Journal of Life Sciences Volume 6 Number (2)
J LS
Journal of Life Sciences
Volume 6, Number 7, July 2012 (Serial Number 51)
Contents
Biochemistry and Medical Science
Lipid Oxidation and Histamine Production in Atlantic Mackerel (Scomber scombrus) Versus Time and Mode of Conservation
Hanane Oucif, Smail Ali-Mehidi and Sidi-Mohamed El-Amine Abi-Ayad
In Vitro Regeneration of Commercial Sugarcane (Saccharum spp.) Cultivars in Nigeria
Sani Lawan Abdu, Mustapha Yahaya and Usman Inuwa Shehu
726 Validation of Analytical Method of Irbesartan Plasma in Vitro by High Performance Liquid
Chromatography-Fluorescence
Harmita, Yahdiana Harahap and I. Kadek Arya M.
732 Isolation, Phenotypic Identification and Fingerprint by RAPD-PCR of Oxalotrophoic
Burkholderia cepacia from Egyptian Fertile Soil
Hala Abou Shady, Mona Kilany Abd El Gawad, Qadria Genedi and Wafaa Farouk
740 The Effect of a Dietary Supplement Spirulina and Bifidobacterium adolescentis on the
Cholesterol-Lowering in Vitro and in Vivo
Amel Doumandji, Dahmane Alili and Abderrahmen Benzaiche
Attitudes and Practices of Health Care Providers Regarding the Management of Uncomplicated Malaria in Abidjan, Cote d’Ivoire
Frederic Nogbou Ello, Thomas Yapo Aba, Ismael Ouattara, Didier Koumavi Ekouévi, Offoue Kra, Pierre Aimé Assemian, Amath Wade, Serge-Paul Eholie and Emmanuel Bissagnene
On the Reproduction Number and a Presentation of Results for Infectious Diseases Models
Valeriy Dmitriy Perminov
Botany and Zoology
758 High Temperature and Abscisic Acid Modified the Profile of Anthocyanins in Grape (Vitis
vinifera L.)
Leonor Deis, Maria Inés de Rosas and Juan Bruno Cavagnaro
Substrate Type Affects Growth, Yield and Mineral Composition of Cucumber and Zucchini Squash
Mariateresa Cardarelli, Youssef Rouphael, Salem Darwich, Elvira Rea, Antonio Fiorillo and Giuseppe Colla
Effect of Compost Based Substrate and Mycorrhizal Inoculum in Potted Geranium Plants
Monica Tullio, Federico Calviello and Elvira Rea
The Breeding Status of the Glossy Ibis Plegadis falcinellus in the Lebna Dam in Cap Bon, Tunisia
Aymen Nefla, Ridha Ouni and Saïd Nouira
Survey on Parasites in Sparrows of Amol (Mazandaran Province, Iran)
Sina Faghihzadeh Gorji, Bahareh Shemshadi, Hamid Habibi, Sattar Jalali, Masoud Davary and Mohammadreza Sepehri
Interdisciplinary Researches
786 Incidence and Development of Powdery Mildew (Blumeria graminis f.sp. tritici) in the Czech
Republic in the Years 1999-2010 and Race Spectrum of This Population
Lubomir Vechet
Poverty and Agroforestry Adoption: The Cases of Mucuna pruriens and Acacia auriculiformis in Godohou Village (Southern Benin)
Emile N. Houngbo, Anne Floquet and Brice Sinsin
Modelling Stand Dynamics after Selective Logging: Implications for REDD and Carbon Pools Estimations from Forest Degradation
Adrien Njepang Djomo, Gode Gravenhorst, Anthony Kimaro and Marney Isaac
Aqua Forestry and Duck Integration in Tamil Nadu, India
Faroque Rahamathulla Sheriff
826 The Importance of Fiber Production for Conservation of Native Sheep and Goat Breeds and
Silkworm Lines in Turkey
Gursel Dellal, Zeynep Erdogan, Erkan Pehlivan, Feryal Soylemezoglu and Aysem Yanar
Journal of Life Sciences 6 (2012) 713-720
Lipid Oxidation and Histamine Production in Atlantic Mackerel (Scomber scombrus) Versus Time and Mode of Conservation
Hanane Oucif, Smail Ali-Mehidi and Sidi-Mohamed El-Amine Abi-Ayad Lab. of Aquaculture and Bioremediation, Dpt. of Biotechnology, University of Es-Sénia, Oran 31000, Algeria
Received: December 11, 2011 / Accepted: February 15, 2012 / Published: July 30, 2012.
Abstract: Lipids oxidation, histamine production and quality loss were studied according to storage time and temperature (ambient temperature (T amb ) 26 °C, 4 °C and –18 °C) in Atlantic mackerel (Scomber scombrus). Muscle pH, hydrolysis of phospholipids, content of primary (hydroperoxides), secondary lipid oxidation products (TBARS) and histamine were determined and compared with a sensory assessment. Atlantic mackerel is sensory acceptable, less than 24 hours at T amb , for up to 3 days at 4 °C and 3 months at –18 °C. Evolution of biochemical parameters with storage time and temperature showed significant differences (P < 0.05). Muscle pH increased from 5.99 to 6.13 at T amb , to 6.23 at 4 °C and to 6.04 at –18 °C. The highest content of TBARS is associated with a decrease in phospholipids and hydroperoxides contents and highest levels of sensory alteration. Histamine content exceeded the limit recommended by the Trade Algerian Ministry (10 mg/100g), after 24 hours at T amb , 5 days of storage at 4 °C only. Therefore, freezing storage has a preserving effect on lipid damage and histamine production and seems the best means of storage; if these species are not consumed during the two days following capture. Moreover, monitoring histamine production is more useful as sanitary index rather than spoilage parameter and the strategy used for measuring kinetic of lipid oxidation appear pertinent for determining the degree of oxidation.
Key words: Conservation, histamine, hydroperoxides, phospholipids, temperature, TBARS, Scomber scombrus.
