Journal of Experimental Botany, Vol. 49, No. 324, pp. 1139–1146, July 1998
The protein of rolB gene enhances shoot formation in
tobacco leaf explants and thin cell layers from plants in
different physiological stages
M.M. Altamura1, S. D’Angeli and F. Capitani
Dipartimento di Biologia Vegetale, Università ‘La Sapienza’, P. le A Moro n.5, 00185 Rome, Italy
Received 12 September 1997; Accepted 27 February 1998
Abstract
The objective was to determine whether the protein
of rolB affects shoot formation and whether this
potentiaI relationship depends on the developmental
stages of the plant and/or on the culture conditions.
Thin cell layers (TCL) and leaf explants were excised
from tobacco plants in the vegetative and flowering
stages and cultured under various hormonal conditions. In TCLs of vegetative-stage plants, the expression of rolB enhanced the formation of the shoot buds
under hormone-free conditions and with specific concentrations of auxin and/or cytokinin. Histological
examination showed that the induction of the shoot
meristemoids was particularly enhanced by rolB protein and that meristemoid growth was accelerated. In
leaf explants from vegetative-stage plants, the expression of rolB increased the formation of shoot buds in
the presence of 1 mM IAA plus 1 or 10 mM cytokinin.
With BA alone, at a 0.1 mM concentration, shoot formation occurred in the transgenic explants only, whereas
with concentrations ranging from 0.5 to 10 mM, it was
higher in these explants than in controls.
RolB protein enhanced the formation of shoot buds
in TCLs from flowering plants under all hormonal conditions. In the presence of 1 mM IAA and kinetin, the
protein also increased the flowering response. In leaf
explants from flowering plants, the expression of rolB
increased the number of shoot buds in the presence
of 1 mM IAA with 10 mM BA.
In conclusion, rolB protein promotes shoot formation; it seems to have a positive interaction with cytokinin and an effect on the induction of the meristematic
condition.
Key words: Shoot formation, in vitro culture, leaf explants,
rolB protein, tobacco thin cell layers.
Introduction
Using thin cell layers (TCL) from the floral branches of
Nicotiana tabacum L., it is possible to induce the formation of flowers, shoot buds, roots or callus, depending on
the concentration and balance of hormones in the culture
medium ( Tran Thanh Van et al., 1974; Meeks-Wagner
et al., 1989). Moreover, using the hormonal conditions
for flowering suggested by Tran Thanh Van et al. (1974)
(i.e. 1 mM indole acetic acid [IAA] and 1 mM kinetin
[ Kin], referred to as ‘flowering medium’), TCLs from
flowering plants form only flowers from the pedicels and
flowers associated with shoot buds from the rachis
(Altamura et al., 1989). Thorpe et al. (1978) and
Altamura et al. (1995) have shown that shoot buds, but
not flowers, can be obtained from TCLs of the stems of
plants at the vegetative stage cultured in the presence of
1 mM IAA and 10 mM benzyl-aminopurine (BA) (‘shootforming medium’, according to Tran Thanh Van et al.,
1974).
Various types of organogenesis in vitro are also obtainable from leaf explants. An auxin concentration of 10 mM
is able to induce rhizogenesis in tobacco leaf explants
with the main veins (Brossard-Chriqui, 1980), whereas
the presence of a cytokinin, in the same or at a higher
concentration than auxin, is required for obtaining shoot
formation (e.g. in Saintpaulia ionantha Wendl. and
Lycopersicon esculentum Mill.) (Dodds and Roberts,
1986). Shoot bud formation has also been obtained in
the presence of various concentrations of cytokinin alone
1 To whom correspondence should be addressed. Fax: +39 6 4463865. E-mail: [email protected]
Abbreviations: BA, benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; HF, hormone-free medium; IAA, indole acetic acid; Kin, kinetin; TCL, thin
cell layer.
© Oxford University Press 1998
1140
Altamura et al.
(e.g. from tobacco leaves) (Martin-Tanguy et al., 1988;
Attfield and Evans, 1991). Moreover, as shown in Crepis
capillaris L. Wallr., leaf explants are able to produce
shoot buds when excised from plants at the vegetative
stage, and flowers when excised from flowering plants
(Brossard, 1979). Thus both TCLs and leaf explants
acquire the competence to produce flowers in vitro only
when they are excised from flowering specimens.
However, the two types of explants are different in terms
of both tissue composition and organ histogenesis in vitro
( Tran Thanh Van and Dien, 1975; Brossard, 1979; Wilms
and Sassen, 1987; Altamura et al., 1994, 1995; Capitani
et al., 1995; Bellincampi et al., 1996).
