ORIGINAL PAPER
Anatomy and morphology of photinia (Photinia 3 fraseri Dress)in vitro plants inoculated with rhizobacteria
Ezequiel E. Larraburu • Nancy M. Apostolo •
Berta E. Llorente
Received: 11 May 2009 / Revised: 3 March 2010 / Accepted: 16 March 2010 / Published online: 6 April 2010
� Springer-Verlag 2010
Abstract Photinia 9 fraseri Dress (photinia) is a woody
plant with high ornamental value. The anatomy and mor-
phology of micropropagated photinia inoculated with the
plant growth-promoting rhizobacteria Azospirillum brasi-
lense and Azotobacter chroococcum, in combination with
pulses of 49.2 lM indole-3-butyric acid during rhizogen-
esis, were characterized using light and electron micros-
copy. Leaves of inoculated in vitro plants showed better
development than those subjected to auxin control only. All
inoculated treatments, independent of the bacterial strain
used, had leaves with two layers of palisade parenchyma, a
thick cuticle and linear unicellular trichomes. There was no
proliferation of undifferentiated tissue in any treatment and
the plants showed shoot–root vascular connections. Ex
vitro leaves and in vitro plants inoculated with Azospiril-
lum brasilense Cd and Azotobacter chroococcum 42 had
large stomata with elliptic aperture radially surrounded by
small stomata on the abaxial foliar surface. In addition,
plants of these treatments had a large root hair zone over
the root surface. Bacteria were only observed on surfaces
of root hairs. The results suggest that the structural changes
induced by bacterial inoculation of photinia in vitro plants
could lead to better adaptation to ex vitro conditions after
transplanting.
Keywords Azospirillum brasilense � Azotobacter
chroococcum � Photinia � Micropropagation �Plant growth-promoting rhizobacteria
Introduction
Photinia 9 fraseri Dress (photinia) is an ornamental shrub
of the Rosaceae family. It reaches 3–5 m in height and is
widely planted in green areas. Its perennial foliage is very
striking during sprouting due to the new bright-red leaves
among the dark green older ones. Like most woody plant
species, photinia is reproduced by rooting apical cuttings
with high concentration pulses of phytohormones
(Bonaminio and Blazich 1983). This species is considered
difficult to propagate by traditional cultural techniques,
limiting their commercial utilization (Bonaminio and
Blazich 1983; Ramırez-Malagon et al. 1997; Larraburu
et al. 2007). Therefore, the use of in vitro culture tech-
niques that allow mass production of cloned plants inde-
pendent of time and season is recommended. However,
plants that have grown in vitro have been continuously
exposed to a unique microenvironment that has been
selected to provide optimum conditions for plant propa-
gation. These special conditions during in vitro culture,
e.g., low level of light, high relative humidity, sugar and
nutrients, often cause harmful abnormal structures and
physiological behavior (Hazarika 2006) and so it is very
important to find methods that avoid these alterations.
Biotechnological techniques, such as the application of
plant growth-promoting rhizobacteria (PGPR), have been
effective in reducing the biochemical, anatomical and
morphological modification of in vitro propagules in
diverse species (Frommel et al. 1991; Ait-Barka et al.
2002; Nowak and Shulaev 2003; Larraburu et al. 2007;
Communicated by D. Treutter.
E. E. Larraburu � N. M. Apostolo � B. E. Llorente (&)
Plant Tissue Culture Laboratory (CULTEV),
Department of Basic Sciences, National University of Lujan,
C.C. 221, 6700 Lujan, Buenos Aires, Argentina
e-mail: [emailprotected]
123
Trees (2010) 24:635–642
DOI 10.1007/s00468-010-0433-x
Rodrıguez-Romero et al. 2008; Russo et al. 2008). PGPR
are a group of rhizosphere-colonizing bacteria that induce
changes in inoculated plants, which can improve plant
growth. The N2-fixing and phytohormone-producing PGPR
Azospirillum brasilense and Azotobacter chroococcum
interact beneficially with numerous plant species. Field
trials have demonstrated that inoculation with these PGPR
enhances plant growth and yield in a wide range of eco-
nomically important crops in different soils and climatic
regions (Bashan and Levanony 1990; Mrkovacki and Milic
2001; Bashan et al. 2004; Dalton and Kramer 2006).
