Volume 8, Number 12, December 2014 (Serial Number 80)
3. Results and Discussion
means of cyclic voltammetry, impedance The corrosion of iron and its alloys causes severe
measurements and potential monitoring the economical loss resulting in a yearly cost of billions of
electrochemical behaviour of a new type of dollars or euros. The use of heavy metals and heavy
anti-corrosive biopolymers has been studied, which metal containing compounds, such as chromate, has to
can be deposited upon metal surfaces as layers.
be reduced in coatings for some are known to be very Besides this electrochemical characterisation, toxic, even carcinogenic, and cause great electrochemical measurements were used to select environmental damage. Prevention of or reduction in
optimal biologically manufactured and chemically the rate of corrosion may be accomplished by the use
modified polymers.
of a biological, environmentally friendly anti Strain L. fermentum Ts was cultivated in a media corrosive layer at the metal interface. The presence of
containing 10% sucrose, 10% fructose, and 10% EPS associated with bacterial cells can be recognized
maltose for 12 h. The steel samples were placed in by the formation of colonies in mucous solid medium
seawater as control probe and a dilution (3: 100) of the [11]. Therefore, the presence of a translucent or
cultural media of the studied strain was added as creamy material involving a mucoid colony is
inhibitor of the corrosion. The received results are indicative of EPS production potential. When presented in Table 1. cultivated in a media with high content of saccharides
In our previous studies [14-17], it was shown that at such as 10% sucrose solutions, 10% fructose solutions,
the presence of high concentration of lactose (5% to and 10% maltose solutions, strain L. fermentum Ts
15%), high concentration of sucrose 4%, mixed synthesizes exopolysaccharides (Fig. 1).
sucrose 4% and 2% maltose and mixed sucrose 5% and 5% maltose, mixed 5% sucrose and 5% fructose
and mixed 5% sucrose and 5% fructose the strains Lactobacillus delbrueckii B5, L. delbrueckii K27, L. delbrueckii B8, L. delbrueckii O43, L. delbrueckii K3, L. delbrueckii K17, and L. delbrueckii K15 synthesized exopolysaccharides which have inhibitory properties. It is well known that some lactobacillus strains such as genus Leuconostoc secreted trans glucosidases after cultivation in the presence of sucrose. The structure of the layer over the steel plates
Fig. 1 EPSs (exopolysaccharides) produced by L. fermentum Ts cultivated in a media containing 10% sucrose,
was analyzed by Scanning electron microscopy.
which are secreted in the culture medium.
The results from this procedure are shown in Fig. 2.
Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors
Table 1 Characterization of the protective properties in seawater with added supernatant.
No Media
The quantity of the
γ 1 10% sucrose* 3.0 0.21 74.07 3.86
supernatant in seawater, % K × 10 -5 , g/cm 2 · h Z, %
sample
2 10% maltose 3.0 0.25 69.14 3.24 3 10% fructose 3.0 0.71 12.35 1.14 4 control 0.81 -
- *The steel plates were photographed after washing; results are mean ± SEM of three separate trails.
(a) (b)
Fig. 2 Biofilm formed by L. delbrueckii B5 on the surface of mild steel, visualized using SEM. (a) Steel plates after corrosion in seawater with inhibitor supernatant obtained of mixed 10% sucrose; (b) control—steel plates after corrosion in seawater.
The biofilm makes it not easily corrodible in The ability of EPS to bind specific metal ions seawater, supplemented with cultivated ambient from
strongly influences its adhesion to metal surface and the same strain grown in a composite of 10% sucrose
its ability to concentrate metal ions from surfaces and (Fig. 2a). Fig. 2b shows a picture of a steel surface
bulk media. Binding of metals may be important in sample treated directly with seawater. The observed
both passivation and activation reactions. The observed
inverse relationship between EPS and the corrosion the corrosion.
lamellaes are most probably FeCl 2 crystals, product of
rates of mild steel suggests that similar reactions may Microscope techniques provide information about
be occurring in the natural environment leading to the the morphology of microbial cells and colonies, their
formation of a protective film on the metal surface. distribution on the surface, the presence of EPS (Fig.
Biofilm [18] of a polysaccharide producing culture. 2a) and the nature of corrosion products (crystalline or
Delta marina was found to act as a strong corrosion amorphous; Fig. 2b). They can also reveal the type of
inhibitor with almost complete passivation of mild attack (e.g., pitting or uniform corrosion) by steel, reducing the corrosion rate by 95%. From this, it visualizing changes in microstructure and surface
is evident that some microorganisms and/or their features after removal of the biofilm and corrosion
polysaccharides can act as a strong corrosion products (Fig. 2b).
inhibitors.
Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors
The corrosion of mild steel starts with generation of
References
ferrous ions by anodic oxidation at the surface because
[1] Arrage, A. A., Vasishtha, N., Sundberg, D., Bausch, G., of the reaction (Fe → Fe + 2e ) which may
Vincent, H. L., and White, D. C. 1995. “On-Line
undergo further oxidation producing Fe +2 species (Fe Monitoring of Antifouling and Fouling-Release Surfaces → Fe +3 +e - ). Ferric ions are particularly deleterious
Using Bioluminescence and Fluorescence Measurements during Laminar-Flow.” Journal of Industrial
for mild steel as they tend to accelerate corrosion by
Microbiology 15: 277-282.
the reaction (Fe → Fe +e ). If ferric ions are [2] Breur, H. J. A. 2001. “Fouling and Bioprotection of immobilized then it may be possible to control the
Metals: Monitoring and Control of Deposition Processes corrosion of mild steel. Some polysaccharides are
in Aqueous Environments.” Ph.D. thesis, Technische reported to exhibit the strongest stability constant for Universiteit Delft. [3] Christensen, B. E., and Characklis, W. G. 1990. Physical
Fe 3+ ions [18]. Such a complex may serve as a and chemical properties of biofilms. New York: John
corrosion inhibitor. The observed inverse relationship
Wiley & Sons.
between EPS and the corrosion rate of mild steel [4] Costerton, W. J., Cheng, K. J., Geesey, G. G., Ladd, T. I., suggests that such a metal-polysaccharide complex Nickel, J. C., Dasgupta, M., and Marrie, T. J. 1987.
“Bacterial Biofilms in Nature and Disease.” Annual was probably involved in developing a protective film
Review of Microbiology 41: 435-464. on the metal surface in natural sea water.
[5] Ford, T. E., Maki, J. S., and Mitchell, R. 1988. The data suggest that biofilm EPS inhibits the
“Involvement of Bacterial Exopolymers in corrosion of mild steel in natural marine waters. Biodeterioration.” Biodeterioration 7: 378-384. [6] Gómez, J. 2006. “Caracterización de los Exopolisacaridos
4. Conclusions
Producidos por Microorganismo Shalófilos Pertenecientes a los Géneros Halomonas, Alteromonas,
From the received results it was evident that a Idiomarina, Palleronia y Salipiger.” Ph.D. thesis, mixture of 10% sucrose, or 10% maltose stimulated Universidad de Granada. [7] Ignatova-Ivanova, Ts., Ananieva, M., Ivanov, R., Iliev, I.,
the formation of microbial biofilm inhibiting the and Ivanova, I. 2014. “Biodiversity of Lactic Acid
corrosion of steel. The present research confirms the Bacteria in Bulgarian Wheat and Rye Flour.” Journal of result of the pilot project [2] that polysaccharides
BioScience and Biotechnology 101-105. made by microorganisms show anti-corrosive [8] Ignatova-Ivanova Ts., Ivanov, R., Iliev, I., and Ivanova, I.
2009. “Study Anticorrosion Effect of EPS from Now properties. Especially, homopolysaccharides showed Strains Lactobacillus Delbruecii.” Biotechnol & interesting results for the protection of steel.
Biotechnol EQ Special edition/on line 705-708. Measurements indicate that it takes some time for
[9] Ignatova-Ivanova, Ts., Ivanov, R., Iliev, I., and Ivanova, I. layers of biopolymers on the metal to build a complete
2011. “Study of Anticorrosion Effect of Exopolysaccharides Produced Lactobacillus Delbrueckii
protective layer. The data showed that L. fermentum b5 Cultivated on Different Carbohydrates.” Biotechnol &
Ts produce EPS, which serve as corrosion inhibitor for Biotechnol EQ Special edition/on line 224-227. mild steel.
