47S.M. Jain et al. (eds.), Date Palm Biotechnology, DOI 10.1007/978-94-007-1318-5_4, © Springer Science+Business Media B.V. 2011

Abstract During recent years, different approaches have been designed for the micropropagation of elite date palm cultivars. The most commonly used techno-logy approach is somatic embryogenesis which presents a great potential for the rapid propagation and genetic resource preservation of this species. Considerable progress has been made in the development and optimization of this regeneration pathway through the establishment of embryogenic suspension cultures. However, several problems still need to be solved and are currently under study, such as the abnormal differentiation of somatic embryo, the proliferation of endophytic bacte-ria within in vitro cultured material and the occurrence of somaclonal variants in regenerated offspring. The present review is aimed at providing updated and inno-vative information on recent progress, applications and prospects for somatic embryogenesis in the date palm.

Keywords  Conservation  •  Date  palm  •  Micropropagation  •  Somatic  embryo­genesis

L. Fki (*) • R. Masmoudi • A. Mahjoub • B. SghaierLaboratoire des Biotechnologies Végétales Appliquées à l’Amélioration des Cultures,   Université de Sfax­Faculté des Sciences de Sfax, Route de Sokra Sfax­Tunisiee-mail: lotfi.fki@fss.rnu.tn; maswkri@yahoo.fr

W. Kriaâ • N. DriraLaboratory of Plant Biotechnology, Department of Biology, Faculty of Sciences Sfax, Road Soukra BP. 1171, Sfax 3000, Tunisiae-mail: maswkri@yahoo.fr; drira_riadh@yahoo.fr

R. Mzid • A. MlikiLaboratoire de Biotechnologie Végétale, Centre de biotechnologie de Borj Sedria,  Borj Sedria Tunis­Tunisiee-mail: rmzid@yahoo.fr

A. RivalUMR DIADE, CIRAD, F­34398, Montpellier, France

Chapter 4Date Palm Micropropagation via Somatic Embryogenesis

L. Fki, R. Masmoudi, W. Kriaâ, A. Mahjoub, B. Sghaier, R. Mzid, A. Mliki, A. Rival, and N. Drira

48 L. Fki et al.

4.1 Introduction

The date palm, Phoenix dactylifera L., is one of the most economically important plants  in  arid  and  hot  regions,  especially  in  the  Middle  East  and  southern Mediterranean countries. Conventionally, this plant is propagated from offshoots which availability is often limited: indeed a given palm produces about 20 offshoots during its lifetime. Seed-derived palms show variable field performance because of their genetic heterogeneity. Using classical breeding methods, the genetic improve-ment of date palm is hampered by its long life cycle. More than 30 years are necessary to complete three backcrosses and to obtain the first offshoots from a given inter-varietal cross.

Bayoud disease caused by Fusarium oxysporum fungus has destroyed two thirds of the Moroccan palm plantations (more than ten million trees) and more than three millions palms  in Algeria,  causing  considerable  economic,  ecological  as well  as social damage (Fernandez et al. 1995). Today a new lethal threat, brittle leaf disease, is spreading at a fast pace, as more than 36,000 trees are infected in Tunisia (Triki et al. 2003). In view of this critical situation, the integration of biotechnology in date palm propagation, breeding and conservation strategies has become essential.

Plant regeneration through tissue culture is able to provide technologies for the large-scale propagation of healthy true-to-type plants. Plant tissue culture is also an essential tool for plant breeding programs (Parveez et al. 2000) and the conservation of plant genetic resources (Engelmann and Dussert 2000). Somatic embryogenesis, which leads to embryo differentiation from somatic cells and not from fertilized ovules, is often employed because of its numerous advantages (Carlos and Martinez 1998).  Embryogenic  cell  suspension  cultures  have  shown  higher  morphogenetic capacity when compared with other methods of in vitro propagation. Well devel-oped somatic embryos from efficient embryogenic cultures are highly critical to produce synthetic seeds (Prewein and Wilhelm 2003). In addition, embryogenic cells are considered as choice material for (i) the isolation of protoplasts with a highly regeneration capacity (Ling and Iwamasa 1994), (ii) genetic engineering (Cabrera et al. 1996) and (iii) the cryopreservation of plant genetic resources (Engelmann 2004).