Each of the rol genes (i.e. rolA, rolB, rolC, and rolD)
from Agrobacterium rhizogenes Conn. has been shown to
modify the growth and development of the plant in
peculiar ways (Sinkar et al., 1988; Schmülling et al., 1988;
Mariotti et al., 1989; Mauro et al., 1996); however, only
the protein of rolB gene (when expressed as a single gene)
is able to induce root formation (Capone et al., 1989a,
b). RolB transformed tobacco leaf discs exhibit increased
sensitivity to auxin (Spanò et al., 1988). Moreover, membrane fragments from transformed plants are able to bind
a higher amount of auxin than those of untransformed
plants, and this increase in activity is completely and
selectively abolished by anti-rolB antibodies (Filippini
et al., 1994). Estruch et al. (1991) assigned to the rolB
protein a b-glycosidase activity on indole-glycosides; however, at the normal plant level, IAA-glycosides were
shown not to be a substrate for rolB protein (Nilsson
et al., 1993). Recently, the rolB protein has been suggested
to have a tyrosine phosphatase activity (Filippini et al.,
1996).
The organogenic role of the protein coded by rolB gene
on tobacco leaf explants has been shown on rhizogenesis
and is demonstrated by the appearance of the rooting
response on hormone-free medium, a response which is
absent in the untransformed explants (Cardarelli et al.,
1987), and by the enhancement of this response in the
presence of exogenous auxin (Spanò et al., 1988;
Bellincampi et al., 1996). It has also been shown that the
protein highly enhances the genesis of root meristemoids
in the callus of auxin-cultured leaf explants (Bellincampi
et al., 1996).
The effects of rolB protein on the organogenesis from
tobacco TCLs have been investigated by analysing root
and flower formation. Altamura et al. (1994) have shown
that the presence of rolB protein enhances the capacities
both of the pedicel tissues to form floral meristemoids
and of the stem tissues to produce root meristemoids
under the hormonal conditions suitable for flowering and
rhizogenesis, respectively ( Tran Thanh Van et al., 1974).
Two hypotheses may explain the results reported in the
literature. Since it is well known that auxin is a general
inducer of rhizogenesis (Blakesley, 1994), and that it is
highly involved also in the control of flower formation
from TCLs (Smulders et al., 1990a, b), the effect of rolB
protein on the formation of root and floral meristemoids
might result from the positive interaction of the protein
with auxin. Alternatively, rolB protein might be able to
promote meristem formation either per se or in combination with hormones (not necessarily auxin). In this case,
it could also be involved in organogenic programmes that
are not auxin-mediated, such as shoot formation, a programme mediated by cytokinin (Skoog and Miller, 1957;
Dodds and Roberts, 1986).
The aim of the present study was to investigate the
possible role of rolB protein on shoot formation obtainable from explants with different tissue compositions and
organogenic patterns ( leaf explants with the main veins
and TCLs), excised from plants in different physiological
stages (vegetative and floral ) and cultured under various
hormonal conditions, including the absence of exogenous
hormones.
It was observed that protein coded by rolB enhances
shoot formation, promoting the organization of shoot
meristemoids. The possibility of a positive interaction of
this protein with cytokinin in shoot formation, and the
possibility that the protein exhibits a meristem-inductive
function acting in combination with exogenous and endogenous hormones are discussed.
Materials and methods
Constructs and gene fusions
Nicotiana tabacum cv. Petite Havana SR1 plants (Maliga et al.,
1973) harbouring two constructs were used: (i) the rolB gene
under the control of a 1185-bp segment of its 5∞ upstream noncoding region, and (ii) the b-glucuronidase (GUS) gene fused
to the rolB promoter (i.e. pMCSHp15 and p1CB1185-GUS
constructs, respectively, described in Altamura et al., 1994).
These transgenic plants, named ‘BpB-GUS’ by Altamura et al.
(1994), are the source of the explants reported as ‘rolB-explants’
in this paper. Furthermore, in this paper, Petite Havana
SR1-BpB-GUS and Petite Havana SR1 genotypes are referred
to as ‘rolB-genotype’ and ‘SR1 genotype’, respectively.
Plant growth
RolB plants (i.e. BpB-GUS plants) and wild type (SR1) (used
as control ) were obtained from seeds germinated in vitro at
25±2 °C under a 16 h illumination period and then transferred
to a thermostatically controlled greenhouse. The presence of
the constructs in rolB plants was tested by the histochemical
staining of the plant leaves in a X-Gluc solution (Jefferson
et al., 1987), and by the rooting response of the leaves on
Murashige and Skoog (1962) (MS ) medium without hormones
(Cardarelli et al., 1987).
The plants positive to both the screening tests (i.e. staining
with X-Gluc and rooting response) and the untransformed
controls were used in two different stages: the vegetative stage,
in which the shoot apex was histologically vegetative in structure
and ten leaves ≥5 cm in length were present on the stem, and
the full flowering stage, in which the inflorescence was fully
developed (almost all the flowers at anthesis).
RolB protein enhances shoot formation 1141
Explants and in vitro culture
Leaf explants with the major veins (1×2 cm) were excised from
the 3rd and 4th leaves starting either from the apex (vegetative
plants) or from the lowest bract (flowering plants), then
sterilized with a 10% dilution of a commercial bleach (0.6%
active Cl ) and rinsed three times for 10 min in sterile
distilled water.