[10] Ignatova-Ivanova, Ts., and Ivanov, R., 2013. Further studies are needed to evaluate the potential
“Anticorrosion Effect of Biofilm Forming by of the biofilm exopolysaccharides as anticorrosive Lactobacillus Strains on Metal Surfaces.” Bulgarian Journal of Agricultural Science 19 (2): 83-85.
agents. [11] Ignatova-Ivanova, Ts. V., and Ivanov, R. I. 2014. “Study of Biofilm Formed by Lactic Acid Bacteria on the
Acknowledgments
Surface of Mild Steel.” Journal of Life Sciences 8: The authors would like to express our gratitude for 799-804. doi: 10.17265/1934-7391. [12] Jayaraman, A.,·Earthman, J. C., and Wood, T. K. 1997.
the support of this work to research grant FSI “Corrosion Inhibition by Aerobic Biofilms on SAE 1018 08-213/10.03.2014 of Shumen University.
Steel.” Appl. Microbiol. Biotechnol. 47: 62-68.
Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors
945
[13] Marshall, K. C. 1992. “Biofilms: an Overview of for Laboratorial Exercises.” Sofia. Bacterial Adhesion, Activity, and Control at Surfaces.”
[16] Sutherland, W. 1972. “Bacterial Exopolysaccharides.” ASM News 58: 202-207.
Advances in Microbial Physiology 8: 143-213. [14] Nicolaus, B., Kambourova, M., and Oner, E. T. 2010.
[17] Sutherland, W. 1982. “Biosynthesis of Microbial “Exopolysaccharides from Extremophiles: from
Exopolysaccharides.” Advances in Microbial Physiology
Fundamentals to Biotechnology.” Environmental
23: 79-150.
Technology 31 (10): 1145-1158. [18] Geel-Schutten, G. H. van, 2000. “Exopolysaccharide [15] Raychev, R., Fachikov, L. and Zaprjanova, V. 2002.
synthesis by Lactobacillus reuteri.” Ph.D. thesis, “Corrosion and Protection of the Materials—Handbook
University of Groningen.
Journal of Life Sciences 8 (2014) 946-954
doi: 10.17265/1934-7391/2014.12.004 DAVID PUBLISHING
Morphogenesis of Oil Palm (Elaeis guineensis Jacq.) Fruit in Seed Development
1 1 2 Hermine Bille Ngalle 1 , Joseph Martin Bell , Georges Franck Ngando-Ebongue , Hernild Eman-Evina , Godswill
Ntsefong Ntsomboh 2 and Armand Nsimi-Mva 3
1. Department of Plant Biology, Faculty of Science, University of Yaoundé I, Yaoundé, P.O. Box 812, Cameroon 2. Specialized Oil Palm Research Centre (CEREPAH of La Dibamba), IRAD, Douala, P.O. 243, Cameroon 3. Ekona Regional Research Centre, IRAD, Buéa, P.O. Box 25, Cameroon
Received: November 14, 2014 / Accepted: December 2, 2014 / Published: December 30, 2014.
Abstract: The place of the oil palm, Elaeis guineensis Jacq., in the market for fats of vegetable commodities makes it a strategic plant which requires continuous improvement. In this context, it seems appropriate to better describe the effects of the Sh gene in the developing fruit. This study aims to set a benchmark for the development of the seed in the natural palm (Elaeis guineensis var. dura). Thus the growth and development of the two major seed tissues were monitored every two weeks from pollination to maturity of the fruit. The results show that the endosperm is still liquid six weeks after pollination. It then begins an accelerated development which leads it, 11 weeks later, to completely fill the seed cavity, with an average mass of 0.81 g. This mass remains stable until the maturity of the fruit. The embryo is only visible when the endosperm is gelatinous, around 70 DPP (days post-pollination). It then has an average length of 1.00 mm. At 126 DPP, the embryo has finished growing and measures 2.82 mm on average. This length also remains stable until 168 DPP (3.04 mm). In perspective, a detailed follow-up of the development of the zygote from the pollination to 100 DPP is proposed. In parallel, the analysis of the chemical composition of the endosperm between 100 DPP and 168 DPP is necessary. These two complementary studies will allow to better specifying the benchmark of seed development in Elaeis guineensis var. dura.
Key words: Elaeis guineensis Jacq., embryo, endosperm, seed, development.
1. Introduction place in the consumption of fats of vegetable origin. Oil palm appears as a strategic plant for the economy of
Since 2006, palm oil, extracted from the mesocarp of
numerous producing countries.
the fruit of the oil palm (Elaeis guineensis Jacq.), Breeding programs and genetic improvement of this became the first source of vegetable fat on the world species are primarily focused on the development of market [1]. With a world production of 57.3 million planting material more efficient in terms of production tons in 2013 [2], this oil also ranks first in terms of of palm oil and kernels [6]. However, the history of production. Palm oil reaches this performance thanks selection in this plant is of recent [4, 7-10]. It to its exceptional yield, with world average around four consisted up to here in indirectly valuing a natural tons of palm oil per hectare [3]. This productivity of the mutation that occurred on the shell (Sh) gene, which oil palm is much greater than that of all oilseed crops. It specifically controls the thickness of the endocarp in is ten times higher than that of soybean [3-5]. With a this species [11, 12]. At this locus, the wild palm dura production of 6.8 million tons in 2013 [2], palm kernel having the genotype Sh + Sh + with thick endocarp and oil extracted from the seed also holds an important large seed is distinguished from the mutated palm - -
Corresponding author: Joseph Martin Bell, Ph.D., named pisifera having the genotype Sh Sh without
associate professor, research fields: genetics and plant breeding. endocarp and with a tiny seed, and the hybrid palm E-mail: [email protected].
Morphogenesis of Oil Palm (Elaeis guineensis Jacq.) Fruit in Seed Development
tenera descended from the cross [ ♀ dura × ♂ pisifera],
Table 1 Genitors used and controlled pollinations made at
having the genotype Sh Sh with thin endocarp and Matings normal seed [13, 14].
CEREPAH (Specialized Oil Palm Research Centre).
♂ Genitors The Sh gene seems to have a direct impact on the
Date of pollination ♀ Genitors
× LD2272 P endocarp and an indirect effect on the survival of the
A98D 22 14
× LD2272 P seed and thus on the female infertility in E. guineensis
A98D 23 21
× LD1568 P [6, 15]. This pleiotropy of the Sh gene has not yet
B91D 29 07
× LD2272 P been clarified. A very interesting orientation and a
C19D 14 08
× LD2272 P source of significant progress would be to develop a
C19D 24 17
D: Elaeis guineensis Jacq. var. dura; P: Elaeis guineensis Jacq. pisifera planting material thus producing fruits var. pisifera.
without endocarp, but in which the indirect effect of development, 30 fruits were taken from the whole the Sh gene mentioned above would be reduced or
bunch, that is 150 fruits for five bunches. eliminated. In other words, it would be a matter to
Fruits sampled underwent longitudinal or transverse identifying candidate genes for the restoration of
sections, which were observed with the naked eye female fertility in E. guineensis Jacq. var. pisifera.
and/or by means of a EUROMAX optical microscope To better analyse the pleiotropy of this gene, it is
with a micrometre. These observations were essentially necessary to have a precise benchmark for the
aimed at assessing the shape of the lodge of the seed, development of oil palm fruit at a time when the
determining the time of onset of the endosperm and mutated allele is not present. A recent study has
embryo. Different measurements on these tissues can already described the development of the pericarp of
track, from pollination fruit maturity, the evolution of the fruit of the oil palm [16]. The general objective of
the:
the present work is to describe the development of the equatorial diameter of the lodge of the seed, seed of E. guineensis Jacq. var. dura. Specifically, the
measured microscopically for the early stages of fruit study assesses changes in the seed lodge; determines
development (0-42 DPP). In later stages, this parameter the deadlines of appearance of the endosperm and the
is measured using a ruler;
embryo, as well as the pace of development of these consistency of the endosperm, appreciated with two tissues, from pollination to the fruit maturity.
the naked eye and the touch;
mass of the endosperm. For young phases of fruit
2. Materials and Methods
development (0-70 DPP), this parameter is estimated The plant material is freely obtained from the
by the formula M en =M wf -M hf , where M en ,M wf and M hf CEREPAH (Specialized Oil Palm Research Centre) of
represent respectively the masses of the endosperm, the La Dibamba, one of the stations of the IRAD (Institute
whole fruit (with the seed) and the hollowed fruit (fruit of Agricultural Research for Development) in freed of the seed). At the advanced stages, this Cameroon. It consists of fruits collected from maturing
parameter was measured using a precision balance 0.1 oil palm bunches.
mg brand RADWAG (series AS/X), minimum and Five assisted pollinations were made between dura
maximum capacity estimated at 10 mg and 220 g (female parent) and pisifera genitors (Table 1).
respectively;
Fruits were sampled on bunches, every two weeks length of the embryo, measured microscopically from the first DPP (day post-pollination) to the for young stages of development (0-98 DPP) and with a maturity of bunches. Maturity is substantiated by the
graduated ruler for the advanced stages (over 112 natural detachment of the first fruits. At each stage of
DPP).
