1995). Because
regenerated organs are unipolar, two distinct organogenic induction
signals – one to induce shoots and the other to induce roots – are
required to regenerate a whole plant. In contrast, bipolar somatic
embryos are induced by a single induction signal.
Fundamental Aspects of IN VITRO MORPHOGENESIS
The two primary morphogenic pathways leading to whole plant
regeneration – which is a prerequisite for most plant breeding,
genetic and transgenic applications of in vitro biology – involve
either somatic embryogenesis, or shoot organogenesis followed by
root organogenesis. Both developmental pathways can occur either
directly without a callus intermediate stage, termed adventitious; or
indirectly following an unorganized callus stage, termed de novo
(Gamborg and Phillips, 1995). Few plant species have been shown
to regenerate by both organogenic and somatic embryogenic
pathways, but many plant species can regenerate by one or the other
of these pathways.
Somatic embryogenesis may be the best example of totipotency
expressed among a large number of plants (Thorpe, 2000). Various
culture treatments can be manipulated to optimize the frequency and
morphological quality of somatic embryos, which are bipolar
structures containing both shoot and root apices, and developing in a
manner parallel to that of zygotic embryos. Typical treatment factors
include the plant growth regulator sources and concentrations
(especially the auxin), choice of explant, nutrient medium
composition (especially inorganic vs. organic nitrogen sources,
carbohydrate sources and concentrations), culture environment
(including the physical form of the medium, e.g. liquid or semi-solid;
*Author to whom correspondence should be addressed: Email gphillips@
astate.edu
REPRINTED FROM: Phillips, G. C. In vitro morphogenesis in plants –
recent advances. In: Goodman, R. M., ed. Encyclopedia of plant and crop
science, vol. 1. New York: Marcel Dekker, Inc.; 2004: 579–583. http://www.
dekker.com/servlet/product/DOI/101081EEPCS120010554; by courtesy of
Marcel Dekker, Inc.
Genetic Components of Morphogenesis
One of the most exciting advances in recent years has been the
discovery of specific genes involved in plant regeneration in vitro.
Such genes are being explored for use to increase transformation
efficiency and to develop marker-free transgenic plants (Zuo et al.,
342
343
IN VITRO MORPHOGENESIS IN PLANTS
2002b). Because a primary factor involved in optimizing somatic
embryogenesis and organogenesis is the use of phytohormone
models, it is of interest that receptors for each of the major
phytohormone classes have now been identified and many of the
corresponding genes have been cloned (Møller and Chua, 1999; Zuo
et al., 2002b). Examples of specific genes involved in the major
plant morphogenesis pathways are summarized in Table 1.
Genetic aspects of somatic embryogenesis. Transgenic expression
of the LEC2 (leafy cotyledon) gene (Table 1) is sufficient to initiate
somatic embryogenesis with high viability but some abnormalities
persist in morphology (Zuo et al., 2002b). Several genes appear to
be involved in the vegetative-to-embryogenic transition, such as
WUS (wuschel; or PGA6, plant growth activator), LEC1 (Zuo et al.,
2002a), SERK (somatic embryogenesis receptor kinase) (Zuo et al.,
2002b), and PT1 (primordial timing) (Harada, 1999). SHR (short
root) establishes the ground tissue through the first asymmetric cell
division (von Arnold et al., 2002), CLV (clavata) and WUS interact
to determine stem cell fate, and CLV and STM (shoot meristemless)
regulate development of the shoot apical meristem (Fletcher, 2002;
von Arnold et al., 2002). LEC1, ABI3 (abscisic acid-insensitive) and
FUS3 (fusca) are involved in somatic embryo maturation (von
Arnold et al., 2002).
Genetic aspects of shoot organogenesis. CYCD3 is involved in
the acquisition of competence for shoot regeneration (Sugiyama,
1999; Fletcher, 2002), as is SRD3 (shoot redifferentiation)
(Sugiyama, 1999, 2000) (Table 1). Shoot redifferentiation also
involves SRD1 and SRD2 (Sugiyama, 1999, 2000). The vegetativeto-shoot organogenesis transition is promoted by ESR1 (enhancer of
shoot regeneration) (Zuo et al., 2002b). Two genes representing
potentially independent pathways involved early in shoot
organogenesis signal transduction are CRE1 (cytokinin receptor)
and CKI1 (cytokinin independent) (Sugiyama, 2000; Zuo et al.,
2002b). The shoot apical meristem stem cell identity is regulated by
CLV and WUS (Fletcher, 2002), parallel to that observed in the
shoot apical meristem of somatic embryos (above). STM and KN1
(knotted) are involved in the function of the shoot apical meristem,
and overexpression leads to the formation of ectopic shoot
meristems (Sugiyama, 1999; Fletcher, 2002). Other regulators
involved in shoot apical meristem organization and lateral shoot
formation include multiple SHO (shoot organization) and MGO
(mgoun) genes (Fletcher, 2002).
