Effect of Pectimorf® - A traditional growth regulator on the development and distribution in clones 'CMC-40' and 'Señorita' of Cassava (Manihot esculenta Crantz) stomata

Article Citation:
Lorenzo Suárez Guerra and Ideoleydis Álvarez Bello.
Effect of Pectimorf® - A traditional growth regulator on the development and
distribution in clones 'CMC-40' and 'Señorita' of Cassava (Manihot esculenta Crantz)
stomata
Journal of Research in Biology (2015) 5(6): 1820-1828
JournalofResearchinBiology
Effect of Pectimorf®
- A traditional growth regulator on the development
and distribution in clones 'CMC-40' and 'Señorita' of Cassava
(Manihot esculenta Crantz) stomata
Keywords:
Cassava, medium, plant anatomy, stoma, oligosaccharides.
ABSTRACT:
The development of more efficient and sustainable technologies in the
production of materials in ‘in vitro’ cassava (Manihot esculenta Crantz), favor the
improvement of seed quality and sanitation of the plant material. The purpose of the
research is to evaluate the effectiveness of Pectimorf®
(mixed oligo-galacturonide), it’s
safe and natural availability in Cuba. It is used as a possible complement or substitute
for growth regulators traditionally used in the culture medium for the propagation of
this crop in vitro. In this study, the results obtained indicate that, at least, under the
experimental conditions, the Pectimorf®
, altered patterns of development and
distribution of stomata in the leaves of cassava plants, where the effect was most
evident when the product is added to the culture medium. The new results contribute
to the elucidation of the mechanisms of action of this substance.
1820-1828| JRB | 2015| Vol 5 | No 6
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www.jresearchbiology.com
Journal of Research in Biology
An International
Scientific Research Journal
Authors:
Lorenzo Suárez Guerra1
and Ideoleydis Álvarez
Bello2
.
Institution:
1. Investigador Agregadodel
Departamento de Genética y
Mejoramiento Vegetale
2. Investigador Auxiliar del
Departamento de Fisiología
y Bioquímica del Instituto
Nacional de Ciencias
Agrícolas (INCA), Carretera
San José-Tapaste, km 3½,
Gaveta Postal 1, San José de
las Lajas, CP 32 700,
Mayabeque, Cuba.
Corresponding author:
Lorenzo Suárez Guerra
Email:
Web Address:
http://jresearchbiology.com/
documents/RA0514A.pdf
Dates:
Received: 3 March 2015 Accepted: 19 March 2015 Published: 30 September 2015
Journal of Research in Biology
An International Scientific Research Journal
Original Research
ISSN No: Print: 2231 –6280; Online: 2231- 6299

INTRODUCTION
Cassava (Manihot esculenta Crantz) is a very
versatile crop planted by small farmers in more than 100
countries (FAO, 2013). Therefore many clones are
existing in this plant species (Alves et al., 2011; Montero
et al., 2011). It is either used for human consumption or
dehydrated and stored for several years as a reserve food
(Nassar et al., 2009). In recent years, the potential of this
crop as an efficient source of raw material for the
production of biofuels (Cortés et al., 2010) is greatly
increased.
The development of efficient and rapid methods
of plant regeneration by culturing in vitro cassava tissue,
either by somatic embryogenesis (Medina et al., 2003;
Ochoa et al., 2012) or organogenesis (Medero et al.,
2001; Mapayi et al., 2013; Fan et al., 2011) ensures the
production of high quality plantlets basically needed for
producers in the expansion of cultivation. However these
techniques are still demanding about the composition of
the culture medium, especially with regard to the use of
growth regulators.
The introduction of bioactive substances
produced domestically in the methodology of in vitro
propagation of cassava (Manihot esculenta), could be a
promising alternative to improve the economic efficiency
of the process with the use of simple techniques and
reliable domestic inputs. These bioactive products can be
obtained with the brand name Pectimorf®
, produced by
the Department of Plant Physiology and Biochemistry,
National Institute of Agricultural Sciences (INCA),
Mayabeque, Cuba. This product is natural, harmless and
obtained from citrus rind and is constituted by a mixture
of oligo-galacturonide with high degree of
polymerization between 7 and 16 (Cartaya et al.,
2011). The Pectimorf®
is considered as a potent elicitor
of plant defense (Hernández et al., 2010; Galletti et al.,
2011). This product stimulates cell growth and
differentiation of different plant species (Hernández et
al., 2009; Hernández et al., 2010).