Morphogenesis of Oil Palm (Elaeis guineensis Jacq.) Fruit in Seed Development
For each stage, the mean and standard deviations of from the 42nd DPP shows that of the 1,350 fruits tested, the parameters were calculated. The curves showing
997 (74%) each contained a lodge, 293 (22%) with two the evolution of these parameters over time are built
lodges each and 60 (4%) with more than two lodges using the Microsoft Office Excel 2010 software. A
(three or four).
digital camera HP PhotoSmart M425 allowed setting
3.2 Evolution of Endosperm
the observed structures.
3. Results The endosperm is present from 17 DPP. It is in liquid
form until 42 DPP. Between 42 DPP and 70 DPP, it
3.1 Evolution of the Seed Lodge becomes gelatinous (Fig. 2a). From 84 DPP, it is
During the early stages of development (0-17 DPP), cartilaginous (Fig. 2b). Beyond 126 DPP, it has already the cutting of the fruit generally presents three small
acquired the final solid consistency (Fig. 2c) of a cavities, arranged in the form of a clover (Fig. 1a).
mature endosperm (Fig. 2d).
Gradually, as the fruit grows, the number of cavities
3.3 Evolution of the Embryo
tends to decrease on average towards two lodges (Fig. 1b). And from the 42nd DPP, the fruit usually has a
In the mature fruit, it is located in the linear single cavity (Fig. 1c), supposed to contain the unique
“extension” of the germ pore (Fig. 3a) and its final seed (Fig. 1d). However, some fruits reach maturity
average size (at 168 DPP) is 3.04 ± 0.15 mm (Fig. 3b). with two or more seeds (Figs. 1e and 1f).
Overall, the study shows that the growth in mass of Evaluation of fruits with regard to existing lodges
the endosperm really starts after 28 DPP, as it is 0.09
(a) (b) (c)
(d) (e) (f)
Fig. 1 Sections of fruits of Elaeis guineensis var. dura. (a): fruit with three cavities (17 DPP); (b): fruit with two cavities (28 DPP); (c): fruit with one cavity (42 DPP); (d): 1-seeded fruit (168 DPP); (e): 2-seeded fruit (168 DPP); (f): 3-seeded fruit (168 DPP); DPP: days post-pollination; arrows indicate the location of cavities (a, b and c) or seeds (d, e and f).
Morphogenesis of Oil Palm (Elaeis guineensis Jacq.) Fruit in Seed Development
Fig. 2 Evolution of the endosperm consistency of Elaeis guineensis var. dura. (a): gelatinous (70 DPP); (b): cartilaginous (112 DPP); (c): solid (154 DPP); (d): solid (168 DPP); DPP: days post-pollination.
Fig. 3 Embryos of Elaeis guineensis var. dura. (a): location of the embryo within the seed; (b): mature embryo; Gp: germ pore; Em: embryo; En: endosperm.
± 0.00 g at 42 DPP. This tissue of the seed grows that the maximum diameter of the seed lodge is steadily and reaches its maximum average mass (0.81
reached, dressing peaks from 1.80 ± 0.07 mm (28 DPP) ± 0.04 g) at 126 DPP. The embryo, which is noticeable
to 10.07 ± 0.60 mm (126 DPP). Fig. 4 shows average on the 56th DPP begins its growth from the 70th DPP
parameters of seed’s growth, including the diameter of and almost achieves its maximum average length
the seed lodge, the length of the embryo and the mass (2.82 ± 0.14 mm) at 126th JPP. It is also at this time
of the endosperm.
Morphogenesis of Oil Palm (Elaeis guineensis Jacq.) Fruit in Seed Development
Fig. 4 Average parameters for growth of the seed of E. guineensis var. dura.
The early seed development of E. guineensis var. Arbequina and Sevillano cultivars of the olive tree dura is characterized by the stabilization of the number
develop two-seeded fruits at respective frequencies of of lodges of the seed. Indeed, the setting of this
14%, 4% and 1% [19]. In Rubiaceae, gender parameter at 42 DPP allows the endosperm to pass
Cosmocalyx and species such as Zizyphus vulgaris and from a liquid to a gelatinous state. And from 70 DPP,
Murraya koenigii, fruits generally carry two seeds the solidification of the endosperm engages in the
[20-22]. Moreover, some species of which Detarium development of the embryo, whose growth seems to
microcarpum and Orbignya oleifera, produce drupes require prior jellification of the endosperm. At 126
containing more than three seeds [23, 24]. DPP, both seed tissues reach their maximum growth.
From 42 DPP, the growth of the endosperm is During the last six weeks of fruit development
evidenced by a quantity of liquid, increasingly (126-168 DPP), these two tissues do not grow
important, as reported elsewhere [25]. This reflects substantially but certainly enter their maturation stage.
continued growth of this tissue, resulting in numerous cell divisions of the primary endosperm nucleus
4. Discussion
taking place at the beginning of fruit formation [26, The first manifestation of the development of the
27]. The liquid consistency of the endosperm of oil seed in E. guineensis var. dura is the stabilization of
palm, which lasts until 56 DPP, corresponds to the the number of lodges that must shelter one or several
coenocytic phase specific to the formation of the future seeds of the mature fruit. Among examined
nuclear type endosperm [28, 29]. After 70 DPP, this fruits, 26% contain at least two seeds. This supports
tissue acquires a gelatinous consistency. These results previous data that set the frequency of such oil palm
corroborate previous data, which place the transition fruits between 20% and 25% [15, 17, 18]. Hojiblanca,
from liquid state to semi-gelatinous of the endosperm
Morphogenesis of Oil Palm (Elaeis guineensis Jacq.) Fruit in Seed Development
of oil palm, between eight and ten weeks post-anthesis This correlation is necessary nutritionally but [30]. This change in consistency is between 70 and
especially mechanically. It is the solidified endosperm 190 days after anthesis in Pritchardia remota [31]. As
which must maintain the embryo positioned opposite for Cocos nucifera, a part of its endosperm remains
the germ pore. In Pritchardia remota, the embryo liquid (coconut milk) within the mature fruit [32, 33].
remains microscopic until 70 days after anthesis [31]. After 70 DPP the mass of the endosperm increases
As for embryos of Actinidia chinensis and Lindera and it passes from gelatinous to cartilaginous. This
melissifolia, they remain in the two-cell stage until 60 development, which coincides with the stunting of the
days post-anthesis [38, 39].
diameter of the lodge of the seed, may be associated After 70 DPP, the embryo grows exponentially with the accumulation of fats. Indeed, the beginning of
through a significant cell magnification [40]. lipogenesis in the palm oil seed is between the tenth
According to some authors, the weight of the embryo and the twelfth week post-anthesis [30, 34].
increases from 80 days after anthesis [37]. This would From 84 DPP endosperm continues to solidify
be due to the fact that the endosperm of probably through the synthesis of lipids. These last up
gelatinous/cartilaginous consistency, fully meets with to twenty weeks post-anthesis [34, 35]. This it, the role of feeder tissue [27, 40, 41]. The embryo lipogenesis in the endosperm, combined with thrust
then lengthens, by its basal pole and almost reaches at dehydration and a departure of K and Ca, according to
126 DPP its final size and shape [42, 43]. Published several authors [31, 36, 37], would contribute to
data place the maximum growth of the embryo at 90, increasing the mass of this tissue and to solidifying it.
