SOMATIC EMBRYOGENESIS AND MASS MICROPROPAGATION OF DATE PALM(Phoenix dactylifera L.). CHARACTERIZATION AND GENETIC STABILITY OF

REGENERATED PLANTLETS BY RAPD MARKERS.

R. Letouze (*),F. Daguin (*),P. Satour (*)L. Hamama (*)and F. Marionnet (U)

ABSTRACT

In the last few decades plant regeneration of date palm (Phoenix dactylifera L.) throughtissue culture has been succeeded, but mass production tI~ue-to-typefrom the mother plant is stilla long and difficult process. Since 1985our laboratory has successfully regenerated date palm ofmore than 50 cultivars through somatic embryogenesis. We report here, a protocol for massregeneration of date palm through somatic embryogenesis and the use of molecular technique forplant material characterization and genetic stability. Thirteen date palm cultivars were screenedfor polymorphic RAPD (Random Amplified Polymorphic DNA) markers with five arbitrary 10-mer primers (A02, A08, B07, BIO and E07). This set of five primers provided fourteenpolymorphic markers that can be used to distinguish the thirteen date palm cultivars. A set of sixprimers was used for Barhee (E07, A02, A05, A08, Al3 and A20) and Mejool (EIS, A02, A03,A05, A08 and A13) to evaluate the genetic stability of somatic embryo-derived plants. Theutilization of RAPD markers both for the identification of date palm cultivars and for theassessment of genetic stability through the process of somatic embryogenesis is reported.

Key words: date palm, plant tissue culture, somatic embryogenesis, RAPD markers.

INTRODUCTION

Date palm (Phoenix dactylifera L.) is an essential plant crop for several million people in theMediterranean region and in the south of Maghreb countries. The crop has been for centuries oneof the key elements in the oasis economy. The date palm is a dioecious and heterozygote speciesand has to be propagated by vegetative means. The multiplication of the species is carried out bythe means of offshoots. This method of propagation is limited and reduces considerably the

. expansion of the existing palm groves, especially in the north of Africa. This method ofpropagation is very slow and cannot solve two important problems :

the lack of material of high quality for the expansion and development ofsome of the existing palm groves, especially in the north of Africa;

the Bayoud disease, the most lethal disease known to date palm causedby a soil fungus (Fusarium oxysporum f. sp. albedinis) in the south ofMorocco and Algeria (Djerbi 1988).

.. For the last 20 years, results have shown that date palm can be propagated by tissue cultureboth by organogenesis (Poulain et aI., 1979; Beauchesne, 1983; Drira, 1983) or by somaticembryogenesis (Tisserat, 1979; Reuveni, 1979;Sharma et al., 1984;Daguin and Letouze, 1988;and Letouze and Daguin, 1989).

"(:") La'boratoirede Recherches en Physiologie Vegetale des Pays de la Loire (L.R.P.V.)'16, bd Lavoisier F-49045 ANGERS Cedex 01 - FRANCE-

(**) AI-Wathba Marionnet L.L.C. p.a.Box 41522 Abu Dhabi - UNITED ARAB EMIRATES-

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Currently, the process of somatic embryogenesis seems to be more attractive for an industrialproduction but the date palms derived from somatic embryos must be true-to-type. Proteins andisoenzymes have been used in date palm cultivar identification and somac1onal variabilityanalysis during the past years. (Torres and Tisserat, 1980;Baaziz and Saadi, 1988; Stegemann etaI., 1987; and Bendiab et aI., 1998).This method presents some inconvenients: the products ofgene expression may vary at different developmental stages or under environmental conditions.The advent of molecular techniques DNA-based procedures have been proposed for cultivaridentification. Williams et al. (1990) and Welsh and McClelland (1990) reported a techniquebased on the amplification of random DNA sequences by polymerase chain reaction (PCR) witharbitrary primers. RAPD (Random Amplified Polymorphic DNA) markers are now used foridentification (Hu and Quiros, 1991; Koller et aI., 1993; and Aruna et aI., 1995) to evaluategenetic integrity (Isabel et aI., 1993)or analysis of somac1onalvariants (Hashmi et aI., 1997).

In 1976, our Laboratory first succeeded in the regeneration of date palm by tissue culturethrough the process of organogenesis. Since 1985our laboratory successfully regenerated datepalm of more than 50 cultivars through somatic embryogenesis. For about 10 years ourLaboratory is working by contract with an industrial Laboratory which is now established inUAE : Al Wathba-Marionnet L.L.C. Now, in relation with this industrial partner, a capacity ofproduction more than one hundred thousand plantlets per year is obtained.

This research reports 13years of work on the regeneration / micropropagation of date palmthrough somatic embryogenesis for industrial propagation purpose as well as the use of RAPDmarkers to characterize the plant material and to study the genetic stability of somatic embryo-derived plants.