1. Introduction most difficult obstacles to be avoided; not only during handling, but also during storage, even in frozen state
Seafood has attracted considerable attention as a and a major cause of sensory changes (rancidity), source of high amounts of important nutritional nutritional (loss of vitamins and essential fatty acids) components to human diet such as proteins of high and technology changes, without neglecting possible biological quality; it is also exceptionally rich in role of oxidation products, highly reactive and toxic polyunsaturated long chain fatty acids, in particular compounds, in some pathogenesis [2]. In addition, the omega 3 (PUFAs- ω3) and minerals [1]. consumption of fish from around the world is often However, it is well known to deteriorate rapidly, implicated in causing the consumer to several especially in hot countries which lack adequate pathological processes, which include histamine infrastructure of conservation. Indeed, immediately poisoning. The condition of food borne disease can after his capture, fish undergoes a natural process of vary; affecting several bodies functions and responds decomposition as a result of successive enzymatic and to ingestion of fish muscle with a high concentration chemical reactions and bacterial infections. Hydrolysis of histamine, formed by bacterial decarboxylation of and lipid oxidation in particular, is certainly one of the free histidine, while those fish appear somewhat
Corresponding author: Hanane Oucif, Ph.D., research field: altered. In addition, histamine is heat; no industrial
quality control. E-mail: [email protected].
Lipid Oxidation and Histamine Production in Atlantic Mackerel
(Scomber scombrus) Versus Time and Mode of Conservation
process can destroy [3]. These two process changes and unacceptable quality (C). This sensory assessment represent a real danger for consumer. Therefore it is
evaluate freshness by analyse certain aspect of general necessary to impose development of modern and
appearances (skin, external odour, gills, consistency traditional methods to ensure the quality of food.
and flesh odour).
The present experiment was carried out on Atlantic
2.3 Chemical Composition
mackerel (Scomber scombrus), one of abundant pelagic fatty fish species, from Scombridae family,
Evolution of pH values in fish muscle along the often implicated in cases of histamine poisoning and
storage time is carried out according to the method in the present work, biochemical and sensory changes
described by Özogul et al. [5]. The lipid fraction is that take place during storage of Atlantic mackerel are
extracted from 5 g of sample after homogenisation in investigated by evaluated lipid damage and histamine
chloroform/methanol (2:1, v/v), according to the value during storage period at –18 °C, 4 °C and
Folch et al. [6] method. Then phospholipids content is ambient temperature and complemented by sensory
measured by colorimetric method of Stewart [7] based analysis.
on formation of a complex between phospholipids and The objective is to identify the critical points of
ammonium ferrothiocyanate. A standard curve is made different treatments of preservation, at low with standard phosphatidylcholine in chloroform and temperatures (4 °C and –18 °C) and ambient
results are expressed as g phosphatidylcholine temperature (26 ± 3 °C) and to develop guidelines and
equivalents per kg of wet weight (g Eq. PC/kg W.W.). index that reflect freshness and can be measured,
2.4 Lipid Damage Measurements using simple and rapid methods. Lipid hydroperoxides content expressed as mmoles
2. Materials and Methods
of cumene hydropeoxide equivalents (CuOOH Eq) per
2.1 Raw Material, Sampling and Processing kg of wet weight is determined according to the
method of Eymard & Genot [8]. The secondary Fresh Atlantic mackerel (Scomber scombrus,
compounds of lipid oxidation (TBARS) are 140.51 ± 14.10 g) are bought from fishing port of
determined by a method of Genot [9]. Results are Oran and kept on ice till arrival to the laboratory (6
expressed as mg of malonaldehyde equivalents per kg hours). Part of the fish is directly packaged in
of wet weight.
polyethylene bags and immediately frozen at –18 °C, and the other is divided into two batches, one is stored 2.5 Histamine Analysis
at ambient temperature (26 ± 3 °C, and at 4 °C at night) Histamine quantification in fish muscle is carried and the second one is stored at 4 °C (24 h/24 h).
out by a colorimetric method of Patange et al. [10], Samples are taken for analysis at day 1, 2, 3, 5, 7 and
based on the interaction between the imidazole ring
9 from storage at 4 °C and ambient temperature and at and p-phenyldiazonium sulfonate. Concentration of day 15, 30, 60, 90 and 120 from storage at –18 °C. In
histamine in sample is obtained from the standard each batch at both temperatures, analyses are carried
curve and expressed as mg per 100g of wet weight out on five mackerels.
(mg/100g W.W.).
2.2 Sensory Analysis
2.6 Statistical Analysis
Sensory analysis is conducted according to the All data are expressed as the mean ± SD and are traditional guidelines [4]. Four categories are ranked:
statistically compared by one-way variance analysis highest quality (E), good quality (A), fair quality (B)
(ANOVA) and Tukey HSD test (for homogeneous
Lipid Oxidation and Histamine Production in Atlantic Mackerel
(Scomber scombrus) Versus Time and Mode of Conservation
variance) and by non-parametric variance analysis of
Table 1 Comparative sensory acceptability of mackerel’s
Kruskal-Wallis and Mann-Withney U test (for non
lots.
homogeneous variance), after verification of variance Days
Storage
homogeneity by Hartley test. 1 2 3 5 7 9 15 30 60 90 120 At 26 °C/4°C E C C
3. Results and Discussion
3.1 Sensory Assessment E: highest quality; A: good quality; B: fair quality; C: unacceptable quality.
At ambient temperature (26 °C)/chilled (4 °C), sensory evaluation of mackerel indicates that the
was proposed in mackerel stored at –20 °C, preceded keeping time is less than 24 hours. Comparable times
by freezing at –80 °C for 24 hours [27]. have been reported within 24 hours [11], 7 hours at
3.2 The pH Evolution
30 °C [12] and 23 hours at 21-27 °C [13] in sardine and 12 hours at 20-25 °C, with anchovies [14]. Upon
Initial pH value of muscle is 5.99. In mackerel, red storage, fish has a rancid smell increasingly strong; as
muscle is well developed and contains more glycogen
a result of biochemical changes such as degradation of than white muscle [28]. It appears that the breakdown ATP [15] and formation of propanol, pentanol and
of glycogen to lactic acid makes pH of fish after ethanol; products of lipid peroxidation [16, 17]. These
capture, acid, especially in mackerel [29]. Evolution of rancid odors become sulfuric smells, following the
pH is dependent on mode of storage (P < 0.05) (Fig. 1).
