Australian Journal of Basic and Applied Sciences, 8(7) May 2014, Pages: 257-264
AENSI Journals
Australian Journal of Basic and Applied Sciences
ISSN:1991-8178
Journal home page: www.ajbasweb.com
Anatomical analyses of
acclimatization process
Tabebuia
roseo-alba
(Bignoniaceae)
seedlings
during
1
Jorge Marcelo Padovani Porto, 2Patrícia Duarte de Oliveira Paiva, 3Francyane Tavares Braga, 4Paulo Augusto Almeida Santos, 5Renato
Paiva, 5Evaristo Mauro de Castro
Department of Agriculture, Universidade Federal dos Vales do Jequitinhonha e Mucuri-UFVJM, Diamantina-MG – Brazil
Department of Agriculture, Universidade Federal de Lavras-UFLA, Lavras-MG - Brazil
3
Department of Education, Universidade Estadual da Bahia – UNEB, Paulo Afonso-Bahia –Brazil
4
Department of Chemistry, Universidade Federal de Lavras-UFLA,Lavras-MG – Brazil
5
Department of Biology, Universidade Federal de Lavras-UFLA, Lavras-MG – Brazil
1
2
ARTICLE INFO
Article history:
Received 2 February 2014
Received in revised form
8 April 2014
Accepted 28 April 2014
Available online 25 May 2014
Keywords:
leaf anatomy, epidermis, stomata,
substrates.
ABSTRACT
The knowledge on morphological changes of plants grown in vitro is essential for
establishing effective protocols for the survival of plants from controlled environments
under natural conditions. This study aimed to examine the acclimatization of ipê-branco
seedlings produced in vitro and also to compare the anatomical structure between
leaves of seedlings grown in vitro and those acclimatized to different substrates. For
acclimatization, ipê-branco seedlings were taken from in vitro cultivation and
transferred to tubes containing Plantmax TM, vermiculite, sand and Plantmax TM +
vermiculite + sand mixture. About 100% of fixation was verified, and seedlings
acclimatized in Plantmax TM were those showing the best development. For the
anatomical study, transversal and paradermal sections were performed in the leaf blades
of seedlings derived from in vitro cultivation and seedlings already acclimatized to the
different substrates. Anatomical differences were observed in the leaf blades of
seedlings from the in vitro environment when compared to those acclimatized to
different substrates. The stomatal density and polar and equatorial diameter of leaves
derived from the in vitro environment were higher than those acclimatized to different
substrates. The PD / ED ratio was lower for leaves derived from the in vitro
environment, and comparing the different substrates, it was greater in seedlings
acclimatized with vermiculite. The mesophyll and adaxial and abaxial epidermises were
smaller in plants grown in vitro and among substrates, seedlings acclimatized to
Plantmax TM were higher, with increased thickness of palisade and spongy parenchyma.
© 2014 AENSI Publisher All rights reserved.
To Cite This Article: Jorge Marcelo Padovani Porto, Patrícia Duarte de Oliveira Paiva, Francyane Tavares Braga, Paulo Augusto Almeida
Santos, Renato Paiva, Evaristo Mauro de Castro, Anatomical analyses of Tabebuia roseo-alba (Bignoniaceae) seedlings during
acclimatization process. Aust. J. Basic & Appl. Sci., 8(7): 257-264, 2014
INTRODUCTION
Acclimatization is defined as the process of transferring a seedling from the in vitro condition to the natural
environment or to an intermediate environment, like a greenhouse (Debergh & Maeno, 1981; Shin, et al., 2014).
Some species are easy to adapt, showing 100% survival rate, while others have difficulties in this process. The
main cause of low survival rate is the excessive loss of water by plants during this process (Sutter & Lamghans,
1982).
There are various mixtures used in the composition of substrates for plants that are submitted to
acclimatization process, and the selection should take into consideration physicochemical and hydric properties,
as these influences the water / air ratio of the substrate and nutrient availability and absorption (Fernandes &
Corá, 2000; Freitas, et al., 2011).
The use of different substrates with effective results for the acclimatization of various plants has been
verified by several authors (Vichiato, et al., 1998; Brasil, et al., 1999; Sediyama, et al., 2000; Souza, 2001;
Mendonça, et al., 2003), as well as for the development of seedlings (Luz, et al., 2007; Ferreira, et al., 2007;
Almeida, et al., 2008).