Thin cell layers (TCL), composed of epidermis, subepidermal
chlorenchyma and cortical parenchyma (1×10 mm, six cellular
layers in depth), were excised from the stem internodes
(vegetative plants) and from the rachises of the inflorescences
(flowering plants) and sterilized as above.
The explants were cultured on MS medium either with 1 mM
IAA and 10 mM BA (shoot-forming medium, Tran Thanh Van
et al., 1974), or with 1 mM IAA and 1 mM Kin (flowering
medium, Tran Thanh Van et al., 1974), or with BA alone
(range 0.1–10 mM ), or under hormone-free conditions (HF
medium). The pH was adjusted to 5.6 with NaOH or HCl, and
0.8% of agar was added to each medium.
Three hundred leaf explants or TCLs per treatment and
genotype were cultured for 30 d at 26±1 °C under a 16 h
illumination period (irradiance 25 W m−2).
One hundred TCLs excised from vegetative plants were also
cultured in the presence of 5 mM 2,4-dichlorophenoxyacetic acid
(2,4-D) plus 0.1 mM kin (callogenic medium, Tran Thanh Van
et al., 1974) for 50 d under continuous darkness (27±2 °C ),
and then transferred for 60 d onto the shoot-forming medium
(16 h light per day, 25 W m−2, 27±2 °C ). As suggested by
Walker et al. (1979) for callus cultures of alfalfa, the explants
not showing organ formation were transferred (for an additional
period of 27 d ) to HF medium under the same environmental
conditions used for the shoot-forming medium.
Two replicas of each experiment were carried out with similar
results; the data from the second experiment are shown here.
The time-course of organogenesis was scored daily with a
stereomicroscope. At the end of the culture period, the explant’s
productivity was evaluated and expressed as the percentage of
explants with organs and as the mean number (±SE) of organs
per explant.
Histochemical assays
Ten randomly picked TCLs per day and per genotype were
used for the histological analysis; sampling was performed at
day 0, 4, 6, 8, 12, 14, and 30. The histochemical treatment of
the transgenic explants with X-Gluc solution was carried out
before fixation, as previously described (Altamura et al., 1991).
Both the X-Gluc treated transgenic explants and the untreated
controls were fixed in 70% ethanol, dehydrated by the tertiary
butyl alcohol series, and embedded in paraffin (melting point
58 °C ). Sections (10 mm) were cut with a Pabish Topsuper
S–150 microtome, and either placed in xylol for 20 min and
mounted in Eukitt (transgenic explants), or stained with
hematoxylin and eosin using a Topstainer LX–100 (control
explants, Altamura et al., 1994).
The use of X-Gluc procedure for the histological observation
of the meristemoids in the transgenic TCLs was preferred to
the staining procedure used for the controls to avoid counting
meristemoids produced in the explant’s callused areas that
might have been no longer transgenic as a consequence of
somaclonal variation events.
The same level of image resolution was obtained in the
sections from explants of both genotypes independently of the
specific histochemical procedure.
Image analysis and statistical evaluation
Micrographs in radial longitudinal section of TCLs were
acquired with a SONY DXC–101P camera applied to a Zeiss
Axiophot microscope. The images were digitised with the Image
Grabber 2.3 software for Power Macintosh 7100/80 and
analysed using Optilab 2.6.1. software.
The total number of meristemoids present in all of the TCLs
of each sampling date and genotype on the shoot-forming
medium was counted and expressed as mean±SE, and the area
of the meristemoids was calculated as the mean±SE of 30–35
micrographs, randomly selected per sampling date.
Significance of differences between means was evaluated by
the Student’s t-test, and of differences between percentages
(calculated on the responding explants) by the chi-square test.
Inflorescences were counted as single flowers.
Results
Histological analysis of shoot formation on TCLs cultured on
the shoot-forming medium
The results of the histological analysis of TCLs cultured
on the shoot-forming medium are shown in Figs 1, 2.
Both in rolB- and control explants shoot meristemoids
were formed within callused areas. In the transgenic
explants these areas showed GUS expression, as did all
the meristemoids formed in these areas.
At day 4, meristemoids were observed in rolB-explants,
but not in the controls. The average number of
Fig. 1. Average number (continuous line) and average area (dotted line)
of meristemoids per TCL excised from rolB ( X ) and SR1 (D) vegetative
plants, cultured on the shoot-forming (1 mM IAA and 10 mM BA)
medium (**, P<0.01 difference between rolB and SR1). SE ranges
from 0.8 to 2.6 for the mean number of meristemoids, and from 400 to
1600 for their average area. Note that no significant difference is present
between the values of meristemoid areas of both genotypes at the
culture end.
1142
Altamura et al.
meristemoids per rolB-explant increased from day 4 up
to the culture end (day 30), whereas in the controls,
meristemoids appeared later (day 8), and their number
remained quite constant afterwards. At day 6, the bulk
of meristemoids on rolB-explants already showed the
differentiation of the tunica ( Fig. 2), thus revealing their
vegetative (shoot) nature ( Esau, 1965). At the culture
end, a highly significant difference in the number of
meristemoids was observed between rolB- and control
explants (Fig. 1). The meristemoid area continuously
increased for the control explants, while in rolB-explants
it reached its final size earlier (day 12). In fact, the
meristemoid dimensions at the culture end were similar
for both genotypes (Fig. 1). The time necessary for the
transformation of a meristemoid into a shoot primordium
( Fig. 2B, C ) was also decreased by the expression of rolB.
