In Vitro Cellular & Developmental Biology - Plant

https://doi.org/10.1007/s11627-019-10034-2

PLANT TISSUE CULTURE

Impacts of photoautotrophic, photomixotrophic,

and heterotrophic conditions on the anatomy and photosystem II
of in vitro-propagated Aechmea blanchetiana (Baker) L.B. Sm.
(Bromeliaceae)
João Paulo Rodrigues Martins 1,2 & Luiz Carlos de Almeida Rodrigues 3 & Elizangela Rodrigues Santos 1 &
Andreia Barcelos Passos Lima Gontijo 2 & Antelmo Ralph Falqueto 1

Received: 12 January 2019 / Accepted: 6 November 2019 / Editor: Charles Armstrong

# The Society for In Vitro Biology 2019

Abstract
In vitro culture conditions have a major impact on the physiology and anatomy of micropropagated plants. Sucrose plays a very
important role in micropropagation protocols, and is frequently employed during in vitro culture. However, it may induce
disorders that negatively interfere with plant growth and development, such as a low photosynthetic rates. The aim of this study
was to analyze the impacts of in vitro conditions on the anatomy and photosynthetic performance of Aechmea blanchetiana
(Baker) L.B. Sm. Plants previously grown in vitro were transferred to culture media containing 0, 15, 30, or 45 g L−1 sucrose.
Two different culture container sealing systems were tested: lids with a filter (permitted gas exchange), and with a PVC-covered
filter (blocking fluent gas exchange). Plants grown with exogenous sucrose displayed anatomical traits that could decrease
mineral and carbohydrate uptake from the medium. Plants cultured under photoautotrophic conditions had a thinner exodermis
and the highest number of metaxylem vessels in the roots. This positively impacted the plant growth and physiological status.
Sucrose induced plants with photosystem II (PS II) disorders, such as a lower number of active reaction centers. Under photo-
autotrophic conditions, there was an increase in absorbed energy flux per cross section (ABS/CSm), and energy transport flux
(ETo/CSm), followed by a decrease in the energy dissipation flux (DIo/CSm). This indicated a high PS II efficiency, according to
the performance index (PI(ABS)). The use of sucrose can induce physiological disorders in A. blanchetiana plants during in vitro
propagation. Photoautotrophic conditions induce plants without anatomical disorders, and positively influence PS II efficiency.

Keywords Bromeliad . Chlorophyll a fluorescence . Plant anatomy . Plant physiology . Plant tissue culture