Since 1970, extensive efforts have been undertaken to mass-propagate date palms using in vitro  techniques  (Al­Khalifa  2000;  Al­Khateeb  2008a,b;  Al­Khayri  and Al­Bahrani 2001; Ammar and Benbadis 1977; Bekheet et al. 2001; Bhaskaran and Smith 1992; Bouguedoura et al. 1990; Daguin and Letouze 1988; Drira 1983; Drira and Benbadis 1985; Eeuwens 1978; El Hadrami et al. 1995; Fki et al. 2003; Masmoudi et al. 1999; Othmani et al. 2009; Poulain et al. 1979; Reuveni 1979; Reuveni et al. 1972; Reynolds and Murashige 1979; Schroeder 1970; Sharma et al. 1980, 1984, 1986; Sharon and Shankar 1998; Taha et al. 2001; Tisserat 1979, 1982, 1984; Tisserat and Demason 1980; Veramendi and Navarro 1996, 1997; Zouine et al. 2005). A number of excellent reviews have also been published (Al­Khayri 2005, 2007; Bhaskaran and Smith 1995; Branton and Blake 1989;  El  Hadrami  and  El Hadrami 2009).

494  Date Palm Micropropagation via Somatic Embryogenesis

To establish aseptic cultures, various different explants have been used, including zygotic  embryos  (Ammar  and  Benbadis  1977;  Reynolds  and  Murachige  1979), shoot tips (Veramendi and Navarro 1996), lateral buds (Bouguedoura et al. 1990; Drira 1983), leaves (Bhaskaran and Smith 1992; Fki et al. 2003) or inflorescences (Bhaskaran and Smith 1992; Drira and Benbadis 1985; Fki et al. 2003). Different types of disinfecting solutions have been used, among them mercuric chloride (Hg Cl

2) was found to be the most efficient (Drira and Benbadis 1985). Some authors

have given more importance to somatic embryogenesis, mainly because of its higher potential for mass propagation (Al­Khateeb 2008a; Al­Khayri and Al­Bahrani 2001; Bhaskaran and Smith 1992; Daguin and Letouzé 1988; El Hadrami et al. 1995; Fki et al. 2003; Masmoudi et al. 1999; Othmani et al. 2009; Poulain et al. 1979; Reynolds and Murachige 1979; Tisserat and Demason 1980). Several research groups have opted for date palm regeneration through adventitious organogenesis, this approach being reputed to be slow, but less risky in terms of somaclonal variation (Al­Khateeb 2008b; Bekheet et al. 2001; Drira and Benbadis 1985; Taha et al. 2001; Tisserat 1984). The simple development of lateral buds has also been explored for the suc-cessful regeneration of whole plants (Drira 1983).

The culture medium devised by Murashige and Skoog (1962) has been commonly used for date palm tissue culture. The 2,4-D auxin is the most popular for callogen-esis (Fki et al. 2003). The use of liquid media for plant regeneration has been widely used (Bhaskaran and Smith 1992; Daguin and Letouze 1988; Fki et al. 2003; Sharma et al. 1986; Veramendi and Navarro 1996; Zouine et al. 2005). The present chapter is aimed at describing research work on date palm somatic embryogenesis. Its potential for large-scale propagation, conservation and breeding is discussed.

4.2 Somatic Embryogenesis for Large-Scale Propagation

Somatic embryogenesis is one of the most important technologies for plant regen-eration. There are two morphogenetic pathways ensuring the production of somatic embryos. The first pathway is direct somatic embryogenesis, which is yet to be fully developed for massive plant regeneration in date palm (Sudhersan et al. 1993). The second pathway is an indirect method which is based on the induction of embryo-genic calli (Al­Khayri 2005).

4.2.1 Initiation of Embryogenic Calli

4.2.1.1 Factors Controlling Callus Induction

Different types of explants have been used to induce embryogenic calli, such as zygotic embryos, shoot tips, leaves, lateral buds or inflorescences. However, the suc-cessful  induction of calli requires very specific physicochemical conditions which 

50 L. Fki et al.

are essential for the dedifferentiation of cells. Previous research work has shown that the 2,4-D auxin is the most suitable plant growth regulator for the initiation of cal-logenesis in date palm; indeed, concentrations such as 5 mg/L and more (El Hadrami and Baaziz 1995; Tisserat 1979) and even lower than 1 mg/L were found to be effi-cient (Fki et al. 2003; Masmoudi et al. 1999). Cytokinins such as 2iP (2-isopentyl adenine) seems not to be necessary, as it is for many monocotyledonous plants (Magnaval et al. 1997). Our studies showed that Picloram (0.2–0.5 mg/L) induced callogenesis although it generated non-embryogenic calli or abnormal somatic embryos (Fki 2005). In date palm, callogenesis is a very slow process which may require 4–8 months. This seems to be a generic characteristic of in vitro cultivated Arecaceae, as it was also described for the coconut palm (Verdeil and Buffard-Morel 1995) and the African oil palm (Elaeis guineensis Jacq.) (Duval et al. 1995). This very slow culturing step has obviously a negative impact on the cost of vitroplants.

