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Chapter 1

Introduction: Date Palm Biotechnology from Theory to Practice D.V. Johnson

Abstract  Date palm has been a cultivated tree crop for at least 5,000 years. Early date palm-specific technologies were developed to enhance crop productivity and fruit quality by means of selecting superior female palms and their propagation by offshoots. Other old innovations included crop and water management, segregation of trees by gender, artificial pollination, naming of cultivars and the characterization of fruit development stages, fruit flesh texture and fruiting seasonality. Modern biotechnology techniques are carrying forward date palm development in attempts to understand the genetic basis of the palm, to produce tissue-cultured plantlets on a large scale to more rapidly expand planting and replanting of date groves and to employ molecular breeding of new cultivars for increased fruit yield and resistance to pests and pathogens. This volume surveys the current state of date palm biotechnology through contributions by leading researchers in the field. Keywords  Cultivar • Domestication • Gender • Genome • Germplasm • Molecular biology • Pathogens and pests • Pollination • Propagation • Tissue culture

1.1 Introduction This comprehensive volume covers a broad range of highly-technical subjects demonstrating how modern scientific theory and practice are being applied to overcome agronomic impediments to achieving the long-term goal of sustained, expanded and enhanced quality of date fruit production worldwide. Disease and pest problems threaten date growing in several countries of North Africa and the Middle East and biotechnology has the potential to make significant contributions

D.V. Johnson (*) 3726 Middlebrook Ave, Cincinnati, OH 45208, USA e-mail: [email protected] S.M. Jain et al. (eds.), Date Palm Biotechnology, DOI 10.1007/978-94-007-1318-5_1, © Springer Science+Business Media B.V. 2011

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to their effective management. DNA studies which are able now to confirm the identity of date palm cultivars, have also provided evidence of genetic resistance to some diseases and pests in certain cultivars. Rather than attempt to describe the technical contents of this book, my intention is to provide some background and context to the chapters which follow. This I believe can be accomplished by sketching major technical breakthroughs associated with the date palm over its long history. Crop domestication is a lengthy process, and we need to remind ourselves that virtually all of the major world crops were brought into cultivation by innovative food providers long before the advent of modern agricultural sciences. The date palm serves as a general example of the necessary sequence of technologies that had to be devised to domesticate, propagate and manage the palm, and to achieve fruit yields of high quantity and quality, sufficient to justify the effort in terms of human inputs. The remainder of this introduction is divided into three sections. First is a discussion of selected aspects of the date palm in terms of its biology, domestication and agronomy. The second section summarizes the technological innovations in early date palm growing, especially in Mesopotamia. Finally, the third section attempts to summarize the rapid technological advances in recent years with regard to tissue culture and molecular biology research. Before proceeding, it is worthwhile to review familiar terminology. The term technology refers to the application of science or the scientific method to achieve a targeted objective. These days we may think of it as referring almost exclusively to intricate sophisticated techniques or to machines that can perform remarkable tasks. But we must remember that the ancient world had its own technological obstacles and breakthroughs, many of them of a mechanical nature, but which had profound importance in their time. The term biotechnology incorporates the use of living organisms to the meaning of technology; it is a relatively new word coined in the early 1940s. A widely accepted definition of biotechnology is: “Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use.” (UN 1993). Science and technology represent, in human history, a hypothetical pyramid constructed such that each successful block laid down becomes a new foundation and provides an opportunity to erect yet another more sophisticated block above it.

1.2 Biology, Domestication and Agronomy The date palm (Phoenix dactylifera L.) is one of the 14 recognized species of the genus Phoenix, which is itself one of the 183 palm genera currently known. Phoenix belongs to the tribe Phoeniceae, the subfamily Coryphoideae and family Arecaceae (Palmae) and has a wide natural distribution in the Old World. Phoenix dactylifera is the type specimen for the genus. Species of Phoenix range in size from stemless to tall, the date palm being the largest, reaching over 30 m in height. Phoenix spp. are solitary or clumping and dioeceous; inflorescences are interfoliar with male and