The shoot primordia were, in fact, histologically visible
on rolB-explants already at day 8 ( Fig. 2B) (1.8±0.3),
and at day 14 ( Fig. 2C ) the quantity of shoot primordia
was nearly three times greater for rolB compared to the
controls (12.5±1.3 and 4.5±0.5, respectively, P<0.01
difference).
Effect of the protein coded by rolB gene on the responses in
vitro of TCLs and leaves from vegetative plants
The percentage of responding explants on the HF medium
was very low, though it was significantly higher in the
rolB genotype, and shoot formation was the only
response, with a significantly greater number of shoot
buds per rolB-explant compared to the control ( Table 1).
On both the shoot-forming and flowering media, the
sole response observed was shoot formation in both
genotypes ( Table 1). However, in both culture conditions,
significant differences were observed between the two
Table 1. Percentage of responding TCLs cultured on hormone
free, shoot-forming (1 mM IAA and 10 mM BA) and flowering
(1 mM IAA and 1 mM Kin) media, and excised from vegetative
plants of rolB- and SR1 genotypes
The mean number of organs produced per explant (±SE) is shown in
parentheses.
Culture medium
Hormone free
Fig. 2. Progressive phases of the formation of a shoot bud on tobacco
TCLs excised from rolB plants and cultured on the shoot-forming
(1 mM IAA and 10 mM BA) medium. (Longitudinal sections). (A),
Meristemoid with a well defined tunica (arrow) attesting to its shoot
nature (day 6; bar=40 mm). (B–C ) Shoot primordia. (B) Onset of the
formation of leaf primordia (day 8; bar=40 mm). (C ) Developed leaf
primordia flanking the shoot apical dome which is shown by the arrow
(day 14; bar=100 mm).
rolB
SR1
Shoot-forming
rolB
SR1
Flowering
rolB
SR1
Shoot buds only
Shoot buds from
mixed response
22%**
(10.2±1.8)*
7%
(2.5±0.8)
63%*
(22.6±2.8)**
43%
(10.9±1.9)
93%*
(16.4±1.3)**
67%
(5.2±0.6)
0
0
0
0
0
0
**, P<0.01 and *, P<0.05 differences between rolB- and SR1
explants cultured on the same medium and showing the same response.
RolB protein enhances shoot formation 1143
genotypes both in terms of percentage of explants with
shoot buds and, mainly, in terms of number of buds per
explant ( Table 1).
At 0.1 mM BA alone, both rolB-explants and controls
were able to produce shoot buds, though the shoot
response was greater for rolB genotype (43% of the
explants with a mean number of 9.8±1.6 shoot buds per
explant compared to 25% of the control explants with
5.0±1.0 shoot buds per explant). With a 100 times higher
concentration of BA alone, the rolB-explant showed
widespread necrosis and no shoot response. In the controls, necrosis was significantly (P<0.01) lower, and the
explants that were still alive did not produce organs, but
callus only.
Thin cell layers were also cultured under callogenic
conditions followed by shoot-forming conditions (see
Materials and methods) with the aim of verifying whether
the rolB protein was able to enhance shoot formation
under these conditions.
Under callogenic conditions for 50 d, no organogenic
response was produced by either genotype. Thirty-five
days after the transfer to the shoot-forming medium,
similar percentages of rolB and control TCLs showed
shoot buds (38% and 35%, respectively). After another
25 d, the explants still showing callus only (62% for rolB
genotype and 65% for the control ) were transferred to
HF medium. After 27 d of culture on HF, the percentage
of rolB-TCLs with shoot buds (48%) was significantly
(P<0.01) higher than that for the controls (26%).
Under HF medium, almost all of the rolB leaf explants
produced roots as the only response, whereas the control
explants showed no response. On the shoot-forming
medium, the sole response obtained in both genotypes
was shoot formation ( Table 2). On this medium, in
rolB-explants, compared with the controls, significant
(P<0.01) increases were observed in the number of
explants with shoot buds, mainly during the first 10 d of
culture (72% and 35%, respectively, at day 10). At
the culture end, highly significant differences were
observed between the genotypes in the number of shoot
buds produced per explant ( Table 2).
Under the flowering medium, leaf explants of rolB
genotype produced many shoot buds and some roots
( Table 2). The percentage of explants with the former
response (pure plus mixed programmes) was significantly
higher for rolB than SR1 plants, and the mean number
of shoot buds per explant was also higher in the rolBexplants (Table 2).