Some histological studies have demonstrated that vegetative organs of plants grown in vitro environments
have poorly differentiated tissues and structures when compared to plants cultivated in greenhouses (Louro, et
al. 2003; Apóstolo, et al. 2005; Saez, et al., 2012). Moreover, the number and shape of stomata are also
affected, which can lead to a greater or lesser photosynthetic efficiency of plants (Osorio, et al., 2005). In ipêCorresponding Author: Jorge Marcelo Padovani Porto, Department of Agriculture, Universidade Federal dos Vales do
Jequitinhonha e Mucuri-UFVJM, Rodovia MGT 367 - Km 583, nº 5000, Alto da Jacuba CEP
39100-000, Diamantina-MG – Brazil.
Tel: +553835328583
E-mail: [email protected]
258
Jorge Marcelo Padovani Porto et al, 2014
Australian Journal of Basic and Applied Sciences, 8(7) May 2014, Pages: 257-264
branco leaves grown in vitro, cuticle and sclerenchyma are absent. However, plants grown ex vitro show not
only these structures, but also higher thickest in relation to the leaf limb, midrib, the abaxial and adaxial
epidermises and palisade and spongy parenchyma, as well as fewer stomata and higher number of trichomes
when compared to plants grown in vitro (Abbade, et al., 2009).
This study aimed to evaluate the acclimatization of ipê-branco seedlings produced in vitro and also to
compare the anatomical characteristics of leaves grown in vitro with those acclimatized.
MATERIAL AND METHODS
Plants grown in vitro were obtained by seed germination on MS medium (Murashige & Skoog, 1962) added
of 0.5 mg L-1 GA3. After 30 days, they were transferred to polyethylene tubes with capacity of 288 cm3, and
filled with the following substrates: sand, PlantmaxTM and vermiculite, and a mixture of PlantmaxTM:
vermiculite: sand (P.V.S.) at a ratio of 1:1:1. Seedlings were kept in growth room and covered with transparent
plastic bags and tubes were partially immersed in distilled water. The plastic covering was gradually perforated
during 30 days, being then fully removed. The plants were kept under 16-hour photoperiod and photon
irradiance of 55 µmol m-2 s-1and temperature of 27 ± 2 º C.
To perform the anatomical analysis, fully expanded leaves were collected from the upper third of ipêbranco seedlings after 30 days of acclimatization and from plants grown in vitro. Both were fixed in 70% FAA
(formaldehyde - glacial acetic acid - 70% ethyl alcohol) for 72 hours and then preserved in 70% ethanol
(Johansen, 1940). The anatomical study of leaves was based on microscopic examination of cross sections,
obtained with manual microtome, and of paradermal sections from abaxial and adaxial surfaces of leaves,
obtained by hand, both from the median region of leaves.
The cross sections were clarified with 50% sodium hypochlorite, rinsed in distilled water, stained with astra
blue and safranin and mounted in 50% glycerol, according to methodology described by Kraus & Arduin
(1997). Measurements of tissue thickness were performed with ocular micrometer coupled to light microscope.
Slides with paradermal sections of abaxial and adaxial surfaces of leaves were mounted with a dye solution
of 1% safranin in glycerol-water solution. Counting the number of stomata was performed on OLYMPUS CBB
microscope. The stomatal density was expressed as the number of stomata per mm2, according to technique of
Labouriau, et al. (1961). The thickness of palisade and spongy parenchyma, the adaxial and abaxial epidermises
of leaves, as well as the polar and equatorial diameter of the stomata and the stomatal density of ipê-branco
leaves were determined.
The thickness, stomatal density and polar and equatorial diameter measurements of the stomata were
conducted using completely randomized design with five replicates per treatment. Each replication consisted of
three measurements for thickness, four for stomatal density and four for polar and equatorial diameter.
After 30 days of acclimatization, number of leaves, root length and shoots we assessed. Data were analyzed
using the SISVAR software (Ferreira, 2003) and results were compared by the Tukey test with 5% probability.
For microscopy analyzes fifteen measurements were performed, the values were analyzed by Skott Knott test
with 5% probability.
Results:
Acclimatized plants showed 100% survival rate in all substrates, which did not affect the shoot length of
plants, but affected the root length and number of leaves (Table 1).
Table 1: Effect of different substrates on the shoot length, root length and number of leaves of ipê-branco at 30 days of acclimatization.
Substrates
Survival rate (%)
Shoot length (cm)
Root length (cm)
Number of leaves
Sand
100 a
5.46 a
5.51 b
9.0 a
PlantmaxTM
100 a
6.30 a
19.73 a
7.6 ab
Vermiculite
100 a
5.76 a
15.57 a
6.4 b
P.V.S.1
100 a
6.82 a
17.67 a
7.4 ab
Means followed by same letter in columns do not differ significantly by Tukey test at 5%.