Genetic aspects of root organogenesis. Competence to regenerate root organs is affected by SRD2 (Sugiyama, 1999, 2000)
(Table 1). The transition of embryonic root cells to initiate
TABLE 1
EXAMPLES OF GENES INVOLVED IN VARIOUS PLANT MORPHOGENESIS PATHWAYS
Gene
Somatic embryogenesis
LEC2
WUS (PGA6), SERK, LEC1
SHR
CLV, WUS
CLV1, CLV3, STM
LEC1, FUS3, ABI3
Shoot organogenesis
CYCD3
SRD3
SRD1, SRD2
ESR1
CRE1
CKI1
CLV, WUS
KN1, STM
SHO, MGO
Root organogenesis
SRD2
PKL
RML
CYCD4;1
RAC
Floral organogenesis
LFY
AP1
UFO
WUS
AG
SEP
Putative function
References
Initiates ectopic somatic embryogenesis
Involved in the vegetative-to-embryogenic transition
Establishes ground tissue via asymmetric cell division
Regulate stem cell fate
Regulate shoot apical meristem development
Regulate embryo maturation
Zuo et al., 2002b
Harada, 1999; Zuo et al., 2002a, b
von Arnold et al., 2002
Fletcher, 2002; von Arnold et al., 2002
Fletcher, 2002; von Arnold et al., 2002
von Arnold et al., 2002
Involved in acquisition of competence for organogenesis
Competence for shoot organogenesis
Competence for redifferentiation of shoots
Enhances shoot regeneration, vegetative-to-organogenic transition
Cytokinin receptor
Cytokinin perception
Preserve stem cell identity in shoot apical meristem
Initiate ectopic shoot meristems, shoot apical meristem function
Modifiers of the shoot apical meristem involved in
leaf founder cell recruitment, lateral organ primordial
Sugiyama, 1999; Fletcher, 2002
Sugiyama, 1999, 2000
Sugiyama, 1999, 2000
Zuo et al., 2002b
Zuo et al., 2002b
Fletcher, 2002; Zuo et al., 2002b
Fletcher, 2002
Fletcher, 2002
Fletcher, 2002
Competence for root organogenesis
Transition of embryonic root cells to grow vegetatively
Root apical meristem function
Involved in lateral root formation
Involved in adventitious root formation and auxin transduction
Sugiyama, 1999, 2000
Harada, 1999
Sugiyama, 2000; Anderson et al., 2001
De Veylder et al., 1999
Sugiyama, 2000
Switch to reproductive development, floral meristem identity
A-class gene involved in establishing the first floral whorl: petals
Interacts with LFY by providing regional specificity within floral
meristems and to control B-class signals which establish the
second floral whorl: sepals
Interacts with LFY to control C-class genes
C-class gene typifying the class; interacts with B-class signals to
produce the third floral whorl: stamens; C-class genes acting alone
produce the fourth floral whorl: carpels
Co-factors for A-, B-, and C-class genes to convert vegetative
leaves into floral organs
Fletcher, 2002; Lohmann and Weigel, 2002
Lohmann and Weigel, 2002
Lohmann and Weigel, 2002
Lohmann and Weigel, 2002
Lohmann and Weigel, 2002
Lohmann and Weigel, 2002
344
PHILLIPS
vegetative growth is controlled by PKL (pickle) (Harada, 1999).
RML1 (root meristemless) and RML2 play specific roles in the
root apical meristem (Sugiyama, 2000), and interact with
components of the apical dominance system (Anderson et al.,
2001). The RAC (rooting auxin-cascade) gene is involved in an
early stage of auxin perception specific to the formation of
adventitious roots (Sugiyama, 2000), and CYCD4;1 is directly
involved in lateral root primordia formation (De Veylder et al.,
1999).
Genetic aspects of floral organogenesis. Floral organs arise as
determinate structures out of the indeterminate shoot apical
meristem (Fletcher, 2002). The concept of floral organs being
specified by the A-, B-, and C-class genes is well established
(Lohmann and Weigel, 2002). LFY (leafy) is a key gene involved in
the switch to reproductive growth and in establishing floral
meristem identity (Fletcher, 2002; Lohmann and Weigel, 2002)
(Table 1). LFY activates the key A-class gene AP1 (apetala),
establishing the petals or outermost whorl of the floral organ
(Lohmann and Weigel, 2002). LFY and UFO (unusual floral organs)
interact to control the B-class genes, with UFO providing regional
specificity within meristems and thereby establishing the sepal
whorl. LFY and WUS interact to control the C-class genes typified
by AG (agamous), and the C-class genes interact with B-class genes
to establish the stamens in the third whorl. C-class genes also act
alone to establish the fourth or innermost whorl comprised of
carpels, because AG suppresses the action of WUS thereby resulting
in a suppression of the B-class components. Three MADS-Box SEP
(sepellata) genes act as cofactors with the A-, B- and C-class genes
to convert vegetative leaves into floral organs.
Technical Innovations
In the past decade, many of the technical improvements resulting
in improved in vitro plant regeneration systems have been related to
manipulation of the gaseous and/or physical environment of the
cultures. In addition, a few other innovations have been noteworthy
that fall outside this category, pertaining to thin cell layer
techniques and synthetic seeds.