110, 120 and 250 days post-anthesis, respectively for In all cases, the endosperm of E. guineensis var. dura
E. is completely solid at 126 DPP, that is 18 weeks
Lindera melissifolia,
Actinidia chinensis,
guineensis var. tenera and Pritchardia remota [31, post-pollination, against 17 weeks post-anthesis 37-39].
reported for the endosperm of E. guineensis var. From 126 DPP, the embryo of E. guineensis does tenera [30] and more than 48 weeks post-anthesis
not grow any more. It is the same with Pritchardia (340 days) for Pritchardia remota [31]. As for the
remota, for which there is no difference between seeds of Actinidia chinensis and Lindera melissifolia,
embryos of 250 days and those of 400 days they reach their maximum growth respectively in 80
post-anthesis, in terms of dry mass [31]. And due to and 90 days post-anthesis [38, 39]. It is also at 126
its location and its space in the seed, the embryo of oil DPP that this tissue reaches its maximum growth in
palm belongs to the category of rudimentary embryos terms of mass, with an average of 0.81 ± 0.04 g. This
[44]. Embryos of Cocos nucifera, Oryza sativa L. spp. value is widely below those observed in Cocos
japonica [33], as well as those of certain nucifera, the mass of the seed of which can range
Caprifoliaceae, which occupy a space within the seed from 354.5 g to 1,107 g [32].
being less than 1/3, even 1/10 [45], are also During the first 69 days after pollination, the zygote
rudimentary. On the other hand, in Prunus serotina (future embryo), is the centre of many divisions that
and Orozoa paniculosa for example, the embryo increase the number of cells [40]. But the endosperm,
grows by invading most of the major part of the fruit which is in its coenocytic phase (liquid consistency),
dedicated to the seed [46, 47].
can not play its role of feeder tissue with the embryo
5. Conclusions
[28, 41]. So it remains invisible before 70 DPP would
be primarily related to the synchronization between This study shows that the evolution of the seed of E. the formation of the endosperm and the embryo [17].
guineensis var. dura consists of three main phases.
Morphogenesis of Oil Palm (Elaeis guineensis Jacq.) Fruit in Seed Development
Within seven weeks, cells from double fertilization d'Amélioration du Palmier à Huile à l'IRHO.” Oléagineux probably undergo qualitative changes though still 27 (1): 1-12. [8] Jacquemard, J. C., Baudouin, L., and Noiret, J. M. 2001.
microscopic. Afterward, and during 11 weeks, the “Oil Palm.” In Tropical Plant Breeding, edited by
endosperm and the embryo move from insignificant Charrier, A., Jacquot, M., Hamon, S., and Nicolas, D. mass or length to maximum values, which are 0.83 g
Montpellier: CIRAD, Science Publishers Inc. and 3 mm respectively. During the last seven weeks,
[9] Cochard, B., Amblard, P., and Durand-Gasselin, T. 2005. “Oil Palm Genetic Improvement and Sustainable
dimensions of the various tissues of the seed do not Development.” Oilseeds & fats Crops and Lipids 12 (2):
evolve any more. The seed is located in the maturation 141-147. doi:10.1051/ocl.2005.0141. phase which prepares it for future germination.
[10] Cochard, B., Adon, B., Rekina, S., Billote, N., de Chenon, To follow-up this work, it seems appropriate to
R. D., Koutou, A., Nouy, B., Omoré, A., Purba, A. R., analyze in detail what happens to the zygote within
Glazsmann, J.-C., and Noyer, J.-C. 2009. “Geographic and Genetic Structure of African Oil Palm Diversity
the seven weeks post-fertilization. It is also important suggests New Approaches to Breeding.” Tree Genetics &
to monitor the chemical composition of the endosperm Genomes 5 (3): 493-504. doi:10.1007/s11295-009- from 100 DPP, in order to clarify the implementation
0203-3.
of the seed within the fruit of E. guineensis var. dura. [11] Beirnaert, A., and Vanderweyen, R. 1941. Contribution à Later studies will allow identifying the direct primary
l'Étude Génétique et Biométrique des Variétés d'Elaeis guineensis Jacquin. East African Standard. Bruxelles:
effect of Sh gene on the development of the fruit of E. Institut National pour l'Étude Agronomique du Congo
guineensis Jacq. var. pisifera.
belge, Série scientifique n 27. [12] Moretzsohn, M. C., Nunes, C. D. M., Ferreira, M. E., and
References
Grattapaglia, D. 2000. “RAPD Linkage Mapping of the [1] SoyStats. 2007. “World Vegetable Oil Consumption
Shell Thickness Locus in Oil Palm (Elaeis guineensis 2006.” Accessed January 13, 2010.
Jacq.).” Theoretical and Applied Genetics 100 (1): 63-70.
http://www.soystats.com/2007/default.htm.
doi:10.1007/s001220050009.
[2] SoyStats. 2014. “World Vegetable Oil Consumption [13] Jannot, C. 2002. “Les Déterminants Historiques du 2013.” Accessed January 5, 2015. http://soystats.
Commerce de l'Huile de Palme. Entre Filière Artisanale com/wp-content/uploads/SoyStats_2014.pdf.
et Agro-industrie: Quel Avenir pour le Palmier en [3] Jacquemard, J. C. 2011. Le Palmier à Huile. Versailles,
Afrique?” Presented at the Conference on The Future of Wageningen, Gembloux: Editions Quæ, CTA, Presses
Perennial Crops. Investment and Sustainability in the Agronomiques de Gembloux.
Humid Tropics, Yamoussoukro, Côte d'Ivoire. [4] Durand-Gasselin, T., Blangy, L., Picasso, C., De
[14] Corley, R. H. V., and Tinker, P. B. 2003. The Oil Palm. Franqueville, H., Breton, F., Amblard, P., Cochard, B.,
4th editon. Oxford: Blackwell Science Ltd. Louise, C., and Nouy, B. 2010. “Sélection du Palmier à
[15] Hartley, C. W. S. 1988. The Oil Palm (Elaeis guineensis Huile pour une Huile de Palme durable et responsabilité
Jacq.). 3 rd edition. New York: Longman Scientific & sociale”. Oléagineux, Corps Gras, Lipides 17 (6):
Technical.
385-392. doi:10.1051/ocl.2010.0343. [16] Ngalle, H. B., Bell, J. M., Ngando-Ebongue, G. F., Nyobe, [5] Skurtis, T., Aïnaché, G., and Simon, D. 2010. “Le
L., Ngangnou, F. C., and Ntsomboh, N. G. 2013. Financement du Secteur de l'Huile de Palme: Pourquoi
“Morphogenesis of Oil Palm Fruit (Elaeis guineensis les Institutions Financières de Développement Doivent
Jacq.) in Mesocarp and Endocarp Development.” Journal Continuer à Investir en Afrique.” Oléagineux, Corps
of Life Sciences 7 (2): 153-158. doi:10.17265/1934-7391/ Gras,
Lipides 17 (6): 400-403.
doi:10.1051/ocl.2010.0346. [17] Hussey, G. 1958. “An Analysis of the Factors Controlling [6] Demol, J. 2002. Amélioration des Plantes. Application
the Germination of the Seed of the Oil Palm, Elaeis aux Principales Espèces Cultivées en Régions Tropicales.
guineensis (Jacq.).” Annals of Botany 22: 259-284. Gembloux (Belgique): Les presses agronomiques de
[18] Bell, J. M. 2000. Evaluation de l'Offre en Graines de Gembloux.
Palmier à Huile Sélectionné au Cameroun. Vol. I: [7] Meunier, J., and Gascon, J. P. 1972. “Le Schéma Général
Rapport principal. Yaoundé: Association Française des
Morphogenesis of Oil Palm (Elaeis guineensis Jacq.) Fruit in Seed Development
Volontaires du Progrès/Ministère de l’Agriculture
S0960258511000523.
(Cameroun). [32] Zizumbo-Villarreal, D., and Piñero, D. 1998. “Pattern of [19] Cuevas, J., and Oller, R. 2002. “Olive Seed Set and its
Morphological Variation and Diversity of Cocos nucifera Impact on Seed and Fruit Weight.” Acta Horticulturae
(Arecaceae) in Mexico.” American Journal of Botany 85 586: 485-488.
(6): 855-865.
[20] Delprete, P. G. 1998. “Notes on Calycophyllous [33] Xu, L., Ye, R., Zeng, Y., Wang, Z., Zhou, P., Lin, Y., and Rubiaceae. Part III, Systematic Position of the Monotypic
Li, D. 2010. “Isolation of the Endosperm-specific Mexican Genus Cosmocalyx and Notes on the
LPAAT Gene Promoter from Coconut (Cocos nucifera L.) Calycophyll Development.” Brittonia 50 (3): 309-317.
and its Functional Analysis in Transgenic Rice Plants.” doi: 10.2307/2807774.
Plant Cell Reports 29: 1061-1068. doi:10.1007/s00299- [21] Koné, B., Kalinganire, A., and Doumbia, M. 2008. La
010-0892-y.