Calli generally appear on either upper or lower surfaces of the leaves; the latter side is clearly more productive and generates the major part of the calli population (Fki et al. 2003). When an immature inflorescence is used as a primary explant, only calli which originated from the proliferation of floral tissues showed embryogenic competency. Comparatively, calli deriving from axial tissues bearing flower primor-dia showed a lower embryogenic potential. The callogenic capacity of inflorescences was generally found to be higher than that of leaves (Drira and Benbadis 1985; Fki et al. 2003).

Mature and immature zygotic embryos produce calli with a low embryogenic capacity (Fki 2005; Reynolds and Murashige 1979). This was an unexpected result considering the immense morphogenetic capacity of this explant in other species like the African oil palm (Teixeira et al. 1994). Calli resulting from zygotic embryos could not produce a high number of embryos, and since date palm is a dioecious and heterozygotic plant, calli cannot be used to propagate desired cultivars. On the other hand, the combination of this propagation method, in vitro flowering (Ammar et al. 1987; Masmoudi­Allouche et al. 2010) and in vitro fertilization technologies may be an innovative approach for date palm breeding.

Furthermore, Al­Khayri (2001) reported that biotin and thiamine could improve the quality of the embryogenic calli. Silver nitrate was also found to promote somatic embryogenesis (Al­Khayri and Al­Bahrany 2001, 2003). In a study to determine the effects of date palm syrup on somatic embryogenesis induction, Al­Khateeb (2008a) found that such a natural extract could be used at a 6% concentration as a replace-ment for sucrose. Date palm meristematic tissues extract also enhanced date palm somatic embryogenesis (El­Assar et al. 2004).

The genotype has a strong influence on callus induction. Indeed, using the same concentration of 2,4­D (1 mg/L), the frequencies of callus induction obtained from juvenile  leaves  from  cvs.  Klasse  and  Barhee  were  found  to  reach,  respectively, 10% and 40% (Fki 2005).

The fragmentation of explants, which cuts morphogenetic correlations and enables a direct contact of totipotent cells with the culture medium, improved rates in the initiation of embryogenic calli (Fki 2005). Nevertheless  it was  found  that explant fragmentation often led to an early proliferation of endophytic bacteria.

514  Date Palm Micropropagation via Somatic Embryogenesis

The massive release of phenolic compounds, which was found in many date palm cultivars, can be overcome by culturing the explant in media supplemented with activated charcoal (Fki 2005). Other anti-oxidative substances, such as citric acid or ascorbic acid, have been successfully used for the reduction of excessive browning and eventual necrosis of date palm tissues (Zaid and Tisserat 1984).

The physiological state of the donor plant, the period in which primary explants were sampled and their position on the culture medium are also impor-tant. For example, in the case of callogenesis from leaves, the period ranging from September to January seems to be the most favorable under the Tunisian climate (Fki 2005).

Recent basic studies by Gueye et al. (2009a) have shown that callus initiation by 2,4­D  requires  polar  auxin  transport  and  is  characterized  by  the  reactivation  of fascicular parenchyma cells followed by the dedifferentiation of perivascular sheath cells. Both callogenesis and rooting are initiated from perivascular sheath cells, the ultimate developmental fate depending upon auxin concentration (Gueye et al. 2009b).

4.2.1.2 Characterization of Embryogenic Calli

In vitro propagation of date palm is a very slow process. Finding cyto-morphological, biochemical and molecular markers characterizing embryogenic cultures would be a  gain.  At  the  cyto­morphological  level,  embryogenic  calli  are  friable,  showing small (<2 mm) white nodules, slightly connected to the rest of the whitish callus matrix (Fig. 4.1). Non­embryogenic cells are prenchymatous with a small nucleus 

Fig. 4.1 Date palm embryogenic callus

52 L. Fki et al.

and a large centrally-located vacuole. In contrast, embryogenic cells are small with a large centrally­located nucleus and dense cytoplasm (Veramendi and Navarro 1997). Morphological and cytological analyses are often insufficient to characterize embryogenic calli; it is then of paramount interest to find biochemical and molecular markers.