Introduction exchange, and artificial temperature and luminosity conditions

In vitro techniques are well known in the clonal propagation of
horticultural crops, which includes bromeliads. The microen-
vironment of conventional in vitro culture consists of closed
containers, with a high relative humidity, reduced gas
* João Paulo Rodrigues Martins
jprmartinss@yahoo.com.br
1
Plant Ecophysiology Laborato, Federal University of Espírito Santo,
Litorâneo, São Mateus, Espírito Santo 29932-540, Brazil
2
Plant Tissue Culture Laboratory, Federal University of Espírito
Santo, Litorâneo, São Mateus, Espírito Santo 29932-540, Brazil
3
Federal University of Alfenas, Centro, Alfenas, Minas
Gerais 38900-000, Brazil
(Rodrigues et al. 2014). Inside the containers, high variations
in CO2 and ethylene accumulation may occur throughout the
day (Kozai 2010; Martins et al. 2015b). These conventional
in vitro propagation techniques are carried out by adding
sugars to the medium as the main carbon source (Xiao et al.
2011), which characterizes this method of in vitro culture as
heterotrophic.
Sugars are required as a carbon source for in vitro culture to
replace the carbon that plants would normally fix from the
atmosphere by in vivo photosynthesis for growth and devel-
opment. The heterotrophy phenomenon results from the low
photosynthetic activity of in vitro plants, and is considered to
be one of the major limiting factors to improve
micropropagation efficiency (Yaseen et al. 2013). These con-
ditions may also induce other physiological alterations and
 MARTINS ET AL.
anatomical disorders in plants cultivated in vitro (Rodrigues
et al. 2014; Martins et al. 2015b, 2016b). Sucrose has been an
extensively utilized carbohydrate in the vast majority of work
on in vitro cultivation, but its effects on plant development and
growth remain highly varied (Yaseen et al. 2013; Lembrechts
et al. 2017).
Sucrose is closely related to stomatal density and chloro-
phyll content, and morphogenetic behavior in some plant tis-
sues, such as vascular and support tissues (Mohamed and
Alsadon 2010; Iarema et al. 2012; Martins et al. 2015b). An
exogenous sucrose supply increases the endogenous levels of
carbohydrate stocks (starch, sucrose, fructose, and glucose) in
micropropagated plants, and may help plants to acclimatize
and accelerate their physiological adaptations (Jo et al. 2009).
During in vitro culture, sucrose may also alter the osmotic
potential of the medium, which increases when sucrose is
hydrolyzed into glucose and fructose by enzymes such as
invertase (Martins et al. 2016b).
P l a n t s c a n a l s o b e p r o p a g a t e d i n v i t ro u s i n g
photomixotrophic conditions, in which sugars are not the only
carbon source, as CO2 is added to containers with filters that
allow free gas exchange, or even using a CO2-enriched atmo-
sphere (Martins et al. 2015b, 2016b; Arencibia et al. 2017;
Hoang et al. 2017). Compared with heterotrophic conditions,
the use of in vitro culture with photomixotrophic conditions
has been shown to provide improved rooting formation, fewer
physiological disorders, and high growth rates after the accli-
matization phase (Martins et al. 2015b; Hoang et al. 2017).
In vitro growth can also be performed without the addition
of sugars to the medium. The use of photoautotrophic condi-
tions with a sugar-free medium has advantages over both het-
erotrophic and photomixotrophic conditions, because healthy
plants with enhanced photosynthesis are normally produced
(Iarema et al. 2012; Martins et al. 2015b; Assis et al. 2016).
There has been a steady increase in the application of tech-
niques that allow for photoautotrophic growth because under
these conditions, in vitro physiology resembles ex vitro phys-
iology and enables fast acclimatization (Saldanha et al. 2014;
Tisarum et al. 2018).
In general, the success of micropropagation systems can be
effectively measured by the percentage of plants that survive
when transferred from in vitro culture vessels, to greenhouse
or field conditions. This survival rate is directly related to
anatomical traits and the photosynthetic performance of the
micropropagated plants (Sáez et al. 2015). During in vitro
culture of the bromeliad Billbergia zebrina (Herbert),
Martins et al. (2015b, 2016b), verified that plants grown under
photoautotrophic conditions displayed anatomical traits and
high endogenous carbohydrate stocks, which allowed higher
growth rates during the acclimatization period. On the other
hand, during in vitro culture with sugar (heterotrophic and
photomixotrophic conditions), the plants had a lower hydrau-
lic conductance induced by thinner xylem vessels, and
carbohydrate stock concentrations increased as a function of
sucrose supplementation. In addition, those plants showed
lower growth rates after transfer to the ex vitro conditions.
According to Mohamed (2008), a low survival rate in addition
to a slow growth rate of tissue culture plants are critical prob-
lems that impact the success of tissue culture as a commercial
technique.
Comparative studies of in vitro conditions (gas exchange
and exogenous sucrose in the medium) have been performed
for some plant species (Iarema et al. 2012; Saldanha et al.
2014; Assis et al. 2016), including a bromeliad (Martins
et al. 2015b). Most of these studies focused on growth traits
and leaf anatomy of in vitro plants, and did not address ana-
tomical changes that could happen in roots or at a deeper
physiological level, such as how the in vitro conditions affect
the performance of the photosynthetic apparatus of plants.
Therefore, it remains to be established how photoautotrophic,
photomixotrophic, and heterotrophic conditions can interfere
with responses in the photosynthetic apparatus, particularly
photosystem II (PS II). Chlorophyll a fluorescence is a very
sensitive, non-invasive, and rapid procedure used to evaluate
changes in the photosynthetic apparatus, and can be useful
when investigating PS II (Kalaji et al. 2014).
In vitro culture conditions can interfere significantly with
the regulation of in vitro growth and development, including
the quality and physiological state, and the anatomical traits of
micropropagated plants. The aim of the present study was to
analyze the impacts of photoautotrophic, photomixotrophic,
and heterotrophic conditions on the anatomy and photosyn-
thetic performance of in vitro-propagated Aechmea
blanchetiana.
Materials and Methods
Plant material and culture conditions Aechmea blanchetiana
plants were previously established in vitro from seeds collect-
ed in São Mateus, Espírito Santo, Brazil, and multiplied in
268 mL glass containers containing 50 mL stationary liquid
MS medium (Murashige and Skoog 1962), supplemented
with 30 g L−1 sucrose (Neon Comercial®, Suzano, Brazil),
and 10 μM 6-benzylaminopurine (Sigma-Aldrich®, St. Louis,
MO) for 60 d. All establishment and multiplication procedures
were completed according to Rosa et al. (2018). The obtained
buds were subcultivated for 60 d in 268 mL glass containers
containing 50 mL stationary liquid MS medium with 30 g L−1
sucrose, to induce the growth of side shoots. The multiplica-
tion and subcultivation procedures were carried out three
times on the side shoots, to achieve the necessary number of
microshoots with similar morphology. All media were adjust-
ed to pH 5.8 and autoclaved at 121°C for 20 min. The in vitro
plant material was kept in a growth room, at 26 ± 2°C with a
16-h photoperiod, with fluorescent tube lamps (Empalux®
 PHOTOAUTOTROPHIC PHOTOMIXOTROPHIC HETEROTROPHIC PROPOGATED BROMELIACEAE
FT8 HO, 36 W/6400 K) that provided 90 μmol m−2 s−1 of
photosynthetically active radiation (PAR).
Sucrose and gas exchange during the in vitro propagation
Microshoots approximately 3 cm in length were individual-
ized (4 to 7 side shoots per seedling), using a scalpel. After
mixing, the side shoots were transferred to 280 mL polypro-
pylene containers (Microbox Combiness®, Nevele, Belgium),
containing 50 mL MS medium solidified with 6 g L−1 agar
(Vetec®, Darmstadt, Germany), and 0, 15, 30, or 45 g L−1
sucrose. This phase was carried out with five shoots per con-
tainer, and the pH was adjusted to 5.8, before autoclaving at
121°C for 20 min. Two different sealing systems were tested:
lids with an XL yellow filter (permitted gas exchange sys-
tem—13.09 gas replacements per day), and lids with an XL
yellow filter covered with three layers of polyvinylchloride
(PVC) transparent film (blocking fluent gas exchange). After
sterile inoculation, the material was kept for 45 d in a growth
room, at 26 ± 2°C with a 16-h photoperiod, under fluorescent
tube lamps (Empalux® FT8 HO, 36 W/6400 K), which pro-
vided 90 μmol m−2 s−1 of PAR.
Growth traits of in vitro plantlets To evaluate growth, 20
plants from each treatment were sampled randomly at 45 d
of growth and divided into four parcels. The fresh weight of
the aerial parts and the number of roots were determined.
Anatomical analysis Anatomical characterization of the
leaves and roots was performed on four plants from each
treatment. Samples were randomly collected at 45 d of
growth, and fixed in formaldehyde, acetic acid, and etha-
nol (FAA; 50%, 0.5/0.5/9, v/v) for 48 h, followed by stor-
age in 70% (v/v) ethanol (Johansen 1940). In the leaves,
paradermal and cross sections were made in the median
region of the first completely expanded leaf in the rosette
central region, using a double-edge razor. Cross sections
were also made at the root base (0.3–0.8 cm from the
shoot). Sections were cleared using sodium hypochlorite
10% (v/v), followed by staining with safranin and astra
blue solutions (Kraus and Arduin 1997), and assembled
on slides using 50% (v/v) glycerine. The sections were
viewed using a light microscope (Leica DM 1000, com-
bined with a Leica ICC50 HD camera; Leica, Wetzlar,
Germany), and two cross sections and four paradermal
section fields from each slide were photographed. The pho-
tomicrographs were used to measure the anatomical char-
acteristics using UTHSCSA-Imagetool® software calibrat-
ed with a microscopy ruler. For the leaves, measurements
were taken of the thickness of the adaxial and abaxial epi-
dermis (μm) and chlorenchyma (μm), and number of xy-
lem vessels. For the roots, the thickness of cell walls in the
exodermis (μm) and endodermis (μm) and the number of
metaxylem vessels were measured.
Measurement and analysis of in vitro chlorophyll a fluores-
cence At 45 d of growth, chlorophyll a fluorescence measure-
ments were made, using the third completely expanded leaves
from 12 in vitro-cultured plants per treatment, using a portable
Handy PEA fluorimeter (Hansatech®, King’s Lynn, Norfolk,
UK), between 8:00 and 9:00 am. Before the measurements
were made, the leaves were dark-adapted for 30 min using a
leaf clip (Hansatech®). The irradiance during the measure-
ment was 3000 μmol (photons) m−2 s−1, which was sufficient
to generate maximal fluorescence for all treatments. The fast
fluorescence kinetics (F0 to FM) were recorded from 10 μs to
1 s. The fluorescence intensities at 20 μs (considered F0),
100 μs, 300 μs, 2 ms (FJ), 30 ms (FI), and 300 ms (FM) were
recorded and analyzed as described previously (Strasser et al.
2004; Stirbet and Govindjee 2011).
Statistical analysis The experiment was performed in a
completely randomized design in a factorial arrangement (four
sucrose concentrations and two different sealing systems).
The data obtained were submitted to analysis of variance
(ANOVA), and the averages were compared using the
Scott–Knott test (Scott and Knott 1974). All statistical analy-
ses were performed using the SISVAR® software, version 5.6.
Results
Growth traits Growth trait responses were modulated by both
the sucrose concentration and sealing system. Rooting was
verified in all treatments; however, plants grown using exper-
imental gas exchange conditions (containers with a filter)
displayed a higher number of roots with 0, 30, and 45 g L−1
sucrose, compared to the same sucrose concentrations when
cultured in containers with no free gas exchange. In containers
with blocked gas exchange, the plants cultured with 15 g L−1
sucrose had the highest number of roots (Fig. 1).
The fresh weight of the aerial parts was higher in plants
grown in filtered containers, than in containers with a blocked
gas exchange, at each sucrose level. Plants cultured in con-
tainers with a filter and 0 (photoautotrophic condition), or
15 g L−1 sucrose (photomixotrophic condition), exhibited
the highest aerial fresh weight. In containers with a blocked
gas exchange, plants grown with 15 g L−1 sucrose (heterotro-
phic condition) had the highest aerial fresh weight (Fig. 2).
Analysis of plant anatomy Root anatomical structures differed
among the treatments (Fig. 3). Increasing the sucrose concen-
tration induced thickening of the exodermis cell walls, regard-
less of sealing system. However, there was no change in the
endodermis as a function of the treatments (Figs. 3, 4).
Plants cultured in containers without a filter exhibited a
similar number of metaxylem vessels at all sucrose concentra-
tions. However, when grown in containers with a filter, a
 MARTINS ET AL.