The leaf explants were also cultured in the presence of
various concentrations (range 0.1 to 10 mM ) of cytokinin
(BA) alone ( Table 3). Shoot formation prevailed over
rhizogenesis in both genotypes, and the highest shoot
formation occurred at 10 mM BA. At 0.1 mM BA, the
control explants produced callus without organs, whereas
rolB-explants expressed both rooting and shoot formation
at similar levels ( Table 3). In the transgenic system, shoot
Table 3. Percentage of the shoot bud (S) and rooting (R)
responses obtained from rolB- and SR1 leaf explants excised
from vegetative plants and cultured under various concentrations
of BA alone
The mean number of organs produced per explant (±SE) is shown in
parentheses.
rolB
S
R
BA (0.1 mM )
BA (0.5 mM )
BA (1 mM )
BA (10 mM )
48.8%
(1.4±0.2)
41.9%
(1.6±0.3)
60.4%
(7.3±0.8)**
37.4%
(2±0.3)
98%
(12±0.8)**
56%
(1.6±0.2)
100%
(37±4.5)**
0
80%
(6.2±1.1)
5%a
80%
(16.7±1.7)
1.9%a
SR1
S
0b
R
0b
60%
(4.4±0.6)
8%a
**, P<0.01 differences between the means of shoot buds of the two
genotypes.
a No more than 1 root per explant.
b100% explants with callus only.
Table 2. Percentage of responding leaf explants cultured on the hormone free, shoot-forming (1 mM IAA and 10 mM BA) and flowering
(1 mM IAA and 1 mM Kin) media, and excised from vegetative plants of rolB- and SR1 genotypes
The mean number of organs produced per explant (±SE ) is shown in parentheses. ‘Mixed response’ indicates shoot buds and roots on the same explant.
Culture medium
Shoot buds only
Hormone free
rolB
Shoot-forming
SR1
rolB
SR1
Flowering
rolB
SR1
0
0
100%
(114.1±5.9)**
100%
(68.7±3.3)
25%
(21.0±3.7)**
37.5%
(2.4±0.3)
Shoot buds from
mixed response
0
0
0
0
73%**
(23.4±2.7)**
29.2%
(3.6±0.6)
Roots only
70%
(4.1±1.2)
0
0
0
2%
(7.0±0.5)**
10.4%*
(1.8±0.4)
Roots from
mixed response
0
0
0
0
73%**
(6.6±1.5)
29.2%
(6.8±1.7)
**, P<0.01 and *, P<0.05 differences between rolB- and SR1 explants cultured on the same medium and showing the same response.
1144
Altamura et al.
formation increased with increasing concentrations (from
0.1 mM to 10 mM ), while rhizogenesis was constantly
present up to 1 m. Under each BA treatment, the number
of shoot buds produced was significantly higher in rolBthan control explants ( Table 3).
Effect of the protein coded by rolB gene on the responses in
vitro of TCLs and leaves from flowering plants
In the absence of the exogenous hormones (HF medium),
in both rolB and SR1 the same low percentage of TCLs
produced flowers and shoot buds; however, the number
of shoot buds on rolB-explants was higher than that on
the controls ( Table 4).
On the shoot-forming medium, the types of the organs
produced (shoot buds and flowers) remained the same as
on the HF medium, and for both genotypes; however,
significant differences were observed for the shoot
response, both in the number of explants showing shoot
buds only (pure programme) and the mean number of
shoot buds per TCL (pure and mixed programmes,
Table 4). No significant difference between rolB- and SR1
TCLs was observed for either percentage of explants with
flowers or mean number of flowers per explant ( Table 4).
On the flowering medium, the percentage of TCLs with
shoot buds was higher for rolB- than SR1 explants, and
the response was mainly mixed (shoot and floral buds on
the same explant) ( Table 4). The percentage of rolBexplants with flowers (pure plus mixed programmes)
highly exceeded that for the controls ( Table 4). Significant
differences were also observed in the TCLs of the two
genotypes in the mean number of flowers per explant
(pure plus mixed programmes) ( Table 4).
On HF medium, the leaf explants of both genotypes
showed a poor response, with rooting on rolB-explants
significantly higher than that on the controls ( Table 5).
On the shoot-forming medium, all the leaves of both
genotypes produced shoot buds. They showed shoot buds
only (pure programme), mainly in the case of rolB
genotype, or shoot buds associated with flowers on the
same explant (mixed programme). The mean numbers of
shoot buds (pure and mixed programmes) on rolBexplants were significantly higher than those of the controls ( Table 5).
Using the flowering medium, the main response
obtained by rolB-leaf explants was the formation of roots,
both as percentage of rooting explants and as mean
number of roots per explant ( Table 5). Flower formation
occurred as a mixed response in both genotypes; the
percentage of rolB-explants with flowers was 1.9 times
greater than that of the controls ( Table 5).