At the biochemical level, total soluble protein contents were found to be higher in embryogenic calli  than  in  their non­embryogenenic counterparts  (El Hadrami and Baaziz 1995; Fki 2005; Masmoudi et al. 1999). Some proteins, as revealed by one­dimensional SDS­PAGE, can be used to distinguish between the two types of calli (Fig. 4.2a). According to studies from our group and El Hadrami and Baaziz (1995), peroxydase activity is much higher in embryogenic callus than in the non-embryogenic, and some isoforms of the enzyme can be used to identify embryo-genic calli (Fig. 4.2b).

While describing major trends of carbon metabolism during the initiation and expression of  somatic embryogenesis  in date palm cv. Deglet Noor, Masmoudi et al. (1999)  determined  PEPC  patterns  in  embryogenic  and  non­embryogenic calli. Detection of PEPC activity on polyacrylamide native gels after electropho-resis revealed the presence of three active isoforms in crude extracts from the embryogenic callus strain, whereas only a single band was present in the non- embryogenic one.

Fig. 4.2 Molecular analyses. (a)  SDS­PAGE  of  total  soluble  proteins  extracted  from  non­embryogenic (NE) and embryogenic (E) date palm calli. Lane M: molecular weight markers. (b) In situ  detection  of  peroxydase  activity  after  PAGE  separation  of  Total  Soluble  Proteins extracted from non­embryogenic (NE) and embryogenic (E) date palm calli

534  Date Palm Micropropagation via Somatic Embryogenesis

4.2.2 Multiplication, Development and Maturation of Somatic Embryos

Solid media have been usually and extensively employed to produce somatic embryos from embryogenic calli. This is obviously an important step in the control of  the different stages of somatic embryogenesis  in date palm (El Hadrami et al. 1995; Masmoudi et al. 1999;  Reynolds  and  Murashige  1979; Tisserat 1979). However, solid media could not be used to ensure large-scale propagation. For this reason, extensive efforts have been deployed to establish embryogenic suspension cultures with high morphogenetic potentialities (Bhaskaran and Smith 1992; Daguin and Letouzé 1988; Fki et al. 2003; Sharma et al. 1986; Veramendi and Navarro 1996; Zouine et al. 2005).

An  optimized  protocol  for  plant  regeneration  from  embryogenic  suspension cultures of date palm cv. Deglet Noor has been described by our group (Fki et al. 2003).  Embryogenic  cell  suspensions  established  after  500 mm  mesh  filtration were studied for cell shape, size and cell cluster formation (Fig. 4.3). The result-ing suspensions were highly heterogeneous, containing cells at various stages of differentiation. Chopping  the callus  into pieces  favored  the  formation of PEMs (Fig. 4.4). These results confirmed those obtained by Kreuger et al. (1995) with cultures of Cyclamen persicum. Sané et al. (2006) and Othmani et al. (2009) recently confirmed the positive effect of callus chopping on the differentiation of date palm somatic embryos. A twofold dilution of liquid MS medium (Murashige and Skoog 1962) showed a positive effect on somatic embryo differentiation. Furthermore, the addition of 1 mg/L 2,4-D and the presence of activated charcoal

Fig. 4.3 Microscopic view of a date palm embryogenic suspension

54 L. Fki et al.

at a low concentration (300 mg l–1) in the liquid medium promoted the differentia-tion of somatic embryos. This result is in contrast to a previous report on the Barhee cv. by Bhaskaran and Smith (1992), which described the occurrence of embryogenesis only in the absence of 2,4-D. Furthermore, De Touchet et al. (1991) described for African oil palm the use of a washing step before embryo differentiation,  with  the  aim  of  eliminating  any  traces  of  PGRs  in  the  culture medium. The simultaneous presence of embryos at various differentiation stages in the same suspension culture facilitated the study of somatic embryogenesis. Indeed, various stages of embryo development, namely: spherical, elongated (after the cotyledon appears) and cotyledonary leaf formation could be observed in a given flask (Fig. 4.5).

It is worth noting that mature embryos were sampled continuously at each transfer. By contrast, the protocol described for African oil palm by De Touchet et al. (1991) involves the total conversion of a given culture towards the differentiation pathway at a given time, this step being initiated by a washing treatment and completed with the plating of the culture on a solid medium.