Figure 1. Number of roots of Aechmea blanchetiana (Baker) L.B. Sm.

plants at 45 d of growth, as a function of sucrose (g L−1) in the in vitro
medium and the sealing system of the culture containers. Means (± SE)
followed by the same letter (lower case for plants cultured with a filter
with a PVC cover, and upper case for plants cultured with a filter) are not
significantly different according to a Scott–Knott test, at 5%. Within
sucrose concentration (g L−1) analyzed, means (± SE) followed by an
asterisk are significantly different according to a Scott–Knott test, at 5%.

decrease in metaxylem vessels with increasing sucrose con-

centrations was observed. The highest metaxylem vessel num-
ber was observed in roots grown under photoautotrophic con-
ditions (0 g L−1 sucrose and permitted gas exchange) (Figs. 3,
5).
The treatments also influenced the anatomical structures of
the leaves (Fig. 6). Stomatal density decreased as a function of
sucrose concentration, but the density of the epidermal cells
did not change. The stomatal index also decreased when the Figure 3. Cross sections of Aechmea blanchetiana (Baker) L.B. Sm.
plant roots grown as a function of the sealing system and sucrose con-
sucrose concentration was raised. The sealing system had no
centration. ex, exodermis; ed, endodermis; x, xylem; and ph, phloem.
significant influence on the stomatal density and index Bars = 100 μm.

(Figs. 6, 7). On the other hand, the thickness of the epidermis

and chlorenchyma and the number of xylem vessels were only
influenced by the sealing system. The thickness of the epider-
mis in the adaxial face, the chlorenchyma thickness, and the
number of xylem vessels were higher in plants cultured with a
filter than in unfiltered containers (Figs. 6, 8).

Chlorophyll a fluorescence of in vitro plants The in vitro con-

Figure 2. Fresh weight (mg plant−1) of Aechmea blanchetiana (Baker)
L.B. Sm. plants (aerial parts) at 45 d of growth, as a function of sucrose
(g L−1) in the in vitro medium and the sealing system of the culture
containers. Means (± SE) followed by the same letter (lower case for
plants cultured with a filter with a PVC cover, and upper case for plants
cultured with a filter) are not significantly different according to Scott–
Knott test, at 5%. Within each sucrose concentration (g L−1) analyzed,
means (± SE) followed by an asterisk are significantly different according
to a Scott–Knott test, at 5%.
ditions had a clear impact on the photosynthetic apparatus of
A. blanchetiana. An increase in the J-step fluorescence level
[VJ = (FJ − F0)/(FM − F0)] was the most evident characteristic
of chlorophyll a fluorescence and OJIP transients for plants
cultured with sucrose. In both sealing systems, VJ values were
lower in plants grown in a medium without sucrose. On the
other hand, the I-step fluorescence level [VI = (FI − F0)/(FM −
F0)] decreased only in plants cultured in containers without a
filter and without sucrose added to the medium (Fig. 9).
All JIP test parameters analyzed that were based on fluo-
rescence emission kinetics varied significantly based on the
sealing system and sucrose concentration. When gas exchange
 PHOTOAUTOTROPHIC PHOTOMIXOTROPHIC HETEROTROPHIC PROPOGATED BROMELIACEAE
Figure 4. Thickness of
exodermis cell wall (A) and
endodermis (B) of Aechmea
blanchetiana (Baker) L.B. Sm.
roots at 45 d of growth, as a
function of sucrose (g L−1) in the
in vitro medium. Means (± SE)
followed by the same letter are
not significantly different accord-
ing to a Scott–Knott test, at 5%.
was restricted (filter blocked by PVC cover), the absorption
(ABS/CSm) values per cross section (CS) were similar regard-
less of sucrose concentration, but trapping (TRo/CSm) in-
creased in the 15 and 30 g L − 1 sucrose treatments
(Fig. 10A−D). ABS/CSm and TRo/CSm values for the plants
cultivated in containers with a filter (aerated containers per-
mitting unrestricted gas exchange) resulted in a negative linear
relationship with increasing sucrose concentrations
(Fig. 10E−H). The highest electron transport (ETo/CSm)
values and number of active reaction centers (RC) [CSm/
ABS = RC/CSm = φPo × (V J/M 0) × ABS/CSm] were ob-
served in plants under photoautotrophic conditions
(Fig. 10E). Plants grown in containers with a filter had a lower
Figure 5. Number of metaxylem vessels in Aechmea blanchetiana
(Baker) L.B. Sm. roots at 45 d of growth, as a function of sucrose
(g L−1) in the in vitro medium and the sealing system of the culture
containers. Means (± SE) followed by the same letter (lower case for
plants cultured with a filter with a PVC cover, and upper case for plants
cultured with a filter) are not significantly different according to Scott–
Knott test, at 5%. Within each sucrose concentration (g L−1) analyzed,
means (± SE) followed by an asterisk are significantly different according
to Scott–Knott test, at 5%.
energy dissipation per CS (DIo/CSm), compared to plants
cultured in containers without a filter (Fig. 10A−H).
Plants cultured under photoautotrophic conditions had the
lowest net rate of PS II closure (M0) [= dV/dto = 4(F300μs −
F0)/(FM − F0)]. The sealing system and sucrose concentration
also had a significant effect on quantum yield parameters. The
highest φPo [= 1 − F0/FM = FV/FM] and φEo [= φPo × (1 −
VJ)] values were verified in plants grown without sucrose, and
in containers with permitted gas exchange (photoautotrophic
condition). In contrast, the lowest φDo [= F0/FM] values were
also obtained in the same plants. In general, plants cultured in
containers without a filter had lower φPo and φEo values
(Table 1).
When comparing the values of the PS II performance index
(PI(ABS)), as a function of the sucrose concentration in each
sealing system, the plants cultured in containers with the filter
blocked by a PCV cover had similar values. However, plants
cultured in containers with a filter showed major differences.
Plants had the highest PI(ABS) under photoautotrophic condi-
tions (Fig. 11).
Discussion
Gas exchange and sucrose concentration both played very
important roles in the morphogenetic responses of
A. blanchetiana, as already shown for other bromeliad species
(Martins et al. 2015a, b, 2016b; Lembrechts et al. 2017).
Plants cultivated in ventilated containers had better root for-
mation (Fig. 1). The positive effects of containers with gas
exchange have been attributed to the continuous replenish-
ment of CO2 from the open air and prevention of ethylene
accumulation (Trevisan and Mendes 2005). An increased sup-
ply of CO2 in culture containers can induce improved rooting
 MARTINS ET AL.
Figure 6. Paradermal sections (A–H) and cross sections (I–X) of leaves of
Aechmea blanchetiana (Baker) L.B. Sm. plants at 45 d of growth, as a
function of the sealing system and sucrose concentration. ad, adaxial
Figure 7. Stomatal and epidermal cell density (mm2) and stomatal index
(%) of Aechmea blanchetiana (Baker) L.B. Sm. leaves at 45 d of growth,
as a function of sucrose concentration (g L−1). For each anatomical trait,
average values (± SE) followed by the same letter do not differ signifi-
cantly according to a Scott–Knott test at 5% probability.
epidermis; ab, abaxial epidermis; chl, chlorenchyma; x, xylem; and ph,
phloem. Bars = 200 μm (A–P) and 50 μm (Q–X).
of in vitro plants (Emam and Esfahan 2014). Containers with a
filter also exhibit a low internal atmospheric humidity. The
difference between leaf water demand and the ability of the
roots to meet the demand can be regulated through phytohor-
mones and hydraulic signals transported through the vascular
system (Vandeleur et al. 2008). Improved transport of phyto-
hormones such as auxins, which were induced by increased
plant transpiration, may have increased the number of roots of
in vitro A. blanchetiana. An increased number of lateral roots
can enlarge the capacity of the root system to take up water
and nutrients (Lopez-Bucio et al. 2003; Martins et al. 2018).
This improves the water status of plants, and helps the nutrient
profile advance plant growth and development, thereby in-
creasing the fresh weight of shoots (Wintermans et al. 2016;
Martins et al. 2018).
The anatomy of A. blanchetiana is in accordance with other
bromeliad species of the subfamily Bromelioideae (Martins
et al. 2015b, 2016a). The sealing system of the culture con-
tainers and sucrose concentration both had significant effects
on the root anatomy of A. blanchetiana plants, and altered the
uptake of water and minerals from the medium. The exoder-
mis and endodermis are apoplastic barriers due to the
 PHOTOAUTOTROPHIC PHOTOMIXOTROPHIC HETEROTROPHIC PROPOGATED BROMELIACEAE

