Development of zygotic and somatic embryos of Phoenix dactylifera L. cv. Deglet Nour: Comparative study

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Scientia Horticulturae 116 (2008) 169–175 www.elsevier.com/locate/scihorti

Development of zygotic and somatic embryos of Phoenix dactylifera L. cv. Deglet Nour: Comparative study Besma Sghaier *, Mouna Bahloul, Radhia Gargouri Bouzid, Noureddine Drira Laboratoire des Biotechnologies Ve´ge´tales Applique´es a` l’Ame´lioration des Cultures, Faculte´ des Sciences de Sfax, B.P. 802, 3018 Sfax, Tunisia Received 9 April 2007; received in revised form 22 October 2007; accepted 14 November 2007

Abstract In order to improve somatic embryogenesis production in date palm Phoenix dactilyfera L. cv. Deglet Nour (DN), a comparative study between somatic (SE) and zygotic (ZE) embryos developments was carried out. The data showed that ZE maturation occurred from 10 to 19 weeks after pollination (WAP). During this period, the fresh weight (FW) and the dry weight (DW) of ZE increased progressively to reach a maximum level at 19 WAP. SE development occurred in three distinct stages. The DW remained constant during the two first stages, and declined slightly during the third and final stage. Embryo protein analysis revealed significant differences between ZE and SE. The ZE total protein level was initially low and increased to the maximum at mature stage. However, no significant change in total protein was detected during SE development. SDS-PAGE analysis showed a poor protein profile for SE, compared to that of ZE. In the latter, a 22 kDa protein was identified by N-terminal sequencing as a glutelin. This protein was accumulated rapidly during early development and remained at a relatively constant level during ZE development, and then declined progressively 12 days after embryo germination (DG). This protein seems to be absent in SE. # 2007 Published by Elsevier B.V. Keywords: Zygotic embryo; Somatic embryo; Phoenix dactylifera L. cv. DN; Total protein; Glutelin

1. Introduction Date palm (Phoenix dactylifera L.) is one of the most economically important fruit crop and is cultivated across North Africa and the Middle-East. Its nutritive value and widespread cultivation underscore the need for improving propagation methodologies, especially in vitro techniques (Al-Khayri and Al-Bahrany, 2004). Clonal propagation via somatic embryogenesis – the production of embryos without recourse to sexual reproduction – is the most promising approach to improve propagation in date palm. This technique has been widely and successfully applied to many cultivated plant species (Shah et al., 2000). In vitro protocols are already available for date palm, which permit the production of many synchronous somatic embryos in liquid culture (Fki et al.,

Abbreviations: DG, days of germination; DN, Deglet Nour; DW, dry weight; ELISA, enzyme-linked immunosorbent assay; FW, fresh weight; MW, molecular weight; OPD, orthophenylene diamine; PBS, phosphate buffered saline; PBS-T, phosphate buffered saline tween; SE, somatic embryo; WAP, weeks after pollination; ZE, zygotic embryo. * Corresponding author. Tel.: +216 22 716 844; fax: +216 74 274 437. E-mail address: [email protected] (B. Sghaier). 0304-4238/$ – see front matter # 2007 Published by Elsevier B.V. doi:10.1016/j.scienta.2007.11.009

2003). Date palms derived from somatic embryos are less susceptible to somatic variation compared to those derived from organogenesis (Ammirato, 1987; Merkle et al., 1990; Osuga et al., 1999). Although somatic embryo production is a well-established process for date palm (Al-Khayri, 2005), there are still possibilities for improvement. For example, a number of biochemical approaches have been proposed to distinguish embryogenic calli (Al-Khayri, 2005). The somatic embryo (SE) differs from the zygotic embryo (ZE), as it lacks a dormancy phase, and it also lacks a seed integument and an endosperm, both of which are required for seed survival and germination (Brownfield et al., 2007). This may explain why SE seedlings are less vigorous than those raised from true seed. More knowledge is required concerning SE physiology, and especially of the differences in protein content between SE and ZE, which may improve the quality of SE and SEderived seedlings. The poor vigour of SE-derived seedlings seems to be related to their incomplete maturation under standard in vitro conditions (Roberts et al., 1990). Major changes take place in the latter stages of ZE maturation, including a switch from cell specification over to the accumulation of carbohydrates, thus preparing the embryo for full seed development (Yadegari and Goldberg, 1997).