So far, the effect caused by this new substance is
unknown in the in vitro propagation of cassava (Manihot
esculenta). That is why, this study aims to histologically
evaluate the effect of Pectimorf®
on the two clones of
cassava (Manihot esculenta) 'CMC-40' and 'señorita' in
vitro, before and after acclimatization.
MATERIALS AND METHODS
This work was done at the Laboratory of
Biotechnology, Department of Genetics and Plant
Breeding and the Department of Physiology and
Biochemistry of the National Institute of Agricultural
Sciences (INCA), located in the municipality of San Jose
de Las Lajas, Mayabeque province.
Plant Material
The two clones viz., ‘CMC-40’ and ‘Senorita’
were obtained from the Cuban bank of cassava
germplasm, present at the Institute of Tropical Research
Viandas (INIVIT), Santo Domingo, Villa Clara, which
showed high productivity, short cycle (6-10 months ) and
excellent cooking quality (INIVIT 2014). The latter is a
prescribed amenity of ‘Senorita’ clone.
General Procedure
The plants that had completed their growth
phase in vitro were listed for acclimatization for about 30
days of age and 3 to 5 cm, in height. A total of eight
treatments from the growth phase in vitro (Table I), as
well as plants after 35 days of acclimatization from the
control treatment and the Pectimorf®
treatment were
done systematically after propagation.
The epidermis of the abaxial leaf surface was
scraped from the surface opposite to observe - allowing
not only to obtain an image of distribution of stomata,
but also to observe aspects of their structure. Leaf
samples were always taken from the middle of the road
and away from the edges.
Epidermal sheet sample was placed on a
microscopic slide and one drop of toluidine blue was
added and kept for a period of five minutes. Two washes
Guerra and Bello, 2015
1821 Journal of Research in Biology (2015) 5(6):1820-1828

were done and a drop of glycerine was added and a cover
slip was placed.
The samples were observed under an optical light
microscope (Zeiss, MODEL; PAIS) and photographed
with a camera (Motic) coupled thereto. Measurements
and counts were performed on micrographs with the use
of Image J program. Morphometric and linear
measurement instruments were used for elucidating the
lengths and breadths. For counting stomata and
epidermal cells with a magnification of 400x and
measurements of length and thickness of the guard cell 1
with a magnification of 1000x; six fields per plant were
taken for a total of 60 fields per treatment.
For counting, a stoma is considered when two
guard cells were present and in the case of epidermal
cells, when they were 60% in the image area.
Stomatal Index (SI) was calculated using the formula
suggested by Wilkinson (1979)
IE = (NE * 100) / (EC + NE)
Where, IE = Stomatal Index.
NE = Number of Stomata per field of view.
EC = Number of Epidermal Cells in the field of
observation.
The data obtained were statistically analysed
using Analysis of Variance (ANOVA). Differences
between means were elucidated by Duncan multiple
Range test at 5% significant level.
RESULTS AND DISCUSSION
In Figure I, you can see the stomata on the
abaxial leaf surface of cassava (Manihot esculenta); thus
proved parasitic or rubiaceous which accompany two
adjoining cells that are arranged parallel or occlusive,
that match the description made in other crops such as
common bean plants (Sánchez et al.,1996). They are
randomized and guard cells showed a kidney shape, as is
the characteristic of the dicotyledonous plants (Taiz et
al., 2010). These structures were observed in both the
abaxial and adaxial surface, which is considered as a
anfiestomática species. It was observed in the higher
frequency of stomata on the abaxial surface (ABA) with
respect to the adaxial side (ADA); these results also
match other cassava varieties (Ceballos et al., 2002).
In Table 1, the results of histological analysis of
cassava leaves at the end of micro propagation phase
were given. In general, significant differences between
the treatments were observed in both clones. In 'CMC-
40', stomatal index on the abaxial surface at treatment-2
reached maximum (19.42), whereas the lowest value for
this character is at the control treatment (9.85); no
significant differences were seen on the treatment 8 (0.01
mgL-1
NAA + 15 mgL-1
of Pectimorf®
), while at the
adaxial surface, the maximum values were found in
treatments where the product is used in the presence of
NAA ( 6, 7 and 8); no significant difference were seen
between them but with the rest, except six which did not
differ from the lower value two. Treatment-3 was
significantly different from the rest. It was shown that
Pectimorf®
increased stomatal index in both cultivars,
which could infer photosynthetic activity and water
status in the subsequent acclimatization plantlets.