The use of liquid medium was found to drastically increase the productivity of somatic embryogenesis. Whereas only 10 ± 2 embryos were recovered from 100 mg callus (FW) on solid medium, the same amount of callus produced up to 200 ± 10 embryos in liquid medium after a 1­month cultivation period (Fig. 4.6). Suspension cultures were maintained up to 6 months, thus enabling the continu-ous production of mature embryos. The overall production rate was 10,000 ± 45 typical embryos per liter per month. Bhaskaran and Smith (1992) reported a lower productivity and a high proportion of abnormal embryos for the Barhee cv. De Touchet et al. (1991)  indicated  that when using a PGR­free  liquid medium, 

Fig. 4.4 Mass production of date palm proembryos through liquid suspension culture

554  Date Palm Micropropagation via Somatic Embryogenesis

fully developed somatic embryos exhibiting both a gemmule and a radicule were obtained at  a very  low  frequency;  they did not germinate  and become necrotic after their transfer to a solid medium.

Microscopic examination showed that the embryogenic aggregates initiated adventitious nodules, which separated from each other with agitation. Each culture was made of nodules showing various different sizes. Some nodules continued

Fig. 4.5 Various different stages of date palm somatic embryo development: (a) globular juvenile, (b) Mature, and (c) Beginning of germination

Fig. 4.6 Mass production of date palm somatic embryos from embryogenic suspension culture

56 L. Fki et al.

to  proliferate,  the  others  differentiated  into  embryos.  No  marked  growth  lag was noticed after the transfer of PEMs into a fresh medium. During the first 5 days of  subculture,  the growth  rate was slow. After  that period, a  steady  increase  in fresh weight was measured up to day 25. From that point onwards, there was a decrease in growth. Thus fresh biomass increased about fourfold in 1 month, a performance comparable  to  that previously  reported  for  the African oil  palm  (De Touchet et al. 1991).

In order to determine the origin of somatic embryos, filters of different mesh sizes were used to sieve cell suspensions (Fki et al. 2003). These experiments suggest that somatic embryos seem to originate from cell aggregates and not from single cells.

Some embryogenic callus lines were not able to generate vigorous somatic embryos. This is certainly a consequence of poor accumulation of storage compounds such as proteins, lipids and sugars. In this respect, a comparative study was carried out between date palm somatic and zygotic embryos (Fki 2005; Sghaier et al. 2008; Sghaier-Hammami et al. 2009) in order to improve the quality of the somatic embryos.

In date palm, the zygotic embryo taken from seed is at a developmental stage corresponding to the post-globular stage. The full development of this embryo takes place after germination, which begins with the elongation of the cotyledon. Morphologically, these embryos are longer and thicker than the somatic ones. Besides, no differences exist between somatic embryos and zygotic embryos which were developed on a PGR­free medium (Fki 2005). Total soluble protein contents were much higher in the zygotic embryos taken from seeds a few days after germi-nation than in somatic embryos. These differences reflect on the regenerated plants. Indeed, seed-derived plants are more vigorous than somatic embryo- and zygotic embryo-derived plants (Fig. 4.7). The vigor of the plants resulting from seeds could be explained by the specific role of the endosperm which contains important storage compounds (Besbes et al. 2004).

Fig. 4.7 Developing date palm plantlets from various origins: (a) Seed-derived seedling; (b) zygotic embryo-derived vitroplant; (c) somatic embryo-derived vitroplant

574  Date Palm Micropropagation via Somatic Embryogenesis

Sucrose and ABA were evaluated at different concentrations for their respective effects on somatic embryo maturation (Fki 2005; Sghaier et al. 2009; Sghaier-Hammamia et al. 2009; Zouine et al. 2005). Mature somatic embryos developed on half strength MS medium enriched with high concentration of sucrose (60 g/L) and ABA (2 mg/L) were  found  to be  thicker,  longer  and  richer  in proteins  than  the control (Fki 2005). The promotive effect of abscissic acid (ABA) is mainly exerted during the development of the cotyledon. Indeed, ABA induces the accumulation of storage proteins and prevents precocious germination.

4.2.3 Somatic Embryos Germination

The  duration  of  the  cultivation  period  in  liquid  medium  was  found  to  be  very important for the balanced germination of somatic embryos. Indeed, the cultivation of mature embryos in liquid medium for more than 1 month led to hyperhydration (Fki et al. 2003). We have demonstrated that partial desiccation of mature somatic embryos, corresponding to a decrease in water content from 90 down to 75%, did significantly improve germination rates on modified MS medium deprived of PGRs (from 25% to 90%).