MATERIALS AND METHODS

Plant Material

Plant material (offshoots) were received from USA (Barhi, Khalas, Zahidy, Medjool) UAE(Barhi, Khalas, Nabtha saif, Kowaiz, Owaid, Hasawy, Umm Dahan, Saggai, Abu Man, Rothana)and Morocco (Medjool).

Somatic embryogenesis

Plant regeneration has been established using shoot tips from offshoots through the processof indirect somatic embryogenesis based on the method described by Tisserat (1979). Theinduction of embryogenic callus is achieved using a Murashige and Skoog (MS) medium(Murashige and Skoog, 1962)supplemented (Table 1) with plant growth regulators (2,4-D, 2-iP).Two other culture media (Table 1) are used for the multiplication/elongation and rooting stages.

In the culture room, plant material were exposed under photoperiodism (light period:15h/i5 !lmol m-2S-Ior 15h/50 !lmol m-2S-l/28 :i:0.50 C - dark period: 9 h/24 :i:0.50 C).

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DNA extraction

Plant genomic DNA samples were extracted from the offshoots, vitroplants and acclimatizedplants following the CTAB method (Doyle and Doyle, 1990). DNA concentration was measuredwith the use of a TKO 100 minifluorometer (Amersham Pharmacia Hoffer, Uppsala).

Table 1. Composition of the culture media for date palm somatic embryogenesis (mg per liter).Basal medium of Murashige and Skoog (1962) with mQdifications.

DNA amplification

Decanucleotide primers from Operon Technologies (Alameda, California) were used for theRAPD analysis based on the protocol of Williams et al. (1990) with minor modifications.Amplification reactions were performed in a 25-IlI reaction mixture consisting of 10mM Tris-HCI pH 8.3 ; 2 mM MgCh; 100 IlM each of dNTP ; 0,2 IlM of primer; 25 ng of DNA and 0,5unit of Taq DNA Polymerase (Perkin Elmer Cetus). The mixture was covered with 25 IIIofmineral oil. For the DNA amplification a Perkin Elmer Cetus 480 DNA thermal cyder wasprogrammed for 4 min at 94°C for 45 cycles, each consisting of a denaturation step (1 min at 94°C), followed by an annealing step (1 min at 36°C) and of an extension step (2 min at 72°C).The last cycle was followed by 10 min at 72°C to ensure that primer extension reactionsproceeded to completion. After the amplification was completed, amplified samples were loadedand electrophoresed on 1.4 % agarose gel, followed by staining with ethidium bromide. The gelswere photographed under UV with Polaroid films (Polaroid 572). The pictures were used for theanalysis of the amplification products. DNA amplifications were repeated three times.

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Component Stage of the CultureInduction Multiplication / Rooting

ElonqationMacronutrientsOligonutrients Murashige and Skoog (1962)Fe-EDTA nutrientand vitaminsaltsVitaminsSucrose 45 000 50 000 50 000Meso-Inositol lOO 100 100GellingAgent (Agar)7 000 (Karraghenan)6500 .

ActivatedCarcoal 3000 500 1000Glutamin 100 200 lOOAscaral!:in lOO - -di-Hydrogeno 100 lOO 50ammoniumcitrateKH2P04 - lOO lOONaH2P04,2H2O - 170 1702,4-D 100 - -2-iP 3 - -

RESULTS AND DISCUSSION

Plant regeneration

Apical tissues of offshoots were fral0 explants. A callus induction medium containingMS salts, supplemented with organic nitrogen (glutamin 100 mg/L, asparagin 100 mg/L, di-ammonium citrate 100 mg/L). and with an enriched sugar concentration (sucrose 45 g/L) provedto be effective to initiate somatic embryogenesis in date palm tissues. Microbial contaminationsof the cultures occurred in only few cases less than 3 to 5 %. Cultures were maintained in thedark during the first three months, the pro-embryogenic calli appeared about two to three monthslater and embryogenic calli were formed within four months of culture. After being maintainedin the dark, the cultures were placed under low intensity light (15 Ilmol m-2S-I).

Embryos development began when the embryogenic calli were transferred on a hormonefree medium. At this stage, the tissue culture was maintained under a light intensity of 50 Ilmolesm-2 S.I. Embryo development could be improved if they were transferred in a liquid mediumcontaining half strengh MS salts, 30 g/L sucrose, 3 g/L activated charcoal and placed on a rotaryagitator. After 30 days in the liquid medium the embryos larger than 5 mm were placed on asolid medium for embryo maturation while embryos between 50 Ilm and 5 mm in size wereplaced again in a fresh suspension medium (Daguin and Letouze, 1988).This process led to abetter individual development of embryos.

At the last stage of the in vitro culture, plandets were transferred for further plantdevelopment on a rooting agar-free medium (Table 1). After two or three subcultures, theplantlets were well formed with strong roots. The acclimatization of the plantlets was performedin the glasshouses at the industrial (GFA-Marionnet, France) with a success above 90 %.

The process of plant regeneration through somatic embryogenesis led to 10 000 to 12000date palm trees regenerated per offshoot within 16 to 20 months for the complete in vitro steps.Such results were assessed for more than 30 different cultivars and for several lines per cultivar.

