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Molecular Analysis of In Vitro Shoot Organogenesis

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DOI: 10.1080/07352680490484569

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Critical Reviews in Plant Sciences, 23(4):325–335 (2004)
Copyright C Taylor and Francis Inc.
ISSN: 0735-2689 print / 1549-7836 online
DOI: 10.1080/07352680490484569
Molecular Analysis of In Vitro Shoot Organogenesis
Shibo Zhang∗ and Peggy G. Lemaux
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA
Referee: Dr. William Gordon-Kamm, Pioneer Hi-Bred International Inc., 7300 N.W. 62nd Avenue, Johnston, IA 50131, USA
Shoot organogenesis is one of the in vitro plant regeneration
pathways. It has been widely employed in plant biotechnology for
in vitro micropropagation and genetic transformation, as well as in
study of plant development. Morphological and physiological as-
pects of in vitro shoot organogenesis have already been extensively
studied in plant tissue culture for more than 50 years. Within the
last ten years, given the research progress in plant genetics and
molecular biology, our understanding of in vivo plant shoot meris-
tem development, plant cell cycle, and cytokinin signal transduction
has advanced significantly. These research advances have provided
useful molecular tools and resources for the recent studies on the
genetic and molecular aspects of in vitro shoot organogenesis. A
few key molecular markers, genes, and probable pathways have
been identified from these studies that are shown to be critically
involved in in vitro shoot organogenesis. Furthermore, these stud-
ies have also indicated that in vitro shoot organogenesis, just as in
in vivo shoot development, is a complex, well-coordinated develop-
mental process, and induction of a single molecular event may not
be sufficient to induce the occurrence of the entire process. Further
study is needed to identify the early molecular event(s) that triggers
dedifferentiation of somatic cells and serves as the developmental
switch for de novo shoot development.
Keywords shoot organogenesis, in vitro, shoot meristem, cell cycle,
cyclin-dependent kinase, cyclin, cytokinin
INTRODUCTION
The in vitro regeneration of a plant can proceed through two
pathways, somatic embryogenesis and shoot organogenesis. In
the latter pathway an adventitious shoot forms first, followed by
development of adventitious roots from the shoot and finally an
entire plant develops. The process of in vitro shoot induction and
development via organogenesis has been reviewed in detail from
the perspective of developmental biology (Hick, 1994) and from
a physiological, biochemical, and molecular viewpoint (Joy and
Thorpe, 1999). Here only the most recent studies detailing the
molecular aspects of in vitro shoot organogenesis are reviewed.
The initiation of in vitro shoot organogenesis differs markedly
from in planta shoot development during embryogenesis. Dur-
ing in vitro shoot organogenesis, the shoot meristem initiates
∗ Corresponding author: E-mail: shibo@nature.berkely.edu
from differentiated somatic cells, not from embryonic cells as
it does during embryogenesis. The process of in vitro shoot
organogenesis usually involves three main aspects: hormonal re-
sponse of somatic cells, cell division of responding cell(s), and
initiation and development of new shoots from the newly divid-
ing cell(s), either directly or indirectly through a callus stage.
When somatic cells respond appropriately to exogenously ap-
plied plant hormones and the cell cycle is accelerated, these cells
can be reprogrammed to assume the de novo developmental fate
of shoot organogenesis.
Cytokinin, sometimes in concert with auxin, is the most ef-
ficient plant growth regulator to induce in vitro shoot organo-
genesis. Recently, genetic and molecular analyses have identi-
fied the important genes involved in the cytokinin signal trans-
duction pathway in arabidopsis (Haberer and Kieber, 2002).
The histidine protein kinases (AHKs) serve as cytokinin recep-
tors, and the histidine phosphotransfer proteins (AHPs) transmit
the signal from AHKs to nuclear response regulators (ARRs),
which can activate or repress transcription. In the study of cell
cycle, a few key regulatory genes in plant species have now
been documented to be involved, and these include the p34 cdc2
and the cyclin genes (Shaul, 1996). In shoot meristem devel-
opment, several regulatory genes have been identified that in-
clude maize KNOTTED1 (KN1) (Vollbrecht et al., 1991), Ara-
bidopsis SHOOT MERISTEMLESS (STM ) (Long et al., 1996),
WUSCHEL (WUS) (Laux et al., 1996; Mayer et al., 1998), and
CLAVATA 1-3 (CLV1-3) (Leyser and Furner, 1992; Clark et al.,
1993, 1995; Kayes and Clark, 1998; Fletcher et al., 1999). Evi-
dence has also been presented that interactions exist at the molec-
ular level among these three processes: cytokinin reception, cell
cycle, and shoot meristem development (Riou-Khamlichi et al.,
1999; Rupp et al., 1999).
With the identification of these critical genes from plant ge-
netic and molecular studies has come the opportunity to use
these as useful molecular markers to understand in vitro shoot
organogenesis at the molecular level. Genetic and molecular
approaches have also been directly used to define the molec-
ular elements more directly involved in shoot organogenesis
in vitro. For example, using this type of approach led to the
identification of the arabidopsis gene, ENHANCER OF SHOOT
325
326 S. ZHANG AND P. G. LEMAUX
REGENERATION 1 (ESR1), and its overexpression appears to
induce adventitious shoot formation from root explants, even
in the absence of the exogenous addition of plant hormones
(Banno et al., 2001). Genomic approaches utilizing arabidopsis
GeneChips have been employed recently to perform global gene
expression profiling analysis during in vitro shoot development
from root explants (Che et al., 2002). Studies such as these are
essential in defining the genetic and molecular mechanisms in-
volved in the process of in vitro shoot morphogenesis. Before
the details of these studies are discussed, the current state of
our molecular understanding of in planta shoot meristem devel-
opment, cell cycle regulation, and cytokinin signal transduction
will be described briefly.
