M. Baaziz

Polymorphism of natural populations of Pinus halepensis Mill. in Morocco as revealed by morphological characters

In Morocco, Aleppo pine (Pinus halepensis Mill.) is the most used species in reforestation. However, no data is available about its intraspecific variation and adaptability. Morphological traits of 15 natural populations located on a North-East South-West orientation, representing its natural distribution in Morocco, were used to assess geographic variation of this species. Seed size (length, width, depth, weight and 1000 seed weight), wing length and width, cone length and width and needle length were measured for 760 trees (43–65 trees per population). A correlation matrix between morphological characters and geographic parameters (altitude, latitude north and longitude west) exhibited negative and positive correlations of seed size with the latitude and longitude parameters, respectively. Also cone characters correlated positively with altitude. Thus, populations originating from the South-West region (High Atlas and a part of Middle Atlas) showed high values of Aleppo pine characters. Factorial component analysis (FCA) and hierarchical classification of all populations lead to the formation of two major groups (South-West and North-East) containing, each one, two minor groups. Geographic pattern of Aleppo pine distribution and its adaptability in Morocco are discussed in this study.

Somatic embryogenesis and plant regeneration from shoot-tip explants inPhoenix dactylifera L.

For maximum avoidance of somaclonal variation risks, the commonly used medium for somatic embryogenesis inPhoenix dactylifera has been lowered in growth regulators and activated charcoal. When initially cultured on MS basal medium containing only 150 mg dm−3 charcoal, 5 mg dm−3 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 mg dm−3 benzylaminopurine (BAP), 10 to 20% of shoot-tip explants developed into embryogenic calli. The embryogenic potential has been maintained for over 24 months with no decline. In addition, this medium has been found to be more efficient than conventionaly one containing 3 g dm−3 charcoal, 100 mg dm−3 2,4-D and 3 mg dm−3 2-isopentyladenosine (2IP). Plantlet regeneration was achieved when somatic embryos were subcultured to medium with 0.1 mg dm−3 2,4-D and 0.5 mg dm−3 BAP or without growth regulators.

Correlation of isoenzyme polymorphism and Bayoud-disease resistance in date palm cultivars and progeny

SummarySeedlings of date palm (Phoenix dactylifera L.), obtained from seven cultivars crossed with two males, were analyzed by polyacrylamide gel electrophoresis for esterase (EST), glutamate oxaloacetate transaminase (GOT), endopeptidase (ENP) and alcohol dehydrogenase (ADH) polymorphisms. Eleven, eight, five and two phenotypes were revealed for the enzymes tested, respectively. Seedlings of F1 populations derived from Bayoud (Fusarium)-resistant and low fruit quality cultivars were characterized by a high electrophoretic polymorphism, when compared with progenies of Bayoud-susceptible and high fruit quality cultivars. In almost all cases, the most frequent electrophoretic phenotypes scored for each enzyme in different F1 populations, were similar to those of the corresponding parent cultivars. Heterozygous phenotypes have been found for, at least, 3 loci; Got-2, Est-1 and Enp, indicating that such loci could be used to screen for hybrid seedlings. The expected Mendelian segregation of allozymes has been observed for these 3 loci, in many F1 populations. It seems that progenies of Bayoud-resistant cultivars are characterized by a high level of electrophoretic polymorphism. The estimation of this index and the search for genetic linkage with segregating allozymes, may be biochemical criteria useful as an aid in distinguishing date palm seedling populations derived from Bayoud-resistant cultivars and suitable for breeding programs.