In the same way, cutting back the cotyledon leaf to about half its length was found to stimulate embryo germination. This kind of physiological response has been observed on zygotic embryos from various plant species. In our experiments, embryos with a shortened cotyledon leaf showed an 80% germination rate, compared to 25% with those with an intact cotyledon leaf. The cotyledon leaf seemed to inhibit the apical meristem development of some embryos, but did not influence the growth of the root meristem. The transfer of germinated embryos onto a medium supplemented with 1 mg/L NAA and 1 mg/L BAP enabled  the production of vigorous plantlets showing a balanced shoot and root development. The positive effect of BAP on the germination  of  African  oil  palm  somatic  embryos  derived  from  embryogenic  suspensions was also reported by Aberlenc­Bertossi et al. (1999).

Al­Khayri (2003) showed a positive effect of IBA (0.2–0.4 mg/L) on germination rates of somatic embryos which were produced on solid medium. Media containing an additional source of inorganic phosphate (170 mg/L sodium dihydrogen phos-phate + 100 mg/L potassium dihydrogen phosphate) resulted in faster germination (Sharon and Shander 1998).

4.2.4 Physiology of the Somatic Embryo-Derived Plants and Their Acclimatization

Acclimatization is the last stage of micropropagation; when not properly controlled, it leads to high loss rates. Plants transplanted into the greenhouse should progres-sively resist to: (i) higher luminosity; (ii) lower relative humidity; (iii) fluctuation of

58 L. Fki et al.

temperature and (iv) biotic stresses. For a better control of acclimatization, we have studied several factors, including the physiology of vitroplants and the physico-chemical conditions of acclimatization (Fki 2005). Our anatomical analyses showed that in vitro grown roots have a structure similar to those sampled from acclimatized plants. This proves that roots of vitroplants are virtually functional at all develop-mental stages (Fig. 4.8). With regard to the photosynthetic capacity of vitroplants, we found that only after an in vitro hardening period of 12 months does the photo-chemical activity of Photosystem II become close to that measured in an already acclimatized plant.

Scanning  electron  microscopy  (SEM)  examination  of  detached  leaf  surfaces showed the regulation of stomatal aperture, preventing excess transpirational vapor loss in 12-month-old vitroplants. Indeed, all stomata were closed in this material, which was not the case for 3-month-old vitroplants (Fig. 4.9). In this respect, Zaid and Hughes (1995) reported that a polyethylene glycol treatment of vitroplants increased the amount of wax deposition on leaf surfaces and as a consequence it was able to decrease water losses which were observed during acclimatization.

When exploring the role of carboxylases in date palm acclimatization (Masmoudi et al. 1999), it was found that the PEPC/RubisCO ratio decreased (from 17.7 to 0.2) throughout the in vitro development of plantlets, due to a substantial depletion of PEPC activity. Concomitantly, RubisCO activity assumed greater importance and became the main route for inorganic carbon fixation. Western blot analysis using polyclonal  antibodies  raised  against  PEPC  and  RubisCO  purified  from  tobacco leaves confirmed this trend in terms of relative enzyme abundance.

Fig. 4.8 Cross section of in vitro grown primary root from a 3 month-old plantlet derived from suspension-cultured somatic embryo

594  Date Palm Micropropagation via Somatic Embryogenesis

According to our experiments, the optimal conditions for acclimatization of date palm vitroplants, ensuring high survival rates (70%) for 12-month-old plantlets, can be listed, as follows: (i) temperature: 25–30°C (ii) moderate light intensity (iii) a draining substrate and (iv) a plant by plant instead of whole batch acclimatization procedure. In order to promote plant growth in the greenhouse, Awad (2008) sug-gested the utilization of a 5-aminolevulinic acid-based fertilizer.

4.2.5 Genetic Stability and In-Field Behavior of Somatic Embryo-Derived Plants

Several strategies have been used to assess the genetic integrity of somatic embryo-derived plants. In the case of date palm, reports about somaclonal variation are still controversial. Using flow cytometry, we have estimated the nuclear genome size of cv. Deglet Noor and analyzed the stability of this parameter in regenerated plants (Fki et al. 2003). Our experiments showed that all the analyzed adult plants and in vitro regenerated plantlets were diploid. No variation in genome size that could be linked to the micropropagation protocol could be detected. Our estimation of 2 C DNA content of cv. Deglet Noor was 1.96 ± 0.05 pg and therefore approximately 1.77 × 109 bp. Date palm was found to have a much smaller genome than the African oil palm, which was reported to be 2C = 3.76 ± 0.09 pg; 3.4 × 109 bp by Rival et al. (1997).