MOLECULAR ANALYSIS OF SHOOT MERISTEM
DEVELOPMENT IN PLANTA
In a plant, the shoot meristem initiates and develops during
embryogenesis within an embryo that derives from a zygote.
Because of its position at the growing tip of the plant, this ini-
tial shoot meristem becomes the shoot apical meristem (SAM)
during later development. All lateral organs, including the axil-
lary shoot meristems, derive from the flanks of the SAM, except
for the root. Based on histological analyses, the SAM can be
subdivided into three distinct zones: peripheral, central, and rib
zones (Figure 1). Lateral organs originate from the peripheral
zone and stem tissue from the rib zone. The central zone serves
as a type of stem cell source, replenishing both peripheral and
rib zones while continuing its role as the source of stem cells.
Genetic and molecular analyses of shoot meristem develop-
ment in maize, arabidopsis, and other plants have led to the
identification of genes critical to these two processes. Maize
KN1 was the first gene to be identified as specifically expressed
in the SAM during embryo development (Smith et al., 1995)
and in the growing tip, but not in the leaf primordia (Jackson
FIG. 1. Diagram of a SAM indicating the central zone (CZ), peripheral zone
(PZ), and rib zone (RZ) (from Trigiano and Gary, 2004).
et al., 1994). Consistent with these observations, expression pat-
terns of KN1-homologs in other cereals are identical, e.g., in
barley (Figure 2). The KN1 gene encodes a homeodomain pro-
tein (Vollbrecht et al., 1991). Especially interesting to shoot
organogenesis, the ectopic expression of maize KN1 in tobacco
or its homologs in arabidopsis caused induction of adventitious
shoot formation from the transgenic leaf tissues (Sinha et al.,
1993; Lincoln et al., 1994). Loss-of-function mutations of KN1
in maize are defective in shoot meristem maintenance (Kerstetter
et al., 1997). In arabidopsis, the STM gene encodes a KN1-type
homeodomain protein, and its expression is similarly restricted
to the SAM (Long et al., 1996). Loss-of-function mutations of
STM result in failure of shoot meristem development during em-
bryogenesis in arabidopsis.
Besides STM, studies have also identified a few other genes
in arabidopsis that are involved in shoot meristem development
and maintenance. For example, WUS codes for a novel subtype
of homeodomain transcription factor (Mayer et al., 1998). In
wus mutants, the development of the shoot meristem stops af-
ter the formation of a few leaves during embryogenesis (Laux
et al., 1996). WUS is expressed first in subepidermal cells of the
apex at the 16-cell stage during embryogenesis; gradually its ex-
pression is limited to the subepidermal cells in the central zone
of the postembryonic SAM (Mayer et al., 1998). Consideration
of these results suggests that WUS is responsible for initiating
and maintaining the stem cells in the central zone of the SAM.
WUS and STM are activated independently in arabidopsis; how-
ever, STM expression is absent in wus mutants and vice versa
(Lenhard et al., 2002).
Aside from the role of STM and WUS in promoting shoot
meristem activity, a different group of genes in arabidopsis,
CLV1-3, has been shown to be responsible for restricting cell
proliferation in the SAM. Loss-of-function mutations in these
genes cause an enlargement of the SAM and floral meristem
(Leyser and Furner, 1992; Clark et al., 1993, 1995; Kayes and
Clark, 1998). Genetic analyses suggest that the products of CLV1
and CLV3 act in the same pathway to regulate cell proliferation in
the SAM (Clark et al., 1995). Although the product of CLV2 may
act in the same meristem control pathway as CLV1 and CLV3,
its role in plant development appears to be broader (Kayes and
Clark, 1998).
The CLV1 gene encodes a leucine-rich repeat (LRR) trans-
membrane receptor serine/threonine kinase, and it is expressed
in the central region of the SAM and floral meristem (Clark et al.,
1997). Given the nature of its protein-binding domain, the CLV1
receptor likely binds to an extracellular protein or peptide ligand.
The CLV3 encodes a small, likely extracellular protein expressed
at the apex of the SAM and floral meristem, predominantly in
the L1 and L2 cell layers (Fletcher et al., 1999). Genetic, molec-
ular, and biochemical studies provide strong evidence that CLV3
acts as, or in the production of, the ligand for the CLV1 receptor
kinase (Rojo et al., 2002).
Based on these studies in arabidopsis, the CLV1-3 suite of
genes and WUS are proposed to act in a feedback loop to maintain
 MOLECULAR ANALYSIS OF IN VITRO SHOOT ORGANOGENESIS 327

FIG. 2. Expression of KNOTTED1-homolog in a SAM of barley. (a) Scanning electron micrograph of a barley shoot apex including the SAM and two leaf
primordia (P1, P2). (b) Immunolocalization of KNOTTED1-homolog in barley shoot apex using KNOTTED1 antibody, showing expression of KNOTTED1-
homolog only in the SAM, not in leaf primordia (P0, P1, and P2) (from Trigiano and Gary, 2004).

