In vitro Morphogenesis in Plants: Recent Advances

Author(s): Gregory C. Phillips

Source: In Vitro Cellular & Developmental Biology. Plant, Vol. 40, No. 4 (Jul. - Aug., 2004),
pp. 342-345
Published by: Society for In Vitro Biology
Stable URL: http://www.jstor.org/stable/4293752
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 DOI: 10.1079/1VP2004555
In Vitro Cell. Dev. Biol.-Plant 40:342-345, July/August 2004
? 2004 Society for In Vitro Biology
1054-5476/04 $18.00+0.00

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.

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;
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.

*Author to whom correspondence should be addressed: Email gphillips@ GENETIC COMPONENTS OF MORPHOGENESIS
astate.edu
REPRINTED FROM: Phillips, G. C. In vitro morphogenesis in plants - One of the most exciting advances in recent years has been the
recent advances. In: Goodman, R. M., ed. Encyclopedia of plant and crop
discovery of specific genes involved in plant regeneration in vitro.
science, vol. 1. New York: Marcel Dekker, Inc.; 2004: 579-583. http://www.
dekker.com/servlet/product/DOI/101081EEPCS120010554; by courtesy of Such genes are being explored for use to increase transformation
Marcel Dekker, Inc. efficiency and to develop marker-free transgenic plants (Zuo et al.,

342

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 IN VITRO MORPHOGENESIS IN PLANTS 343

Genetic
2002b). Because a primary factor involved inaspects of shoot
optimizing organogen
somatic
embryogenesis and organogenesis is the
the use of phytohormone
acquisition of competence for sho
1999; Fletcher,
models, it is of interest that receptors for each of2002), as is SRD3 (s
the major
phytohormone classes have now been(Sugiyama,
identified1999, 2000)of
and many (Table
the 1). Sh
corresponding genes have been clonedinvolves
(MollerSRD1 and SRD2
and Chua, 1999;(Sugiyama,
Zuo 1
et al., 2002b). Examples of specificto-shoot
genes involved in thetransition
organogenesis major is pro
shoot regeneration)
plant morphogenesis pathways are summarized (Zuo et al., 2002b
in Table 1.
potentially
Genetic aspects of somatic embryogenesis. independent
Transgenic pathways
expression
of the LEC2 (leafy cotyledon) gene (Table 1) is sufficient
organogenesis signalto initiate
transduction are
and CKII
somatic embryogenesis with high viability but(cytokinin independent) (S
some abnormalities
2002b). The
persist in morphology (Zuo et al., 2002b). shoot
Several apical
genes meristem
appear to stem
CLV and WUS
be involved in the vegetative-to-embryogenic (Fletcher,
transition, 2002),
such as parall
WUS (wuschel; or PGA6, plant growthshoot apical meristem
activator), LEC1 (Zuoof et somatic
al., emb
2002a), SERK (somatic embryogenesis receptor
(knotted) arekinase)
involved(Zuo et al.,
in the function o
and
2002b), and PT1 (primordial timing) overexpression
(Harada, 1999). SHRleads to the fo
(short
meristems
root) establishes the ground tissue through (Sugiyama,
the first 1999;
asymmetric cell Fletche
involved
division (von Arnold et al., 2002), CLV in shoot
(clavata) apical
and WUS meristem org
interact
to determine stem cell fate, and CLV formation include
and STM (shoot multiple SHO (sh
meristemless)
(mgoun)
regulate development of the shoot apical genes (Fletcher,
meristem 2002).
(Fletcher, 2002;
von Arnold et al., 2002). LEC1, ABI3 Genetic
(abscisic aspects of root organogenesi
acid-insensitive) and
ate root
FUS3 (fusca) are involved in somatic embryoorgans is affected
maturation (von by SRD
Arnold et al., 2002). (Table 1). The transition of embry

TABLE 1

EXAMPLES OF GENES INVOLVED IN VARIOUS PLANT MORPHOGENESIS PATHWAYS

Gene Putative function References

Somatic embryogenesis
LEC2 Initiates ectopic somatic embryogenesis Zuo et al., 2002b
WUS (PGA6), SERK, LEC1 Involved in the vegetative-to-embryogenic transition Ha
SHR Establishes ground tissue via asymmetric cell division von Arnold et al., 2002
CLV, WUS Regulate stem cell fate Fletcher, 2002; von Arnold et al., 2002
CLV1, CLV3, STM Regulate shoot apical meristem development Fletcher, 2002; von Arnold et al., 2002
LEC1, FUS3, ABI3 Regulate embryo maturation von Arnold et al., 2002
Shoot organogenesis
CYCD3 Involved in acquisition of competence for organogenesis Sugiyama, 1999; Fletcher, 2002
SRD3 Competence for shoot organogenesis Sugiyama, 1999, 2000
SRD1, SRD2 Competence for redifferentiation of shoots Sugiyama, 1999, 2000
ESRI Enhances shoot regeneration, vegetative-to-organogenic transition Zuo et al., 2002b
CRE1 Cytokinin receptor Zuo et al., 2002b
CKII Cytokinin perception Fletcher, 2002; Zuo et al., 2002b
CLV, WUS Preserve stem cell identity in shoot apical meristem Fletcher, 2002
KN1, STM Initiate ectopic shoot meristems, shoot apical meristem function Fletcher, 2002
SHO, MGO Modifiers of the shoot apical meristem involved in Fletcher, 2002
leaf founder cell recruitment, lateral organ primordial
Root organogenesis
SRD2 Competence for root organogenesis Sugiyama, 1999, 2000
PKL Transition of embryonic root cells to grow vegetatively Harada, 1999
RML Root apical meristem function Sugiyama, 2000; Anderson et al., 2001
CYCD4;1 Involved in lateral root formation De Veylder et al., 1999
RAC Involved in adventitious root formation and auxin transduction Sugiyama, 2000
Floral organogenesis
LFY Switch to reproductive development, floral meristem identity Fletcher, 2002; Lohmann and Weigel, 2002
API A-class gene involved in establishing the first floral whorl: petals Lohmann and Weigel, 2002
UFO Interacts with LFY by providing regional specificity within floral Lohmann and Weigel, 2002
meristems and to control B-class signals which establish the
second floral whorl: sepals
WUS Interacts with LFY to control C-class genes Lohmann and Weigel, 2002
AG C-class gene typifying the class; interacts with B-class signals to Lohmann and Weigel, 2002
produce the third floral whorl: stamens; C-class genes acting alone
produce the fourth floral whorl: carpels
SEP Co-factors for A-, B-, and C-class genes to convert vegetative Lohmann and Weigel, 2002
leaves into floral organs

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 344 PHILLIPS

vegetative growth is
improve yields of shoot proliferation cultures, microtubers
RML1 (root somatic meristemles
embryos, as well as improve the quality and vigor of the
root apical propagules
meristemwith reduced frequencies of abnormalities and
components of
hyperhydricity. the
2001). The RAC Another interesting development is(rooting
the use of perfluorochemicals
early stageand commercially-stabilized
of auxin
bovine hemoglobin as gas carriers to
adventitious enhance cell performance
roots in liquid culture systems such (Sugias
involved in bioreactors.
lateral Perfluorochemicals are recyclable (canrootbe used to
1999). deliver gases, then recovered from the culture and recharged), and
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 API (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. Cul-
tured 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
and con
(S
apic
per
p
emulsion with the surfactant Pluronic F-68? 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 techni-
ques. 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 organogen-
esis, 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

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 IN VITRO MORPHOGENESIS IN PLANTS 345

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