As a result, pH values are lower at low temperatures. at ambient temperature [18]. In addition, Cheuk et al.
growth of microorganisms producing H 2 S particularly
The increase in pH is due to production of volatile [19] report that there is a strong correlation between
bases (NH 3 , TMA and DMA) and an accumulation of productions of volatile nitrogenous bases, responsible
peptides and amines [30]. It has been reported in for unpleasant odors and bacterial growth. Advanced
sardine [31] and mackerel [16]. However, the decrease decay in some cases is due to the relative content of
in pH is recorded in early days of storage under lipid species (fatty fish deteriorates quickly) [20].
refrigeration (4 °C). This is not observed at ambient During storage at 4 °C, mackerel is considered unfit
temperature because bacterial multiplication is rapid for consumption beyond three days. These delays are
with a short or no lag phase [13]. The small difference consistent with other results, including 3 days in
in pH in terms of absolute values, from the beginning sardine [21], 4 days in Trachurus mediterraneus [22].
Delays a little longer were reported: 5 or 6 days in T. trachurus [23, 24], 5 days [11, 25] in sardine and 4-6 days in anchovy [14]. These relatively short periods of acceptability can be explained by the predominance of psychrotrophic bacteria and gram-negative psychrophilic in fish caught in cold and temperate waters [26]. These differences in shelf life can be attributed to fishing areas that influence the character of psychrophilic or mesophilic natural flora of fish
[16]. By way of freezing (–18 °C), the samples are Fig. 1 Temporal evolution of pH of Atlantic mackerel (S.
scombrus) of lots A, B and C. Histogram’s (same storage
very good for 2 months and unacceptable beyond 3
conditions) with different indices are significantly different
months (Table 1). A preservative period of 5 months
(P < 0.05).
Lipid Oxidation and Histamine Production in Atlantic Mackerel
(Scomber scombrus) Versus Time and Mode of Conservation
to the end of different modes of preservation; the same way so; phospholipases. Lipolytic activity, incentives Laghmari & El Marrakchi [32] to conclude
although small, continues during storage in the frozen that pH cannot be considered as reliable for state [37] and can become the limiting factor for assessment of spoiled fish.
duration of freezing, this is especially true for fatty fish [38].
3.3 Lipid Damage Analysis Hydroperoxides record a maximum on day 2
Initially, phospholipids content (PL) is 260.72 g Eq. (propagation phase) (P < 0.05) at ambient temperature PC/kg W.W.). Hydrolysis of PL is the main factor of
and 4 °C, respectively. During the first three days, lipids degradation [17, 33] and particularly for
TBARS (Thiobarbituric acid reactive substances) are mackerel stored at ambient temperature/chilled and
relatively low and stable; this observation was also refrigerated at 4 °C. Indeed, at ambient temperature,
reported by Simeonidou et al. [24], during ice storage we are witnessing a breakdown of weak bonds [34].
of fatty fish. Followed by, a decrease of However, in mackerel, phospholipases seem inactive
hydroperoxides, due to their interactions with from the seventh day at 4 °C (Fig. 2). Indeed, it
biological components or their decomposition [39]. appears that the activity of phospholipases decreases
Indeed, after 5 days, spread of TBARS is launched (P after 7 days of storage of mackerel at 2-3 °C [35],
< 0.05) (Figs. 3, 4). These secondary products of lipid which probably the explanation of a sudden increase
Hydroperoxides
in the amount of PL. However, she found the initial
(mmol Eq. CuOOH/kg W.W.)
PL value at Day 9; this suggests that it is likely due to interindividual variations within the species [36] or possibly an accumulation of phospholipids after total inactivation of phospholipases. Moreover, there is any significatively difference between storage at 26 °C and at 4 °C (P ≥ 0.05) During freezing at –18 °C, the decrease (P < 0.05) then stability (from 2 months) of
the PL content of mackerel are likely due to excessive Fig. 3 Temporal evolution of hydroperoxides content
(mmol Eq. CuOOH/kg W.W.), of Atlantic mackerel (S.
lowering of temperature, which has the effect of
scombrus) of lots A, B and C. Histogram’s (same storage
slowing down and braking hydrolysis of PL and in
conditions) with different indices are significantly different (P < 0.05).
Phospholipids (g Eq. PC/kg W.W.)
TBARS (mg Eq. MDA/kg W.W.)
Fig. 2 Temporal evolution of phospholipids content (g Eq. Fig. 4 Temporal evolution of TBARS content (mg Eq. PC/kg W.W.), of Atlantic mackerel (S. scombrus) of lots A, B
MDA/kg W.W.), of Atlantic mackerel (S. scombrus) of lots A, and C. Histogram’s (same storage conditions) with different
B and C. Histogram’s (same storage conditions) with indices are significantly different (P < 0.05).
different indices are significantly different (P < 0.05).
Lipid Oxidation and Histamine Production in Atlantic Mackerel
(Scomber scombrus) Versus Time and Mode of Conservation
oxidation interact with other molecules, including the synthesis and activity of histidine decarboxylase proteins, and/or degraded to other substances not
(from 2.5 to 6.5), reached rapidly in the flesh of accessible to analysis [40], which would explain the
mackerel [16]. At 4 °C, accumulation of histamine shift of TBARS values observed at day 9 at 4 °C. Such
exceeds the threshold, only after 5 days of storage and decrease in amounts of TBARS was also observed
never at –18 °C (Fig. 5). Indeed, effectiveness of icing after 9 days of storage of sardines on ice [25]. To this
in control of histamine production has already been end, we can say that time course of lipid peroxidation
proven on sardine [13], mackerel [16, 44], bonito [45] in mackerel follows the general pattern of the
and other species. Several studies agree that kinetics of lipid oxidation, in other words; TBARS
histamine-producing bacteria are mesophilic and at content increases or decreases in parallel with the
temperatures between 0 and –20 °C, microorganisms decreasing or increasing of hydroperoxides content,
are in a slowed down state of life and can not multiply respectively. This pattern of evolution is also observed
and invade fish muscle [46]. However, other authors during freezing, where lipid oxidation persists. [47] have shown that histamine is synthesized at Moreover, hydroperoxides and TBARS contents are
significant levels at temperatures as low as 2-5 °C. not dependent on mode of storage (P ≥ 0.05). In
Photobacterium histaminum and agreement with Rodriguez et al. [41], it seems that the
Indeed,
Photobacterium phosphorum show a decarboxylase refrigerated temperature storage is not sufficient to
activity at 4 °C and even at –20 °C [48]. However, in inhibit or slow formation of oxidation products in
frozen mackerel, we see a decrease in histamine small pelagic fish. Probably, the acidic pH of these
content after 2 months of freezing. According to Sato fish would promote activation of haem proteins and
et al. [49], histamine content depends on release of iron that are pro-oxidant agents [42]. Then,
histamine-producing bacteria as well as on degrading freezing is a good way to increase shelf life of
bacteria (bacteria with histaminase activity) [50]. products, because lipid oxidation rate and pro-oxidant
4. Conclusions
effect of haem pigments are reduced at low temperatures. However, it is necessary to reach
Finally, freezing storage has a preserving effect on temperatures of –40 °C to completely stop oxidation,
lipid damage and histamine production and seems the because at a temperature of –15 °C, formation of
best means of storage; if these species are not peroxides stays possible [43].