the integrity of the SAM as a continuous source of stem cells
during plant development (Figure 3). Besides STM, WUS, and
CLV1-3, more genes have been identified in arabidopsis that are
involved in shoot meristem development and maintenance, e.g.,
CUC1, and CUC2 (Aida et al., 1997; Takada et al., 2001). Both
the CUC1 and CUC2 genes encode NAC domain-containing
proteins. They are expressed as a stripe at the apex of the globu-
lar stage embryo between two cotyledon primordia, suggesting
CUC1 and CUC2 are essential genes for embryonic shoot api-
cal meristem formation and cotyledon separation (Aida et al.,
1997, 1999; Takada et al., 2001). The identity and details of
these two genes with others have already been discussed in re-
views (e.g., Fletcher and Meyerowitz, 2000; Lenhard and Laux,
1999).
Taken together, it can be seen that shoot meristem develop-
ment and maintenance in planta involve a large number of genes
that orchestrate a complicated but well-regulated process. At
the present time certain aspects of the molecular mechanisms
involved in this complex process have been identified; how-
ever, full elucidation of this pathway still requires additional
research.
MOLECULAR ANALYSIS OF PLANT CELL DIVISION
There appear to be a number of functional similarities be-
tween plants and animals with regard to the molecular mech-
anisms controlling cell division. These similarities include the
cyclin-dependent kinases (CDKs) and cyclins (CYCs), the key
regulatory gene products that function during the plant and ani-
mal cell cycle (Shaul, 1996).

FIG. 3. Diagram showing feedback loop controlling stem cell maintenance in
shoot meristem. CLV3 signals through the CLV1 receptor complex, restricting
the WUS-expressing stem cell population. WUS provides a feedback signal to
the overlying cell population to maintain the CLV3-expressing cell population
in the superficial layers (from Trigiano and Gary, 2004).
Cyclin-Dependent Kinases
The first CDK gene to be identified, cdc2, was found in
Schizosaccharomyces pombe (Hindley and Phear, 1984). Sub-
sequently, plant homologues were identified and cloned from
pea (Feiler and Jacobs, 1990), alfalfa (Hirt et al., 1991), maize
(Colasanti et al., 1991), rice (Hashimoto et al., 1992), petunia
(Bergounioux et al., 1992), soybean (Miao et al., 1993), ara-
bidopsis (Ferreira et al., 1991; Hirayama et al., 1991; Imajuku
et al., 1992), and antirrhinum (Fobert, 1994). Functional equiva-
lence of cdc2 homologues from alfalfa, maize, rice, and soybean
was demonstrated by their ability to complement, at least in part,
yeast cell-cycle mutants; however, complementation was possi-
ble only for mutations affecting certain phases of the yeast cell
division cycle. That this occurred was because, unlike yeast,
where the product of a single cdc2 functions in all phases of
328 S. ZHANG AND P. G. LEMAUX
the cell cycle, plants have distinct cdc2 genes active in different
phases of the cell cycle.
The expression of one cdc2 gene in arabidopsis, cdc2aAt, was
extensively analyzed and its expression correlated with actual
cell division and with competence for cell division (Ferreira
et al., 1991; Martinez et al., 1992; Hemerly et al., 1993). Based
on in situ hybridization studies, expression of cdc2aAt was found
to occur in root and shoot apical meristems, in developing first
leaves, and at the root–shoot junction from which adventitious
roots initiate. Its expression was nearly undetectable in fully
expanded leaves. In the vegetative shoot apex, the expression of
cdc2aAt corresponded precisely to the pattern of mitotic activity.
In the root, except for the apical meristem, cdc2aAt expression
was restricted to division-competent parenchymal and pericycle
cells. Similar expression patterns of cdc2Zm were observed in
maize (Colasanti et al., 1993).
Cell-cycle genes might interact at some level with plant
growth regulators that play a role in mediating their effects. Us-
ing a cdc2aAt promoter-uidA (gus) fusion, expression of GUS
was studied during dedifferentiation in leaf mesophyll proto-
plasts from transgenic tobacco plants (Hemerly et al., 1993).
Cultivation of the protoplasts in the presence of either auxin or
cytokinin caused cdc2aAt-mediated induction of GUS expres-
sion. Division competence, despite the lack of observable cell
division, was presumably due to effects of the hormone treatment
on the endogenous cdc2aAt gene. Consistent with these results
it was observed in wounded transgenic arabidopsis leaves that
there was a rapid induction of cdc2aAt promoter-driven GUS
expression that occurred around damaged surfaces, which de-
spite the lack of significant cell proliferation might represent
increased division competence. A correlation between cdc2 tran-
script abundance and an increased proliferating state of the tissue
was also observed in maize (Colasanti et al., 1993) and alfalfa
(Hirt et al., 1991). Although cdc2 expression appears to be in-
volved in the competence to proliferate, it is likely that additional
factors are required for cell division, e.g., an activating cyclin
subunit.
Cyclins
The first cyclins were identified in marine invertebrate em-
bryos (Evans et al., 1983). Since that time eight different groups
of cyclins, A–H, have been found in mammals. A and B types,
which are required for cells to enter mitosis, are termed mitotic
cyclins. The C, D, and E type cyclins are identified as G1 cyclins
(Lew et al., 1991). The D type is believed to be involved in the
exit from the quiescent state (G0 ) and re-entry into the cell cycle
(Quelle et al., 1993); E-type cyclins are required for progression
from G1 to S phases (Koff et al., 1992).