Discussion
The tissue composition of explants such as leaves, TCLs,
and pith, the physiological stage of the donor plant from
which they are excised ( Tran Thanh Van et al., 1974;
Brossard, 1979; Altamura et al., 1995), and the genetically
determined responsiveness ( Kamate et al., 1981) are
important factors in organ formation in vitro. The results
of this study show that rolB protein can affect organ
formation independently of such factors. However, rolB
seems to interact positively with the inherent competence
of the explant, another factor that can affect organogenesis (George, 1993). In fact, in the absence of exogenous hormones, TCLs excised from vegetative plants
produce shoot buds only, thus showing a strictly shoot
competence independent of the genetic transformation
with rolB, and the presence of rolB protein enhances this
response. In the same culture conditions, leaf explants
show a rhizogenic competence, with explants from normal
plants forming root meristemoids and those from rolB
Table 4. Percentage of responding TCLs cultured on hormone free, shoot-forming (1 mM IAA and 10 mM BA) and flowering (1 mM
IAA and 1 mM Kin) media, and excised from flowering plants of rolB- and SR1 genotypes
‘Mixed response’ indicates shoots and floral buds on the same explant. The mean number of organs produced per explant (±SE ) is shown in
parentheses.
Culture medium
Hormone free
Shoot buds only
rolB
SR1
Shoot-forming
rolB
SR1
Flowering
rolB
SR1
10.2%
(11.8±1.2)**
7.5%
(1.8±0.6)
54%**
(8.5±1.1)**
12%
(2.4±1.4)
5.5%
(6.2±1.6)
0
Shoot buds from
mixed response
0
0
24%
(12.8±1.8)**
23%
(6.3±0.4)
45.3%**
(6.5±0.8)*
6.1%
(3.3±1.4)
Flowers only
9%
(2.2±0.9)
4.8%
(1.8±0.5)
0
0
34.7%
(4.6±0.7)
37.6%
(3.7±0.5)
Flowers from
mixed response
0
0
24%
(2.5±0.5)
23%
(5.4±1.2)
45.3%**
(5.3±0.6)*
6.1%
(3.1±0.9)
*, P<0.01 and *, P<0.05 differences between rolB- and SR1 explants cultured on the same medium and showing the same response.
RolB protein enhances shoot formation 1145
Table 5. Percentage of responding leaf explants cultured on the hormone free, shoot-forming (1 mM IAA and 10 mM BA) and flowering
(1 mM IAA and 1 mM Kin) media, and excised from flowering plants of rolB- and SR1 genotypes
The mean number of organs produced per explant (±SE ) is shown in parentheses. ‘Mixed response’ indicates either shoot and floral buds on the
same explant, or roots, shoot and floral buds.
Culture
medium
Hormone free
Shoot buds only
rolB
SR1
Shoot- forming
rolB
SR1
Flowering
rolB
SR1
6%
(1.7±0.5)
3%
(1.4±0.4)
40%**
(47.4±3.1)**
9.5%
(17.8±3.1)
0
20%
(2.0±0.3)
Shoot buds from
mixed response
0
0
60%
(50.8±3.3)**
90.5%
(28.9±2.8)
38%**
(2.7±0.4)
20%
(1.8±0.4)
Flowers from
mixed response
0
0
60%
(1.7±0.3)
90.5%
(1.6±0.4)
38%**
(4.0±0.8)
20%
(4.4±0.3)
Roots only
27%**
(1.3±0.3)
6%
(1.2±0.5)
0
0
62%**
(22.0±4.4)**
7%
(2.7±1.0)
Roots from
mixed
response
0
0
0
0
38%**
(12.1±1.5)**
20%
(2.5±0.70)
**, P<0.01 differences between rolB- and SR1 explants cultured on the same medium and showing the same response.
plants forming macroscopic roots (Bellincampi et al.,
1996; this paper).
The role of rolB protein becomes more evident when
organ formation is controlled by exogenous hormones. Its
promotion of exogenous auxin-induced rhizogenesis is
known for both TCLs and leaf explants (Altamura et al.,
1994; Bellincampi et al., 1996) and confirmed here. Its
promotive effect on flowering, another programme under
exogenous auxin control (see Introduction), is also known
for TCLs (Altamura et al., 1994), and has, in the present
study, also been observed for the leaf explants. These results
show that the protein coded by the rolB gene also exerts a
consistent, promotive effect on shoot formation. In shootforming medium and excising the explants from vegetative
plants, the promotion of shoot formation induced by rolB
protein is very high and totally independent of the explant
type (Tables 1, 2). This promotion is also evident under
suboptimal conditions: explants excised from vegetative
plants and cultured on the flowering medium (Tables 1, 2)
and explants excised from flowering plants and cultured on
the shoot-forming medium (Tables 4, 5).
It is known that in the transgenic tissues, rolB enhances
cell responsiveness to exogenous auxin (Spano’ et al., 1988).
Since auxin is present in the shoot-forming and flowering
media, and considering that it may also increase in the
explants as a consequence of excision, as observed in the
wound reaction of sweet potato tubers (Tanaka and
Uritani, 1979), it is possible that the effect of rolB on shoot
formation is indirect, resulting from the interaction between
its protein and (exogenous and endogenous) auxin.
However, the enhanced shoot response of rolB-leaves
observed under a wide range of concentrations of cytokinin
alone, and the sensitivity of the transgenic tissues to a very
low cytokinin concentration suggest the presence of a positive interaction between rolB protein and exogenous cytokinin in the promotion of shoot formation.