consumed during the two days following capture. The extent of primary lipid oxidation products such
3.4 Histamine Production
A few hours after capture, initial histamine content in mackerel is already high (6.39 mg/100g). It exceeds the toxic level of histamine of 10 mg/100g muscle rapidly after 24 h of storage at ambient temperature. In agreement with Mendes [44], Ababouch et al. [13] and Chaouqy & El Marrakchi [14], this excessive production is probably due to a high amount of histidine in red muscle of fish (especially the
Scombridae), a large proliferation of mesophilic
Fig. 5 Temporal evolution of histamine content (mg/100g) of Atlantic mackerel (S. scombrus) of lots A, B and C.
bacteria capable of histidine decaboxylation and an
Histogram’s (same storage conditions) with different
optimum temperature (26 °C) and a suitable pH for
indices are significantly different (P < 0.05).
Lipid Oxidation and Histamine Production in Atlantic Mackerel
(Scomber scombrus) Versus Time and Mode of Conservation
as hydroperoxides is considered to be one that would accurate estimate of the keeping time of the species provide the most relevant information on the intensity
studied and to develop a representative index of the of lipoperoxidation, as long as precautions are taken to
real fish freshness.
prevent their rapid decomposition [51]. In addition,
Acknowledgments
quantifying secondary lipid oxidation products (TBARS), more stable compounds, is considered
This work was supported by National Agency for a more reliably to estimate the level of lipid oxidation.
development of research in Health (A.N.D.R.S.). The By cons, given the bell-shaped curve, it does not
authors express their gratitude to all members of determine promptly the level of lipid oxidation [17].
Laboratory of Aquaculture and Bioremediation Nevertheless, it seems that the peaks recorded in
(Department of Biotechnology, University of Es-Sénia, TBARS correspond to a decrease in hydroperoxides
Oran, Algeria).
content and very advanced sensory impairments.
References
Therefore, the strategy used, measuring kinetic of
phospholipids hydrolysis and lipid oxidation products, F. Medale, Caractéristiques nutritionnelles des poissons et facteurs de variations, Aquaculture 79 (2005) 87-93.
appear pertinent for determining; degree of fish lipids [2] J. Cillard, P. Cillard, Mécanismes de la peroxydation oxidation.
lipidique et des anti-oxydations, Oléagineux, Corps Gras, Accumulation of histamine is significantly slowed
Lipides 13 (2006) 24-29.
when icing occurs but does not correlate with sensory C. Boulay, Intoxications histaminiques: Poissons en cause, conditions de survenue et mécanismes
impairment. By cons, under conditions of storage at pathogéniques actuellement connus, Thèse de Doctorat, ambient temperature, the correlation is good. Indeed,
Ecole Nationale de Vétérinaire de Nantes, 1999. spoilage flora mainly formed by Shewanella
[4] Council Regulation, Official Journal of the European putrefaciens Communities, No, C84, 1990, p. 69. and Pseudomonas that are mesophilic and [5] Y. Özogul, G. Özyurt, F. Özogul, E. Kuley, A. Polat,
psychrophilic, but little or no histidinolytic. At low Freshness assessment of European eel (Anguilla anguilla) temperatures, the activity is still possible, so there are
by sensory, chemical and microbiological methods, Food many sensory impaired but histamine accumulates in
Chemistry 92 (2005) 745-751.
[6] flesh at low levels as long as the Enterobacteriaceae J. Folch, M. Lees, G.H.S. Sloane Stanley, A simple method for the isolation and purification of total lipids
(mesophilic), leading producers of this amine are from animal tissues, Journal of Biological Chemistry 226 inhibited [52]. According to Sato et al. [53], Mendes
(1957) 497-509.
[44] and Chaouqy & El Marrakchi [14], the lack of [7] J.C. Stewart, Colorimetric determination of phospholipids correlation between accumulation of histamine and with ammonium ferrothiocyanate, Analytical
Biochemistry 104 (1980) 10-14.
sensory impairment, said that monitoring histamine [8] S. Eymard, C. Genot, A modified xylenol orange method
production is more useful as sanitary index rather than to evaluate formation of lipid hydroperoxides during spoilage parameter.
storage and processing of small pelagic fish, European
For each analysis, the number of specimens Journal of Lipid Science and Technology 105 (2003) 497-501.
collected (n = 5) was not sufficient to avoid changes in
C. Genot, Some factors influencing TBA test, Report of biochemical composition between individuals
Diet-Ox Project (AIR III-CT-92-1577), 1996. manifested by high standard deviations. It would be
[10] S.B. Patange, M.K. Mukundan, K. Ashok Kumar, A preferable and very appropriate to increase number of
simple and rapid method for colorimetric determination of histamine in fish flesh, Food Control 16 (2005)
sample analyzed and make the first analyze on fishing
465-472.
boats to monitor the formation of these compounds [11] M. Barhoumi, Essai de conservation de la sardine par la from T 0 (from capture). This could lead us to a more
glace et l’eau de mer refroidie, Institut Scientifique des
Lipid Oxidation and Histamine Production in Atlantic Mackerel
(Scomber scombrus) Versus Time and Mode of Conservation
Pêches Maritimes, Travaux et Documents N°26,
288-292.
V. Losada, C. Piñeiro, J. Barros-Velázquez, S.P. Aubourg, [12] M.A. Afilal, H. Daoudi, S. Jdaini, A. Asehraou, A. Bouali,
Casablanca, Morocco, 1980.