Manipulation of the gaseous and/or physical environment. Cultured plant tissues are known to interact with the culture medium
and gaseous environment. Forced ventilation and use of ventilated
culture vessels, for example, have facilitated optimization of in vitro
morphogenesis systems, and high CO2 treatments have permitted
establishment of photoautotrophic cultures (Buddendorf-Joosten
and Woltering, 1994). Control of the amount of ethylene released by
the cultured tissues into the head space of the culture vessel, or
alternatively, inhibition of ethylene synthesis or action have led to
improved morphogenic responses (Kumar et al., 1998).
Efforts to improve bioreactor designs to facilitate economical
large-scale production of plants or plant products have continued.
Key issues that must be addressed with bioreactor designs for plant
cell and tissue growth include aeration and minimization of shear
damage. Advances in automation and computer-controls have
rendered bioreactor performance more reliable (Paek et al., 2001).
One of the most exciting developments in bioreactor design has
been the temporary immersion system, which alternates immersion
of the plant tissues in the liquid culture medium with exposure to
the air space at timed intervals (Etienne and Berthouly, 2002).
Temporary immersion bioreactors have been demonstrated to
improve yields of shoot proliferation cultures, microtubers and
somatic embryos, as well as improve the quality and vigor of the
propagules with reduced frequencies of abnormalities and
hyperhydricity.
Another interesting development is the use of perfluorochemicals
and commercially-stabilized bovine hemoglobin as gas carriers to
enhance cell performance in liquid culture systems such as
bioreactors. Perfluorochemicals are recyclable (can be used to
deliver gases, then recovered from the culture and recharged), and
emulsion with the surfactant Pluronic F-68w appears to
synergistically enhance effectiveness. These gas carriers have
been shown to improve cell division rates, stimulate biomass
production, improve yields of cellular products, and enhance
morphogenic totipotency (Lowe et al., 1998, 2003). A technical
innovation with more of a physical impact on the culture
environment is the use of semi-permeable cellulose acetate
membranes to enhance citrus somatic embryogenesis and
particularly to normalize somatic embryo development (Niedz
et al., 2002).
Applications of thin cell layer and synthetic seed techniques. Thin cell layer culture, an approach mainly involving the
manipulation of explant size to induce and optimize regeneration,
has been used for many years with dicotyledonous species to study
in vitro morphogenesis. Thin cell layer cultures can be manipulated
for rigorously controlled programming of different morphogenic
responses: callus formation, shoot organogenesis, root organogenesis, floral organogenesis, or somatic embryogenesis (Nhut et al.,
2003). In recent years the thin cell layer technique has been
extended to a variety of species formerly considered to be
recalcitrant to in vitro morphogenesis. Evidence also is gathering
that thin cell layer techniques can be useful for recovering
transgenic plants from species heretofore considered recalcitrant to
genetic transformation.
There continues to be interest in developing synthetic seed
technology based on artificial encapsulation of somatic embryos
suitable for direct field sowing with reliable conversion into viable
plants. The most important technical advances in this area involve
the use of automated bioreactors to improve yields, combined with
the use of computer-imaging to sort out the somatic embryos
possessing sufficient quality for encapsulation and subsequent
conversion (Ibaraki and Kurata, 2001). Even more exciting are the
advances in using non-embryogenic (unipolar) structures for
encapsulation as synthetic seed (Standardi and Piccioni, 1998).
There seems to be a lower risk of somaclonal variation using
unipolar structures such as microbulbs, microtubers, rhizomes,
corms, shoots or nodes containing either apical or axillary buds,
meristemoids and bud primordia for encapsulation, and the
synthetic seed technology can be extended to a wider variety of
genotypes.
Conclusions and Future Prospects
Basic research has begun to dissect the complex genetic
pathways involved in various aspects of plant morphogenesis,
including all of the major pathways leading to in vitro plant
regeneration. A number of candidate genes are being identified that
can be expressed transgenically to enhance or even to initiate plant
regeneration from cultured cells and tissues. Such genes are being
explored for potential use in developing marker-free transgenic
IN VITRO MORPHOGENESIS IN PLANTS
systems as well as to potentially enhance the frequencies of
transgenic plant recovery (Zuo et al., 2002b). These advances, as
well as advances in specific culture systems such as thin cell layers
(Nhut et al., 2003), offer the prospects of extending more efficient in
vitro plant regeneration techniques to previously recalcitrant crops,
and of developing more efficient genetic transformation methods.
Such advances in controlling in vitro morphogenesis should play
important roles at the applied level in developing new crop cultivars
and reducing the cost of micropropagation, and in furthering basic
research in the area of functional genomics by testing of transgenes
in a wider array of plant species.
Acknowledgments
The author thanks Dr. Oluf Gamborg, Dr. Trevor Thorpe, and Dr.
Prakash Lakshmanan for helpful comments regarding topics
appropriate to this review.
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