In clone 'Señorita', stomatal index on the abaxial
surface reached the highest value in treatments 2 and 7,
which did not differ significantly from each other and
Journal of Research in Biology (2015) 5(6):1820-1828 1822
Treatments
NAA
(Mg.L-1)
Pectimorf®
(Mg.L-1)
1(Control) 0.01 -
2 (absolute control) - -
3 - 05
4 - 10
5 - 15
6 0.01 05
7 0.01 10
8 0.01 15
Table 1. Origin of plantlets of cassava (Manihot
esculenta) used in the experiments in the growth
phase in vitro

neither for the treatments 3, 5 and 6. The lower values
for this character were found in 1, 4 and 8 due to the
control treatment 10 mgL-1
of Pectimorf®
and the
combination of 0.01 mgL-1
NAA + 15 mgL-1
Pectimorf®
respectively, without significant differences between
them. However, in the adaxial side treatments of 5, 6, 7
and 8; they showed no significant differences between
them or with treatment 2. The lowest value was in
treatment 4, which did not differ significantly with 1 and
3. Also in 'señorita' the application of Pectimorf®
has
produced increased stomatal index.
The increased stomatal index caused by the
Pectimorf®
when added to the culture medium surely
have a positive impact on the development of plantlets in
the acclimatization phase influencing photosynthetic
activity and avoiding excessive perspiration. The results
coincide with Altamura et al. (1998), who noted that the
treatment with mixtures of biologically active oligo-
galacturonide increase the formation of stomata on leaf
explants snuff (Nicotiana tabacum L.) grown in culture
media with the specific concentrations of auxins.
The length of stomata in 'CMC-40' (table – 2)
showed significant differences between treatments. The
maximum values were in 1 and 2 for the control medium
and absolute control, which did not differ significantly
between them. Treatment 2 and 3 did not differ
significantly but differs with the addition of, Pectimorf®
in the presence of NAA (treatments 6, 7 and 8). The
lowest values were observed in the treatments 4 and 5
corresponding to the concentrations of 10 and 15 mgL-1
respectively without NAA and Pectimorf®
, with no
significant differences between them. These results
indicate that the modified length of stomatal cells,
resulted in less conduction, minimized loss of water by
evapo-transpiration and thus better survival of plants in
the acclimatization phase.
In the clone 'Señorita', there were also significant
differences between treatments (Table 2). Longest
stomata were those that developed in treatments 1 and 7,
which did not statistically differ from each other. The
analysis also differed in treatments 6 and 8
corresponding to the media where Pectimorf®
was
employed in the presence of NAA. The lowest values
for the length of stomata were present in the treatments 2
and 3, corresponding to the medium without the
regulator 5 mg.L-1
Pectimorf®
(T3), with no significant
differences between them. As in 'CMC-40' clone, results
indicate that the product also decreased in stomatal cell
length for providing less water wastage.
As the width of the guard cells in 'CMC-40'
showed significant differences between the treatments
(Table 2), the best treatment in ‘Senorita’ was 6 which
differed significantly from the rest. The control treatment
did not differ significantly from the treatments 7 and 8
corresponding to the media where Pectimorf®
was used
in the presence of NAA. Treatments 3, 4 and 5 did not
differ between them and showed intermediate values for
this character and corresponded to the media where the
product as a substitute for NAA was added. The above
table showed that the clone 'señorita' differences were
significant but contrary to what happened in 'CMC-40',
Control treatments, 5, 7 and 8 were higher without
Figure 1. Photomicrograph of the leaf epidermis of the
abaxial surface of plantlets of cassava (Manihot escu-
lenta). This Image is a representative for both
clones. EC Epidermal Cells, Ca– Adjoining Cells, Co–
Guard Cells and E-Stomata (1 000x).

significant differences between them, and for the control
medium where 15 mgL-1
of Pectimorf®
was added in the
absence/ presence of NAA and for treatment 6, it did not
differ in the presence of 10 mgL-1
of Pectimorf®
in
NAA. The lowest values of this character were for
treatments 2, 3 and 4, which showed significant
differences between them.