The mitotic-type cyclins (A and B) were isolated from sev-
eral plant species, including soybean (Hata et al., 1991), carrot
(Hata et al., 1991), alfalfa (Hirt et al., 1992), antirrhinum (Fobert
et al., 1994), maize (Renaudin et al., 1994), and tobacco (Qin
et al., 1995). The A- and B-type cyclins in arabidopsis (Hemerly
et al., 1992), soybean (Hata et al., 1991), and maize (Renaudin
et al., 1994) are believed to be the functional homologues of
animal mitotic-type cyclins because of their functional ability
to induce maturation of xenopus oocytes. In situ hybridization
studies of roots of arabidopsis seedlings, designed to uncover the
spatial and temporal expression patterns of the A- and B-type
cyclins, particularly cyc1At, revealed that the cyc1At transcript is
restricted primarily to the root apical meristem (Hemerly et al.,
1993). In zones of lateral root initiation, expression was ob-
served in pericycle cells only in zones of lateral root initiation,
and this induction occurred even before morphological change
was observed. This result was similar to that obtained in ara-
bidopsis plants containing the cyc1At promoter fused to GUS
(Ferreira et al., 1994), in which a close correlation was found
between GUS expression and mitotic activity in the shoot apical
meristem, developing flowers, and embryos. During dedifferen-
tiation of mesophyll protoplasts, GUS expression driven by the
cyc1At promoter was induced only when cell division occurred
as a result of treatment with appropriate combinations of auxins
and cytokinins. However, it is perhaps pertinent that various cy-
clins are expressed during the cell cycle, and each cyclin might
play a different role during cell division (Ferreira et al., 1994;
Fobert et al., 1994; Hirt et al., 1992).
Based on the work on cyc1At and cdc2aAt in arabidop-
sis, Shaul et al. (1996) proposed a simplified model for the
role of cyclins during plant development and dedifferentia-
tion. In this model, cell cycle genes are highly expressed in
young, dividing tissues; however, during the process of differ-
entiation a reduction, but not elimination, in cdc2aAt expres-
sion occurs, followed by cessation of cyc1At expression. In
differentiated tissues, the level of cdc2aAt expression reflects
the degree of division competence. Dedifferentiation and reac-
quisition of the potential to divide involves cdc2aAt activation;
however, other activation signals are likely needed to trigger cell
division.
D-type cyclins in arabidopsis (Soni et al., 1995) are the most
closely similar to mammalian D-type cyclins, which are known
to mediate exit from the quiescent state (G0 ) and re-entry into the
cell cycle. In order to determine timing of D3 cyclin expression
in arabidopsis during the cell cycle, hydroxyurea or colchicine
was used to treat suspension culture cells in order to block them
at early S phase or metaphase, respectively (Soni et al., 1995). A
dramatic reduction in D3 cyclin expression was observed as a re-
sult of both treatments. When the block caused by hydroxyurea
was removed, expression of D3 cyclin recommenced, slightly
before the induction of expression of histone H4. Expression
remained high until the end of the S phase, and no significant
induction of the mitotic cyc1At gene occurred during this time.
The profile of transcription of the D3 cyclin genes in response
to plant growth regulators was studied in arabidopsis suspen-
sion cultures by depleting the medium of hormones and carbon
source. After restoring these compounds, transcript levels of D
cyclin genes varied, depending on the plant mitogenic signal. In
contrast to the expression of the A- and B-type mitotic cyclins,
 MOLECULAR ANALYSIS OF IN VITRO SHOOT ORGANOGENESIS
expression of D-type cyclins is not restricted to dividing cells
and is expressed earlier in the cell cycle.
CYTOKININ SIGNAL TRANSDUCTION AND ITS
RELATION TO CELL DIVISION AND SHOOT
MERISTEM DEVELOPMENT
Of the plant growth regulators, cytokinin is the most effec-
tive one at inducing in vitro shoot organogenesis. However,
as demonstrated by Skoog and Miller (1957) the critical fac-
tor in triggering developmental events is probably the ratio of
cytokinin to auxin, rather than the absolute amount of either
hormone. Work completed over the last few years has led to sig-
nificant progress in understanding cytokinin metabolism, trans-
port, perception and signal transduction (for review, see Haberer
and Kieber, 2002) and in revealing the relationship of cytokinin
action with cell division and shoot meristem development (Riou-
Khamlichi et al., 1999; Rupp et al., 1999; Werner et al., 2003).
Cytokinin Signal Transduction
The first step in the cellular response to cytokinin involves
the cytokinin receptor. Using an activation-tagging mutagen-
esis scheme, a putative cytokinin receptor in arabidopsis, the
gene of cytokinin-independent1 (CKI1) was cloned (Kakimoto,
1996). Its overexpression resulted in a typical cytokinin response
in vitro; that was rapid cell division and shoot formation in the
absence of exogenous cytokinin application. The CKI1 encodes
a protein similar to the two-component regulators (Chang and
Stewart, 1998). It has a histidine kinase domain, and a receiver
domain, and therefore resembles a possible cytokinin receptor.
However, its gain-of-function mutation did not lead to a defini-
tive conclusion about its function as a cytokinin receptor.