from the medium (Figs. 3, 4). High sucrose concentrations

Figure 8. Anatomical structures of Aechmea blanchetiana (Baker) L.B.
Sm. leaves at 45 d of growth, as a function of the sealing system of the
culture containers. Means (± SE) followed by the same letter, in each
anatomical structure, are not significantly different according to Scott–
Knott test, at 5%.
formation of Casparian bands in their cell walls. Casparian
bands consist of a water-impermeable lignin polymer that
seals the extracellular spaces between neighboring exodermal
and endodermal cells (Nakayama et al. 2017). Lignification
and suberization of the cell walls in these tissues both occur
naturally in bromeliads (Martins et al. 2016a). However, root
radial conductivity decreases with the increasing formation of
these apoplastic barriers (Vetterlein and Doussan 2016). An
increment in cell wall thickness of the exodermis in plants
grown in a medium supplemented with 30 and 45 g L−1 su-
crose may decrease the uptake of carbohydrates and minerals
may not be totally consumed by in vitro plants, and decrease
the osmotic potential of the culture medium (Martins et al.
2016b). During the in vitro culture, plants may also use inver-
tase to hydrolyze sucrose into glucose and fructose, and fur-
ther decrease the osmotic potential of the medium (Karhu
1997; Martins et al. 2016b). Thicker exodermal wall cells
can act as effective first apoplastic barriers against high con-
centrations of molecules (carbohydrates), which can induce
some type of stress (Cheng et al. 2012; Martins et al. 2015b,
2016a), including osmotic stress. A greater suberization of cell
walls of the exodermis can lower conductance by reducing the
apoplastic inflow of ions that occur near the root surface, as
previously verified in bromeliads (Martins et al. 2016a).
These authors also verified that there are not any differences
in endodermis thickness, even when the bromeliads were un-
der stress conditions.
In roots, the number and diameter of the metaxylem vessels
can also indicate the potential water conductance at axial po-
sitions along the root. Improved water conductance distribu-
tion is indicated by a higher number of metaxylem vessels
(Steinemann et al. 2015; Vejchasarn et al. 2016). A large
water conductance from the roots is important to meet the
demands of photosynthesis. It also improves mineral uptake
and can be reflected in the growth and physiology of plants, as
verified in plants grown under photoautotrophic conditions
(Martins et al. 2015b). Mineral ions are carried by water and
enter the cytoplasm of epidermal cells, after which they move
across the cortex and are ultimately secreted into the xylem for
transfer to the shoot (Nakayama et al. 2017). During photo-
synthesis, a large quantity of water is necessary because plants
use the water-splitting and oxygen-evolving reactions

1.2
-1
0 g L sucrose
-1
Filter + PVC cover a -1
0 g L sucrose
-1
Filter b
15 g L sucrose 15 g L sucrose
1 -1
30 g L sucrose 30 ms
-1
30 g L sucrose 30 ms
-1 -1
45 g L sucrose 45 g L sucrose
VOP = (Ft - F0) / (FM - F0)

2 ms 2 ms
0.8

0.6 1.2 1.2

VJ VI VJ VI
1 A 1 A* A A A
A A
0.8 B a 0.8 a a a
b a a b
0.4 0.6 0.6
0.4 0.4
0.2 0.2
0.2
0 0
0 15 30 45 0 15 30 45
-1 -1
Sucrose (g L ) Sucrose (g L )
0
0.01 0.1 1 10 100 1000 0.01 0.1 1 10 100 1000
Time (ms) Time (ms)
Figure 9. Relative variable fluorescence between F0 and FM (VOP) and and upper case for VI) are not significantly different according to a Scott–
relative variable fluorescence at phase J (VJ) and phase I (VI) of the Knott test, at 5%. Within each sucrose concentration (g L−1) analyzed in
fluorescence induction curve at 45 d of growth, as a function of sucrose each relative variable fluorescence phase, means (± SE) followed by an
(g L−1) in the in vitro medium and the sealing system of the culture asterisk are significantly different according to a Scott–Knott test, at 5%
containers. Means (± SE) followed by the same letter (lower case for VJ
 MARTINS ET AL.

Figure 10. Summary of phenomenological energy fluxes per excited
cross section approximated by F M (CSm = P step), in Aechmea
blanchetiana (Baker) L.B. Sm. leaves at 45 d of growth, as a function
of the sealing system of the culture containers. ABS/CSm, absorption flux
per cross section; TRo/CSm, trapped energy flux per cross section (CS);
ETo/CSm, electron transport flux per CS; DIo/CSm, dissipated energy
flux per CSm. CSm/ABS, active reaction centers (RCs), are indicated by
black circles. In each parameter, means followed by the same letter (up-
per case for sucrose concentration in each sealing system and lower case
for sealing system in each sucrose concentration) are not significantly
different according to a Scott–Knott test, at 5%.

catalyzed by PS II (Shen 2015). This is in agreement with
these results, because the highest number of metaxylem ves-
sels was found in plants under photoautotrophic conditions,
which had the highest PS II performance.
During in vitro culture, a very common concern is stomatal
shape (Martins et al. 2015b; Monja-Mio et al. 2015; Asayesh
et al. 2017). Open stomata are related to low stomatal func-
tioning, which is an important feature because plants with
dysfunctional stomata are unable to avoid water deficiency
in an ex vitro environment (Martins et al. 2015b; Hoang
et al. 2017). In this study, there were no apparent stomata with
open pores (Fig. 6). Nevertheless, the density and index of
stomata decreased with increasing sucrose concentration in
the medium. This could be linked to a low photosynthetic
activity in those plants grown under high sucrose concentra-
tions. Plants may present a short-term plastic response to the
high relative humidity of the in vitro conditions, which indi-
cates the low need to assimilate CO2 once the sucrose input is