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Therefore maturation is considered as a critical step in embryo development. Histocytological observations indicated that storage protein accumulation in SE is poor compared to ZE (Aberlenc-Bertossi et al., 1999; Sane´ et al., 2006; Zouine et al., 2005). In Eucalyptus nitens, the cells of SE and ZE contain similar lipidrich globular bodies, except in the meristematic regions. SE have slightly lower levels of storage proteins in their cotyledonary cells compared to ZE (Saumitra and John, 2000). These carbohydrate reserves play a vital role in seedling survival and development, as they sustain the seedling until photosynthesis (Galau et al., 1986). The storage proteins of dicots are predominantly albumins and globulins, whereas prolamin and glutelin are the major ones in monocots (Derbyshire et al., 1976). In the case of rice seeds, glutelin is the predominant storage protein, reaching 80% of total endosperm proteins (Takaiwa et al., 1999). In the present report, a comparative study between protein content of date palm ZE and SE is given. The evolution of the glutelin as an important known storage protein during embryo’s development was also investigated. This latter protein may be a useful biochemical marker. 2. Materials and methods 2.1. Plant material and embryo collection ZE were collected from date palm kernels (cv. Deglet Nour), beginning 10 weeks after pollination (WAP) when the embryo endosperm had hardened and finishing after 19 weeks. Fifty ZE were collected at each harvest. Mature seeds were imbibed in water to induce germination and raised at 25  1 8C in the dark for 25 days. Every 3 days 20 seeds were collected. Somatic embryos were obtained from embryogenic suspension cultures derived from the DN variety as previously described (Fki et al., 2003). Before being structured, samples of 0.5 g of embryos were removed from their maintenance media (M3) and transferred on a fresh medium. Embryos were subcultured weekly until maturation (structured embryos). Three development stages of SE were identified (Fig. 1B). 2.2. Protein extraction The embryos were frozen in liquid nitrogen and ground into a fine powder and mixed with 50 mM Maleate Tris Buffer (pH 8.3) containing 2% SDS, 0.5 mM EDTA, 2 mM PMSF, 1 mM DTT and 2 mM b-mercaptoethanol. After centrifugation, the quantity of soluble proteins in the supernatant was estimated using Bradford’s Method (Bradford, 1976). Proteins were then separated by SDS-PAGE according to Laemmli (1979) and stained with Coomassie Brilliant Blue-R250. The molecular weights (MW) were obtained by comparison to standard protein markers (SDS-PAGE Standards, 161-0304, Bio-Rad). The protein N-terminal sequencing was performed using an Applied Biosystems Procise 492 equipped with 140 C HPLC systems.

Fig. 1. Date palm seed cv. DN incised in the middle showing the position of the ZE (A). The three stages of date palm SE (B): stage 1: ovoid embryo; stage 2: longed embryo and stage 3: structured embryo.

2.3. Immunological analysis After SDS-PAGE electrophoresis, the glutelin protein band was cut from the gel and used as antigen for rabbit immunization. The gel slice was dissolved in 0.5 ml of PBS and the mixture was injected into a rabbit in presence of complete Freund’s Adjuvant (F-5881, SIGMA). Four subsequent injections were performed every 10 days in presence of incomplete Freund’s Adjuvant (F-5506, SIGMA). The rabbit immunological reaction was tested by ELISA using an antirabbit serum (1/1000) conjugated to the peroxidase. The optical density was measured at 492 nm. The obtained rabbit antiserum was used for protein gel blot analysis as follows: after electrophoresis in SDS-PAGE, the proteins were electroblotted on a nitrocellulose membrane using 20 mM Tris Buffer containing 150 mM glycine (pH 8.5) at 70 V for 1 h. The fresh membrane was blocked for 2 h with PBS buffer 3% milk containing rabbit polyclonal antiserum (1/1000) of date palm glutelin. The membrane was then incubated with an anti-rabbit IgG conjugated with peroxidase for 2 h. The reaction was revealed using the ECL Plus Western Blotting Detection Reagents from Amersham Biosciences, as described by the Supplier. Glutelin protein was quantified in crude protein extract by indirect ELISA. Proteins (20 ng) extracted from embryos were diluted in PBS buffer. Immunoplate wells were coated with 100 ml of protein solution for 24 h at 4 8C. The plate wells were then blocked with 3% milk in PBS for 2 h at 37 8C. Polyclonal