Inhibition of chemical changes related to freshness loss Study of the histamine production in the red flesh fish
during storage of horse mackerel (Trachurus trachurus) (Sardina pilchardus) and a with flesh fish (Dicentrarchus
in slurry ice, Food Chemistry 93 (2005) 619-625. punctatus ), Turkish Journal of Fisheries and Aquatic
[24] S. Simeonidou, A. Govaris, K. Vareltzis, Quality Sciences 6 (2006) 43-48.
assessment of seven Mediterranean fish species during [13] L.H. Ababouch, L. Souibri, K. Rhaliby, O. Ouahdi, M.
storage on ice, Food Research International 30 (1998) Battal, F.F. Busta, Quality changes in sardines (Sardina
479-484.
pilchardus) stored in ice and ambient temperature, Food [25] M.L. Nunes, I. Batista, R. Morao de Campos, Physical, Microbiology 13 (1996) 123-132.
chemical and sensory analysis of sardine (Sardina [14] N.E. Chaouqy, A. El Marrakchi, Aspects chimiques et
pilchardus ) stored in ice, Journal of the Science of Food bactériologiques de l’anchois (Engraulis encrasicholus)
and Agriculture 59 (1992a) 37-43. entreposé sous glace et à moyenne température
A. El Marrakchi, R. Azlaf, M. Barhoumi, Quelques (20-25 °C), Revue de Médecine Vétérinaire 156 (2005)
aspects bactériologiques de la sardine (Sardina 341-349.
pilchardus ) récemment capturée et conservée aux [15] R.B. Hughes, N.R. Jones, Measurement of hypoxantine
températures de réfrigération, Consultation d’Experts de concentration in canned herring as an index of the
la FAO Sur la Technologie du Poisson en Afrique, freshness of the raw material, with a comment on the
Casablanca, Maroc, FAO, Rapport des Pêches, N° 268, flavor relations, Journal of the Science of Food and
1982, pp. 45-53.
Agriculture 17 (1966) 434-436. [27] L. Stodolnik, A. Stawicka, G. Szczepanik, S.P. Aubourg, [16] M. Bennour, A. El Marrakchi, N. Bouchriti, A. Hamama,
Rancidity inhibition study in frozen whole mackerel M. El Ouadaa, Chemical and microbiogical assessments
(Scomber scombrus) following flaxseed (Linum of mackerel (Scomber scombrus) stored in ice, Journal of
usitatissimum ) extract treatment, Grasas y Aceites 56 Food Protection 54 (1991) 789-792.
(2005) 198-204.
[17] S. Eymard, Mise en évidence et suivi de l’oxydation des [28] Y. Shimizu, H. Toyohara, T.C. Lanier, Surimi production lipides au cours de la conservation et de la transformation
from fatty and dark-fleshed fish species, in: T.C. Lanier, du chinchard (Trachurus trachurus): Choix des procédés,
C.M. Lee (Eds.), Surimi Technology, Marcel Dekker Inc., Thèse de Doctorat, Université de Nantes, 2003.
New York, 1992, pp. 181-207.
[18] H.H. Huss, Le poisson frais: Qualité et altérations de la [29] M. Sainclivier, L’Industrie Alimentaire Halieutique, Tech. qualité, Manuel de Formation Préparé Pour le Programme
and Bull. Scien., De l’École Nationale de Rennes, 1983, de Perfectionnement F.A.O./DANIDA Sur la
pp. 27-263.
Technologie du Poisson et le Contrôle de Qualité, [30] J.M. Ryder, D.H. Buisson, D.N. Scott, G.G. Flether, Collection FAO, Pêche, Rome, Italie, 1988.
Storage of new Zeland jack mackerel in ice: Chemical, [19] W.L. Cheuk, G. Finne, R. Nickelson, Stability of
microbiological and sensory assessment, Journal of Food adenosine deaminase and adenosine monophosphate
Science 49 (1984) 1453-1456.
deaminase during ice storage of pink and brown shrimp
A. El Marrakchi, N. Bennour, N. Bouchriti, A. Hamama, from the Gulf of Mexico, Journal of Food Science 44
H. Tagafait, Sensory, chemical and microbiological (1979) 1625-1628.
assessments of Moroccan sardines (Sardina pilchardus) [20] S.P. Aubourg, A. Rodriguez, J.M. Gallardo, Rancidity
stored in ice, Journal of Food Protection 53 (1990) development during frozen storage of mackerel (Scomber
600-605.
scombrus ): Effect of catching season and commercial
H. Laghmari, A. El Marrakchi, Appréciation presentation, European Journal of Lipid Science and
organoleptique et physico-chimique de la crevette rose Technology 107 (2004) 316-323.
Parapenaeus longirostris (Lucas, 1846) conservée sous [21]
F. Özogul, Y. Özogul, Biogenic amine content and glace et à température ambiante, Revue de Médecine biogenic amine quality indices of sardines (Sardina
Vétérinaire 156 (2005) 221-226.
pilchardus) stored in modified atmosphere packaging and [33] J.B. Rossell, Measurement of rancidity, in: J.C. Allen, R.J. vacuum packaging, Food Chemistry 99 (2006) 574-578.
Hamilton (Eds.), Rancidity in Foods, Elsevier, New York, [22] R. Mbarki, S. Sadok, I. Barkallah, Quality changes of the
1989, pp. 23-52.
Mediterranean horse mackerel (Trachurus mediterraneus)
H. Chan, Autooxidation of Unsaturated Lipids, Academic during chilled storage: The effect of low-dose gamma
Press, New York, 1987.
irradiation, Radiation Physics and Chemistry 78 (2009) [35] K.T. Hwang, J.M. Regeinstein, Characteristics of
Lipid Oxidation and Histamine Production in Atlantic Mackerel
(Scomber scombrus) Versus Time and Mode of Conservation
mackerel minces lipid hydrolysis, Journal of Food temperatures, Journal of Food Biochemistry 23 (1999) Science 58 (1993) 79-83.
33-43.
[36] S.P. Aubourg, Damage detection in horse mackerel [45] M.A. Mazorra-Manzano, R. Pacheco-Aguilar, E.I. (Trachurus trachurus) during chilled storage, Journal
Diaz-Rojas, M.E. Lugo-Sanchez, Post mortem changes in of the American Oil Chemists Society 78 (2001a)
black skipjack muscle during storage in ice, Journal of 857-862.
Food Science 65 (2000) 774-779.
[37] A.J. De Koning, T.H. Mol, Rates of free fatty acid [46] M.P. Doyle, L.R. Beuchat, T.J. MontVille, Food formation from phospholipids and neutral lipids in frozen
Microbiology: Fondamentals and Frontiers, ASM Press, cape hake (Merlussius spp) mince at various temperatures,
Washington DC, 1997, p. 94.