Azospirillum brasilense (Cd and Sp7 strains) and Azo-
tobacter chroococcum 42, in combination with indole-3-
butyric acid (IBA 49.2 lM) induction pulses, improved in
vitro rhizogenesis and the quality of micropropagated
photinia shoots (Larraburu et al. 2007). In this study, we
describe the structural characteristics of micropropagated
photinia inoculated with these rhizobacteria, using light
and electron microscopy.
Materials and methods
Plant material and culture conditions
Tissue culture plants of Photinia 9 fraseri Dress derived
from the rooting stage of the micropropagation protocol of
Larraburu et al. (2007) were utilized. In brief, shoot tips from
6-year-old mature plants were used as initial explants. They
were cultured on Murashige and Skoog (MS, 1962) medium
with B5 vitamins (Gamborg et al. 1968), 100 mg L-1
myoinositol, 3% sucrose, N6-benzyladenine (BA: 11.1 lM),
gibberellic acid (GA3: 1.3 lM) and 0.7% agar. The shoot
multiplication stage was achieved by cultivating them for
4 weeks in the same basal medium supplemented with
4.4 lM BA. The rooting medium (RM) contained half-
concentration MS salts, 2% sucrose and 0.6% agar and the
root induction medium (RIM) was prepared using RM sup-
plemented with 49.2 lM IBA. Media pH was adjusted to 5.8
before autoclaving at 121�C for 20 min. One explant per tube
was cultured in RIM for 6 days. Each explant was then
transferred to auxin-free RM until the end of the experiment
(20 days). Inoculation was done when transferring induced
explants to RM medium, by adding 0.1 mL of bacterial
culture (108 cfu mL-1) at the base of each explant. Treat-
ments without bacterial inoculation were controls. Cultures
were incubated in a growth chamber at 24 ± 2�C under
Philips fluorescent daylight tubes (55 lmol m-2 s-1) for a
16-h photoperiod.
Twenty plants each of inoculated and control in vitro
treatments and five green leaves of young shoots of mature
plants growing in a natural stand (6–7 years) were collected
for structural and ultrastructural studies. Leaves from the third
node from the apex, roots (3 cm in length from the apex) and
the shoot–root transition region (3 cm in length) of in vitro
plants were treated for anatomical studies.
Bacterial strains and culture conditions
Azospirillum brasilense Cd (American Type Culture Col-
lection; ATCC 29710) and Sp7 (ATCC 29145) and Azo-
tobacter chroococcum 42 (locally isolated) were used
according to Larraburu et al. (2007). Bacteria inocula were
grown in 250-mL Erlenmeyer flasks with 150 mL of
medium for Azospirillum brasilense (Okon et al. 1977) or
Azotobacter chroococcum (Brown et al. 1962). Strains
were incubated at 32 ± 1�C on an orbital SontecTM shaker
(140 rpm); Azospirillum brasilense for 72 h and Azoto-
bacter chroococcum for 6 days.
Stereoscopic microscopy
Five roots of all in vitro treatments were washed with distilled
water. Root surfaces were observed and photographed using
Zeiss STEM 2000C stereoscopic microscopy (Microscopy
Laboratory, National University of Lujan, Argentina).
Light microscopy (LM)
Five in vitro plants of each treatment and five ex vitro leaves
were fixed in FAA (ethanol 96�:distilled water:formalde-
hyde:acetic acid, 10:7:2:1). Leaves, shoot–root transition
regions and roots were dehydrated and embedded in paraffin.