In both the cultivars, the lowest values to match
Pectimorf®
inclusion in the culture medium, resulted in
reduced width of the guard cells; and this modification
positively contributed in the acclimatization of plants.
If the value of size of stomata influenced by the
length and width of the guard cells on the abaxial surface
is taken into consideration, we could say that again on
treatment 4 (10 mgL-1
Pectimorf®
as a substitute for
NAA), under these culture conditions, caused changes in
the structure of the guard cells, with smaller length and
width compared to the control treatment. This could
promote acclimatization stage in seedlings which
achieved the conditions of ex vitro, therefore the
potential to become stressed because of decrease in
response to water deficit for getting transition to the new
conditions of autotrophism.
Stomata morphology plays an important role in
controlling water loss, which can be adapted to many
plant species at varying environmental conditions
(Hetherington et al., 2003). The variation of the
characteristics of stomata could play an important role in
the process of acclimation of a species into that
environment.
Treat-
ments
Stomatal
Index
Stomata
Length
(Microns)
Guard cells
Width
(Microns)
Stomatal Index
Stomata
Length
(Microns)
Occlusive
cell widths
(Microns)
CMC-40 Senorita
ABA ADA ABA ABA ABA ADA ABA ABA
1 09.85 c
1.13 c
109.41 to
29.10 b
12.02 c
0.68 cd
101.45 to
27.86 to
2 19.42 to
1.45 bc
99.47 Ab
22.76 d
20.04 to
0.99 abc
86.35 d
18.22 e
3 12.41 b
0.41 d
95.31 bc
24.39 cd
16.44 Ab
0.88 bcd
83.35 d
23.68 c
4 12.75 b
0.64 cd
78.57 d
25.49 c
14.23 bc
0.14 d
92.09 c
19.84 d
5 12.02 b
1.08 c
80.88 d
24.38 cd
17.51 Ab
1.91 to
91.59 c 27.34 Ab
6 12.14 b
2.20 Ab
83.43 cd
33.33 to
16.77 Ab
1.72 Ab
93.59 bc
26.18 b
7 12,92 b
2.31 to
89.40 bcd
27.85 b
18.80 to
1.54 abc
97,95 Ab
27.74 to
8 9.89 c
2.96 to
87.82 bcd
29.06 b
12.06 c
1.35 abc
95.45 bc
27,59 Ab
9 6.04 2.28 23.35 07.94 06.60 1.71 09.34 05.90
Table 2. Influence of applying Pectimorf®
on stomatal index, stomatal length and width of the stoma guard cells
in the cassava (Manihot esculenta), clones 'CMC-40' and 'Señorita' at the end of micropropagation
Legend: ADA: Adaxial (beam) ABA: Abaxial (underside) NAA: Naphthalene Acetic Acid
(Treatment1: Control Environment : 0.01mgL-
1 NAA, Treatment 2: Absolute Control : combination
(without regulators), Treatment 3:5 mgL-1
Pectimorf®, Treatment 4: mgL -1
Pectimorf®, Treatment 5: 15 mgL-
1
Pectimorf®, Treatment6 : Combination (0.01 mgL-1
NAA + 5 mgL-1
Pectimorf®), Treatment 7 : Combination
(0.01 mg L -1
NAA + 10 mgL-1
Pectimorf®)’ Treatment 8 : Combination (0.01 mgL-1
NAA + 15 mgL-1
Pectimorf®))
Means with different letters differ statistically according to Duncan test p ≤ 0.05. (* significant at p <0.1; **
significant at p <0.01; *** significant at p <0.001)

Similar results were shown in the cultivation of
beans (Phaseolus vulgaris) and the effect of
Pectimorf®
on morphology and distribution of stomata
was studied. Álvarez et al. (2012) reported that oligo-
galacturonide mixture resulted in alterations in the
density and size of stomatal guard cells and adaxial
surface differences were observed in terms of length of
the stoma.
The results of histological analysis in the leaves
of cassava plantlets at the end of the acclimatization
phase revealed that the use of ‘Pectimorf®’
modify some
of the traits evaluated (Table 3). In both cultivars
'Señorita' and ‘CMC-40', the stomatal was index showed
no significant differences between treatments, however
modified the size of the stomata since it altered the
length of stomata with highly significant differences
from the control. In the 'CMC.40', the clone stomatal
average length was 240.84 microns and in 'Señorita' it
was 238.51 microns; these plants were grown in the in
vitro medium with 10 mgL-1
Pectimorf®
. The rest of the
characters were not significantly different.
