بسم الله الرحمن الرحيم

Introduction Date Palm (Phoenix dactylifera L.) is a salt and drought tolerant fruit crop mainly cultivated in the Middle East. The date palm (Phoenix dactylifera L.) is today a major world fruit crop in the Middle East and North of Africa countries. Number of other countries became interested in date palm such as China, USA, Turkey, Albania, Spain, Mexico, Benin, Kenya, Namibia, Peru, Cameron, Swaziland and Colombia where it has been introduced for commercial production. Population of date palms in the world now exceeded 100 million. Egypt, Iran, Saudi Arabia, UAE and Pakistan are the major countries of the date palm population. Adel A. Abul-Soad

Statistics of Dates Fruit (FAO, 2008) Worldwide, date fruit production stands at about 6.8 million mt per year. Ten countries of Middle East and North Africa produce about 90% of the world’s date production. There are 3000-5000 different cultivars in the world. Adel A. Abul-Soad

Background Shoot tip explants of offshoots were used traditionally for various micropropagation protocols on research or commercial levels. However, its main disadvantage was the scarification of the entire plant. Subsequently, this hinders the micropropagation of male and female recalcitrant individuals with no offshoots or the interesting cultivars with a limited population. Consequently, current study is a step wise protocol for commercial production of tissue cultured palms. Adel A. Abul-Soad

Date Palm (Phoenix dactylifera L.) Micropropagation by Inflorescence Adel Ahmed Abul-Soad Horticulture Res. Inst., Agricultural Res. Center, Egypt adelaboelsoaud@gmail.com

Adel A. Abul-Soad Why are we using inflorescence explants ? Wide Objectives: Micropropagation of superior productive female lines. Micropropagation of evaluated and powerful male lines. Micropropagation of resistant palms to epidemic diseases (Fusarium oxysporum albdenis) and pests (Rhynchophorus ferrugineus F.).

Adel A. Abul-Soad Specific Objectives: The lowest percentage of contamination. No browning was noticed. High amount of meristematic tissues. Short term of the starting stage (6m). Getting direct organized tissues. Reducing the overall production cycle.

Adel A. Abul-Soad How to sustain that?

Adel A. Abul-Soad Surface sterilization of inflorescence explants

Adel A. Abul-Soad Culturing the explants onto the starting nutrient medium

Adel A. Abul-Soad Inflorescence explants

Adel A. Abul-Soad Schematic illustration of a whole female inflorescence (floral bud) components of date palm, 1- inflorescence base; 2- spike base; 3- spikes; 4- protective sheath.

Adel A. Abul-Soad 1- inflorescence base 2- spike base 3- spikes 4- protective sheath

Adel A. Abul-Soad Spikes of inflorescence during starting stage

Adel A. Abul-Soad Direct conversion of florets into direct somatic embryos with minimal callus appearance.

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Adel A. Abul-Soad Differentiation of embryogenic callus

Shoot proliferation Adel A. Abul-Soad

Rooting of date palm plantlets Adel A. Abul-Soad

Acclimatization Adel A. Abul-Soad

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Genetic Stability

Adel A. Abul-Soad DNA Extraction from the plant materials (Walbot, 1988): Homogenization of the starting material. Lysis of starting material. Removing proteins and RNAs from the samples. Precipitation of DNA.

Adel A. Abul-Soad Gel electrophoresis and DNA visualization: The agarose gel (electrophoresis grade) was suspended in an appropriate amount of electrophoresis buffer in a flask (1 gm/100 ml of electrophoresis buffer). The suspension was boiled until the agarose had dissolved. The agarose solution was cooled to 60 ºC, inserting a slot-forming comb (2 mm) and the agarose was poured into the gel mold to make thickness about 5 mm. The gel mold was inserted into an electrophoresis apparatus filled with buffer. Slowly the samples of DNA were loaded into submerged slots by using micropipette. Power supply of the electrophoresis apparatus was turned on, and samples were allowed to migrate toward the anode at 65 volts for 1-2 hours. The amplification products were analyzed by electrophoresis in 2% agarose in TBE buffer, stained with 0.2 g/ml ethidium bromide and in TBE buffer, stained with 0.2 g/ml ethidium bromide and photographed under UV light.

Adel A. Abul-Soad Fig. 15. RAPD-based DNA fingerprints of in vitro propagated date palm shoots (lanes 3-5), tissue culture-derived date palm plantlets (lanes 6-8) and control donor mother tree (lanes 1-2). RAPD fingerprints were generated by PCR amplification using the random primer OPD2. M is 1 kb plus DNA marker of Gibco.

Adel A. Abul-Soad Fig. 16. RAPD-based DNA fingerprints of in vitro propagated date palm shoots (lanes 3-5), tissue culture-derived date palm plantlets (lanes 6-8) and control donor mother tree (lanes 1-2). RAPD fingerprints were generated by PCR amplification using the random primer OPB18. M is 1 kb plus DNA marker of Gibco.

Adel A. Abul-Soad Fig. 17. RAPD-based DNA fingerprints of in vitro propagated date palm shoots (lanes 3-5), tissue culture-derived date palm plantlets (lanes 6-8) and control donor mother tree (lanes 1-2). RAPD fingerprints were generated by PCR amplification using the random primer OPC14. M is 1 kb plus DNA marker of Gibco.

Adel A. Abul-Soad Recommendations: The best method to surface sterilize the female spathe of date palm was using Hg Cl2 solution at 0.1% for 5 min. Date palm spikes at 2.5 cm in length need to be oriented to somatic embryogenesis as a main target.

Adel A. Abul-Soad Explants of small spikes cultured onto the basal nutrient medium supplemented with 2.5 mg l-1 2, 4-D + 0.5 mg l-1 IBA + 0.2 mg l-1 2ip, the small florets which existed on the spikes formed a white callus.

Adel A. Abul-Soad The RAPD analysis indicated that all DNA patterns of in vitro plantlets; the control donor mother tree, from which the explants were removed and the established tissue culture plantlets were similar. Subsequently, no variation was occurred during micropropagation process.

Adel A. Abul-Soad Thank you for your attention