Transverse sections (15–20 lm) were made with a rotative
microtome MICRON HM325. After paraffin elimination by
immersion in xylol, sections were stained with safranin-
fastgreen and mounted on synthetic resin PMYRTM
(Instrumental Pasteur, Buenos Aires, Argentina) using
standard procedures (Ruzin 1999). The observations, mea-
surements and micrographs were carried out using a LM
Zeiss HBO50 (Microscopy Laboratory, National University
of Lujan). Leaf thickness, outermost palisade parenchyma
thickness, spongy parenchyma thickness and ad- and abaxial
epidermis thickness were measured in five randomly chosen
optical microscope fields on five cross-sections from each
leaf. The anatomical characteristics of roots were observed.
The vascular connection was studied in 15–20 lm longitu-
dinal sections of the shoot–root transition region and treated
with the same methodology.
Environmental scanning electron microscopy (ESEM)
Three fresh leaves of each treatment were directly collected
from the tubes. Bacteria growing in the culture media and
both surfaces of ex vitro and in vitro leaves were observed
and photographed by Electroscan Microscope (ESEM
636 Trees (2010) 24:635–642
123
Service, CITEFA, Buenos Aires, Argentina). Stomatal
density (stomata mm-2) was determined on five digitized
ESEM micrographs of each leaf. Stomata length and width
were measured on five randomly selected stomata in each
micrography of 0.05 mm2. Stomatal length was the dis-
tance between the ends of guard cells and width was the
transverse distance across them.
Scanning electron microscopy (SEM)
Three roots of inoculated in vitro plants were sectioned into
small pieces and placed in perforated Eppendorf tubes. The
sectioned material was fixed in FAA for 24 h and treated with
an ascending ethanol gradient (30�, 50�, 75� and 100�) for
5–10 min per step. Finally, roots were dried using the critical
point method. Roots were mounted and metalized with gold.
Root surfaces, root hair distribution and bacteria localization
were observed and photographed using Philips XL-30 SEM
(SEM Service, MACN, Buenos Aires, Argentina).
Transmission electron microscopy (TEM)
Small pieces (1 mm3) of three in vitro leaves and roots
from each treatment and controls were sectioned and fixed
in 4% glutaraldehyde in 0.1 M sodium cacodilate at pH
7.4. Then, the material was rinsed in the same buffer, post-
fixed in osmium tetroxide and dehydrated with acetone.
Samples were embedded in epoxy resin Spurr (Union
Carbide International Co) and thin transverse sections
(60 nm) were sliced using an ultramicrotome with a dia-
mond knife. The sections were mounted in copper grids of
200 mesh and stained with 2.5% uranyl acetate and lead
citrate. Observations and micrographs were carried out using
a TEM Philips EM301 (Advance Microscopy Center, Exact
and Natural Sciences Faculty, Buenos Aires University).
Ultrastructure of palisade parenchyma cells and cuticle of
leaves, cortex and epidermal cells of roots and bacteria
localization were observed. Cuticle thickness was measured
on ten digitized electron micrographs per treatment.
Morphometry and statistical analysis
Images were viewed on a monitor and analyzed for morpho-
metric features using the OPTIMA 6.5 software (Media
Cybernetics, USA). For data analysis, ANOVA with Tukey’s
multiple range tests (P B 0.05) was used. All data were
evaluated using SPSS v.12.0 (SPSS Inc, Chicago, IL, USA).
Results
Photinia 9 fraseri Dress ex vitro had two types of leaves,
green (mature leaves) and red (young leaves) (Fig. 1a, b).
Both had a dorsiventral mesophyll with 3–5 palisade
parenchyma layers, but red leaves had two upper layers of
palisade cells with red pigments. Spongy parenchyma of
mesophyll contained abundant intercellular spaces (Fig. 1c,
d). The stomata and unicellular trichomes were localized
on the abaxial epidermis but were absent from the adaxial
epidermis (hypostomatic leaves) (Fig. 1e–g).
Leaves of the in vitro control treatment showed one
layer of palisade parenchyma with elongated cells (44 lm
long) (Fig. 2a, b; Table 1). However, independent of bac-
teria type, all inoculated treatments had leaves with two
layers of palisade parenchyma cells, the outermost palisade
cells being longer (20–23 lm) than those of the second
layer (16–20 lm) (Fig. 2c–h; Table 1).