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antibodies were diluted to a suitable concentration in blocking solution (1% milk-PBS) and incubated in the plate wells for 2 h at 37 8C. The plate wells were then washed with PBS-T (1%) solution. The second antibody (anti-rabbit IgG conjugated to peroxidase previously diluted to 1:5000 in PBS milk) was added to each well and incubated for 1 h at 37 8C. Plates were washed with PBS-T and then 100 ml of OPD (SIGMA, P-5412), prepared in substrate buffer (Bio-Rad, 2J0431), was added to each well. After incubation for 30 min at room temperature, the absorbance was measured at 492 nm. 3. Results 3.1. Fresh, dry weights and water content of zygotic and somatic embryos The ZE (Fig. 1A) were extracted from seeds of date palm cv. DN after the transition stage (see section 2). The regular determination of the dry and fresh weights, at different times after pollination showed that they increased until maturation (Fig. 2A). At 12 WAP the water content started to decrease from an initial level of 76.25% to reach 28% in the mature embryo. SE showed rapid in vitro-development, with morphological and shape changes occurring within 1 month. The embryos were then classified according to their developmental stages: globular (stage 1), elongated (stage 2) and structured (stage 3) (Fig. 1B). The dry weight of the SE remained constant during

Fig. 2. Variations in dry weight and water content during development of zygotic (A) and somatic (B) embryos. Data represent means  standard error for three replicates from different extracts.

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stages 1 and 2, then decreased slowly at stage 3 while the water content remained high during all stages reaching 90% at the latest one (Fig. 2B). In contrast, the ZE water content is low at the end of seed development. 3.2. Comparison of the protein content in mature zygotic and somatic embryos The quantitative analysis of date palm ZE protein content showed that it was approximately 20-fold higher than SE, with 120.4 and 5.28 mg g 1 FW, respectively. This important difference in protein content can be explained by the presence of a much higher number of proteins in ZE than in SE, as shown by SDS-PAGE analysis (Fig. 3). The ZE major bands with MW ranging from 16 to 82 kDa were named P1 to P13. The P1, P2 and P3 protein bands of high MW ranged between 55 and 82 kDa, showed the most darkly stained bands. The comparison of protein profiles between zygotic and somatic embryos showed that they share five common bands (P3, P4, P5, P8 and P9). The other bands P1, P2, P6, P10, P11, P12 and P13 were only present in the ZE profile. However, a 30 kDa band was detected only in the SE protein profile (Fig. 3). The P3 protein band was more abundant in the ZE profile. To investigate further the differences between the ZE and SE protein profiles, the three protein bands corresponding to P1,

Fig. 3. SDS-PAGE analysis of total proteins from mature zygotic and somatic embryos of date palm (cv. DN). MM: molecular mass standards, numbers indicate molecular weight of protein markers in kDa; ZE: zygotic embryos; SE: somatic embryos. Proteins were resolved in a 12% polyacrylamide gel and stained with Coomassie Brilliant Blue.

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Table 1 Comparison of amino acid sequences of P1, P11 and P12 bands with those from other species (EMBL data base)

The N-terminal sequences of bands P1, P11 and P12 are in bold.