1
P.V.S. = Plantmax TM + vermiculite + sand.
In plants acclimatized in substrates Plantmax TM and P.V.S., larger leaves were formed, as can be seen in
Figure 1A and B. In plants acclimatized in vermiculite, the occurrence of necrosis in leaves was observed
(Figure 1C).
The root length was significantly affected for plants acclimatized in sand; however, this substrate showed
the formation of higher number of leaves (9), in cases similar to that observed in plants acclimatized in
substrates Plantmax TM and P.V.S. In addition, despite the smaller size of the root system, acclimatization in
sand did not affect the shoot length. Plants acclimatized in vermiculite showed lower leaf formation. However,
the substrate did not influence the shoot length or root length, which did not differ from plants grown in
substrates Plantmax TM and P.V.S.
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Jorge Marcelo Padovani Porto et al, 2014
Australian Journal of Basic and Applied Sciences, 8(7) May 2014, Pages: 257-264
Fig. 1: Appearance of ipê-branco plants after 30 days of acclimatization.
Anatomical Analyses:
Analyzing the paradermal sections of ipê-branco leaves, the presence of stomata was verified only on the
abaxial epidermis of leaf blades, both for plants grown in vitro, and for those acclimatized to different
substrates, featuring ipê-branco as a hypostomatic species.
However, they show differences in shape. Figure 2 shows that the stomata of acclimatized plants showed
elliptical shape (BCDE), while stomata of leaves produced in vitro (A) showed more circular shape.
Fig. 2: Photomicrograph of paradermal sections of ipê-branco leaves grown in vitro (A) and acclimatized in
sand (B), PlantmaxTM (C), P.V.S. (D) and vermiculite (E). Bar = 10 μm.
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Jorge Marcelo Padovani Porto et al, 2014
Australian Journal of Basic and Applied Sciences, 8(7) May 2014, Pages: 257-264
Comparing the paradermal sections of plants produced in vitro with those acclimatized to different
substrates, higher stomatal density was observed in plants grown in vitro, with average of 345.33 stomata per
mm2. Among the acclimatized plants, the stomatal density was low, and substrate sand provided the formation
of large number of stomata per leaf area, as shown in Figure 3.
400
a
Stomatal density (mm-2)
350
300
250
200
b
c
150
c
c
100
50
0
In vitro
Sand
Vermiculite Plantmax
P.V.S.
P.V.A.
Fig. 3: Stomatal density of abaxial epidermal tissue of ipê-branco leaf blades grown in vitro and acclimatized to
different substrates for 30 days. Means followed by same letter do not differ significantly by the ScottKnott test at 5% significance level.
The polar diameter of stomata was greater in seedlings grown in vitro and acclimatized in vermiculite. The
equatorial diameter was also greater in stomata of seedlings grown in vitro, and no difference between stomata
of acclimatized plants was observed. The highest (PD / ED) ratio occurred in plants acclimatized in vermiculite,
1.83 μm, while the lowest was observed in plants grown in vitro (1.26 μm) (Figure 4).
35
a
Diameter stomata (μm)
30
25
a
b b
c
a
In vitro
20
b b b
Vermiculite
b
Plantmax
15
Sand
P.V.A.
P.V.S.
10
5
c a
b b b
0
Polar diameter
Equatorial diameter
Relationship PD/ED
Fig. 4: Polar diameter (PD) and equatorial diameter (ED) and (PD / ED) ratio of stomata of abaxial epidermal
tissue of ipê-branco leaf blades grown in vitro and acclimatized to different substrates for 30 days.
Means followed by same letter for each group of bars do not differ significantly by the Scott-Knott test
at 5% significance level.
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Jorge Marcelo Padovani Porto et al, 2014
Australian Journal of Basic and Applied Sciences, 8(7) May 2014, Pages: 257-264
Analyzing the cross sections, both leaves grown in vitro and those acclimatized to different substrates
showed dorsiventral organization, as shown in Figure 5.
The cross sections showed that in all culture conditions, both epidermises consisted of only one cell layer,
being characterized as unistratified epidermis (Figure 5).
Fig. 5: Photomicrograph of cross sections of ipê-branco leaves acclimatized in Plantmax
vermiculite (C), and sand (D) and those grown in vitro (E). Bar = 10 μm.