Table 1. Net rate of photosystem II (PS II) closure (M0) and quantum yields based on fluorescence emission kinetics as a function of the sealing system
of the culture containers and sucrose concentration

Sucrose M0 φPo φEo φDo

(g L−1)
Sealing system

Filter Filter with PCV Filter Filter with PCV Filter Filter with PCV Filter Filter with PCV
cover cover cover cover

0 1.82 ± 0.06Bb 2.18 ± 0.02Aa 0.64 ± 0.02Aa 0.51 ± 0.01Bb 0.23 ± 0.01Aa 0.18 ± 0.01Ab 0.36 ± 0.02Ab 0.49 ± 0.01Ba
15 2.05 ± 0.07Aa 2.02 ± 0.05Aa 0.56 ± 0.01Ba 0.60 ± 0.02Aa 0.16 ± 0.01Ba 0.19 ± 0.01Aa 0.44 ± 0.01Ba 0.41 ± 0.02Aa
30 1.96 ± 0.05Aa 2.15 ± 0.03Aa 0.62 ± 0.01Aa 0.56 ± 0.01Ab 0.18 ± 0.01Ba 0.17 ± 0.01Aa 0.38 ± 0.01Ab 0.44 ± 0.01Aa
45 2.07 ± 0.03Aa 2.15 ± 0.08Aa 0.58 ± 0.01Ba 0.54 ± 0.01Bb 0.16 ± 0.01Ba 0.16 ± 0.01Aa 0.42 ± 0.01Bb 0.46 ± 0.01Ba