The size of the stomata is a key factor in the
process of acclimatization, for having an inverse
relationship between the size of the stoma and resistance
to water stress (Aasaman et al., 2001). Also authors have
commented that the size of the stomata and stomata
index appear to be the most sensitive to altered
environmental conditions; avoiding excessive
perspiration and allowing better adaptation of plants to
conditions of greater water demand.
In this sense, the results obtained indicate that, at
least under these conditions, the Pectimorf®
probably
changed the patterns of development and distribution of
stomata in cassava plants. The effect was more evident
when the product is added to the culture medium, which
may be due to the controlled conditions and is a way of
evading the effects of the substance in these conditions in
vitro.
Also in Mikania laevigata ex Baker Shultz, it is
said that a reduction of perspiration may be associated
with high stomatal density, which is often observed in
the conditions of more radiation or less water availability
(Souza et al., 2007). Álvarez et al. (2011) and Yin et al.
(2006) have shown that oligo-galacturonide affect the
growth and development of plant cells and organs and
differentiation of stomata and pericycle cells.
Treatments
Stomatal
index
Length of
stomata
(microns)
Width of
guard
cells
(microns)
Stomatal index
Length of
stomata
(microns)
Width of
guard cells
(microns)
CMC-40 Senorita
ABA ADA ABA ABA ABA ADA ABA ABA
I 18.37 0.14 224.22 b
64.65 16.70 0.35 227.56 b
63.71
II 18.92 0.14 240.84 to
64.85 17.38 0.32 238.51 to
63.00
Result NS NS 0.11 *** NS NS NS 0.30 *** NS
Table 3. Pectimorf® influence on stomatal index, stomatal length and width of the stoma guard cells in the
leaves of cassava (Manihot esculenta) clones'CMC-40' and 'Señorita', at 35 days of acclimatization
ADA: Adaxial; ABA: Abaxial; NAA: Naphthalene Acética Acid
1: Control : In vitro plants from the control medium 0.01 mg.L-1
NAA, 2 : In vitro Plants Provenientes medium with
10 mgL-1
Pectimorf®
. Means with different letters differ statistically according to Duncan test p ≤ 0.05. (*significant at
p <0.1; ** significant at p <0.01; *** significant at p <0.001)

The results indicate that the changes occurred in
both the cultivars of cassava on addition of Pectimorf®
in
the in vitro phase, had no influence on stomatal index,
but if the size of the guard cells are modified by the
length of the stomata, the conditions transfer from
in vitro to ex vitro. Castro et al. (2009) noted that the
polar diameter or length of the stomata is directly related
to the size of the stomata and that this could be varied in
response to a hybrid deficit.
So far there are no literature about the possible
role of Pectimorf®
on the histology of cassava
plants. Furthermore, these results constitute the basis for
future research, where we can understand the possible
effects of this substance in plant histology. The use of
Pectimorf®
helped reducing the stress of plants
transferring from in vitro condition to ex vitro condition,
which may be associated in including the amendments
that produced this compound on leaf anatomy. However,
there is a need to dwelve into the mechanisms of action
of this oligo-galacturonide to determine the exact way
with which it exerts its action and determine whether the
product may or may not induce genetic variability on the
materials spread.
CONCLUSIONS
The Pectimorf®
incorporation resulted increased
stomatal index and size of stomata in the in vitro growth
phase and acclimatization of plantlets of the cassava
(Manihot esculenta), clones 'CMC-40' and 'Señorita'.
ACKNOWLEDGEMENTS
We thank the Department of Physiology and
Biochemistry of the National Institute of Agricultural
Sciences (INCA), Province of Mayabeque to facilitate
the Pectimorf®
, especially Dr. Inés Ma Reynaldo Escobar
and technical assistant, Mr. Miladys Sanchéz Quintana
from Biotechnology Laboratory, Department of Genetics
and Plant Breeding belonging to the same institution.
.
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Effect of Pectimorf® - A traditional growth regulator on the development and distribution in clones 'CMC-40' and 'Señorita' of Cassava (Manihot esculenta Crantz) stomata

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