Identification of date palm cultivars with RAPD markers

Figure 1 reports the amplification of genomic DNA of different date palm cultivars in thesame PCR run. A key was developed for 13cultivars (Table 2) using five primers (A02, A08,B07, B10 and E07). These primers were selected after preliminary experiments. A total of 48fragments were generated by the selected primers and could be used as potential genetic markersbut only 14 were used as polymorphic markers. The primer A02 (Fig. 1) amplified four RAPDbands (A02-930, A02-1020, A02-1150 and A02-1230). Three bands were amplified by theprimer A08 (A08-500, A08-630 and A08-870). Four bands were amplified by the primer B07(B07-630, B07-830, B07-91O and B07-1020), two bands by the primer BlO (BI0-830, BlO-1740)and only one fragment was amplified by the primer E07 (E07-1480).

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These 14 RAPD fragments produced by the five primers permit a theoreticaldifferentiation of 16 384 combinations. This technique provides a faster and easierapproach to detect extensive polymorphisms and needs very small amounts of genomicDNA. Amplifications were reproducible over time and gave the same RAPD profiles.The technique can be routinely applied for varietal identification (Isabel et aI., 1993).

Table 2 : Differentiation of 13 date palm cultivars using 5 primers (A02, A08, B07, BIO andE07). This set provided 14 polymorphic markers that can be used to distinguish the 13cultivars.

(+) presence of the chosen RAPD band(-) absence of the chosen RAPD band

Genetic stability of somatic embryo-derived plants

The genetic stability of somatic embryo-derived plants was evaluated with the aid of RAPDmethod (Isabel et aI., 1993). We report here only the work done on two high economic valuecultivar : Barhee and Medjool. Genomic DNA amplification was performed using a set of 6primers: E07, A02, AOS,A08, A13 and A20 for the cultivar Barhee and EIS, A02, A03, AOS,A08 and A 13 for the cultivar Medjool to evaluate the genetic stability of somatic embryo-derived plants.

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Cultivars

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1: A02-1230 + + + + + + + + - - + + +2: A0211S0 + + + - - + + + + - + - -3: A021020 - - - + + - - - - + - + +4: A02-930 + + + + + + + + + + + - +5: A08-870 + + - + + + + - + - + - +6: A08-630 + + + + + + + + + + + + +

en'"' 7: A08-S00 + + + +Q) - - - - - - - - -a8: B071020 + + + + + +.t:: - - - - - - -

p..9: B07-91O + - + - + - - + + - - + -10: B07-830 + + - + - - + + - + + +11: B07-630 - + + - - - + + - + + + +12: B1O-1740 - + + - + + + + + + + - +13: B 10-830 + + + + + - - + + - + + +14:E07-1480 - + - + + + + - - + + - +

For each cultivar, three stages were evaluated (Fig. 2): offshoot, embryogenninating andacclimatized plantlets. The results (Fig. 3) show that the polymorphism revealed by RAPDmarkers differed towards cultivars and primers used but that for a cultivar and a given primerchosen, no genetic change was detected regardless of the stage.

CONCLUSION

During the last five years in our laboratory, somatic embryo-derived plants led to true-to-type date palms from the mother plant and no variation was detected within clones. For the pastten to twenty years isoenzyme systems have been used in cultivar identification (Weeden andLamb, 1985) but these markers were limited and gave no direct assessment of the potentialvariation in the genome. The use of RAPD marker analysis for characterization and for varietalidentification is a technique of high efficiency to distinguish cultivars by comparing theiramplified DNA patterns.

The utilization of RAPD markers can now be used for cultivar identification and forassessing the genetic stability of somatic embryogenesis-derived materials.

ACKNOWLEDGEMENTS

Plant material was provided by Marionnet G.F.A. France, we thank C. Talion, assistant engineer, for his.appreciatehelp in tissue culture. This research was supported in part by EU-STD Contract TS2A 0097 F.

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Fig.1 : Amplification of genomic DNA of 13 different Date Palm cultivarsin the same PCR run using five primers (A02,A08, 807, 810 and E07).Thearrows(-to) indicatethe RAPDmarkers.

Date Palm cultivars - 1 ; Zahidi 2: Barhee 3: Nebusief 4: Sukkari 5: Khalass 6: Howaiz 7: Owaid8: Hasawi 9: Medjoul 10: Oum Dahan 11: Saggai 12: Abu Maan 13: Rothana

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Fig.2 : Stages of plant material evaluatedfor genetic stability of somaticembryo-derived plant.

Staqe 1 : Offshoot Staqe 2 : Embryo germinating Staqe 3 : Acclimatized plant

Fig.3 : Banding patterns of two cultivars (Barhee and Medioul) at threestages using a set of six primers.

-Sarhee: PrimersOPE-O?,OPA-02,OPA-05,OPA-08,OPA-13andOPA-20.

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