In each JIP test parameter, means (± SE) followed by the same letter (lower case for sucrose concentration in each sealing system, and upper case for
sealing system in each sucrose concentration) are not significantly different according to a Scott–Knott test, at 5%
 PHOTOAUTOTROPHIC PHOTOMIXOTROPHIC HETEROTROPHIC PROPOGATED BROMELIACEAE
Figure 11. PI(ABS) of Aechmea blanchetiana (Baker) L.B. Sm. at 45 d of
growth, as a function of sucrose (g L−1) in the in vitro medium and the
sealing system of the culture containers. Means (± SE) followed by the
same letter (lower case for plants cultured with a filter with a PVC cover,
and upper case for plants cultured with a filter) are not significantly
different according to a Scott–Knott test, at 5%. Within each sucrose
concentration (g L−1) analyzed, means (± SE) followed by an asterisk
are significantly different according to a Scott–Knott test, at 5%.
artificially guaranteed (Rodrigues et al. 2014). High sucrose
concentrations in the vascular tissue may result in a decreased
sink demand and down-regulated transporter activity. When
sugar levels are increased in mesophyll cells, photosynthesis
is down-regulated (Lemoine et al. 2013). In vitro plants cul-
tured with sucrose may have high stocks of sucrose and starch
in the leaves, and low photosynthetic rates (Iarema et al. 2012;
Martins et al. 2016b; Hoang et al. 2017). This could be
reflected in a low investment in stomatal cells due to this
low photosynthetic demand. In contrast, plants grown without
any sucrose supplementation had the highest stomatal density,
which suggested an adaptation to improve the photosynthetic
rate. An increased stomatal density enhances photosynthetic
capacity by modulating gas diffusion into the mesophyll
(Tanaka et al. 2013).
Containers with a filter present have a lower atmospher-
ic humidity compared to regular (hermetically sealed) cul-
ture containers, which can induce anatomical responses by
leaves to their microenvironment (Iarema et al. 2012;
Martins et al. 2015b). Leaves cultured under non-
ventilated conditions may be thinner and have poor meso-
phyll differentiation, compared to leaves under ventilated
conditions (Mohamed and Alsadon 2010). The thicker epi-
dermis observed in containers with a filter is a response to
the low humidity of the culture environment, and protects
the plant against water loss (Shibuya et al. 2015). In addi-
tion, plants grown in ventilated containers may have
thicker, better-formed chlorenchyma due to the availability
of the adequate CO 2 levels required for in vitro
propagation with non-accumulation of other gases, such
as O 2 and ethylene (Mohamed and Alsadon 2010;
Martins et al. 2015b).
Even though the increment in the number of xylem vessels
in the leaves was small, this could be responsible for the in-
crease in chlorenchyma thickness, and in the fresh weight of
the aerial parts of plants cultured in containers with a filter
(Figs. 2, 8). The number and diameter of xylem vessels appear
to be key factors that influence the hydraulic conductance. A
raised hydraulic conductance can enhance the water potential,
and consequently, shoot growth increments (Tombesi et al.
2010). In addition, an improved hydraulic conductance could
improve the uptake and distribution of minerals and carbohy-
drates from the medium to the aerial parts of plants. This
increment in the number of vessels was probably stimulated
by the difference in water potential between the medium and
humidity in the container, because the plants under filter con-
ditions may have a higher water demand in their leaves.
The in vitro conditions also had a high impact on the phys-
iological status of A. blanchetiana in vitro. Under in vitro
culture with sucrose, electron transference in PS II beyond
quinone A (QA) was delayed, and this physiological event
resulted in an increase in the J-step level (VJ). An increased
VJ is interpreted as lower rates of electron transport between
QA and quinone B (QB) (Chen et al. 2015; Cai et al. 2016).
Next, the J-step implied that sucrose affected electron flow
from QA to QB or QB−, which resulted in the fast accumulation
of QA−. In this regard, A. blanchetiana plants grown without
sucrose (added to the medium) did not have the ability to
undertake electron transport from QA to QB (Fig. 9A, B).
However, the I-step level (VI) was decreased in plants without
sucrose that were cultured in containers with a blocked gas
exchange (Fig. 9A). This is consistent with a photoinhibitory
effect, and over-reduction of the plastoquinone pool (and/or
the QA site), driven by the CO2-depleted/anoxic conditions
(Joët et al. 2002a, b). Plants grown without any exogenous
carbon source during in vitro culture may present a limited
photosynthesis rate, but can survive for a short time by con-
suming carbohydrates stocked in their tissues. However, after
that, the plants start a senescence process (Martins et al.
2016b). According to Wang et al. (2016), a decrease in VI
values indicates that inhibition of the donor side of PS II is
possibly greater than that of the acceptor side. This indicates
that the electron transfer capability of both the donor and
acceptor sides of the PS II reaction center may be altered by
natural senescence. The reduction in VI may be related to a
low net photosynthetic rate, which suggests an accumulation
of reduced plastoquinone produced in the luminous phase of
photosynthesis, which was not capable of reaching the dark
phase reactions (Gravano et al. 2004).
The results of the present study indicated that in vitro con-
ditions can drastically alter the efficiency of the energy fluxes
per CS. These results show low electron transport (ETo/CSm)
 MARTINS ET AL.
and high dissipation (DIo/CSm), when A. blanchetiana plants
were cultured in closed containers and/or with an exogenous
supply of sucrose (Fig. 10). This could indicate a physiolog-
ical disorder due to the conversion of active RCs into inactive
RCs, which reduces the electron transport from PS II (Mehta
et al. 2010; Gururani et al. 2017). Reducing the number of
active RCs indicates that less energy is used to drive electron
transport. This energy must therefore be dissipated by
nonphotochemical mechanisms (Zushi and Matsuzoe 2017).
This dissipation refers to the loss of energy through the trans-
fer of heat, fluorescence, and energy to systems other than
electron transport (Strasser et al. 2000; Eullaffroy et al.
2009). Plants under photoautotrophic conditions presented
the most efficient photosynthetic apparatus, with a very high
ABS/CSm, ETo/CSm, TRo/CSm, and a low DIo/CSm. This is
evidence of a greater PS II performance, as shown by PI(ABS)
(Fig. 11). Much of the absorbed energy was harnessed in the
biochemical processes by the plants, rather than being lost.
High photosynthetic efficiency may be expressed by PI(ABS),
which is a multiparameter value associated with the perfor-
mance of PS II reaction centers, and is responsible for the
efficiency of light energy absorption, trapping, and transfer
(Gururani et al. 2017).
M0 denotes the maximum closure rate of PS II, and an
increase in this parameter indicates an inhibition on the accep-
tor side, with electron transport between QA and QB (Wang
et al. 2016). A significant increase in M0 suggests a decrease
in the number of active RCs, and therefore an increase in the
number of closed reaction centers (Einali and Shariati 2015).
Based on this, plants grown under photoautotrophic condi-
tions had the highest number of active RCs, and did not show
any damage in the PS II process (Fig. 10, Table 1).
The φPo values were altered as a result of the in vitro
conditions. The efficiency by which light is absorbed by
the antenna, trapped by the RC, and used to reduce to QA−
is affected by the treatment condition (Perales-Vela et al.
2016). High sucrose concentrations added during in vitro
growth may cause a decline in φPo due to down-
regulation of photosynthesis, as the high sucrose content
causes a low sink demand in the plants (Matysiak and
Gabryszewska 2016). A decreasing φPo may also be
related to a decrease in the D1 protein content (Perales-
Vela et al. 2016). Reduction in φPo is consistent with the
ratio of active/inactive RCs; a decrease in active RCs may
also reduce φPo (Yusuf et al. 2010). The excess excita-