Journal of the Science of Food and Agriculture 50 (1990)
C. Grau, D. Sanchez, A. Zerpa, O. Vallenilla, O. Berti, 391-398.
Study of microflora associated with histamine formation [38]
G. Özyurt, A. Polat, B. Tokur, Chemical and sensory in sardines (Sardinella aurita), Revista changes in frozen (–18 °C) wild sea bass (Dicentrarchus
Cientifica-Facultad de Ciencias Veterinarias 13 (2003) labrax ) captured at different fishing seasons, International
199-204.
Journal of Food Science and Technology 42 (2007) [48] M. Okuzumi, S. Okuda, M. Awano, Isolation of 887-893.
psychrophilic and halophilic histamine-forming bacteria [39] S.P. Aubourg, Review: Recent advances in assessment of
from Scomber japonicas, Bulletin of the Japanese Society marine lipid oxidation by using fluorescence, Journal of
of Scientific Fisheries 47 (1981) 1591-1598. the American Oil Chemists Society 76 (1999) 409-419.
[49] T. Sato, T. Fujii, T. Masuda, M. Okuzumi, Changes in [40] S.P. Aubourg, I. Medina, Influence of storage time and
numbers of histamine-metabolic bacteria and histamine temperature on lipid deterioration during cod (Gadus
content during storage of common mackerel, Fisheries morhua ) and haddock (Melanogrammus aeglefinus)
Science 60 (1994) 299-302.
frozen storage, Journal of the Science of Food and [50] S.L. Taylor, Histamine food poisoning: Toxicology and Agriculture 79 (1999) 1943-1948.
clinical aspects, Critical Reviews in Toxicology 17 (1986) [41]
A. Rodríguez, N. Carriles, J.M. Cruz, S.P. Aubourg,
91-128.
Changes in the flesh of cooked farmed salmon [51] M. Laguerre, L.J. Lopez-Giraldo, J. Lecomte, M. Pina, P. (Oncorhynchus kisutch) with previous storage in slurry
Villeneuve, Outils d’évaluation in vitro de la capacité ice, LWT-Food Science and Technology 41 (2008)
antioxydante, Oléagineux, Corps gras, Lipides 14 (2007) 1726-1732.
278-292.
[42] C. Genot, A. Meynier, A. Riaublanc, J.M. Chobert, [52] L.H. Ababouch, S.C. Rafaramandimy, V.C. Rahanjavelo, Protein alterations due to lipid oxidation in multiphase
A. Razafindramonjy, A.R. Rabesalama, R. Ducaud, systems, in: A. Kamal-Eldin (Ed.), Lipid Oxidation
Construction et expérimentation de conteneurs et couffins Pathways, AOACS Press Champaign, 2003, pp.
isothermes pour la pêche artisanale à Madagascar, Les 265-292.
actes de l’Institut Agronomique et Vétérinaire Hassan II [43] P.J. Ke, R.G. Ackman, B.A. Linke, D.M. Nash,
12 (1) (1992) 29-35.
Differential lipid oxidation in various parts of frozen [53] T. Sato, M. Okuzumi, T. Fujii, Evaluation of polyamines mackerel, Journal of Food Technology 12 (1977) 37-47.
of common mackerel during storage as indicators of [44] R. Mendes, Changes in biogenic amines of major
decomposition, Journal of the Food Hygienic Society of Portuguese bluefish species during storage at different
Japan 36 (1995) 743-747.
Journal of Life Sciences 6 (2012) 721-725
In Vitro Regeneration of Commercial Sugarcane (Saccharum spp.) Cultivars in Nigeria
1 2 Sani Lawan Abdu 3 , Mustapha Yahaya and Usman Inuwa Shehu 1. College of Engeneering Science Technology, Federal Polytechnic Kazaure, PMB 5004, Kazuare, Nigeria
2. Department of Biological Sciences, Bayero University Kano, PMB 3011, Kano, Nigeria 3. Department of Plant Science, Institute for Agricultural Research, Ahmadu Bello University, PMB 1044, Zaria, Nigeria
Received: September 11, 2011 / Accepted: November 04, 2011 / Published: July 30, 2012.
Abstract: The effect of different concentrations of 6-benzylaminopurine (BA) with or without 0.2 mg/L NAA on in vitro regeneration of sugarcane (Saccharum spp.) cultivars SP726180, B47419, M1176/77 and M2119/88 were evaluated. Leaf base explants were cultured on Murashige and Skoog (MS) basal medium supplemented with 3.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) for 4 weeks. Thereafter, induction of somatic embryogenesis was observed following the transfer of resulting calli to 2,4-D-free medium for another 4 weeks. Regeneration was achieved by transfer of the embryogenic calli to regeneration media fortified with different concentrations of BA ± α-naphthylacetic acid (NAA). The number, length and vigor of shoots produced in all the genotypes were highest on media supplemented with 1.0 and 1.5 mg/L BA with and without 0.2 mg/L NAA. Among the genotypes tested, B47419 and M1176/77 recorded highest number of shoots, while maximum shoot length and crop vigor was obtained with M1176/77. Induction of callus with 3.0 mg/L 2,4-D and its subsequent incubation on 2,4-D-free media, followed by regeneration on media supplemented with 1.0 or 1.5 mg/L BA with 0.2 mg/L NAA was found to be efficient for in vitro regeneration of the sugarcane genotypes used in this study. This protocol could be applied for micropropagation of other elite genotypes.
Key words : Saccharum spp., leaf explant, 2,4-D, callus, BA, NAA, regeneration.