The thicknesses of both ab- and adaxial epidermis were
not significantly different between treatments (Fig. 2;
Table 1). However, PGPR inoculation significantly
increased the cuticle thickness compared with the controls
(Fig. 3; Table 1). Linear unicellular trichomes occurred in
all inoculated treatments but not in the controls (Fig. 4c).
Only small stomata with rounded apertures were found
on the abaxial epidermis of control leaves (Fig. 4a, e).
Fig. 1 Light microscopy (LM) and environmental scanning electron
microscopy (ESEM) of photinia ex vitro leaves. a Mature green
leaves, general view; b young red leaves, general view; c mature
green leaves, transverse section (LM); d young red leaves, transverse
section (LM); e adaxial epidermis (ESEM); f abaxial epidermis
(ESEM); g large stomata detail (ESEM). ad adaxial epidermis, pppalisade parenchyma, sp spongy parenchyma. Bars a, b 40 mm, c, d50 lm, e, f 100 lm; g 20 lm
Trees (2010) 24:635–642 637
123
Ex vitro leaves and plants inoculated with Azospirillum
brasilense Cd and Azotobacter chroococcum had large
stomata radially surrounded by small stomata on the
abaxial foliar surface (Figs. 1f; 4b, d). All stomata of
inoculated plants had an elliptic aperture like those of ex
vitro leaves (Figs. 1f, g; 4f–h). There were significant
differences (P B 0.05) in stomata length and width in
plants inoculated with Azospirillum brasilense Cd com-
pared with the controls (Table 1). The stomatal density of
in vitro plants inoculated with Azospirillum brasilense Cd
and Azotobacter chroococcum was higher (532 and
502 mm-2, respectively) than the controls and those
inoculated with Azospirillum brasilense Sp7 (465 and
444 mm-2, respectively), although the differences were
not significant (Table 1).
Chloroplasts of all in vitro plant treatments had intact
structure with large grana and small starch grains (data not
shown).
Roots of all plants, including controls, were tetrarch
with Casparian bands in the endodermis and they had a
thick cortex (Fig. 5i). Plants inoculated with Azospirillum
brasilense Cd and Azotobacter chroococcum had a large
root hair zone over their root surface (Fig. 5c, d, g, h, j)
which was more notable than in the control treatment
(Fig. 5a, b), while those treated with Azospirillum brasi-
lense Sp7 had a small, concentrated root hair zone in the
middle of the root (Fig. 5e, f). There was no proliferation
of undifferentiated tissue in any treatment (Fig. 6a). Con-
trol and inoculated plants showed shoot–root vascular
connections which were evident due to the continuity of
tracheary elements between organs (Fig. 6b).
The strains of Azospirillum brasilense used formed
microaggregates around the roots (Fig. 7a–c), as observed
in bacterial culture (Fig. 7d). When the root–bacteria
interaction was observed using TEM, a bacterial aggre-
gated complex was seen in the amorphous material layers
surrounding the rhizodermis surface (Fig. 7c). Bacteria
were not found in the cortical intercellular spaces (Fig. 7e,
f). Bacterial colonization was only observed on root hairs
and epidermal surfaces. Azospirillum brasilense Sp7 and
Cd strains contained large granules in their cytoplasm
(Fig. 7a, c). The morphology of these granules was in
accordance with the poly-b-hydroxybutyrate (PHB) bodies
described in Azospirillum (Grilli-Caiola et al. 2004;
Levanony et al. 1989). Chemical analysis should still be
performed to confirm the presence of PHB.
Discussion
Stomatal aperture is controlled by a number of factors that
allow water to be conserved and maintain the capacity of
the mesophyll to fix carbon dioxide (Willmer and Fricker
1996). In heterotrophic micropropagation of several spe-
cies using sugar-containing culture medium and high rel-
ative humidity, SEM studies indicated that stomatal
structure differed markedly from that of plants grown in
greenhouses or in the field (Hazarika 2006; Frommel et al.