Culture du Jujubier: Manuel pour l'Horticulteur Sahélien. [34] Crombie, M. 1956. “Fat Metabolism in West African Oil ICRAF Technical Manual N 10. Nairobi: World
Palm (Elaeis guineensis). Part I. Fatty Acid Formation in Agroforetry Centre.
the Maturing Kernel.” Journal of Experimental Botany 7 [22] Handral, H. K., Pandith, A., and Shruthi, S. D. 2012. “A
(2): 181-193. doi:10.1093/jxb/7.2.181. Review on Murraya koenigii Multipotential Medical
[35] Boatman, S. G., and Crombie, M. 1958. “Fat Metabolism Plant.” Asian Journal of Pharmaceutical and Clinical
in the West African Oil Palm (Elaeis guineensis).” Research 5 (4): 5-14.
Journal of Experimental Botany 9 (1): 52-74. [23] Balick, M. J. 1979. “Amazonian Oil Palms of Promise: a
doi:10.1093/jxb/9.1.52.
Survey.” Economic Botany 33 (1): 11-28. [36] Prevot, P. 1962. Données Récentes sur la Physiologie du doi:10.1007/BF02858207.
Palmier à Huile. Physiologie des Plantes Tropicales [24] Kouyaté, A. M., Van Damme, P., de Meulenaer, B., and
Cultivées. Paris: ORSTOM.
Diawara, H. 2009. “Contribution des Produits de [37] Aberlenc-Bertossi, F., Chabrillange, N., Corbineau, F., Cueillette dans l'Alimentation humaine. Cas de Detarium
and Duval, Y. 2003. “Acquisition of Desiccation microcarpum.” Afrika Focus 22 (1): 77-88.
Tolerance in Developing Oil Palm (Elaeis guineensis [25] Rusfiandi, H., Sitorus, A., Forster, B. P., Nelson, S. P. C.,
Jacq.) Embryos in planta and in vitro in Relation to Sugar and Caligari, P. D. S. 2011. “Oil Palm Fruit
Content.” Seed Science Research 13 (2): 179-186.
Development.” In Proceedings of the Malaysian
doi:10.1079/SSR2003135.
International Palm Oil Congress (PIPOC) 161-166. [38] Hopping, M. E. 1976. “Structure and Development of [26] Percie du Sert, C., and Durrieu, G. 1988. “Edification de
Fruit and Seeds in Chinese Gooseberry (Actinidia l'Akène et de la Graine de Tournesol (Helianthus annus).”
chinensis Planch.).” New Zealand Journal of Botany 14 Informations Techniques du C.E.T.I.O.M. 103: 12-20.
(1): 63-68. doi:10.1080/0028825X.1976.10428651.
[27] Heller, R., Esnault, R., and Lance, C. 2004. Physiologie [39] Connor, K., Schaefer, G., Donahoo, J., Devall, M.,
Gardiner, E., Hawkins, T., Wilson, D., Schiff, N., Hamel, [28] Vallade, J. 2001. Structure et Développement de la Plante:
Végétale. 2. Développement. 6 e édition. Paris: Dunod.
P., and Leininger, T. 2007. “Development, Fatty Acid Morphogenèse et Biologie de la Reproduction des
Composition and Storage of Drupes and Seeds from Angiospermes. Paris: Dunod.
Pondberry (Lindera melissifolia).” Biological [29] Judd, W. S., Campbell, C. S., Kellog, E. A., and Stevens,
137 (4): 489-496. doi:10.1016/ P. 2011. Botanique Systématique: une Perspective
Conservation
j.biocon.2007.03.011.
Phylogénétique. Paris: De Boeck. [40] Natesh, S., and Rau, M. A. 1984. “The Embryo.” In [30] Oo, K. C., The, S. K., Khor, H. T., and Ong, A. S. H.
Embryology of Angiosperms, edited by Johri, B. M. 1985. “Fatty Acid Synthesis in the Oil Palm (Elaeis
Berlin: Springer-Verlag.
guineensis): Incorporation of Acetate by Tissue Slices of [41] Laberche, J. C. 2004. Biologie Végétale. 2 e édition. Paris: Developing Fruit.” Lipids 20 (4): 205-210. doi:
Dunod.
10.1007/BF02534189. [42] Anonymous. 1980. La Culture du Palmier à Huile. [31] Pérez, H. E., Hill, L. M., and Walters, C. 2012. “An
Fascicule I : Classification, Morphologie et Biologie. La Analysis of Embryo Development in Palm: Interactions
Mé: Institut de Recherche pour Les Huiles et Oléagineux Between Dry Matter Accumulation and Water Relations
(France).
in Pritchardia remota (Arecaceae).” Seed Science [43] Ataga, C. D., and van der Vossen, H. A. M. 2007. “Elaeis Research 22 (2): 97-111. doi:10.1017/
guineensis Jacq.” In Ressources Végétales de l'Afrique
954
Morphogenesis of Oil Palm (Elaeis guineensis Jacq.) Fruit in Seed Development
Tropicale 14. Oléagineux, edited by Mkamilo, G. S., and 104 (2): 253-276. doi:10.1093/aob/mcp131. van der Vossen H. A. M. Wageningen: Fondation
[46] Labrecque, M., and Barabé, D. 1984. “Développement PROTA, Backhuys Publishers, CTA.
du Fruit de Prunus serotina (Rosaceae).” Revue [44] Meyer, D. J. L. 2005. “Seed Development and Structure
Canadienne de Botanique 62 (2): 195-206. in Floral Crops.” In Flower seeds: Biology and
doi:10.1139/b84-033.
Technology, edited by McDonald, M. B., and Kwong F. [47] Teichman, I. V. 1993. “Development and Structure of the Y. Wallingford (UK): Elsevier.
Seed of Ozoroa paniculosa (Anacardiaceae) and [45] Jacobs, B., Lens, F., and Smets, E. 2009. “Evolution of
Taxonomic Notes.” Botanical Journal of Linnean Society Fruit and Seed Characters in the Diervilla and Lonicera
111 (4): 463-470. doi:10.1111/j.1095-8339.1993. Clades (Caprifoliaceae, Dipsacales).” Annals of Botany
tb01915.x.
Journal of Life Sciences 8 (2014) 955-966
doi: 10.17265/1934-7391/2014.12.005 DAVID PUBLISHING
Characterization in Greenhouse Conditions of Two Salt Tolerant Citrumelo (Citrus paradisi Macf. x Poncirus trifoliata (L.) Raf.) Cultivars
1 2 3 Anas Fadli 1 , Ouiam Chetto , Abdelhak Talha , Rachid Benkirane , Raphaël Morillon and Hamid Benyahia
1. Department of Plant Breeding and Germplasm Conservation, National Institute for Agricultural Research (INRA), Kenitra 14000,
Morocco 2. Department of Biology, Faculty of Science, Ibn Tofail University, Kenitra 242, Morocco 3. Department of Genetic Improvement and Adaptation of Mediterranean and Tropical Plants, Agricultural Research Center for
International Development (CIRAD), Montpellier 34398 Cedex 5, France
Received: November 20, 2014 / Accepted: December 6, 2014 / Published: December 30, 2014.
Abstract: Increasing salinity in Mediterranean soils and the wide spread of citrus tristeza virus have challenged the use of sour orange (Citrus aurantium) and have accelerated the process of seeking alternative rootstocks. In the present study, nine cultivars of citrumelo (Citrus paradisi Macf. x Poncirus trifoliata (L.) Raf.) were evaluated for salt tolerance. Two month-old seedlings were raised under greenhouse conditions and irrigated with a half strength Hoagland solution supplemented with different concentrations of NaCl, i.e., 0 mM, 35 mM and 85 mM. Tolerance was assessed after two months of stress by measuring stem growth, number of leaves, fresh and dry weight organs and leaf water, chlorophyll and chloride contents. A differential behavior was noticed among the seedlings we studied. When using increased concentration of salt in irrigation water, all the parameters were significantly reduced except for leaf chloride content which highly increased in response to stress. At 85 mM, the cultivar SC2 showed a high tolerance resulting in less apparent leaf symptoms, higher growth and higher leaf chlorophyll content when compared to other seedlings. Similarly, the cultivar C4475-C was shown to be a strong root chloride excluder with less than 2.6% DW (dry weight) chloride accumulation at leaf level. By contrast, our results suggest that C4475-A and C4475-B are salt sensitive cultivars regarding to all the parameters studied while the other citrumelos were considered as moderately tolerant.
Key words: Citrus, rootstock, salinity, growth, NaCl, chloride, screening.