tion energy is dissipated as heat through inactive RCs, as
indicated by the increase in DIo/RC and φDo. Dissipation
results in a decrease in the quantum yield of electron
transport, and such a decrease in φEo suggests it is prob-
able that electron transport beyond QA− was decreased
(Sampaio et al. 2016). The highest φEo in plants under
photoautotrophic conditions indicated that light absorp-
tion and photosynthetic electron transport became much
more efficient, which was also indicated by the perfor-
mance index, PI(ABS).
Conclusions
In vitro conditions had a high impact on the anatomical and
photosynthetic performance of A. blanchetiana plants. Plants
grown with sucrose displayed anatomical responses to the
exogenous carbohydrates, which decreased the radial conduc-
tivity of the roots. Sucrose also induced physiological disor-
ders in the photosynthetic apparatus of the plants. The number
of active RCs was directly related to in vitro culture condi-
tions, and it was increased in photoautotrophic conditions rel-
ative to photomixotrophic and heterotrophic conditions.
Photoautotrophic conditions produced A. blanchetiana plants
without anatomical disorders, and it positively enhanced the
efficiency of PS II.
Funding information The authors would like to acknowledge the schol-
arship awarded by the CNPq (Brazilian National Council for Scientific
and Technological Development), the CAPES (Coordination for the
Improvement of Higher Education Personnel), and the FAPES (Espírito
Santo State Research Foundation).
References
Arencibia AD, Gómez A, Poblete M, Vergara C (2017) High-
performance micropropagation of dendroenergetic poplar hybrids
in photomixotrophic temporary immersion bioreactors (TIBs). Ind
Crop Prod 96:102–109. https://doi.org/10.1016/j.indcrop.2016.11.
065
Asayesh ZM, Vahdati K, Aliniaeifard S, Askari N (2017) Enhancement of
ex vitro acclimation of walnut plantlets through modification of
stomatal characteristics in vitro. Sci Hortic 220:114–121. https://
doi.org/10.1016/j.scienta.2017.03.045
Assis ES, Rubio-Neto A, Lima LR, Silva FG, Rosa M, Vasconcelos-Filho
SC, Leite MS (2016) In vitro culture of Mouriri elliptica (Mart.)
under conditions that stimulate photoautotrophic behavior. Aust J
Crop Sci 10:229–236
Cai W, Gao X, Hu J, Chen L, Li X, Liu Y, Wang G (2016) UV-B radiation
inhibits the photosynthetic electron transport chain in
Chlamydomonas reinhardtii. Pak J Bot 48:2587–2593
Chen S, Kang Y, Zhang M, Wang X, Strasser RJ, Zhou B, Qiang S (2015)
Differential sensitivity to the potential bioherbicide tenuazonic acid
probed by the JIP-test based on fast chlorophyll fluorescence kinet-
ics. Environ Exp Bot 112:1–15. https://doi.org/10.1016/j.envexpbot.
2014.11.009
Cheng H, Tam NFY, Wang Y, Li S, Chen G, Ye Z (2012) Effects of
copper on growth, radial oxygen loss and root permeability of seed-
lings of the mangroves Bruguiera gymnorrhiza and Rhizophora
stylosa. Plant Soil 359:255–266. https://doi.org/10.1007/s11104-
012-1171-1
Einali A, Shariati M (2015) Effects of propyl gallate on photosystem II
efficiency in Dunaliella bardawil under high illumination as inves-
tigated by chlorophyll fluorescence measurements. Theor Exp Plant
Phys 27:61–73. https://doi.org/10.1007/s40626-015-0032-8
Emam M, Esfahan EZ (2014) Effect of chemical (enriched-CO2 and
sucrose-free medium) and physical factors (light period and
 PHOTOAUTOTROPHIC PHOTOMIXOTROPHIC HETEROTROPHIC PROPOGATED BROMELIACEAE
temperature) on rooting and hardening of Sorbus aucuparia L.
plantlets. Int J Biosci 4:176–181
Eullaffroy P, Frankart C, Aziz A, Couderchet M, Blaise C (2009) Energy
fluxes and driving forces for phytosynthesis in Lemna minor ex-
posed to herbicides. Aquat Bot 90:172–178. https://doi.org/10.
1016/j.aquabot.2008.09.002
Gravano E, Bussotti F, Strasser R, Schaub M, Novak K, Skelly J, Tani C
(2004) Ozone symptoms in leaves of woody plants in open top
chambers: ultrastructural and physiological characteristics. Physiol
Plant 121:620–633. https://doi.org/10.1111/j.1399-3054.2004.
00363.x
Gururani MA, Venkatesh J, Ghosh R, Strasser RJ, Ponpandian LN, Bae H
(2017) Chlorophyll-a fluorescence evaluation of PEG-induced os-
motic stress on PSII activity in Arabidopsis plants expressing SIP1.
Plant Biosyst 1–8. https://doi.org/10.1080/11263504.2017.1403392
Hoang NN, Kitaya Y, Morishita T, Endo R, Shibuya T (2017) A compar-
ative study on growth and morphology of wasabi plantlets under the
influence of the micro-environment in shoot and root zones during
photoautotrophic and photomixotrophic micropropagation. Plant
Cell Tissue Organ Cult 130:255–263. https://doi.org/10.1007/
s11240-017-1219-2
Iarema L, Cruz ACF, Saldanha CW, Dias LLC, Vieira RF, Oliveira EJ,
Otoni WC (2012) Photoautotrophic propagation of Brazilian gin-
seng [Pfaffia glomerata (Spreng.) Pedersen]. Plant Cell Tissue
Organ Cult 110:227–238. https://doi.org/10.1007/s11240-012-
0145-6
Jo EA, Tewari RK, Hahn EJ, Paek KY (2009) In vitro sucrose concen-
tration affects growth and acclimatization of Alocasia amazonica
plantlets. Plant Cell Tissue Organ Cult 96:307–315. https://doi.org/
10.1007/s11240-008-9488-4
Joët T, Cournac L, Peltier G, Havaux M (2002a) Cyclic electron flow
around photosystem I in C3 plants. In vivo control by the redox state
of chloroplasts and involvement of the NADH-dehydrogenase com-
plex. Plant Physiol 128:760–769. https://doi.org/10.1104/pp.
010775
Joët T, Genty B, Josse EM, Kuntz M, Cournac L, Peltier G (2002b)
Involvement of a plastid terminal oxidase in plastoquinone oxida-
tion as evidenced by expression of the Arabidopsis thaliana enzyme
in tobacco. J Biol Chem 277:31623–31630. https://doi.org/10.1074/
jbc.M203538200
Johansen DA (1940) Plant microtechnique, 2nd edn. Mc Graw-Hill, New
York, p 523
Kalaji HM, Schansker G, Ladle RJ, Goltsev V, Bosa K, Allakhverdiev SI,
Brestic M, Bussotti F, Calatayud A, Dąbrowski P, Elsheery NI,
Ferroni L, Guidi L, Hogewoning SW, Jajoo A, Misra AN,
Nebauer SG, Pancaldi S, Penella C, Poli D, Pollastrini M,
Romanowska-Duda ZB, Rutkowska B, Serôdio J, Suresh K, Szulc
W, Tambussi E, Yanniccari M, Zivcak M (2014) Frequently asked
questions about in vivo chlorophyll fluorescence: pratical issue.
Photosynth Res 122:121–158. https://doi.org/10.1007/s11120-014-
0024-6
Karhu S (1997) Sugar use in relation to shoot induction by sorbitol and
cytokinin in apple. J Am Soc Hortic Sci 122:476–480
Kozai T (2010) Photoautotrophic micropropagation—environmental
control for promoting photosynthesis. Prop Ornam Plants 10:188–
204
Kraus JE, Arduin M (1997) Manual básico de métodos em morfologia
vegetal. EDRU, Seropédica, p 198
Lembrechts R, Ceusters N, De Proft M, Ceusters J (2017) Sugar and
starch dynamics in the medium-root-leaf system indicate possibili-
ties to optimize plant tissue culture. Sci Hortic 224:226–231. https://
doi.org/10.1016/j.scienta.2017.06.015
Lemoine R, Camera SL, Atanassova R, Dédaldéchamp F, Allario T,
Pourtau N, Bonnemain JL, Laloi M, Coutos-Thévenot P,
Maurousset L, Faucher M, Girousse C, Lemonnier P, Parrilla J,
Durand M (2013) Source-to-sink transport of sugar and regulation
by environmental factors. Front Plant Sci 4:1–21. https://doi.org/10.
3389/fpls.2013.00272
Lopez-Bucio J, Cruz-Ramirez A, Herrera-Estrella L (2003) The role of
nutrient availability in regulating root architecture. Curr Opin Plant
Biol 6:280–287
Martins JPR, Martins AD, Pires MF, Braga RA Jr, Reis RO, Dias GMG,
Pasqual M (2016a) Anatomical and physiological responses of
Billbergia zebrina (Bromeliaceae) to copper excess in a controlled
microenvironment. Plant Cell Tissue Organ Cult 126:43–57. https://
doi.org/10.1007/s11240-016-0975-8
Martins JPR, Pasqual M, Martins AD, Ribeira SF (2015a) Effects of salts
and sucrose concentrations on in vitro propagation of Billbergia
zebrina (Herbert) Lindley (Bromeliaceae). Aust J Crop Sci 9:85–91
Martins JPR, Verdoodt V, Pasqual P, De Proft M (2015b) Impacts of
photoautotrophic and photomixotrophic conditions on in vitro prop-
agated Billbergia zebrina (Bromeliaceae). Plant Cell Tissue Organ
Cult 123:121–132. https://doi.org/10.1007/s11240-015-0820-5
Martins JPR, Rodrigues LCA, Santos ER, Batista BG, Gontijo ABPL,
Falqueto AR (2018) Anatomy and photosystem II activity of in vitro
grown Aechmea blanchetiana as affected by 1-naphthaleneacetic
acid. Biol Plant 62:211–221. https://doi.org/10.1007/s10535-018-
0781-8