Fig. 2 Leaf transverse section of photinia in vitro plants (light
microscopy). a, b Plants without bacterial inoculation, c, d Azospir-illum brasilense Cd-inoculated plant, e, f Azospirillum brasilenseSp7-inoculated plant, g, h Azotobacter chroococcum-inoculated plant.
ab abaxial epidermis, ad adaxial epidermis, pp palisade parenchyma,
ppi inner palisade parenchyma cells, ppo outermost palisade paren-
chyma cells, sp spongy parenchyma, st stomata, vb vascular bundle.
Bars a, c, e, g 50 lm; b, d, f, h 25 lm
638 Trees (2010) 24:635–642
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1991; Apostolo et al. 2005; Apostolo and Llorente 2000;
Llorente et al. 2007). Physiological experiments have
shown that this change in the structure of the stomata has
been associated with altered functionality in some of the
species listed (Frommel et al. 1991; Hazarika 2006). In
general, leaves of photinia plants treated with Azospirillum
brasilense Cd and Azotobacter chroococcum had stomata
with elliptical pores, while those in the controls showed
wide-open protruding stomata with rounded pores. The
elliptical shape is characteristic of ex vitro stomata with
normal function (Willmer and Fricker 1996). Some bac-
teria had been effective in avoiding alterations to the sto-
mata structure produced by in vitro culture, for example,
the inoculation of nodal potato explants with Burkholderia
phytofirmans strain PsJN (formerly nonfluorescent Pseu-
domonas sp.) produced in vitro plants with functional
stomata (Frommel et al. 1991). Additionally, the stomatal
distribution of the in vitro plants inoculated with Azospir-
illum brasilense Cd and Azotobacter chroococcum (large
Fig. 3 Cuticle and outermost wall of leaf epidermal cell of photinia
in vitro plants (transmission electron microscopy). a Plants without
bacterial inoculation, b Azospirillum brasilense Cd-inoculated plant,
c Azospirillum brasilense Sp7-inoculated plant, d Azotobacterchroococcum-inoculated plant. cu cuticle, cw cell wall, cy cytoplasm.
Bars a–d 1 lm
Fig. 4 Abaxial epidermis of photinia leaves in vitro plants (environ-
mental scanning electron microscopy). a–d General aspect; e–hstomata, detail; a, e plants without bacterial inoculation; b, fAzospirillum brasilense Cd-inoculated plant; c, g Azospirillumbrasilense Sp7-inoculated plant; d, h Azotobacter chroococcum-
inoculated plant. st stomata, stl stomata length, stw stomata width, utunicellular trichome. Bars a–d 100 lm, e–h 20 lm
Table 1 Morphometric analysis of in vitro photinia leaves: control and PGPR-inoculated treatments
Parameters N Control Azospirillum brasilense Azotobacter chroococcum
Cd Sp7
Cuticle thickness (nm) 30 430.43 ± 46.72 c 887.50 ± 89.92 a 674.50 ± 48.93 b 966.11 ± 35.29 a
Adaxial epidermis thickness (lm) 125 17.87 ± 2.19 a 18.94 ± 2.85 a 19.29 ± 2.31 a 18.40 ± 2.78 a
Abaxial epidermis thickness (lm) 125 12.57 ± 2.75 a 11.51 ± 1.20 a 12.35 ± 1.71 a 12.74 ± 2.78 a
Palisade parenchyma thickness (lm) 125 44.08 ± 3.02 a 36.62 ± 6.42 c 40.40 ± 5.50 bc 42.57 ± 6.47 ab
Length of outermost palisade cell (lm) 125 44.08 ± 3.02 a 20.01 ± 2.35 c 21.66 ± 3.17 bc 22.80 ± 5.02 b
Spongy parenchyma thickness (lm) 125 81.36 ± 12.61 a 67.68 ± 7.12 b 66.82 ± 7.65 b 85.06 ± 8.83 a
Stomata width (lm) 75 14.64 ± 3.96 b 17.10 ± 2.68 a 14.53 ± 3.84 b 13.32 ± 2.41 b
Stomata length (lm) 75 17.75 ± 3.90 b 20.44 ± 3.35 a 19.66 ± 3.61 a 17.47 ± 2.56 b
Stomatal density (mm-2) 15 465.25 ± 92.43 a 531.93 ± 101.16 a 443.64 ± 132.74 a 501.96 ± 101.10 a
Different letters in the same row indicate significant differences between treatments for each parameter by ANOVA and Tukey’s test (P B 0.05)
N number of replicates per treatment
Trees (2010) 24:635–642 639
123
stomata radially surrounded by small stomata) was similar
to that in ex vitro leaves.