1. Introduction which may raise the electrical conductivity up to 3 dS/m, the critical level for citrus production [6]. In
Among the environmental factors which can limit studies carried out in Morocco we showed a high successful production and/or yield of crops worldwide, correlation between soil salinity and severity of salinity is considered to be one of the most important gummosis on sour orange (Cirus aurantium) caused along with water deficit. Salinity represents a serious by Phtophthora sp. [5, 7, 8]. We also observed that threat for salt-sensitive crops such as Citrus sp. [1-3]. increasing salinity inirrigation water predispose sour In Morocco, almost 35% of irrigated land is orange and troyer citrange (Citrus sinensis × Poncirus considered as salt-affected [4, 5]. Moreover, the trifoliata) to root rot caused by Phytophthora prasitica irrigation water from aquifers can often contain
by specific effect of Cl + − .
excessive amounts of soluble salts (Cl − and/or Na ) The detrimental effects of salinity in citrus were
Corresponding author: Hamid Benyahia, Ph.D., research widely reported and have been frequently related to fields: plant physiology, plant breeding and phytopathology.
E-mail: [email protected]. the toxic effect of Cl ions [3]. Indeed, it is well
Characterization in Greenhouse Conditions of Two Salt Tolerant Citrumelo
(Citrus paradisi Macf. x Poncirus trifoliata (L.) Raf.) Cultivars
established that high leaf Cl − concentrations due to Nowadays, the recourse to germplasm banks and root zone salinity may lead to physiological disturbances
the management of genetic resources such as those and eventually growth and yield reduction [9-11].
offered by citrumelo rootstocks are necessary However, it is also known that citrus species differ
considering the critical current situation of citrus in widely in their ability to restrict Na +
the Mediterranean region. Indeed, the recent spread of root level and the translocation of these toxic ions
and Cl − uptake at
quick-decline isolates of CTV in the Mediterranean from roots to shoots [3, 12, 13]. Oppenheimer [14]
basin have limited the use of sour orange which has was the first to report the effect of the rootstock in
been historically the most utilized rootstock in this salt-tolerance of citrus species. His works have shown
area. The latter provides a wide soil adaptability and that mature orange trees on sour orange rootstock
superior horticultural performance, notably under accumulated less Cl − in the scion leaves than trees did
stressed conditions [24, 25]. Thus, new sources of on Palestine sweet lime (C. limettioides Tan.). Later
CTV tolerance with similar or better salinity tolerance on, studies carried out on different rootstocks have
than sour orange are needed.
shown Rangpur lime (Citrus limonia Osbeck), Sunki In the present study, the authors investigated the mandarin (Citrus sunki Hort. ex Tan.) and Cleopatra
tolerance of nine citrumelo accessions to salinity by mandarin (Citrus reshni Hort. ex Tan.) are using a fast standardized and reproducible screening salt-tolerant species, while trifoliate orange (Poncirus
test in order to appraise their suitability in salt-affected trifoliata (L.) Raf.) and its hybrids such as Carrizo and
soils.
Troyer citranges (Citrus sinensis (L.) Obseck x P.
2. Materials and Methods
trifoliata (L.) Raf.) were ranked as salt-sensitive [12,
2.1 Plant Material and Growth Conditions compared the performance of Citrus and trifoliate
13, 15-19]. However, few studies have directly
The experiment was carried out at the Regional orange hybrids under saline conditions. Citrumelos,
Center for Agricultural Research in Kenitra (Morocco) for example, which are hybrids of trifoliate orange and
during the season 2011-2012. Ten rootstock cultivars grapefruit (Citrus paradisi Macf. x Poncirus trifoliata
belonging to the germplasm collection of INRA (L.) Raf.), were largely overlooked as potential
(National Institute for Agricultural Research) Kenitra, rootstocks, until superior performance of Swingle
and including nine citrumelo accessions (Citrus citrumelo was demonstrated in field trials in the
paradisi Macf. x Poncirus trifoliata (L.) Raf.) were 1940’s [20, 21]. Since then, Swingle citrumelo has
investigated for their properties of salt stress tolerance become a popular rootstock in many areas. The
(Table 1). Rangpur lime, which is known to be a current success of citrumelo cultivars can be generally
salt-tolerant rootstock [26] was also included in the attributed to their many desirable characteristics such
experiment to accurately estimate the tolerance of the as tolerance to Phytophthora spp., exocortis and
other rootstocks.
particularly to tristeza disease [20]. Nevertheless, the Healthy mature fruits of all rootstocks were works of Garnsey et al. [22] reported a high tolerance
harvested in the experimental fields of the institute. of some citrumelo cultivars to CTV (citrus tristeza
Seeds were extracted, washed and air-dried in shade, virus) but the response was CTV strain-dependant. In
then germinated in 60 × 40 cm trays filled with peat. this sense, Grisoni et al. [23] investigated the
The experiment was carried out during the late resistance of different rootstocks to a severe strain of
summer in a greenhouse when temperature ranged CTV and found that citrumelo 1452 may have a
from 25 °C to 40 °C and relative humidity varied moderate to susceptible reaction.
between 40% and 60%.
Characterization in Greenhouse Conditions of Two Salt Tolerant Citrumelo
(Citrus paradisi Macf. x Poncirus trifoliata (L.) Raf.) Cultivars
Table 1 List of the rootstock cultivars used in the experiment.
Rootstock accession
ICVN a or SRA b Code Citrumelo 4475 AB6A4
Origin
C4475-B Citrumelo 4475 B2G3
SRA INRA/Cirad Corse
ICVN 0110140/SRA 732
C4475-C Citrumelo 4475 BB6A5
SRA INRA/Cirad Corse
ICVN 0110145/SRA 928
C4475-A Citrumelo 5798502
SRA INRA/Cirad Corse
ICVN 0110141/SRA 733
C502 Citrumelo 5798506
CRC Riverside
C506 Sacaton citrumelo B230057
CRC Riverside
CS Citrumelo winter Haven B231431
SRA INRA/Cirad Corse
ICVN 0110144/SRA 843
CWH Swingle Citrumelo 741
SRA INRA/Cirad Corse
ICVN 0110147
SW2 Swingle Citrumelo F92255
CRC Riverside
SW1 Rangpur lime
CRC Riverside
RL a International citrus variety numbering.
CRC Riverside
b Agronomical research station numbering.
After two months of growth, uniform seedlings symptoms, i.e., chlorosis, wilting and defoliation. presenting 8 to 10 leaves were uprooted from the
2.3.2 Growth Measurement and Number of Leaves nursery and transferred into 0.5 L plastic pots in a
Stem height and the number of leaves were mixture of peat and sterilized sand at 1 : 1 ratio [27].
measured for each plant at initial time (H i , L i ) and The seedlings were then irrigated regularly twice a
after seven weeks of saline treatments (H f , L f ). SGR week using a half-strength Hoagland solution [28].
(stem growth rate) and the PRNL (percent reduction Each plant received 100 mL.
of the number of leaves) were estimated from these parameters according to the following equations:
2.2 Application of Saline Treatment
Hf Hi
(1) Salt stress treatments were carried out for seven
SGR
Hi
weeks. Salt stress was applied by supplementing the dL control dL treated nutrient solution with NaCl at two different
PRNL
avoid osmotic shock, salt was added gradually by
dL
concentrations, 35 mM and 85 mM respectively. To
control
Where dL is the difference between the final three-day intervals until reaching desired levels.
number and the initial number of leaves. Control plants were watered only with half strength
At the end of the experiment, plants were harvested Hoagland solution. The 100 mL we used allowed
and divided into roots, stems and leaves for biomass leaching of the saline solution from the pot and
determination. Fresh weigh of each part was avoiding salt accumulation.
immediately measured, whereas dry weight was determined after oven-drying tissue at 60 °C for 48 h
2.3 Evaluation of Salt Tolerance
2.3.1 Estimation of Leaf Injury
2.3.3 Physiological Analyzes
The response of the seedlings to salt stress was After seven weeks of treatment, leaf chlorophyll determined by recording the occurrence of symptoms
Content was estimated using a portable chlorophyll of leaf injury after seven weeks. All seedlings were
meter (SPAD)-502 device (Minolta, Osaka, Japan). visually evaluated and a 0-6 score was given to each
chloride were extracted from dry leaf tissue using hot plant according to the scale of Goell [29]. The score
water and determined by titration according to the was given on the basis of the severity of injury
method of Cotlove [31], whereas LWC (leaf water
Characterization in Greenhouse Conditions of Two Salt Tolerant Citrumelo
(Citrus paradisi Macf. x Poncirus trifoliata (L.) Raf.) Cultivars
content) was calculated from LFW (leaf fresh water)
3. Results and Analysis
and LDW (leaf dry water) weights as follows:
3.1 Effect of Salt Stress on Leaf Injury
LFW LDW LWC
Leaf symptoms of damages were observed in all
LDW
treated plants 30 to 45 days after the beginning of the Most of the parameters listed above were estimated
experiment. These symptoms began generally with relatively to control using RP (relative percentage)
necrosis at leaf tips then progressed inward towards and PR (percentage of reduction):
petioles. It noted also that injury began at lower leaves
and thereafter progressed to upper leaves. Treated RP
Based on statistical results, a clear difference
Control
regarding the salt tolerance was observed depending
on seedling cultivars and salt levels. At 85 mM NaCl, Control Treated PR
most of C4475-B seedlings showed severe necrosis
Control
and defoliation symptoms which was reflected by an average SSI (symptom severity index) of 5.8 (Table 2),
2.4 Experimental Design and Statistical Analysis whereas the occurrence of injured leaves was much
The experiment was carried out in a split-plot lesser in Rangpur lime which showed the lowest SSI design with six replications by rootstock and treatment.