P11 and P12 in the ZE profile were excised from the gel and their N-terminal were sequenced by EDMAN sequencing method (Hewick et al., 1981). Amino acid sequence comparisons in the EMBL data base (Table 1) showed that the N-terminal sequence of the 22 kDa ZE band (P12) displayed high identity (82%) with an oil palm glutelin (Elaeis guineensis: accession number AAF69015). This known storage protein was then used as a potential marker to follow embryonic maturation. The N-terminal sequences of P11 and P1 bands displayed identities with the 1-cys-peroxyredoxin of Medicago truncatula (68%) and a phenylalanine ammonia-lyase from Populus tremuloides (80%), respectively. 3.3. Analysis of the protein contents during ZE and SE development In order to define specific markers characterizing distinct embryogenesis phases, the accumulation of total proteins was monitored during ZE development, from the moment of solidification of the endosperm (10 WAP) until the maturation stage (19 WAP). The data showed that total protein level increased from an initial of 49.76 mg g 1 FW up to 120.3 mg g 1 FW at embryo maturity. The protein level remained constant from 17 WAP until dormancy (around 110 mg g 1 FW). The investigation of the protein profile evolution during ZE development showed that important changes in protein pattern started after 13th WAP, with the appearance of the P2 band and a number of other proteins ranging between P4 and P12 (Fig. 4A). The P1 and P3 content increased whereas other bands remained at the same level throughout all development stages (particularly P4, P9 and P12). The protein level for SE during the various developmental stages was low and remained steady (5.5 mg g 1 FW), and the SDS-PAGE showed a similar protein composition without any modification in protein profile (Fig. 4C). During the germination process, the protein content started to be similar to that of mature ZE until 9 days of germination (DG). During this period, the protein profile remained stable and similar to that of mature ZE, then decreased abruptly

between the 9th and 12th DG (Fig. 5A). There were notable changes in the number and intensity of protein bands after 12 DG. The P4 band increased in strength while other bands disappeared gradually especially P1, P2, P3, P9 and P12 (Fig. 4B). This decrease in quantity and number of protein bands coincided with the emergence of the cotyledonary leaf (Fig. 5B). The P12 protein remained constant during ZE development and disappeared during germination. This protein could also be a storage protein since it shares 82% of identity with the glutelin of Elaeis guineensis, so we have considered it as a biochemical marker of the mature phase. 3.4. Glutelin as a biochemical marker of the developmental process In order to follow the evolution of glutelin content during development of ZE and SE, a specific antiserum was produced by injection of the purified protein into a rabbit. These antibodies were able to recognize the target proteins (P12) from a crude extract of ZE (Fig. 6). An ELISA test on crude protein extracts obtained from different stages of ZE maturation showed that the glutelin remained constant (OD  0.02). P12 decreased rapidly in germinating embryos and disappeared after 12 DG (Fig. 7). In contrast, no glutelin signal was detected throughout the three stages of SE development, suggesting a lack of this storage protein in this kind of embryo which confirms our previous results. They correlate also with the SDSPAGE analysis showing that glutelin level remained stable during ZE development but it rapidly disappeared during germination. 4. Discussion 4.1. Comparative study between zygotic and somatic embryos The comparison of ZE and SE described in this report showed that ZE reach maturity after 23 WAP, whereas SE

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Fig. 5. Accumulation of total soluble proteins of zygotic embryos of date palm cv. DN during germination. Data represent means  standard error for three determinations from different extracts (A). Date palm seeds during germination (B), DG: days of germination (numbers of days are indicated).

Fig. 4. SDS-PAGE analysis of total proteins of zygotic embryos date palm cv. DN during development (A), germination (B) and during somatic embryos development (C). MM: molecular mass standards, numbers indicate molecular mass of protein markers in kDa; ZE: zygotic embryos. Proteins were resolved in a 12% polyacrylamide gel and stained with Coomassie Brillant Blue.

maturation took 1 month, and can be classified into three stages. These data are in agreement with those reported by Sane´ et al. (2006), who observed such difference between both kinds of embryos. They were also able to distinguish three maturation stages 1, 2 and 3 in date palm SE. ZE water content showed a decrease during maturation, which is known to induce suppression of precocious germination in Arabidopsis. Water depression is required for acquisition of desiccation tolerance and induction of dormancy (Koorneef and Karssen, 1994). However, the high water content of SE contributes to its rapid germination, without undergoing any dormancy process. Consequently, the regenerated plants will be less vigorous than those obtained through zygotic embryogenesis (Dodemann et al., 1997).

Fig. 6. Protein gel blot analysis of zygotic embryo crude extraction with antiglutelin, MM: molecular mass standards, numbers indicate molecular mass of protein markers.