The thin cuticle, indicative of less epicuticular wax
deposition, is consistent with observations of carnation in
vitro leaf surfaces (Majada et al. 2001). Therefore, the
anatomical characteristics seen in the leaves of photinia
in vitro controls might lead to abnormally high rates of
transpiration which could result in significant water loss
in the acclimatization stage. This developmental distor-
tion has been related to ambiental and nutritional
Fig. 5 Roots of photinia in vitro plants. a–h General view of root
apical and root hair zone (stereoscopic microscopy). a, b Plants
without bacterial inoculation, c, d Azospirillum brasilense Cd-
inoculated plant, e, f Azospirillum brasilense Sp7-inoculated plant,
g, h Azotobacter chroococcum-inoculated plant. i Root transverse
section of plants without bacterial inoculation, general aspect (light
microscopy), arrows Casparian bands. j Detail of root hair zone of
Azospirillum brasilense Cd-inoculated plant (environmental scanning
electron microscopy), arrows root hairs. co root cortex, en endoder-
mis with Casparian bands. Bars a, c, e, g 1 mm; b, d, f, h, j 200 lm;
i 50 lm
Fig. 6 Photinia in vitro plants inoculated with Azospirillum brasi-lense Cd. a General aspect of in vitro plant. b Detail of vascular
connection between root and shoot, longitudinal section (light
microscopy). r root, sh shoot, vc vascular connection. Bars a 1 cm,
b 500 lm
Fig. 7 Bacteria–photinia in vitro plants interaction. a, b Azospirillumbrasilense Cd-inoculated plant. a Bacteria on the epidermal cell wall
of root (TEM), b bacteria on root hair surface (SEM). c Azospirillumbrasilense Sp7-inoculated plant, bacteria, amorphous layer and root
cell wall complex (TEM). d Bacteria aggregate growing in culture
(ESEM), e Root cortex intercellular space of Azotobacter chroococ-cum-inoculated plant (ESEM). f Root cortex intercellular space of
Azospirillum brasilense Cd-inoculated plant (TEM). am amorphous
layer, ba bacteria, cw cell wall, cy cytoplasm, ESEM environmental
scanning electron microscopy, is intercellular space, rh root hair, SEMscanning electron microscopy, TEM transmission electron micros-
copy. Bars a, c 1 lm; b, d 10 lm; e, f 25 lm
640 Trees (2010) 24:635–642
123
conditions used in the in vitro culture (Nowak and
Shulaev 2003).
In vitro leaves also had a reduced number of palisade
layers, with only one in the controls and two in inoculated
in vitro plants, while ex vitro plants had 3–5 layers.
Increases in the number of palisade layers indicate
enhanced cellular division that could be related to changes
in the plant hormone balance induced by the bacteria. The
variations in phytohormone concentrations are probably
related to PGPR ability to produce them or to modify
metabolism of plant endogenous regulators (Bashan et al.
2004).
It has been well documented that a functional root
system at the end of the in vitro process is essential for the
ability to manage water and photosynthesize and it deter-
mines the ex vitro performance of propagules (Nowak and
Shulaev 2003; Ziv and Chen 2008). Adventitious root
formation on in vitro plants could be formed directly from
the original tissues, or indirectly, via newly formed callus
(undifferentiated tissue). In the first case, an adequate
vascular connection exists between the root and shoot
ensuring a strong root system. However, in the second case,
the vascular connection was weak or absent (Altamura
1996). Plants of all in vitro photinia treatments had direct
vascular connections at the shoot to root interface and no
undifferentiated tissue was observed.