RAPD and ISSR analyses of leaf genomic DNA reportedly are able to distin-guish between date palm cultivars (Corniquel and Mercier 1994; Sedra et al. 1998; Zahdi et al. 2002) although according to studies from our group, they failed to reveal polymorphism in genomic DNA which could be associated with somaclonal variations in tissue culture-derived date palm plants (Fki 2005;  Mahjoub  2003; Mzid 1999; Othmani 1998). Similarly, Rival et al. (1998) reported that a RAPD approach could not differentiate between normal and variant oil palms, as the frac-tion of the genome explored through this technology was clearly not large enough

Fig. 4.9  SEM observation of leaf epidermis: (a) 3-month-old vitroplant (400x); (b) 12 month-old vitroplant

60 L. Fki et al.

to draw any valuable conclusion. However, Saker et al. (2000) reported that RAPD and  isozyme  analyses  were  reliable  techniques  which  could  be  used  to  detect somaclonal variations in date palm.

The use of juvenile explants and low doses of 2,4­D can minimize the risks of somaclonal variation and generally ensure regeneration of morphologically uniform plants, as proven by the fruit quality of palms that have been planted in the field by our group a few years ago (Fig. 4.10). On the other hand, high concentrations of 2,4-D could induce somaclonal variation and the most genetically unstable cultivar seems to be Barhee, since some somatic embryo derived-plants now produce parthenocarpic fruits (unpublished results). The genetic instability of this cultivar also has been mentioned by Cohen et al. (2004) after a multi-seasonal analysis of fruit setting and by Zivdar et al. (2008) according to isoenzyme analysis.

In the same way, morphological abnormalities in somatic embryo-derived date palm cv. Sukkari have been described by Al­Mazroui et al. (2007). However, it has been reported that date palm cv. Barhee derived from somatic embryos and natural offshoots produced fruits bearing the same characteristics (Smith and Aynsley 1995).

4.2.6 Major Constraints and Remedies

According to our investigations, the three major constraints in date palm somatic embryogenesis are endophytic bacterial contamination, abnormal somatic embryo differentiation and somaclonal variation (Fki 2005). Concerning endophytic bacte-rial contamination, only juvenile explants could be used to establish clean in vitro tissue culture, since antibiotics such as cefotaxim have only a bacteriostatic effect. Immaturity of vascular tissue in these explants may explain the absence of endophytic

Fig. 4.10  Normal fruit bunches from a somatic embryo­derived date palm

614  Date Palm Micropropagation via Somatic Embryogenesis

contaminants (Fki 2005). Both abnormal somatic embryo differentiation and somaclonal variation were especially associated with the utilization of high concen-trations of 2,4­D. Reducing its concentration significantly minimized the number of abnormal somatic embryos and somaclonal variants (Fki 2005).

4.3 Applications of Somatic Embryogenesis

4.3.1 Synthetic Seed Technology

Bio-encapsulation of somatic embryos is most appropriate, especially for dioecious and vegetatively­propagated plant species such as the date palm. As we are able to produce a high number of somatic embryos, our research group is now focusing on the production of date palm synthetic seed. Somatic embryos were placed in sterilized 3% sodium alginate, dissolved in MS medium without calcium. Each one was picked up using a micropipette and dropped into a 75 mM CaCl

2.2 H

2O solution in MS medium.

After 30 min, the calcium chloride solution was decanted and the alginate beads, each containing  a  single  somatic  embryo,  were  rinsed  with  sterile  distilled  water.  Each encapsulated somatic embryo can be considered a synthetic seed, provided it survives further conservation. Furthermore, alginate was not toxic to somatic embryos and the in vitro germination rate was unaffected (Fig. 4.11). This is in accordance with results obtained by Daikh and Demarly (1987) and Bekheet et al. (2002).

4.3.2 Protoplasts

Chabane et al. (2007) reported for the first time the formation of a callus from pro-toplasts in date palm. Protoplasts were isolated from young offshoot leaves and embryogenic calli. Different combinations of enzyme solutions were tested for pro-toplast isolation and a cocktail of 1.5% Cellulase RS (Yakult Pharmaceutical Ind. Co., Ltd, Tokyo Japan), 0.15% Pectolyase (Kyowa Chemical Products Co., Ltd, Osaka  Japan)  and  0.2%  Hemicellulase  (Sigma,  USA)  was  found  to  be  the  most efficient.  Cell  division  was  induced  in  both  liquid  culture  and  nurse  culture. Nevertheless these proto­calli failed to regenerate plants.