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carbohydrate and protein accumulation in SE during maturation. Date palm ZE continually accumulate proteins until maturity. Some of these may be storage proteins, as they rapidly disappear during germination. Protein content changed and declined during germination, especially between 9 DG and 12 DG, which corresponds to the emergence of the cotyledonary leaf. Since this process needs the hydrolysis of storage proteins, we suggest that these proteins could be classified as storage proteins. These results are in agreement with those found by Lai and McKersie (1994), who showed that storage proteins and starch reserves are rapidly hydrolysed following germination of alfalfa embryos. Fig. 7. ELISA tests for ZE during development and germination.

Protein content of date palm ZE and SE showed that it was higher in ZE compared to SE, as was reported by Sane´ et al. (2006), after staining cytoplasm proteins by NBB. They concluded that a fundamental difference between the development of date palm SE and ZE is the very weak accumulation of reserves during SE development. Similar results were also found for alfalfa SE and mature ZE (Lekha and Bewly, 1998). The cotyledonary stage SE displayed less protein content when compared to ZE at the same developmental stage. These differences also concerned protein profiles. The ZE was rich in protein, with the presence of three darkly stained protein bands (P1, P2 and P3) with MW between 55 and 82 kDa. These proteins have their homologues in the oil palm ZE profile (Chandra and Demason, 1988). Differences between ZE and SE were previously described for white spruce (Joy et al., 1991). They are likely due to differences in the embryonic environment. Contrary to the ZE, SE may lack biochemical signals for synthesis and accumulation of storage carbohydrates. Moreover, the composition of the culture medium may not be adequate for such massive synthesis (Joy et al., 1991). These conditions are not in favour of endosperm differentiation and suspensor tissue formation as reported for carrot somatic embryos (Dodemann et al., 1997). ZE protein content showed that the date palm protein profile did not show a significant variation during the first and the last developmental stages. Important changes were evident in the protein profiles during the intermediate phase (13 and 15 WAP). According to these data, it seems that the 13–15th WAP correspond to an important period during which significant quantitative and qualitative changes occurred in ZE protein content. Similar data were reported for avocado ZE (Carolina et al., 2002), where noticeable changes in storage protein content were observed during maturation. Several storage proteins initiated their accumulation at early stages and then remained constant during embryo development, while other proteins seem to be associated with the latest maturation stages (Carolina et al., 2002). In contrast to ZE, the protein level and the SDS-PAGE profile of SE remained fairly constant during the different developmental stages. Similar results were reported by Sane´ et al. (2006). They found little evidence of

4.2. Glutelin as a biochemical marker of the developmental process The immunoblotting results showed that glutelin is present during the first stage of ZE maturation. The presence of the glutelin storage protein at an early developmental stage in ZE of other angiosperms was reported (Rahman et al., 1982). Raghavan (1997) showed that avocado embryos (Persea americana Mill.) accumulated 40, 45 and 48 kDa albumins at early developmental stages. The same patterns were described in carrot, in which the synthesis of storage proteins occurs as soon as embryogenesis is initiated and independently of any maturation background (Dodemann, 1995). However, in a standard developmental medium, date palm SE did not accumulate glutelin at any stage. Thomas (1993) suggested that the expression of seed storage proteins is tissue-specific, since it occurs in embryo and endosperm but never in mature vegetative tissues. The sequential biosynthesis and accumulation of specific storage proteins showed that they are under a strict developmental control (Goldberg et al., 1989). They were then used as indicators of embryo developmental processes. Low rate of protein accumulation observed in somatic embryos could be due to a lack of precursors in the medium (Misra, 1994; Nomuna and Komanine, 1995). It may also be the result of the absence of inducing signals (e.g. hormones and/or desiccation), which are required to stimulate the synthesis of specific molecules. The absence of such storage protein accumulation may explain the lack of maturation phases in SE under the culture conditions used. 5. Conclusion Zygotic and somatic embryogenesis are complex phenomena that have been widely described in the literature. In this paper a comparative study between ZE and SE protein contents was carried out. In ZE, the physical enclosure by the seed coat, the gradual lowering of water content and the qualitative and quantitative changes in protein content lead to the developmental arrest. However, in the case of SE, such programmed and co-ordinate changes do not occur, as the embryonic environment is different from that found in ZE. Thus, it is not surprising to find that SE germinates precociously. The glutelin was considered as a potential marker for ZE embryo development.

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