Greater production of root hairs is an effect cited for
plants inoculated with some PGPR (Ait-Barka et al. 2002;
Nowak and Shulaev 2003; Bashan et al. 2004). These
modifications could be mimicked by local application of
phytohormones (Bashan et al. 2004). All bacterial strains
produced changes in root hair distribution in the inoculated
photinia in vitro plants, consistent with different morpho-
genetic effects on wheat root hairs observed by Jain and
Patriquin (1984). These authors explained that the magni-
tude and direction of root hair response to inoculation
appeared to be determined by the host and bacterial gen-
omes. It has been suggested that the effect of Azospirillum
spp. on root morphology is induced by polysaccharides
from capsular material, as well by phytohormone produc-
tion (Bashan et al. 2004).
Root colonization is the key factor in successful plant–
PGPR interactions. Various studies have suggested that
PGPR colonize root surfaces as well as the root interior
(Levanony et al. 1989; Bashan and Levanony 1990;
Assmus et al. 1995; Puente et al. 1999). The bacteria–root
association depends mainly on the plant species, PGPR
strains and the nutritional quality of the substrate
(Mrkovacki and Milic 2001). Reports on the allocation of
bacterial strains showed that Azospirillum brasilense Sp7
was restricted to the surface of wheat root hairs (Assmus
et al. 1995), while Levanony et al. (1989) observed the
distribution of Azospirillum brasilense Cd on and within
wheat roots. ESEM and TEM studies verified that Azoto-
bacter chroococcum and Azospirillum brasilense (Cd and
Sp7 strains) colonized surfaces of photinia root hairs and
rhizodermis cells; however, the bacteria could not be found
in root cortex intercellular spaces under the in vitro culture
conditions used. These results agree with other reports
(Bashan and Levanony 1990; Assmus et al. 1995; Puente
et al. 1999).
The Azospirillum brasilense strains used for photinia
inoculation formed microaggregates around roots and this
capacity could be of interest in agricultural inoculant pro-
duction. Azospirillum spp. secrete exopolysaccharides and
the involvement of surface polysaccharides in bacterial
aggregation and colonization of roots has been reported
(Burdman et al. 2000). Also, the Azospirillum brasilense
Sp7 and Cd on the photinia root surface contained large
bodies, morphologically coincident with PHB granules
observed by Grilli-Caiola et al. (2004) and Levanony et al.
(1989). If so, their accumulation may favor proliferation
and the competitive ability of bacteria in soil under stress
conditions because PHB is an intracellular carbon and
energy storage compound. This physiological stage with a
high content of PHB and cell flocculates may increase
bacterial survival and promote plant growth (Kadouri et al.
2003).
The morphological changes observed in inoculated
photinia in vitro plants (stomata morphology, vascular
shoot–root connections, thicker cuticle and abundant root
hairs) might lead to improved post-transplanting water
management and resistance to biotic and abiotic stresses.
These results are similar to those found in PGPR inocula-
tion of potato, tomato, grapevine, banana and some Prunus
in vitro cultures (Frommel et al. 1991; Ait-Barka et al.
2002; Nowak and Shulaev 2003; Rodrıguez-Romero et al.
2008; Russo et al. 2008).
The colonization of in vitro photinia microshoots by
PGPR can be used as a model system for better under-
standing how different bacterial strains promote plant
growth. Light and electron microscope analyses showed
that Azospirillum brasilense and Azotobacter chroococcum
enhanced structural properties in inoculated photinia in
vitro plants that improved the in vitro culture-induced
modifications and resembled that of ex vitro plants.
Acknowledgments The present work was supported by grants from
the Basic Sciences Department, National University of Lujan,
Argentina.
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