Electrophoretic patterns of acid soluble proteins and active isoforms of peroxidase and polyphenoloxidase typifying calli and somatic embryos of two reputed date palm cultivars in Morocco

SummaryWhen subjected to micropropagation by tissue culture, the two reputed cultivars of date palm (Phoenix dactylifera L.); Bou-Sthammi noire, resistant to Bayoud disease and Bou-Feggous, of high fruit quality, give rise to three types of calli, called white and root-forming callus, hyperhydric and degenerating callus and friable and embryogenic callus. All explant sources, calli and germinated embryos were analysed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) for acid soluble protein composition. Phenol-oxidizing enzymes; peroxidase and polyphenoloxidase, were also, evaluated and the isoforms separated by polyacrylamide gel electrophoresis. When compared with the explant and germinated embryos, embryogenic calli of the two date palm cultivars could be identified by a concentrated polypeptide of molecular weight 27 500 and polypeptides of molecular weights 70 000 and 11 500. Hyperhydric and degenerating callus contained the polypeptide exhibiting the molecular weight 32 000. Embryogenic calli showed high levels of soluble, ionically and covalently bound peroxidases. The soluble acidic isoperoxidase of Rf 0.60, revealed in these calli and germinated embryos could be a marker of the two tissues. White and root-forming calli of Bou-Feggous cultivar were typified by soluble acidic isoperoxidases with high mobility (Rf0.75) and anodic ionically wall-bound polyphenoloxidases similar to those of the explant sources. Polyphemoloxidase activities detected in calli and embryos were very low when compared with those of explants. Used as an early test to screen embryogenic calli of date palm, acid soluble proteins, peroxidase and polyphenoloxidase data could lead to introduce lightening and economy in the tissue culture technique.

Somatic embryogenesis and analysis of peroxidases inPhoenix dactylifera L

To determine some physiological parameters implicated in somatic embryogenesis in date palm (Phoenix dactylifera L.), peroxidases have been studied. Activated charcoal commonly used in date palm tissue culture as an essential antibrowning factor decreased cellular protein contents and peroxidase activities. During the first months of culture, the conventionally used medium (100 mg dm−3 of 2,4-dichlorophenoxyacetic acid, 3 g dm−3 charcoal) reduces 2 to 3 and 4 to 6 times protein contents and peroxidase activities, respectively, in comparison with the same one containing only 5 mg dm−3 of 2,4-D and with or without 150 mg dm−3 charcoal. In addition, the standard procedure decreased the embryogenic potential which is positively related to the intra- and extracellular (excreted into culture medium) peroxidase activities. In medium with embryogenic calli, extracellular peroxidase activity was three times as high as the activity determined in the same medium with non-embryogenic calli. There were two basic isoforms and four to five acidic bands characterizing the embryogenic calli. It can be suggested that peroxidases play a key role in somatic embryogenesis of date palm and the charcoal used at 3 g dm−3 constitute a perturbating factor for this process.

Genetic diversity of natural populations of Atriplex halimus L. in Morocco: An isoenzyme-based overview

Based on polyacrylamide gel electrophoresis, nine natural populations of Atriplex halimus L., a perennial shrub, collected in different regions of Morocco, were studied for their genetic variation using isoenzyme polymorphism of the highly active enzyme systems: esterases (EST), acid phosphatases (ACP) and glutamate oxaloacetate transaminase (GOT). Different allozyme frequencies from 7 different loci were obtained for all populations of this halophyte species. High levels of genetic diversity were revealed. The mean number of alleles per locus (A = 1.9–2.0), the percentage of polymorphic loci (p = 71.4–85.7) and the mean expected heterozygosity (He = 0.339–0.385) showed an important variability in all populations. Gene diversity was essentially explained by the within population component. The between populations differentiation accounted for 8% of the whole diversity (FST, averaged over all loci, is 0.08). The relationships among the 9 populations were inferred from the Nei’s genetic distances. Four major groups were formed. The northern population ‘Tanger’, forming a unique group, was highly divergent from the other groups. It appeared that the genetic distance between all groups was related to the geographic distance that separates them.