(4.2). By contrast, control plants showed no salt stress The salinity factor was placed in the main plot and the
symptom throughout the treatment period. The
rootstock factor in subplot. Collected data were average SSI ranged at these conditions from 1 to 1.5 transferred to SAS software and subjected to analysis
and no significant difference was found among using a two-way ANOVA. Means were separated by
cultivars. Using 35 mM NaCl solution, an intermediate Duncan’s multiple range test.
response was found in all cultivars we tested. However,
Table 2 Severity of leaf injury symptoms according to the scale of Goell (1969).
Symptom severity index a
C4475-A 1.5 a 3.3 a 5.3 ab C4475-B 1.5 a 3.3 a 5.8 a CWH 1 a 2.3 b 4.7 ab SC1 1 a 3.5 a 5.5 ab CS 1.5 a 3.5 a 5.0 ab SC2 1.3 a 3.5 a 4.5 ab C502 1.2 a 3.8 a 4.8 ab C506 1.2 a 3.7 a 5.3 ab C4475-C 1.3 a 3.5 a 5.5 ab
RL 1.3 a 3.0 ab 4.2 b
Analysis of variance b R *** T * R × T
NS a Means followed by the same letter in same rows do not differ significantly at P ≤ 0.05 (one-way-ANOVA, separated by Duncan
test). b The factors R and T refer respectively to rootstock and treatment. Significant effects are indicated by * = P < 0.05, ** = P < 0.01
and *** = P < 0.001, and NS indicates not significant difference.
Characterization in Greenhouse Conditions of Two Salt Tolerant Citrumelo
(Citrus paradisi Macf. x Poncirus trifoliata (L.) Raf.) Cultivars
at this salt concentration, CWH was found to be more seedlings in their response to salinity although the tolerant than Rangpur lime.
interaction rootstock × salt treatment was not significant. As compared to their respective controls, seedlings
3.2 Effect of Salt Stress on Growth and Number of of SC2 showed the greatest tolerance at both salt
Leaves treatments, whereas those of C4475-A and C4475-B
NaCl caused a significant reduction in all growth were the most sensitive. For instance, under 35 mM parameters we considered. As shown in Figs. 1 and 2,
treatment, SRGR (stem relative growth rate) values
a significant decline in stem growth was found with were respectively 96%, 58% and 57% for SC2, increasing salt concentration in the irrigation water. A
C4475-B and C4475-A. The corresponding values at high genotypic difference was also found between
85 mM NaCl were 52%, 31% and 24%.
Fig. 1 Effect of salt stress on growth of Rangpur lime (a) and Sacaton citrumelo (b) seedlings. (T0) Control; (T1) 35 mM NaCl; (T2) 85 mM NaCl.
Stem growth rate (% of control) 40
Stem growth rate (% of control) 15
(a) (b)
Fig. 2 Effect of salt treatments on stem growth rate in the ten rootstocks studied expressed as % of control plants. (a) 35 mM NaCl; (b) 85 mM NaCl. Means represented by the same letter do not differ significantly at P ≤ 0.05 (one-way-ANOVA, separated by Duncan test). Vertical bars indicate the mean values ± SE (n = 6).
Characterization in Greenhouse Conditions of Two Salt Tolerant Citrumelo
(Citrus paradisi Macf. x Poncirus trifoliata (L.) Raf.) Cultivars
ANOVA analysis showed that both plant FW (fresh 11.6%, 17.8% and 19.3% respectively. By contrast, weight) and DW (dry weight) were significantly
the highest reduction in fresh and dry biomass was decreased in response to salt stress but the impact was
observed in C4475-A and C4475-B cultivars whatever more or less important depending on the cultivar
the organ studied.
(Table 3). Also, the reduction in biomass was quite The number of leaves also considerably declined in variable depending on plant organ. Indeed, a reduction
response to high salt stress (P < 0.001). However, no gradient was observed at high salt concentration which
significant difference was found among cultivars at 35 could be summarized as following from the most
mM NaCl (Fig. 3). By contrast, at high salt affected to the least affected: leaves > roots > stem.
concentration (85 mM), the comparison of PRLN Among rootstocks, SC2 showed less biomass
(percent reduction of the number of leaves) means for reduction at whole plant level. Relatively to its control,
the different cultivars studied revealed the presence of this rootstock displayed a 40.2%, 16.9% and 19.2%
three statistically different groups: reduction in fresh leaf, stem and root weight
Group 1, which included C4475-C that showed respectively and a 9.5%, 19.5% and 22.8% reduction
more than 250% reduction in the number of leaves in dry leaf, stem and root weight. However, at low
when compared to control;
salinity level, RL seedlings were found to be more Group 2, composed of C.4475-B and C506 cultivars tolerant than SC2 seedlings resulting in no fresh
that showed a moderate reduction ranging from 150% weight reduction at stem level (-1%), 4.1% reduction
to 200% relatively to control;
in stem dry weight and 4.3% reduction in leaf fresh Group 3, that included all other seedling cultivars weight. The corresponding values for SC2 were
for which the values of PRNL were less than 150%.
Table 3 Effect of salt treatments on fresh and dry biomass expressed relatively to control values. (T1) 35 mM NaCl; (T2) 85 mM NaCl.
Dry weight a (% lower than control) Rootstock
Fresh weight a (% lower than control)
Stem Roots T1
Leaves Stem Roots
T1 T2 C4475-A 18.7 a 55.4 a 14.0 ab 35.7 ab 29.7 a 49.0 a 16.8 a 39.3 ab 34.7 ab 46.7 a 34.0 abc 51.9 a C4475-B 22.9 a 63.1 a 27.9 ab 46.9 a 34.7 a 45.0 a 21.2 a 41.2 ab 32.2 ab 39.6 abc 39.3 ab 48.1 ab CWH 34.0 a 48.4 a 36.8 a 47.1 a 38.9 a 41.5 a 27.2 a 29.2 ab 39.2 a 42.2 ab 41.6 a 43.6 ab SC1 21.0 a 51.3 a 3.6 b 20.5 b 22.1 a 39.9 ab 1.7 a 21.4 ab 14.4 bc 25.7 bc 25.8 abc 44.3 ab CS 24.5 a 49.2 a 13.0 ab 32.7 ab 25.2 a 41.9 a 10.6 a 24.7 ab 18.8 abc 32.8 abc 26.9 abc 43.7 ab
SC2 19.3 a 40.2 a 11.6 ab 16.9 b 15.9 a 19.2 b 5.5 a 9.5 b 17.8 abc 19.5 c 18.3 c 22.8 c C502 27.5 a 51.5 a 22.4 ab 31.4 ab 23.4 a 41.6 a 18.5 a 31.4 ab 30.3 ab 35.7 abc 28.5 abc 43.4 ab C506 21.2 a 53.5 a 17.5 ab 34.3 ab 14.2 a 40.3 ab 13.3 a 32.0 ab 30.6 ab 41.0 ab 23.3 bc 46.7 ab C4475-C 16.6 a 50.1 a 17.7 ab 28.5 ab 21.5 a 27.9 ab 5.4 a 24.6 ab 27.1 ab 31.8 abc 24.3 abc 30.9 bc RL 4.3 a 40.1 a -1.0 b 33.9 ab 24.9 a 40.5 ab 7.6 a 44.3 a 4.1 c 34.3 abc 25.2 abc 42.0 ab
Analysis of variance b R
NS a Means followed by the same letter in same rows do not differ significantly at P ≤ 0.05 (one-way-ANOVA, separated by Duncan
test). b The factors R and T refer respectively to rootstock and treatment. Significant effects are indicated by * = P < 0.05, ** = P < 0.01
and *** = P < 0.001, and NS indicates not significant difference.