Martins JPR, Verdoodt V, Pasqual P, De Proft M (2016b) Physiological
responses by Billbergia zebrina (Bromeliaceae) when grown under
controlled microenvironmental conditions. Afr J Biotechnol 15:
1952–1961. https://doi.org/10.5897/AJB2016.15584
Matysiak B, Gabryszewska E (2016) The effect of in vitro culture condi-
tions on the pattern of maximum photochemical efficiency of pho-
tosystem II during acclimatisation of Helleborus niger plantlets to ex
vitro conditions. Plant Cell Tissue Organ Cult 125:585–593. https://
doi.org/10.1007/s11240-016-0972-y
Mehta P, Jajoo A, Mathur S, Bharti S (2010) Chlorophyll a fluorescence
study revealing effects of high salt stress on photosystem II in wheat
leaves. Plant Physiol Biochem 48:16–20. https://doi.org/10.1016/j.
plaphy.2009.10.006
Mohamed AA (2008) Promotive effects of a 5-aminolevulinic acid based
fertilizer on growth of tissue culture-derived date palm plants
(Phoenix dactylifera L.) during acclimatization. Sci Hortic 118:48–
52. https://doi.org/10.1016/j.scienta.2008.05.034
Mohamed MA, Alsadon HAA (2010) Influence of ventilation and su-
crose on growth and leaf anatomy of micropropagated potato plant-
lets. Sci Hortic 123:295–300. https://doi.org/10.1016/j.scienta.2009.
09.014
Monja-Mio KM, Pool FB, Herrera GH, EsquedaValle M, Robert ML
(2015) Development of the stomatal complex and leaf surface of
Agave angustifolia Haw. ‘Bacanora’ plantlets during the in vitro to
ex vitro transition process. Sci Hortic 189:32–40. https://doi.org/10.
1016/j.scienta.2015.03.032
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiol Plant 15:473–497.
https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Nakayama T, Shinohara H, Tanaka M, Baba K, Ogawa-Ohnishi M,
Matsubayashi Y (2017) A peptide hormone required for Casparian
strip diffusion barrier formation in Arabidopsis roots. Science 355:
284–286. https://doi.org/10.1126/science.aai9057
Perales-Vela HP, García RV, Juárez EAG, Salcedo-Álvarez MO,
Cañizares-Villanueva RO (2016) Streptomycin affects the growth
and photochemical activity of the alga Chlorella vulgaris.
Ecotoxicol Environ Saf 132:311–317. https://doi.org/10.1016/j.
ecoenv.2016.06.019
Rodrigues SP, Picoli EAT, Oliveira DC, Carneiro RGS, Isaias RMS
(2014) The effects of in vitro culture on the leaf anatomy of
Jatropha curcas L. (Euphorbiaceae). Biosci J 30:1933–1941
Rosa WS, Martins JPR, Rodrigues ES, Rodrigues LCA, Gontijo ABPL,
Falqueto AR (2018) Photosynthetic apparatus performance in func-
tion of the cytokinins used during the in vitro multiplication of
 MARTINS ET AL.
Aechmea blanchetiana (Bromeliaceae). Plant Cell Tissue Organ
Cult 133:339–350. https://doi.org/10.1007/s11240-018-1385-x
Sáez PL, Bravo LA, Latsague MI, Toneatti MJ, Coopman RE, Álvarez
CE, Sánchez-Olate M, Ríos DG (2015) Influence of in vitro growth
conditions on the photosynthesis and survival of Castanea sativa
plantlets during ex vitro transfer. Plant Growth Regul 75:625–639.
https://doi.org/10.1007/s10725-014-9965-1
Saldanha CW, Otoni CG, Rocha DI, Cavatte PC, Detmann KSC, Tanaka
FKO, Dias LLC, DaMatta FM, Otoni WC (2014) CO2-enriched
atmosphere and supporting material impact the growth,
morphophysiology and ultrastructure of in vitro Brazilian ginseng
[Pfaffia glomerata (Spreng.) Pedersen] plantlets. Plant Cell Tissue
Organ Cult 118:87–99. https://doi.org/10.1007/s11240-014-0464-x
Sampaio OM, Lima MMC, Veiga TAM, King-Díaz B, Silva MFGF,
Lotina-Hennsen B (2016) Evaluation of antidesmone alkaloid as a
photosynthesis inhibitor. Pestic Biochem Physiol 134:55–62. https://
doi.org/10.1016/j.pestbp.2016.04.006
Scott AJ, Knott M (1974) A cluster analysis method for grouping means
in the analysis of variance. Biometrics 30:507–512
Shen JR (2015) The structure of photosystem II and the mechanism of
water oxidation in photosynthesis. Annu Rev Plant Biol 66:23–48.
https://doi.org/10.1146/annurev-arplant-050312-120129
Shibuya T, Itagaki K, Wang Y, Endo R (2015) Grafting transiently sup-
presses development of powdery mildew colonies, probably through
a quantitative change in water relations of the host cucumber scions
during graft healing. Sci Hortic 192:197–199. https://doi.org/10.
1016/j.scienta.2015.06.010
Steinemann S, Zeng Z, McKay A, Heuer S, Langridge P, Huang CY
(2015) Dynamic root responses to drought and rewatering in two
wheat (Triticum aestivum) genotypes. Plant Soil 391:139–152.
https://doi.org/10.1007/s11104-015-2413-9
Stirbet A, Govindjee (2011) On the relation between the Kautsky effect
(chlorophyll a fluorescence induction) and photosystem II: basics
and applications of the OJIP fluorescence transient. J Photochem
Photobiol B 104:236–257. https://doi.org/10.1016/j.jphotobiol.
2010.12.010
Strasser RJ, Srivastava A, Tsimilli-Michael M (2000) The fluorescence
transient as a tool to characterise and screen photosynthetic samples.
In: Yunus M, Pathre U, Mohanty P (eds) Probing photosynthesis:
mechanisms, regulation and adaptation. Taylor and Francis,
London, pp 445–483
Strasser RJ, Tsimilli-Michael M, Srivastava A (2004) Analysis of the
chlorophyll a fluorescence transient. In: Papageorgiou GC,
Govindjee (eds) Chlorophyll fluorescence: a signature of photosyn-
thesis. Kluwer Academic Publishers Press, Netherlands, pp 321–
362. https://doi.org/10.1007/978-1-4020-3218-9_12
Tanaka Y, Sugano SS, Shimada T, Hara-Nishimura I (2013) Enhancement
of leaf photosynthetic capacity through increased stomatal density in
Arabidopsis. New Phytol 198:757–764. https://doi.org/10.1111/nph.
12186
Tisarum R, Samphumphung T, Theerawitaya C, Prommee W, Cha-um S
(2018) In vitro photoautotrophic acclimatization, direct transplanta-
tion and ex vitro adaptation of rubber tree (Hevea brasiliensis). Plant
Cell Tissue Organ Cult 133:215–223. https://doi.org/10.1007/
s11240-017-1374-5
Tombesi S, Johnson RS, Day KR, DeJong TM (2010) Relationships
between xylem vessel characteristics, calculated axial hydraulic con-
ductance and size-controlling capacity of peach rootstocks. Ann Bot
105:327–331. https://doi.org/10.1093/aob/mcp281
Trevisan F, Mendes BMJ (2005) Optimization of in vitro organogenesis
in passion fruit (Passiflora edulis f. flavicarpa). Sci Agric 62:346–
350. https://doi.org/10.1590/S0103-90162005000400007
Vandeleur RK, Mayo G, Shelden MC, Gilliham M, Kaiser BN, Tyerman
SD (2008) The role of plasma membrane intrinsic protein aquapo-
rins in water transport through roots: diurnal and drought stress
responses reveal different strategies between isohydric and
anisohydric cultivars of grapevine. Plant Physiol 149:445–460.
https://doi.org/10.1104/pp.108.128645
Vejchasarn P, Lynch JP, Brown KM (2016) Genetic variability in phos-
phorus responses of rice root phenotypes. Rice 9:29. https://doi.org/
10.1186/s12284-016-0102-9
Vetterlein D, Doussan C (2016) Root age distribution: how does it matter
in plant processes? A focus on water uptake. Plant Soil 407:145–
160. https://doi.org/10.1007/s11104-016-2849-6
Wang YW, Xu C, Lv CF, Wu M, Cai XJ, Liu ZT, Song MX, Chen CX, Lv
CG (2016) Chlorophyll a fluorescence analysis of high-yield rice
(Oryza sativa L.) LYPJ during leaf senescence. Photosynthetica 54:
422–429. https://doi.org/10.1007/s11099-016-0185-y
Wintermans PCA, Bakker Peter AHM, Pieterse CMJ (2016) Natural ge-
netic variation in Arabidopsis for responsiveness to plant growth-
promoting rhizobacteria. Plant Mol Biol 90:623–634. https://doi.
org/10.1007/s11103-016-0442-2
Xiao Y, Niu G, Kozai T (2011) Development and application of photo-
autotrophic micropropagation plant system. Plant Cell Tissue Organ
Cult 105:149–158. https://doi.org/10.1007/s11240-010-9863-9
Yaseen M, Ahmad T, Sablok G, Standardi A, Hafiz IA (2013) Review:
role of carbon sources for in vitro plant growth and development.
Mol Biol Rep 40:2837–2849. https://doi.org/10.1007/s11033-012-
2299-z
Yusuf MM, Kumar D, Rajwanshi R, Strasser RJ, Tsimilli-Michael M,
Govindjee Sarin NB (2010) Overexpression of γ-tocopherol methyl
transferase gene in transgenic Brassica juncea plants alleviates abi-
otic stress: physiological and chlorophyll fluorescence measure-
ments. Biochim Biophys Acta 1797:1428–1438. https://doi.org/10.
1016/j.bbabio.2010.02.002
Zushi K, Matsuzoe N (2017) Using of chlorophyll a fluorescence OJIP
transients for sensing salt stress in the leaves and fruits of tomato. Sci
Hortic 219:216–221. https://doi.org/10.1016/j.scienta.2017.03.016