Somatic embryogenesis and plant regeneration from leaf tissue of jojoba

A protocol was developed for the induction, maturation and germination of somatic embryos from leaf tissue of jojoba [Simmondsia chinensis (Link) Schneider]. Explants were placed on their adaxial sides in Petri dishes and maintained in darkness on half-strength Murashige and Skoog basal medium (MS/2). Combinations of 2,4-dichlorophenoxyacetic acid (1.35–4.52 μM) with 6-benzylaminopurine (1.33–4.43μM) and 2 synthetic cytokinins, N-(2-chloro-4pyridyl)-N′-phenylurea (1.21–4.03μM) or (E)-6-[3-(trifluoromethyl)-but-2-enylamino] purine (1.11–3.71μM) resulted in formation of embryogenic cultures and somatic embryos. After two 30-day subcultures, embryogenic cultures were transferred onto MS/2 medium supplemented with different auxins and cytokinins. Somatic embryo maturation, germination and plantlet formation were achieved using 1-naphthaleneacetic acid (3.75μM) or indole-3-butyric acid (3.44μM) in combination with BA (0.44 or 1.33μM) or F3iP (0.37 or 1.11μM). Histology confirmed each stage of development.

Use of two bacteria for biological control of bayoud disease caused by Fusarium oxysporum in date palm (Phoenix dactylifera L) seedlings

The Bayoud, caused by Fusarium oxysporum f. sp. albedinis (Foa), is the most destructive disease of date palm (Phoenix dactylifera L) in Morocco and Algeria, with no effective control strategy yet available. In this work, two bacteria, Bacillus amyloliquefaciens strain Ag1 (Ag) and Burkholderia cepacia strain Cs5 (Cs), were examined for their potential to control this disease. Both bacterial strains inhibited both growth and sporulation of Foa. They released compounds into the culture medium, which resulted into cytological changes in Foas mycelial structure. When Jihel-date palm plantlets, a susceptible cultivar, were induced with these bacteria, the size of the necrosis zone, which reflected the spreading of the pathogen, was reduced by more than 70%, as compared with uninduced controls. To further investigate the mechanisms of such disease reduction, phenolic compounds and peroxidase activity were assessed. One month after inoculation, date palm defense reactions against Foa were different depending on the bacterium used, B. cepacia led to higher accumulation of constitutive caffeoylshikimic acid isomers while B. amyloliquefaciens triggered the induction of new phenolic compounds identified as hydroxycinnamic acid derivatives. Peroxidase activity has also been stimulated significantly and varied with the bacterial strain used and with Foa inoculation. These results add to the promising field of investigation in controlling Bayoud disease.

Arbuscular Mycorrhizal Fungi Limit Incidence of Fusarium oxysporum f.sp. albedinis on Date Palm Seedlings by Increasing Nutrient Contents, Total Phenols and Peroxidase Activities

Date palm seedlings derived from Jihel (JHL), a susceptible cultivar to Bayoud disease (fusariosis caused by Fusarium oxysporum f.sp. albedinis, Foa), were subjected to root inoculation with an arbuscular mycorrhizal fungus (AMF) collected from south Morocco and multiplied on barley as host plant. Successfully colonized plants by mycorrhizal fungi (85 % of treated plants) produced typical intraradical structures (arbuscules, vesicles, hyphae). After ten months of colonization, mycorrhizal plants showed a significant increase in their growth expressed as shoot height, number of leaves per plant, shoot weight, root weight and the total biomass. Mycorrhizal and non-mycorrhizal (controls) date palm seedlings showed great differences in their leaf contents of phosphorus (P), potassium (K + ) and sodium (Na + ). When compared with controls, P increased more than two folds in mycorrhizal plants, while the values of K + and Na + doubled. When inoculated with Foa by injecting roots with a spore suspension, mycorrhizal (M + Foa) and non- mycorrhizal (C + Foa) date palm seedlings showed significant increases in their root total phenols and peroxidase activities during the first month after inoculation. The highest increases were found in mycorrhizal seedlings accompanied by limited plant death. Mycorrhization alone did not affect significantly total phenols and peroxidase activities during the first week of culture. Plant death decrease in plant lots subjected to root inoculation with the he AMF fungus. As revealed by mycorrhization of date palm seedlings, these results supported the hypothesis that induced resistance to Bayoud disease is mediated by high increases in phenolic compounds and peroxidase activities. These results highlight the importance of mycorrhizal fungi as biocontrol agents to combat Bayoud disease and improve date palm culture in infected palm groves.