1. Introduction has failed to keep pace with the growing demand for improved varieties, due to limitations imposed by
Sugarcane (Saccharum spp.) is an important crop cytogenetic complexity of sugarcane [1]. for many tropical and subtropical countries which In vitro propagation offers a faster means of account for aproximately 75% of global sugar producing large number of disease-free plants and is a production. Efficient photosynthesis and biomass viable tool for improvement of genetic traits in
production make this graminae a good candidate for sugarcane using transgenic technologies. These
green energy generation and other industrial technologies have recently complement traditional
processing. It is an important cash crop in Nigeria, breeding by offering the possibilities of transferring
cultivated for both vegetable and industrial uses. single trait while breaking the barrier that limits
Propagation of sugarcane using stem cutting has always been associated with the problem of traditional breeding. An important prerequisite for transmission of pathogens from one generation to
rapid clonal propagation and development of genetic another which results in rapid degradation of transformation system in sugaracane, is the ability to improved varieties. Moreover, conventional breeding
develop a stable morphogenic culture. In sugarcane, this can be achieved through somatic embryogenesis
Corresponding author: Sani Lawan Abdu, M.Sc., research and organogenesis [2]. Since embryogenic culture fields: plant tissue culture and genetic transformation. E-mail: allow for cyclic recovery of more plants and somatic
In Vitro Regeneration of Commercial Sugarcane (Saccharum spp.) Cultivars in Nigeria
embryos are known to be good candidates for genetic
2.3 Callus Induction and Plant Regeneration transformation of sugarcane, regeneration via somatic
Leaf cylinders provided by immature leaf rolls and embryogenesis is an attractive option for both rapid
apical meristem were used for callus induction. Apical clonal propagation and genetic transformation of
portions of healthy shoots were stripped to the sugarcane. Sugarcane regeneration from callus culture
terminal bud and washed with sterilized distilled water in has been widely reported [3-6]. Since genotype and
and surface sterilized by initial dipping in 70% the type and concentration of exogenous hormones
ethanol (v/v) for 2 minutes followed by treatment with used, tremendously affect morphogenesis in sugarcane
20% commercial bleach (containing 3.5% sodium and other species, development of genotype specific
hypochlorite w/v) for 20 minutes, thereafter the protocol is critical for rapid propagation of selected
materials were rinsed 3 times in sterile double distilled sugarcane cultivars. To date no report on successful
water. The outer immature leaf rolls were removed regeneration of sugarcane cultivars used in Nigeria.
under aseptic condition using a sterile forcep and This paper report the effect of BA and NAA on in
surgical blade. Explants were cut into 10 mm vitro regeneration of four commercial cultivars in
segments and planted on sterilized calllus induction Nigeria.
medium consisting of MS basal medium
2. Materials and Methods supplemented with 3.0 mg/L 2,4-D. Ten explants
were cultivated in Petri dish (11 mm diameter)
2.1 Plant Materials containing 10 mL of the callus induction medium and Petri dishes were used per treatment. Cultures were
Four sugarcane genotypes were used in this study, kept in the dark for 4 weeks after which were
two (SP726180 and B47/419) were obtained from tranferred to fresh medium for further callus
experimental site of National Sugar Development proliferation. After eight weeks of culture in the
Company (NSDC), Hadejia. While M2119/88 and presence of 2,4-D, the resulting calli were
M1176/77 were obtained from sugarcane programme transferred to auxin-free media for another four weeks
of Jigawa Research Institute, Jigawa State, Nigeria. to induce embryogesis. After 4 weeks of culture on
Cane setts were established on screening plots at auxin-free media, ramdomized embryogenic callus
Jigawa Research Institute, after basal application of peices (25 ± 1 mg) were transferred to culture vessels Nitrogen fertilizer (100 kg/ha). The plots were
(125 × 25 mm) containing 35 mL of regeneration watered every other two days and growing tips of six
media consisting of MS meduim modified by addition month old plants were used as source of explants. The
of 1.0, 1.5 or 2.0 mg/L of BA with or without 0.2 in viro study was carried out in the Plant mg/L NAA. Ten vessels were used for each treatment
Biotechnology Laboratory, Jigawa Research Institute, and the experiment was laid in a completely Jigawa State, Nigeria
randomized design with 3 replications. Cultures were
2.2 Medium and Culture Condition incubated in growth chamber under 16-hrs photoperiod for eight weeks. The number, length and
The meduim used in this study was Murashige and vigor of the plantlets regenerated for each treatment Skoog (MS) [7] basal meduim and was suplemented
were recorded.
with 30% sucrose, pH was adjusted to 5.8 with 1 M
2.4 Rooting and Acclimatization
KOH and solidified with 8% agar before autoclaving for 15 minutes at 121 °C and 15 psi. All cultures were
Plantlets with length 4-6 cm were succesfully incubated at 29 ± 2 °C.
rooted on rooting media consisting of MS basal
In Vitro Regeneration of Commercial Sugarcane (Saccharum spp.) Cultivars in Nigeria
medium supplemented with 60% sucrose and 1.5
3. Result and Discussion
mg/L of Indole-butyric acid (IBA). Plantlets with well Swelling of explants was observed after 2-3 days of
developed roots were transferred to poly-pots (250 × culture (Fig. 1Aa). Callus initiation started after 2
125 mm) containing sterilized river sand and manure weeks of culture from the cut edges and injured sites
(2:1) under high humidity (> 90%) created by and gradually covered the explants (Fig. 1Ab). A close
covering with transparent polythene sheet. The observation of the callus showed two major types; the
plantlets were watered every day with half-strength compact nodular embryogenic callus which appeard MS macro and micro salts. The humidity was reduced
white to cream in colour (Fig. 1Ac), and the pale gradually by punching hole on the polythene sheet,
yellow nonembryogenic callus (Fig. 1Ab). Such types until it was finally removed after 2 weeks. of calli were also reported in sugarcane by Ch.
Acclimatization operations were carried out in the Gandonuo et al. [9]. The embryogenic callus greenhouse.
eventually differenciated into somatic embryos
2.5 Statistical Analysis following 4 weeks of culture on auxin free medium
and the embryos developed into shoots by given rise The data collected were subjected to analysis of
to coleoptile followed by the elongation of the first variance (ANOVA) and means were saperated using
leaf out of the coleoptile (Fig. 1B). In sugarcane and Duncan Multiple Range Test [8].
other monocots, high concentration of 2,4-D is
Fig. 1 A-D: Stages for in vitro regeneration of sugarcane (Saccharum spp.) via somatic embryogenesis. A: callus induction on inmature leaf in the presence of 3.0 mg/L 2,4-D (EP: explant; NEC: non-embryogenic callus; EC: embryogenic callus); B: plantlets regeneration from embryogenic callus (GSE: germinating somatic embryo); C: in viro plantlets; D: acclimatized seedlings in greenhouse.
In Vitro Regeneration of Commercial Sugarcane (Saccharum spp.) Cultivars in Nigeria
Table 1 Effect of different concentrations of BA ± NAA on in vitro plantlet regeneration in sugarcane.