Recently, a more confirmed cytokinin receptor, the cytokinin
response 1 (CRE1), was identified in arabidopsis as a result
of screening 19,000 plants from an ethyl methanesulfonate-
mutagenized population; the cells from the cre1 mutant dis-
played reduced sensitivity to cytokinin in vitro (Inoue et al.,
2001). The CRE1 was identified to be identical to two other genes
in arabidopsis, AHK4 (Suzuki et al., 2001; Ueguchi et al., 2001)
and WOL (Mahonen et al., 2000). Its amino acid sequence shares
weak homology to CKI1. The CRE1 protein contains an unusual
type of hybrid kinase domain, two C-terminal response regulator
domains, a histidine kinase domain, and two transmembrane re-
gions flanking a novel, putative extracellular domain. Analyses
of a series of experiments in yeast and E. coli provided evidence
that cytokinin acts as a ligand for CRE1/AHK4 (Inoue et al.,
2001; Suzuki et al., 2001; Yamada et al., 2001).
Additional components downstream in the cytokinin signal
transduction pathway have also been identified. The arabidopsis
AHPs act as signaling shuttles between the cytokinin receptors
and downstream nuclear responses (Miyata et al., 1998; Suzuki
et al., 1998, 2000). Nuclear AHP translocation enables acti-
vation of response regulators, Arabidopsis response regulators
(ARRs), which based on structure and cytokinin-inducible ex-
329
pression patterns are classified into two distinct subtypes, type
A with 10 members and type B with 11 members (D’Agostino
and Kieber, 1999; Imamura et al., 1999). B-type ARRs activate
A-type ARRs as a result of their role as transcriptional activa-
tors (Hwang and Sheen, 2001; Sakai et al., 2001). Analysis of
the results of all of the studies described above showed that a
multistep phosphorelay pathway is involved in cytokinin signal
transduction in the following manner: CRE1/AHK4/WOL →
AHPs → ARRs.
The transport of cytokinin through tissues might also play a
role in the in vitro response to cytokinins, because in some cases
tissues or cells that responded during the induction of in vitro
shoot organogenesis were not in direct contact with the hor-
mones in the induction media. For example, during the in vitro
culture of vegetative shoots of maize and barley on a medium
containing a high ratio of cytokinin to auxin, responding cells
were in the axillary shoot meristematic domes (Zhang et al.,
1998) or in the nodal regions of some maize inbreds (Zhang
et al., 2002), not in direct contact with the hormone-containing
culture medium. Cells located at the cut edge of the stem, which
were in direct contact with the induction medium, did not re-
spond. Further suggestion of the existence of a cytokinin trans-
porter is supported by the results of studies of its role during
plant growth; cytokinin exists in the xylem sap and yet the root
tip is the major site of cytokinin biosynthesis. This leads to the
assumption that cytokinins are transported through the xylem in
order for them to exert their effects on aerial parts of a plant. In
fact a putative cytokinin transporter, AtPUP1, was identified in
arabidopsis, based on its functional complementation of a yeast
mutant deficient in adenine uptake (Gillissen et al., 2000).
Cytokinin and Cell Division
Cytokinin was initially identified as being involved in pro-
moting cell division (Miller et al., 1955, 1956); however, the
detailed molecular mechanisms by which this occurs have still
not been fully elucidated. To date, genetic and molecular studies
have identified a few possible links between cytokinin and cell
division. Some studies suggest that cytokinin functions in the
G2 → M transition, although a decisive link has not been demon-
strated. For example, in tobacco protoplasts cytokinin controls
the cell cycle at mitosis by stimulating tyrosine dephosphory-
lation and activation of p34 cdc2 -like H1 histone kinase (Zhang
et al., 1996). In addition cytokinins were shown to induce ex-
pression of cdc2 in arabidopsis roots and other tissues (Hemerly
et al., 1993).
Cytokinin was also shown to regulate G1 → S transition
mediated by a D-type cyclin (CycD3) (Riou-Khamlichi et al.,
1999). Increased steady-state levels of CycD3 mRNA were
achieved by applying cytokinin to both seedlings and in vitro
cultured cells. Also, expression of CycD3 in the intact plant is
localized in meristematic tissues, including the shoot meristem,
leaf primordia, and axillary shoots. In the arabidopsis mutant
amp1 (Chaudhury et al., 1993), there are increased levels of
endogenous cytokinins and higher steady-state levels of CycD3
330 S. ZHANG AND P. G. LEMAUX
mRNA. Perhaps more relevant to in vitro plant development, leaf
explants overexpressing CycD3 form healthy green calli with-
out the addition of cytokinin, in contrast to identical explants
from wild-type plants, which formed such calli only in the pres-
ence of cytokinin. These experiments demonstrate that CycD3
overexpression bypasses the need for cytokinin to turn on the
cell cycle and that cytokinin regulates the cell cycle through
CycD3.
Cytokinin probably works in concert with auxin in regulating
cell division in vitro. The gene PROPORZ1 (PRZ1), isolated
from arabidopsis, was found to be essential for triggering the
developmental switch from cell proliferation to differentiation,
and the response was dependent on the variation in auxin and
cytokinin concentrations (Sieberer et al., 2003). Based on this
observation, it was suggested that PRZ1 acts as a transcriptional
adaptor protein affecting the expression of cell cycle regulators
and thus mediating its effect on the control of cell proliferation.