Biochemistry and molecular biology in Morocco: Overview of scientific research and education

Just after independence in 1956, Morocco had only one university, the Mohamed V University, in Rabat, the capital of the country. In 2008, it boasts of 14 universities including a private institution. From the beginning of university education, programmes in biology at the Faculty of Sciences initially focused on natural sciences, in which biochemistry comprised one of many subjects. Given the general nature of this training, the Moroccan system produced nonspecialized graduates. Indeed, the major objective was to train a native teaching corps for secondary education to replace the mass of foreign teachers, which, until then, had been responsible for this level of education. Furthermore, at the onset of university education, scientific research activities were very limited with respect to biochemistry and molecular biology. Consequently, there was no return effect on the education in these disciplines. The development of biochemistry at each level of education together with scientific research is closely associated with the growth of the university system in Morocco over the last 40 years. This growth can be subdivided into two phases, one from 1957 till 2003 and the other from 2003 until the present day. The first phase was typified by ‘‘education for all’’ with a centralized system of management while the second was characterized by a reformed system, preceded by the appearance in 1999 of the National Charter of Education and pertinent law (01.00) on the organization of higher education. The second phase gave the Moroccan university a broad educational and scientific self-government and favored the development of specific programmes for education by reducing centralization. Higher education is now organized in modules, in which the time devoted to biochemistry and molecular biology may vary from one university to another. In many Moroccan universities, the first cycle includes two modules devoted to biochemistry, called ‘‘Structural Biochemistry – Enzymology’’ and ‘‘Metabolic Biochemistry – Molecular Biology’’. Each module comprises an average of 90 hours, including 20% lab practice. In the second module, molecular biology covers 37% of the time allocated and includes 20% of lab practice. Programmes in the second cycle and the master’s courses increasingly include more biochemistry in their curricula, such as analytical techniques and supplementary training. Scientific research in Morocco is still in its infancy for numerous reasons. Among these, of note is the lack of interest shown by the private sector in educational institutions and the many barriers between the distinct ministries and departments performing scientific research in Morocco. To enhance research opportunities in Morocco, a restructuring of this sector is essential. A survey carried out in 1995 by the National Centre of Scientific and Technical Research showed that only a part of the 4% of research units among a thousand in Morocco performs research in biochemistry and molecular biology. Even before the National Charter of Education in 1999, Morocco showed a clear need to develop scientific research and education in biochemistry and molecular biology. Indeed, in 1992 the educational body of our universities began to contemplate the development of an association. The fruit of this effort emerged in 1995 with the setting up of the Moroccan Society of Biochemistry, currently called the ‘‘Moroccan Society of Biochemistry and Molecular Biology’’ (SMBBM, http://smbbm. org). The 10th anniversary of this association was held in 2005 and was marked by a series of activities. In addition to its association with the Federation of European Biochemical Societies (FEBS) (signed on October 25, 2002), the SMBBM is also a member of the IUBMB. This widened field of international cooperation is the result of perseverance for more than 10 years, and has been consolidated through the organization of the last M. Baaziz, former President of SMBBM. Address correspondence to: M. Baaziz, Laboratoire de Biochimie et Amélioration des Plantes, Université Cadi Ayyad, Faculté des SciencesSemlalia, B.P. 2390, Marrakech 40000, Morocco. E-mail: baaziz@ucam.ac.ma Received 20 February 2008; accepted 20 February 2008