Histological studies have shown that the rolB protein
enhances the formation of floral and root meristemoids
in tobacco TCLs (Altamura et al., 1994). In the present
paper it is shown that the same protein also enhances the
formation of shoot meristemoids and accelerates their
growth, without affecting their final size. Thus, the general
effect of rolB on organogenesis seems to be related to the
early induction and growth of meristemoids.
In conclusion, rolB protein enhances shoot formation
in different tissue types and from plants in different stages,
stimulating the induction and accelerating the growth of
the shoot meristemoids. The protein appears to enhance
shoot formation, rhizogenesis, and flower formation by
positively interacting at the level of meristemoid formation with exogenous and endogenous auxin and cytokinin
concentrations, which are responsible for the different
types of organogenesis.
Acknowledgements
We thank Professor Paolo Costantino of the University of
Rome ‘La Sapienza’ for his suggestions in planning the
experiments, for the generous gift of the transgenic seeds, and
for helpful discussions.
References
Altamura MM, Archilletti T, Capone I, Costantino P. 1991.
Histological analysis of the expression of Agrobacterium
rhizogenes rolB-GUS gene fusions in transgenic tobacco. New
Phytologist 118, 69–79.
Altamura MM, Capitani F, Falasca G, Gallelli A, Scaramagli S,
Bueno M, Torrigiani P, Bagni N. 1995. Morphogenesis in
cultured thin cell layers and pith explants of tobacco. I.
Effect of putrescine on cell size, xylogenesis and meristemoid
organization. Journal of Plant Physiology 147, 101–6.
Altamura MM, Capitani F, Gazza L, Capone I, Costantino P.
1994. The plant oncogene rolB stimulates the formation of
flower and root meristemoids in tobacco thin cell layers. New
Phytologist 126, 283–93.
1146
Altamura et al.
Altamura MM, Monacelli B, Pasqua G. 1989. The effect of
photoperiod on flower formation in vitro in a quantitative
short-day cultivar of Nicotiana tabacum. Physiologia
Plantarum 76, 233–9.
Attfield EM, Evans PK. 1991. Developmental pattern of root
and shoot organogenesis in cultured leaf explants of Nicotiana
tabacum cv. Xanthi nc. Journal of Experimental Botany
42, 51–7.
Bellincampi D, Altamura MM, Zaghi D, Salvi G, Cervone F, De
Lorenzo G, Serino G, Gatz C, Costantino P, Cardarelli M.
1996. Oligalacturonides prevent rhizogenesis in rolB transgenic tobacco explants by inhibiting auxin-induced gene
expression. The Plant Cell 8, 477–87.
Blakesley D. 1994. Auxin metabolism and adventitious root
initiation. In: Davis and Haissing, eds. Biology of adventitious
root formation, 143–54.
Brossard D. 1979. Néoformation de bourgeons végétatifs et
inflorescentiels à partir de disques foliaires du Crepis capillaris
L. Wallr. cultivés in vitro. Zeitschrift für Pflazenphysiologie
93, 69–81.
Brossard-Chriqui D. 1980. Origine et degrés de ploı̈die des
méristèmes raçinaires regénérés in vitro sur des disques
foliaires issus de plantes haploı̈des et tétraploı̈des de Nicotiana
tabacum (variétés Wisconsin 38 et Xanthi). Canadian Journal
of Botany 58, 477–85.
Capitani F, Altamura MM, Calzecchi-Onesti B, Pasqua G,
Monacelli B. 1995. Histological effects of hormones on
caulogenesis and rhizogenesis from normal and transgenic
tobacco leaves. Cytobios 81, 109–17.
Capone I, Cardarelli M, Trovato M, Costantino P. 1989a.
Upstream non-coding region which confers polar expression
to Ri plasmid root inducing gene rolB. Molecular and General
Genetics 216, 239–44.
Capone I, Spano’ L, Cardarelli M, Bellincampi D, Petit A,
Costantino P. 1989b. Induction and growth properties of
carrot roots with different complements of Agrobacterium
rhizogenes T-DNA. Plant Molecular Biology 13, 43–52.
Cardarelli M, Mariotti D, Pomponi M, Spano’ L, Capone I,
Costantino P. 1987. Agrobacterium rhizogenes T-DNA genes
capable of inducing hairy root phenotype. Molecular and
General Genetics 209, 475–80.
Dodds JH, Roberts LW. 1986. Experiments in plant tissue
culture, 2nd edn. Cambridge University Press.
Esau K. 1965. Plant anatomy. J. Wiley & Sons.
Estruch JJ, Schell J, Spena A. 1991. The protein encoded by
the rolB plant oncogene hydrolyses indole glycosides. EMBO
Journal 10, 3123–8.
Filippini F, Lo Schiavo F, Terzi M, Costantino P, Trovato M.
1994. The plant oncogene rolB alters binding of auxin to
plant cell membranes. Plant Cell Physiology 35, 767–71.