In promoting the re-entry of petunia leaf protoplasts into the
cell cycle, both auxin and cytokinin were required to induce
the appearance of cdc2Pet mRNA transcripts prior to S-phase
engagement with auxin likely acting before cytokinin (Trehin
et al., 1998). In some cases, auxin alone is sufficient to induce
cell cycle regulatory genes. For example, CycD1, CycD3, and
CDKB1 were induced by auxin during cell-cycle re-entry, as well
as during bud growth of whole tubers of Jerusalem artichoke
(Helianthus tuberosus L.) (Freeman et al., 2003). Also, auxin
can act to downregulate the cyclin-dependent kinase inhibitor,
KRP2, during pericycle cell activation that is needed for lateral
root initiation in arabidopsis (Himanen et al., 2002).
Cytokinin and Shoot Meristem Development
Cytokinin, or more likely the higher ratio of cytokinin to
auxin, is usually required for the induction of shoot organogen-
esis in vitro. Recent genetic and molecular studies have indicated
that the induction by cytokinin appears to occur through the up-
regulation of expression of certain key regulatory genes.
When a cytokinin biosynthetic gene from bacteria, ipt, was
introduced into arabidopsis, its overexpression led to increased
levels of KNAT1 (an arabidopsis homologue of KN1) and STM
(Rupp et al., 1999). Elevated levels of KNAT1 and STM were
also found in the arabidopsis mutant, amp1, that also had a
higher level of cytokinin. Overexpression of KN1 driven by
the senescence-specific promoter of SAG12 resulted in delayed
senescence in tobacco (Ori et al., 1999). Endogenous cytokinin
levels were 15 times higher in older leaves of SAG:KN1 plants
than in wild-type leaves, suggesting that KN1 inhibits senes-
cence by increasing cytokinin levels. Similar effects on senes-
cence were observed when the cytokinin biosynthetic gene, ipt,
was expressed under the control of the same SAG12 promoter.
When rice plants were engineered to overexpress OSH1 (a rice
KN1 homologue), there were also increased levels of the active
form of cytokinin (Kusaba et al., 1998). Taken together, these
studies suggest that cytokinin levels and KN1-type gene expres-
sion may interact in an interdependent fashion.
The relationship between cytokinin and KN1-like genes was
studied in more detail through the use of a fusion protein be-
tween the homeodomain of KNAT2 (an arabidopsis KN1-like
protein) and the hormone-binding domain of the glucocorticoid
(GR) receptor (Hamant et al., 2002). Upon activation of the
KNAT2-GR fusion following cytokinin treatment, delayed leaf
senescence and a higher rate of shoot initiation were observed,
suggesting that cytokinin and KNAT2 act synergistically to reg-
ulate meristem activity in the shoot meristem.
MOLECULAR ANALYSIS OF SHOOT ORGANOGENESIS
IN VITRO
Use of Specific Genes as Molecular Markers
Until recently, the study of in vitro shoot organogenesis was
limited primarily to the morphological or physiological level.
Now, with the availability of the many cloned plant genes, molec-
ular markers are available that can be used to understand the
in vitro shoot organogenesis process at the molecular level. The
first example of this type was the expression analysis of maize
KN1 and its homolog in barley during in vitro axillary shoot
meristematic cell proliferation and adventitious shoot meristem
formation (Zhang et al., 1998). Vegetative shoot segments iso-
lated from germinated seedlings of maize and barley were cul-
tured in vitro on a medium, containing higher levels of cytokinin
and lower levels of auxin. Following several weeks of culture,
cell division within the axillary shoot meristems of the cultured
shoots changed from its normally well-regulated state into a
proliferating state, such that the relatively small meristematic
dome became an enlarged shoot meristematic dome. Adventi-
tious shoot meristems (ADMs) arose directly from the cells in
that enlarged dome (Figure 4a).
Expression of KN1 in maize or KN1-homologue in barley
was maintained in shoot meristematic cells during in vitro cell
proliferation of the axillary shoot meristems. ADMs appeared
to derive directly from KN1-expressing shoot meristematic cells
(Figure 4b). In addition, the expression pattern of KN1 within the
ADMs in maize is the same as that of KN1 in normal shoot apical
meristems. That is, KN1 is expressed throughout the meristem-
atic dome, except in those cells that are involved in leaf initia-
tion. The results of this study demonstrated both that KN1-type
genes can be used as reliable molecular markers to detect in vitro
shoot meristem formation and in vitro shoot meristem develop-
ment appears to follow the same pathways as in planta shoot
meristem development.
In contrast to the induction of ADMs on the leaf surfaces
of tobacco engineered to overexpress KN1 (Sinha et al., 1993),
however, overexpression of KN1 in the leaf tissues of transgenic
maize did not lead to ADM formation. Based on this differ-
ence between maize and tobacco, it was suggested that other
components besides KN1 expression might be needed to induce
ADM formation in maize (Zhang et al., 1998). In the Zhang
et al. study, it was also shown that the maize p34 cdc2 homolog,
ZmCDC2, can be used as a molecular marker to identify in vitro
cell division activity.
 MOLECULAR ANALYSIS OF IN VITRO SHOOT ORGANOGENESIS
FIG. 4. Expression of KNOTTED1 in the in-vitro–generated ADMs of maize. (a) Multiple ADMs induced from an enlarged shoot meristematic dome in vitro.
(b) Expression of KNOTTED1 (arrows) in the in vitro ADMs (from Trigiano and Gary, 2004).