Characterization in Greenhouse Conditions of Two Salt Tolerant Citrumelo
(Citrus paradisi Macf. x Poncirus trifoliata (L.) Raf.) Cultivars
60 the number of
ab ab of
50 a a the number of
leaves 30 a a leaves (%) b 140 b b Reduction
90 b 10 Percent
Fig. 3 Reduction in the number of leaves in response to salinity expressed relatively to control. (a) 35 mM NaCl; (b) 85 mM NaCl. Means represented by the same letter do not differ significantly at P ≤ 0.05 (one-way-ANOVA, separated by Duncan test). Vertical bars indicate the mean values ± SE (n = 6).
3.3 Effect of Salt Stress on Leaf Physiological Traits much greater tolerance of C4475-C and SC2 cultivars which reached respectively 90% and 85% the control
3.3.1 Water Content values of LCC. By contrast, C4475-A, CWH, CS and
Fig. 4 shows the changes in LWC (leaf water content) with respect to salt treatments. At 35 mM
C502 cultivars showed the lowest values at this level NaCl, almost all genotypes maintained more than 80%
(respectively 76%, 72%, 77% and 77%). At 85 mM LWC as compared to their respective controls, in
NaCl, differences were higher. For instance, RL contrast to 85 mM NaCl treatment which caused an
resulted in 79% LCC of the control, which important desiccation of leaves. However, the leaves
corresponds to three fold the average value obtained in of the cultivar SC1 showed a considerable reduction
the most sensitive cultivar, C4475-B (25%). The of water content even at low salinity (68%). ANOVA
authors should note also that SC2 maintained higher results revealed that both rootstock and salt treatment
leaf chlorophyll content in high salt stress condition factors had significant effects on LWC (P < 0.01) as
even though it was not significantly different from well as their interaction (P < 0.05). The authors should
other cultivars according to Duncan’s multiple range also note that RL showed a different behavior than
test.
other rootstocks tested, resulting in a slight increase in
3.3.3 Chloride Content
LWC (succulence) under salt stress compared to The concentration of Cl in leaves extracted after control condition. The relative LWC values for the
seven weeks of treatment was significantly (P < 0.001) latter were 105% and 109% respectively under 35 mM
increased under saline conditions (Table 4). Indeed, and 85 mM NaCl treatments.
control seedlings of all genotypes showed low levels
3.3.2 Chlorophyll Content of leaf Cl − content which ranged from 0.72% to 1.37% The LCC (leaf chlorophyll content) patterns in
DW, whereas the seedlings treated with 85 mM NaCl response to salt stress were similar to the ones of − showed an accumulation of Cl in their leaves ranging
LWC (Fig. 4). At both salt concentrations, RL from 2.48% to 3.22% DW which indicates a performed better than all citrumelo cultivars. However,
difference of three to four fold between the two when using a moderate salinity treatment, we noted a
treatments.
Characterization in Greenhouse Conditions of Two Salt Tolerant Citrumelo
(Citrus paradisi Macf. x Poncirus trifoliata (L.) Raf.) Cultivars
ll Content bc 80 ab
bc bc
c c c ll Content
50 ab ab ab
control) 75 c control)
Leaf Chlorophy 65
Leaf Chlorophy
Water Content (% of control) 70
Water Content (% of control) 40
Fig. 4 Changes in leaf water and chlorophyll contents expressed relatively to control values in response to saline treatments. (a, c) 35 mM NaCl. (b, d) 85 mM NaCl. Means represented by the same letter do not differ significantly at P ≤ 0.05 (one-way-ANOVA, separated by Duncan test). Vertical bars indicate the mean values ± SE (n = 6).
Similarly to the other traits studied, the comparison intermediate values at low salinity (35 mM). among the seedlings for leaf chloride contents reveals
4. Discussion
significant differences depending on the salt treatment concentration that was applied. As shown in the Table
Salt present in the irrigation solution considerably
4, many cultivars groups were identified using Duncan’s affected seedling growth and physiology in all the multiple range test. Generally, RL and C506 seedling
cultivars even at low concentration. Salt stress cultivars exhibited a lower leaf Cl − accumulation
symptoms are related to cellular toxicity and manifest compared to other cultivars whatever the condition is,
as chlorosis, leaf tip burn and defoliation. Such whereas the greatest accumulation was observed for
symptoms have been reported in earlier studies and CWH. The authors should note also that C506 showed
have been associated with the accumulation of toxic the highest leaf accumulation of Cl − when exposed to
ions such as chloride, sodium and boron in plant tissue high salt level (85 mM), although this cultivar showed − [10]. Cl was reported to be the most harmful element
Characterization in Greenhouse Conditions of Two Salt Tolerant Citrumelo
(Citrus paradisi Macf. x Poncirus trifoliata (L.) Raf.) Cultivars
Table 4 Effect of saline treatments on the accumulation of chloride ions in leaves of the ten rootstocks studied expressed as % of dry weight.
Leaf chloride content a (%DW)
C4475-A 1.37 a 1.82 bc 3.00 ab C4475-B 0.93 bc 2.19 ab 2.70 bcd
CWH 1.05 b 2.46 a 3.13 a SC1 0.94 bc 2.21 ab 2.91 abc CS 0.93 bc 2.05 abc 2.95 abc SC2 0.78 c 2.19 ab 2.88 abc C502 0.84 bc 1.70 bc 2.91 abc
C506 0.83 bc 1.96 abc 3.22 a
C4475-C 0.77 c 1.93 bc 2.58 cd
RL 0.72 c 1.63 c 2.48 d
Analysis of variance b R *** T *** R × T
a Means followed by the same letter in same rows do not differ significantly at P ≤ 0.05 (one-way-ANOVA, separated by Duncan test).
b The factors R and T refer respectively to rootstock and Treatment. significant effects are indicated by * = P < 0.05, ** = P < 0.01 and *** = P < 0.001, and NS indicates not significant difference.
for leaves [32, 33]. In the present study, RL, which accumulation on one hand and to the decrease in was used as a reference seedling, maintained low Cl − number of leaves, leaf water content and leaf
content in leaves and obviously showed less toxicity chlorophyll content on the other hand, given that these symptoms. Conversely, SC2 exhibited the least
last three effects may inevitably affect gas exchange toxicity symptoms among citrumelo cultivars although
and photosynthetic processes [37]. its leaves accumulated moderate amounts of Cl − .
The decrease in chlorophyll content under salt
Simultaneously to symptoms, a considerable stress conditions has been for long time a controversy growth inhibition was observed which was reflected in
for researchers. Different reasons were given, but the decreased plant height and biomass yield. Growth
most probable is the suppression of specific enzymes suppression was more apparent in some cultivars such
that are responsible for chlorophyll biosynthesis and as C4475-A and C4475-B conversely to SC2 and RL
the reduction in magnesium, iron and manganese [38, which showed respectively the greatest tolerance.
39]. On the other hand, the reduction in water content Previous works suggest that there are many was widely reported and had been described as a hypotheses to explain growth inhibition under salt
consequence of a water imbalance between the stress conditions. Most of these reports agree that
apoplast and symplast that leads to turgor decrease, growth reduction may be attributed to Cl − and Na + which in turn may cause growth reduction [40].
inhibitory effects [34] and to disturbance in However, many reports have indicated that tolerant physiological processes of the plant such as species can adjust their osmotic potential when photosynthesis and gas exchange [32, 35, 36]. In our
subjected to salt stress through the accumulation of case, both hypotheses can be accepted as we found
soluble compounds known as osmolytes and/or similar patterns for growth inhibition under saline
osmoprotectants [41, 42]. This hypothesis could be conditions to the ones observed for leaf Cl − valid for RL which maintained high water content and
Characterization in Greenhouse Conditions of Two Salt Tolerant Citrumelo
(Citrus paradisi Macf. x Poncirus trifoliata (L.) Raf.) Cultivars
simultaneously showed high growth rate and biomass Furthermore, other contributory features may enhance yield at the end of the experiment. It is also important
salinity tolerance as well such as the osmotic to note that seedlings of the same rootstock proved to
adjustment mechanism described above and/or the
be successful in maintaining high levels of proline + regulation of Na entry and translocation in plant under salt stress as shown by the findings of Balal et
tissue [51, 52]. The study of Gonzalez et al. [53], for al. [43].
example, have shown a higher capacity for Na + CWH and C506 accumulated much more leaf Cl − sequestration in root tissue vacuoles of Swingle
than the other cultivars, whereas the least citrumelo than in Rangpur lime, which could be a accumulation was observed in the salt-tolerant rational explanation for our results. rootstock RL followed by C4475-C. These data