Filippini F, Rossi V, Marin O, Lo Schiavo F, Terzi M,
Downey PM, Costantino P, Trovato M. 1996. A plant
oncogene as a phosphatase. Nature 379, 499–500.
George EF. 1993. Plant propagation by tissue culture Part. 1.
The technology. Exegetics Ltd, England.
Jefferson RA, Kavanagh TA, Bevan MW. 1987. GUS fusions:
b-glucuronidase as a sensitive and versatile gene fusion
marker in higher plants. EMBO Journal 6, 3901–7.
Kamate K, Cousson A, Trinh TH, Tran Thanh Van K. 1981.
Influence des facteurs génetique et physiologique chez le
Nicotiana sur la néoformation in vitro de fleurs à partir
d’assises cellulaires épidermique et sous-épidermiques.
Canadian Journal of Botany 59, 775–81.
Maliga P, Sz-Breznovits A, Marton L. 1973. Streptomycinresistent plants from callus culture of haploid tobacco. Nature
New Biology 244, 29–30.
Mariotti D, Fontana GS, Santini L, Costantino P. 1989.
Evaluation under field conditions of the morfological alterations (‘hairy root phenotype’) induced on Nicotiana tabacum
by different Ri plasmid T-DNA genes. Journal of Genetics
and Breeding 43, 157–64.
Martin-Tanguy J, Martin C, Paynot M, Rossin N. 1988. Effect
of hormone treatment on growth bud formation and free
amine and hydroxycinnamoyl putrescine levels in leaf explant
of Nicotiana tabacum cultured in vitro. Plant Physiology
88, 600–4.
Mauro ML, Trovato M, De Paolis A, Gallelli A, Costantino P,
Altamura MM. 1996. The plant oncogene rolD stimulates
flowering in transgenic tobacco plants. Developmental Biology
180, 693–700.
Meeks-Wagner DR, Dennis ES, Tran Thanh Van K, Peacok WJ.
1989. Tobacco genes expressed during in vitro floral initiation
and their expression during normal plant development. The
Plant Cell 1, 25–35.
Murashige T, Skoog F. 1962. A revised medium for rapid
growth and bioassay with tobacco tissues cultures. Physiologia
Plantarum 15, 473–97.
Nilsson O, Crozier A, Schmülling T, Sandberg G, Olsson O.
1993. Indole–3-acetic acid homeostasis in transgenic tobacco
plants expressing the Agrobacterium rhizogenes rolB gene.
The Plant Journal 3, 681–9.
Schmülling T, Schell J, Spena A. 1988. Single genes from
Agrobacterium rhizogenes influence plant development.
EMBO Journal 7, 2621–9.
Sinkar VP, Pythoud F, White H, Nester EW, Gordon MP. 1988.
RolA locus of the Ri plasmid directs the developmental
abnormalities in transgenic tobacco plants. Genes and
Development 2, 268–97.
Skoog F, Miller CO. 1957. Chemical regulation of growth and
organ formation in plant tissues cultured in vitro. Symposium
for the Society of Experimental Biology 11, 118–40.
Smulders MJM, Van de Ven EJPM, Croes AF, Wullems GJ.
1990a. Metabolism of 1-naphthaleneacetic acid in explants of
tobacco. Evidence for release of free hormone from conjugates. Journal of Plant Growth Regulation 9, 27–34.
Smulders MJM, Visser EJW, Croes AF, Wullems GJ. 1990b.
The dose of 1-naphthaleneacetic acid determines flower-bud
regeneration in tobacco explants at a large range of
concentrations. Planta 180, 410–15.
Spano’ L, Mariotti D, Cardarelli M, Branca C, Costantino P.
1988. Morphogenesis and auxin sensitivity of transgenic
tobacco with different complements of Ri T-DNA. Plant
Physiology 87, 479–83.
Tanaka Y, Uritani I. 1979. Polar transport and content of
indole-3-acetic acid in wounded sweet potato root tissues.
Plant Cell Physiology 20, 1087–96.
Thorpe TA, Tran Thanh Van M, Gaspar T. 1978. Isoperoxidases
in tobacco epidermal layers. Physiologia Plantarum 44,
388–94.
Tran Thanh Van K, Thi Dien N. 1975. Étude au niveau cellulaire
de la différentiation in vitro et de novo de bourgeons végétatifs,
de racines, ou de cal à partir de couches minces de cellules
de type épidermique de Nicotiana tabacum L. Canadian
Journal of Botany 53, 553–59.
Tran Thanh Van K, Thi Dien N, Chlyah A. 1974. Regulation of
organogenesis in small explants of superficial tissues of
Nicotiana tabacum L. Planta 119, 149–59.
Walker KA, Wendeln ML, Javorski EG. 1979. Organogenesis in
callus tissue of Medicago sativa. The temporal separation of
induction process from differentiation process. Plant Science
Letters 16, 23–30.
Wilms F, Sassen M. 1987. Origin and development of floral
buds in tobacco explants. New Phytologist 105, 57–65.