The utility of the KN1-type and cell cycle-related genes as
molecular markers were further confirmed in the study of the
expression patterns of the Brostm (a KN1-type gene in Bras-
sica oleracea) and Brocyc (a cyclin-type gene also in Brassica)
during in vitro shoot organogenesis (Teo et al., 2001). Adven-
titious shoots were induced directly without intermediate cal-
lus formation from internodal stem segments of brassica, using
in vitro culture in the presence of exogenous cytokinin. Brostm
and Brocyc were used during various stages of shoot induction
as molecular tags to mark, respectively, shoot meristematic cells
and dividing cells. This analysis permitted an investigation of
how cells in the stem segment could directly switch their devel-
opmental fate to become shoot meristematic cells. Analysis of
these results showed that the process of developmental switch-
ing started before any cell division occurred and within groups
of 5–7 phloem parenchyma cells, subtending the vascular bun-
dles in the stem segment. Induction of expression of Brostm
specifically required cytokinin application and occurred within
4 h of treatment, and its expression lasted throughout the cell
proliferation process that gave rise to shoot formation. During
adventitious shoot formation, cells expressing Brostm were dis-
tinct from those expressing Brocyc.
This study also showed that there is a time gap from the
acquisition of cell competence to cell determination. As demon-
strated by the initiation of Brostm expression, developmental
switching of stem segment cells began within 4 h of cytokinin
treatment; however, actual shoot development required 8 h of
cytokinin treatment. This result is consistent with the hypothe-
sis that de novo organogenesis involves three-steps: acquisition
of competence, shoot induction, and organogenesis determina-
tion (Christianson and Warnick, 1983, 1984, 1985). The required
time gap also indicated that expression of Brostm alone is not
sufficient to induce in vitro shoot meristem formation.
Molecular Cloning of Genes Involved in In Vitro
Shoot Organogenesis
Recently, genetic and molecular efforts have been directly
utilized to identify new genes that might either regulate or be
331
involved directly in shoot organogenesis in vitro. For exam-
ple, a novel MADS box cDNA, PkMADS1, was isolated from
a cDNA library of leaf explants from a woody tree species,
Paulownia kawakamii, undergoing adventitious shoot forma-
tion (Prakash and Kumar, 2002). Analysis of the deduced amino
acid sequence of its MADS domain revealed 90% similarity to
the AGL24 (AGAMOUS-like) protein in arabidopsis (Hartmann
et al., 2000) and to the STMADS16 protein in potato, Solanum
tuberosum (Carmona et al., 1998). Expression from PkMADS1
was not detected in callus-forming cultures of paulownia but
was found in shoot-forming cultures. In the plant, transcripts
from PkMADS1 were detected only in shoot apices, not in root
apices, flowers, or initial leaf explants. In the antisense knock-
outs of this gene, shoots were stunted, had altered phyllotaxy
and in some cases, lack of development of the shoot apical
meristem. Shoot regeneration from leaf explants of the anti-
sense transgenic plants was decreased ten-fold compared with
leaf explants from PkMADS1 overexpressing transformants or
wild-type plants. These results suggest that PkMADS1 expres-
sion is essential for both in vitro and in vivo shoot formation.
Because cytokinins are the most efficient plant growth reg-
ulators to induce in vitro shoot organogenesis, genes involved
in cytokinin metabolism or in the signal transduction pathway
are likely to have either a positive or negative effect on shoot
organogenesis. By screening an arabidopsis cDNA library for
a gene that when overexpressed in root explants confers the
ability for cytokinin-independent shoot induction, ESR1 (En-
hancer of Shoot Regeneration) was identified (Banno et al.,
2001). ESR1 encodes a putative transcription factor with an
AP2/EREBP domain (Ohme-Takagi and Shinshi, 1995; Buttner
and Singh, 1997; Hao et al., 1998; Fujimoto et al., 2000).
By overexpression of the ESR1 gene product, the efficiency of
shoot regeneration, following application of cytokinin to root ex-
plants, was greatly enhanced and was coupled with a downward
shift in the optimal cytokinin concentration needed. Also wild-
type root explants, when cultured on cytokinin-containing and
shoot-induction medium, had transiently elevated ESR1 tran-
script levels, which occurred before detectable STM expression
332 S. ZHANG AND P. G. LEMAUX
but after the acquisition of competence for shoot regeneration
on callus-induction medium. Consideration of these results sug-
gests that ESR1 may be a downstream effecter for cytokinin
that enhances shoot regeneration initiation after acquisition of
competence for shoot organogenesis. It would be of interest to
analyze whether endogenous cytokinin levels are unaltered in
the transgenic plants overexpressing the ESR1 product.
Overexpression of the cytokinin biosynthesis gene, ipt, led
to overproduction of cytokinin and also enhanced shoot regen-
eration in tobacco (Hagen and Guilfoyl, 1992). Recently, high
shoot regeneration capacity was also observed in the arabidop-
sis mutant, hoc, which overproduces cytokinin (Catterou et al.,
2002), possibly because of its role in cytokinin metabolism.
Root explants from the hoc mutant developed numerous shoots
when cultured in vitro without addition of exogenous growth
regulators.