PGR Conc. Shoot number Shoot length Crop vigor (mg/L)
Mean ± SD
0.00 8.94 ± 2.54 d 2.69 ± 0.63 c 2.74 ± 0.87 c
0.2 NAA
7.81 ± 0.34 e 2.51 ± 0.14 cd 1.97 ± 0.15 d
1.0 BA
15.10 ± 4.63 b 2.83 ± 0.53 c 2.78 ± 0.71 c
1.5 BA
15.69 ± 3.08 ab 2.50± 0.51c d 2.78 ± 0.70 c
2.0 BA
12.03 ± 2.51 c 2.23 ± 0.42 d 1.97 ± 0.74 d 1.0BA + 0.2 NAA 16.19 ± 2.56 a 4.80 ± 1.34 a 4.09 ± 0.96 a 1.5BA + 0.2 NAA 15.85 ± 2.26 a 4.95 ± 1.01 a 3.62 ± 0.87 b
2.0BA + 0.2 NAA 12.18 ± 2.31 c 3.76 ± 1.22 b 3.06 ± 1.13c
Fig. 2 Response of sugarcane genotypes to in vitro shoot
CV (%)
14.24 18.05 16.39 regeneration.
required for callus induction and development of bilateral symmetry during enbryogenesis [10, 11]. However, reduction in the concentration of 2, 4-D or
its complete removal from the meduim is required for
ber
the retention of bilateral symmetry and subsquent expression of somatic embryogenesis [12].
Shoot num
Regeneration was achieved on both hormone-free medium and media fortified with plant growth regulators indicating that once bilateral symmetry is
established at callus stage, exogenous hormones are not
Fig. 3 Response of sugarcane genotypes to in vitro shoot
essential for plantlets regeneration. However, low
length and crop vigor, with error bars .
regeneration frequency was observed on hormone-free more important with respect to morphogenesis in medium (Table 1). Addition of exogenous BA ± NAA
callus culture of sugarcane. For callus induction and resulted in an increased frequency of regeneration.
embryogenesis high concentration of auxin is required Plantlets regeneration was significantly (P < 0.05)
while high concentration of cytokinins favoured higher when MS was supplemented with 1.0-2.0 mg/L
shoots proliferation from callus culture. Although the BA with or without 0.2 mg/L NAA (Table 1).
mechanism of cytikinins is not clearly understood, Regeneration frequency increased with the increase in
their ability to initiate shoot from callus in tissue the concentration of BA from 1.0 to 1.5 mg/L but,
culture suggested thier role in controlling shoot apical further increased in the concentration of BA to 2.0
meristem development. Kerstetter et al. [15] suggested mg/L caused a decline in the regeneration frequency
that regulation of gene expression is one possible (Table 1). Plantlets regeneration was significantly (P <
mechanism by which cytokinins influence shoot
0.05) higher in both number, length and vigor when MS apical meristem development. Increase in cytokinin was supplented with 1.5 mg/L BA with 0.2 mg/L NAA,
levels in trangenic Arabidopsis resulted in the increase but, addition of 0.2 mg/L NAA alone decreases the
in the expression STM-genes which regulate shoot frequency of regeneration suggesting a possible apical meristem development [16]. synergistic effect of these hormones on morphogenesis
The genotypes used in this study showed variation in sugarcane. Synergistic effect of combination BA and
in their response to in vitro morphogenesis (Figs. 2 NAA on plantlets regeneration from callus culture were
and 3). M1176/77 and B47419 exhibited the highest earlier repoted in sugarcane [13] and wheat [14].
morphogenic potentials, producing significantly (P < The ratio of cytokinins and auxins proved to be
0.05) higher number of well developed shoots than the
In Vitro Regeneration of Commercial Sugarcane (Saccharum spp.) Cultivars in Nigeria
other genotypes tested. Merkle et al. [17] reported that, (Sacharrum officinarum L.), the morphology and ontogeny varation in response to tissue culture among the plant of somatic embryos, Protoplasm 118 (1983) 169-180.
E.A. Brisibe, H. Miyake, T. Taniguchi, E. Maeda, species could be attributed to their physiological
Regulation of somatic embryogenesis in long term callus differences. The variable responses of these genotypes
cultures of sugarcane (Saccharum officinarum L.), New to the exogenous BA and NAA may be explained by
Phytology 126 (1994) 301-307.
A. Khatri, I.A. Khan, M.A. Javed, M.A. Siddiqui, M.K.R. their differences in the levels of endogenous hormones. Khan, M.H. Khanzada, et al., Studies on callusing and
So, when these genotypes are exposed to exogenous regeneration potential of indigenous and exotic sugarcane plant growth regulators, their entire hormone levels
clones, Asian Journal of Plant Science 1 (2002) 41-43.
may be either sub-optimal, optimal or super-optimal A.A. Mir Ali, N. Shagufta, F.A. Saddiqui, J. Iqbal, Rapid clonal multiplication of sugarcane (Saccharum
for morphogenesis. The response of the genotypes to officinarum ) through callogenesis and organogenesis, exogenous plant growth regulators would, therefore,
Parkistan Journal of Botany 40 (2008) 123-138. depend mainly on the level of endogenous of
[7] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures,
hormones in the genotypes which will finally Physiology Plantae 15 (1962) 473-497.
determine their response to in vitro regeneration. [8] Statistical Analysis System, User’s Guide Statistics, Well developed plantlets (Fig. 1C) were transfered
Version 6 Edition, SAS Institute, Inc. Carry N. C., 1998. [9] to rooting media consisting of MS supplemented with Ch. Gandonuo, T. Errabil, J. Abrini, M. Idaomar, F. Chibi, N.S. Senhaji, Effect of genotype on callus induction and
1.0 mg/L IBA. Plantlets with well developed roots plant regeneration from leaf explants of sugarcane were transferred to poly-pots (250 × 125 mm)
(Saccharum spp.), African Journal of Biotechnology 4 containing sterilized river sand and manure (2:1)
(2004) 1250-1255. [10] L. Michalczuk, D.M. Ribnicky, T.J. Cooke, J.D. Cohen,
under high humidity (> 90%) created by covering the Regulation of indole-3-acetic acid biosynthetic pathways in plantlets with transparent polythene sheet. Plantlets
carrot cell cultures, Plant Physiology 100 (1992) 1346-1353. were watered every day with half-strength MS (macro
C. Fischer, G. Neuhaus, Influence of auxin on the and micro salts). Humidity was reduced gradually by establishment of bilateral symmetry in monocots, Plant
Journal 9 (1996) 659-669.