4.3.3 In Vitro Mutagenesis

Mutagenesis is a very interesting approach for crop improvement since many superior genotypes have been obtained using diverse mutagenic agents. In our laboratory, date palm embryogenic  calli were  exposed  to gamma  irradiation  (30 Gy)  and plantlets material. In order to select bayoud-resistant cell lines, a toxin extracted from Fusarium

62 L. Fki et al.

oxysporum f. sp. albedinis was added to the culture media at several concentrations (5, 10 and 20 mg/mL) (Fki 2005). This experiment suggests that a toxin can be used as an in vitro selection agent to screen for resistance. The putatively resistant plantlets are now under investigation at the biochemical and molecular levels and resistance will be field-assessed in bayoud-contaminated zones.

4.3.4 Genetic Transformation

In our laboratory, embryogenic cultures of date palm were bombarded with tungsten particles coated with a plasmid encoding b­glucuronidase  (GUS)  and  neomycin phosphotransferase  (NPTII)  which  results  in  kanamycin  resistance  (Fki  2005). Expression  of  these  proteins  is  under  the  control  of  a  CaMV  35  S  promoter. Expression  of  the  reporter  gene  was  histochemically  confirmed  in  embryogenic  cultures of cvs. Barhee and Deglet Noor (Fig. 4.12). Helium pressure of 2 bars and a distance of 6 cm between the particle launch and the target were found to increase the number of GUS­expressing colonies. The number of GUS­expressing colonies decreased with time and further studies are being carried out to obtain stable transformation.

4.3.5 Cryopreservation

Studies on cryopreservation of date palm embryogenic cultures are scarce. In most of cases, a classical freezing protocol, which is relatively time consuming, has been used. It consists in a slow cooling of plant material down to a temperature range of −30°C to −40°C, followed by the immersion of the vials containing tissues and a cryoprotectant  in  liquid  nitrogen  (Bagniol  and  Engelmann  1991, 1992; Fki 2005; Mycock et al. 1995; Ulrich et al. 1982).

Fig. 4.11 Synthetic seeds of date palm

634  Date Palm Micropropagation via Somatic Embryogenesis

Cryopreservation of date palm tissue cultures  is a major  research  topic  in our group and optimized cryopreservation protocols based on the vitrification process have been established for date palm embryogenic cultures (unpublished results).

4.4 Conclusion and Prospective

Somatic embryogenesis has a tremendous potential for date palm development. This technology can be used for large-scale propagation, thereby opening the way for the production of synthetic seeds. However, the factors controlling callus induction still need to be mastered and the quality of embryogenic calli requires further improvement. 

Fig. 4.12 Transient GUS gene expression in date palm embryogenic cultures. (a) Callus. (b) Suspension culture

64 L. Fki et al.

Moreover,  high­frequency  callus  induction  needs  to  be  obtained  from  explants originating from various organs/genotypes. Further research is needed to find the most appropriate biochemical and molecular markers of embryogenesis in date palm. In date palm, most of the molecular methods used to assess somaclonal variation have shown some limitations. Indeed, cytogenetic analysis proved unable to reveal any alteration in genome structure, and isozyme markers are subject to large ontogenic variation. Molecular markers are able to investigate only a small part of the genome and they are useless in the case of epigenetic changes. Somaclonal variation was found to be from epigenetic origin in oil palm and differ-ences in DNA methylation rates could be linked to the occurrence of epigenetic instability (Jaligot et al. 2000). This research opens new ways for the investigation of somaclonal variation in date palm. To date, field evaluation, even if it is a long and costly process, remains the most reliable strategy to assess the genetic integrity of regenerated date palms.

Very limited work has been carried out on the cryopreservation of date palm embryogenic cultures and therefore the development of innovative procedures is needed for the efficient preservation of genetic resources and the management of commercial propagation. Preliminary studies revealed that embryogenic cultures provide a choice plant material for in vitro mutagenesis experiments and the further selection of useful mutants, the generation of protoplasts and somatic cell hybridiza-tion, and for genetic engineering. Further research is required to overcome problems such as abnormal somatic embryo differentiation, endophytic bacteria proliferation and somaclonal variation.

Acknowledgements This  work  was  supported  by  the  Ministry  of  Higher  Education,  Scientific Research  and  Technology  of  Tunisia,  the  International  Atomic  Energy  Agency  (TC  Project RAF/5/049) and the European Commission (COST Action 871).

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