Genomic Analysis of In Vitro Shoot Organogenesis
In addition to the molecular and genetic approaches discussed
above, a genomic approach utilizing arabidopsis GeneChips was
used to study gene expression profiles during in vitro shoot mor-
phogenesis (Che et al., 2002; Cary et al., 2002). In vitro shoot
induction from root explants was carried out by preincubation
on callus-induction medium (CIM) containing low levels of cy-
tokinin and high levels of auxin for 4 d. This was followed by
transfer to a shoot-induction medium (SIM) containing higher
cytokinin levels for 14–15 d (Valvekens et al., 1988). Under
these conditions, explants acquired competence to respond to
shoot formation signals during preincubation on CIM. Upon
transfer to SIM, explants became committed to shoot formation
after 6 d; shoots emerged 6–8 d later.
During preincubation on CIM, many auxin− responsive
genes, e.g., IAA1, IAA5, IAA9, and IAA10, are upregulated, fol-
lowed by downregulation upon transfer to SIM. Based on the fact
that SIM is known to contain a higher level (5.0 µM) of cytokinin
compared to CIM (0.2 µM), cytokinin is believed to be the major
factor responsible for inducing shoot regeneration from callus.
Message levels of CKI1 and of the cytokinin receptor encoded by
CRE1/AHK4/WOL were increased by ∼three-fold upon transfer
to SIM. It was therefore suggested that upregulation of CRE1
and CKI1 upon transfer from CIM to SIM may be related to the
acquisition of competence.
Two cytokinin primary response genes, ARR4 and ARR5,
were highly upregulated during shoot development; ARR5, in
particular, was upregulated by more than seven-fold. Expression
of ARR5 was observed in callus generated from root explants
and its level increased over subsequent days, rising during the
preincubation period on CIM. The level also increased during
the early stages of incubation on SIM, peaking at 6 d, with the
decline occurring at approximately the time of shoot commit-
ment. This expression pattern indicated that ARR5 expression is
not a simple reporter signal of cytokinin in this system. Rather,
ARR5 expression accompanied early stages of callus formation
and later appears to mark presumptive sites of shoot formation.
Key genes involved in shoot meristem formation, such as
WUS, STM, CLV1, and CUC2 were also examined in the expres-
sion profiling experiment. WUS, STM, and CLV1 were found to
be expressed at relatively low levels during the callus induction
stage on CIM; however, all three were upregulated during incu-
bation on SIM. Relative to the other genes, WUS was expressed
early (6 d on SIM), and its expression level rose nearly 20-fold
from 2 d on CIM to 15 d on SIM; STM and CLV1 expression lev-
els rose later. At about the time of shoot commitment, expression
of STM increased about seven-fold, while expression of CLV1
increased about two-fold. CUC2 was shown to be upregulated
earlier and to higher levels than WUS, STM, and CLV1. In addi-
tion, expression of CUC2 was transient, peaking in cells at 6 d
on SIM and declining thereafter. The CUC1 gene was not repre-
sented on the arabidopsis GeneChip used, and so its expression
was analyzed using quantitative RT-PCR. Analysis of these re-
sults showed that CUC1 was also upregulated, with expression
rising early after 3-6 d on SIM.
The genomic profiling analyses of in vitro shoot develop-
ment demonstrated that different types of genes are expressed at
different developmental stages, from initial hormone response
to late-shoot meristem development. This observation, there-
fore, further supports the model that in vitro shoot development
follows three stages of competence acquisition, induction, and
determination.
SUMMARY
Molecular, genetic, and genomic analyses of in vitro shoot
organogenesis conducted to date support the developmental
model proposed by Christianson and Warnick (1985) in which
shoot organogenesis is conceptually divided into three phases:
competence acquisition, shoot induction, and shoot development
determination. Competence acquisition is the stage before cells
are induced to develop into a shoot. The expression of genes
involved in the cytokinin signal transduction pathway, such as
CKI1 and CRE1, might be good indicators of cell competence to
respond to cytokinin, which is usually required for shoot organo-
genesis. The best candidates for indicators of competence for cell
division are probably the CDC2a-type and cyclin-type genes, as
demonstrated in shoot organogenesis induction from stem tis-
sues in tobacco (Boucheron et al., 2002). For the shoot induc-
tion phase, maize KN1-type homologues are to date the most
definitive molecular markers that can be used to identify early
stages during induction of in vitro shoot organogenesis; their ex-
pression is also maintained during the later shoot development
determination stage.
Cytokinin likely acts through activation of the CycD-3 type
gene to initiate or accelerate the cell cycle. However, the molec-
ular connection between the cytokinin action and CycD-3 acti-
vation has not yet been elucidated. Although molecular insights
already exist into why cytokinin, or more accurately a higher
ratio of cytokinin to auxin, favors induction of shoot organogen-
esis, the actual molecular pathway leading from higher levels
of cytokinin to upregulation of KN1-type gene expression still
 MOLECULAR ANALYSIS OF IN VITRO SHOOT ORGANOGENESIS
needs to be determined. Future study of the genes and regu-
latory pathways involved in dedifferentiation of somatic cells
in vitro, coupled with the elucidation of the role of chromatin
structure changes during the course of dedifferentiation (e.g.,
Zhao et al., 2001; Williams et al., 2003), will provide valuable
insights into our understanding of in vitro shoot organogenesis
and the developmental flexibility of the plant cell.
ACKNOWLEDGEMENTS
We are very grateful to Drs. Michael Christianson and
Malgorzata Gaj for their critical review of this manuscript, and
to Barbara Alonso for help in the preparation of the figures
and the references. We attempted to cite all pertinent references
but apologize to any colleagues whose work was inadvertently
missed.
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