TM
(A), P.V.S. (B),
The cuticle thickening was lower in leaves of plants grown in vitro in relation to acclimatization. The
spongy parenchyma of plants grown in vitro showed 2 to 3 cell layers and smaller intercellular spaces when
compared to tissues of acclimatized plants, where 3 to 4 cell layers were observed. The palisade parenchyma
consists of only one layer, and the cells of acclimatized plants were more elongated and juxtaposed in relation to
that observed in seedlings derived from in vitro environments (Figure 5).
The thickness of the adaxial and abaxial epidermises and the mesophyll (Figure 6), besides the palisade and
spongy parenchyma (Figure 7) were higher in plants acclimatized to Plantmax TM, differing from the other
treaTMents. Smaller thicknesses were observed in plants grown in vitro (Figure 6 and 7).
120
a
100
b
Thickness (μm)
c c
80
d
P.V.A.
P.V.S.
Vermiculite
60
Sand
40
20
Plantmax
In vitro
a b b
c d
a b b
c d
Adaxial epiderm
Abaxial epiderm
0
Mesophyll
Fig. 6: Adaxial and abaxial epidermises and mesophyll of ipê-branco leaves grown in vitro and acclimatized to
different substrates for 30 days. Means followed by same letter for each group of bars do not differ
significantly by the Scott-Knott test at 5% significance level.
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90
a
80
Thickness (μm)
70
b
60
c
c
c
50
P.V.A.
P.V.S.
Vermiculite
40
a
30
Plantmax
Sand
b
b
b
In vitro
c
20
10
0
Palisade parenchyma
Spongy parenchyma
Fig. 7: Thickness of the palisade and spongy parenchyma of ipê-branco leaves grown in vitro and acclimatized
to different substrates for 30 days. Means followed by same letter for each group of bars do not differ
significantly by the Scott-Knott test at 5% significance level.
Discussion:
In the acclimatization of pineapple variety Pérola, Moreira (2006) observed that, in relation to plant height,
root length and number of leaves, substrate Plantmax TM was the best substrate, having been enriched with soil
and manure. In this study, it was observed that substrates Plantmax TM and Plantmax TM + vermiculite + sand
mixture provided better development of shoots, roots and higher number of leaves in acclimatized ipê-branco
plants.
According to Gislerod (1982) and HarTMann, et al. (1990), the substrate must have good water retention
capacity, optimum amount of pore spaces filled with gases and adequate oxygen diffusion rate for root
respiration. Plantmax TM presents physical (porosity, texture, drainage and low compression) and chemical
characteristics (presence of nutrients and pH suitable for plant development) adequate for the growth of
seedlings and emission of adventitious roots (Hoffman, et al., 2001).
El-Bahr, et al. (2003) also found lower PD / ED ratio for stomata of plants grown in vitro when compared
with acclimatized plants. According to Abbade, et al. (2009) circular-shaped stomata and lower DP / DE ratios
were observed in ipê-branco seedlings grown in vitro, explaining the lower stomatal function in plants grown in
vitro.
According to Metcalfe & Chalk (1950), all Bignoniaceae species are dorsiventral, and isobilateral structure
was only recorded in genus Kigelia. According to Alquini, et al. (2003), the cuticle thickening provides a natural
protection against the action of solar radiation. The presence of stellate trichomes was verified on both
epidermises, in all treaTMents.
In yellow-ipe, the same differences were also observed in the palisade parenchyma when plants grown in
vitro were compared to those acclimatized (Dousseau, et al., 2008). According to Lee, et al. (2000), more
elongated palisade cells are an adaptation of plants to the high light intensity, thus justifying the presence of
more cell layers in acclimatized plants.
The lower mesophyll differentiation and smaller thickness of palisade and spongy parenchyma are changes
often observed in plant grown in vitro when compared to ex vitro (Louro, et al., 2003). The tissue differentiation
is an important factor in the light absorption process, especially in the mesophyll structure. It is expected that,
the thicker the palisade parenchyma, the higher the photosynthetic rates (Bolhar-Vordenkampf & Draxler, 1993;
Saez, et al., 2012), which is an essential process to plant growth and development.
Conclusions:
For acclimatization of ipê-branco seedlings, the use of Plantmax TM as substrate is recommended.
The mesophyll, adaxial and abaxial epidermises and palisade and spongy parenchyma were thicker in plants
acclimatized in Plantmax TM when compared to other substrates and to plants grown in vitro.
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Jorge Marcelo Padovani Porto et al, 2014
Australian Journal of Basic and Applied Sciences, 8(7) May 2014, Pages: 257-264
The PD / ED ratio of stomata is higher in leaves acclimatized in vermiculite and lower in leaves grown in
vitro.
ACKNOWLEDGMENTS
We thank the CNPq, FAPEMIG and CAPES, for the financial support.
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