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female flowers borne on separate trees. The leaves are pinnate, induplicate (V-shaped) and erect, with basal leaf spines. In date palm the leaves are 3–6 m in length. Phoenix fruits are ovoid to oblong, smooth and have a fleshy mesocarp. Domesticated date palm fruits have quite variable shapes and range in size from 18 to 110 × 8 to 32 mm with the weight of an individual fruit varying from 2 to 60 g (Zaid 2002); they are significantly larger than in other Phoenix species. Date palm has 36 chromosomes (n = 18; 2n = 36). Worldwide, about 3,000 date-palm cultivar names exist; however, some names are probably synonyms, the result of a local or national name given to a cultivar which also occurs in another country, but under a different name. Apart from being classified by cultivar or variety according to fruit characteristics, two other sets of overriding fruit designations typically are employed: flesh consistency and ripening time. Date cultivars are grouped into three classes: soft, semidry or dry, based upon the texture of the fruit under normal ripening conditions. Cultivars are also classified according to the length of time needed to produce mature fruit, as early, midseason and late. Date palm is one of the world’s first cultivated fruit tree. It is one of the ­classical Old World fruits, and, along with the olive and fig, the three represent an ancient group of fruit trees closely associated with the beginnings of agriculture. Date palm was domesticated in Mesopotamia, modern-day Iraq, 5,000 or more years ago. Until recently, it was believed that there were no wild ancestors of the date palm. However, in recent decades, archaeological research coupled with contemporary botanical field and laboratory studies have revealed that the cultivated date is closely related to wild and feral populations in North Africa and the Middle and Near East. Wild dates are considered to be the same species, can hybridize with the named cultivars, and are morphologically similar to the domesticated form; the main distinguishing feature of wild dates is their much smaller fruits (Zohary and Hopf 2000). The value of the date palm within a subsistence or market economy represents much more than a source of nutritious high-energy fruit which can be eaten fresh, and also is easily stored by sun drying and used as supplementary foodstuff throughout the year. Fruits can be pressed into an easily-transportable date cake, made into syrup and fermented into date wine, vinegar and serve as a source of bioethanol; processing of the latter generates a feedstock by-product. When a tree is felled, the heart can be extracted and eaten. The pits (seeds) are eaten by livestock. At present, the fruits are a source of raw materials for agrofood industries and the production of secondary metabolites are important in human diet. In an oasis, the trees create a microenvironment suitable for other forms of agriculture and livestock raising, and provide much-needed shade. Stems of trees can be split for construction wood, the leaf midribs provide fencing material and the leaves can be woven into baskets, mats, hats and so forth. Date palm indeed is a multipurpose species from the past and of the present. Date palms are commonly distinguished between seed-derived (seedling or khalt) plants, the result of sexual propagation, and those derived from offshoots removed from desirable female or male cultivars. Seedling dates exhibit characters

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inherited from both parents and may be female or male, with gender only revealed at the initiation of flowering at 5–7 years of age. In contrast, an offshoot-derived plant is the gender of the parent plant and in other ways true-to-type. Date palms produce only a limited number of offshoots; estimates range from 20 to 30, most of them when the palm is relatively young. The number of offshoots produced varies with the cultivar and in certain ones is very low or even nil. Date palms reach their full fruit-bearing potential at about 10 years of age (Zaid 2002). On average, a date palm plantation has an economic life of 40–50 years. Some estimates are that the palm can continue to bear fruit until the age of 100 or even older, but no documented long-term studies in this regard appear to have been done. In practical terms, there are strong inducements for replacement planting of very old palms because of the difficulty in climbing ever-taller trees, declining productivity and greater susceptibility, with age, to pests, diseases and blow-down. Date palms cultivated in the typical desert environment flower once a year, in spring in the Northern Hemisphere. This habit appears to be governed by climate. In years of exceptional rainfall in the desert, a second flowering in the same year can occur. In the wild, date palms are pollinated by wind or insects. In extreme northern Chile, date palms grown under ideal stress-free conditions of plentiful irrigation water, high temperatures with very little annual temperature fluctuation, the palms flower and fruit continuously like coconuts. The fact that date palm has the genetic capability to flower and fruit continuously, very likely would not result in a greater annual fruit production per tree than occurs in a single annual flowering season. However, the potential of this genetic trait may be worth exploiting to cultivate dates, where similar ideal environments exist, for off-season marketing. Diseases and pests are, to varying degrees depending upon location, ever-present threats to date fruit production, and in certain cases to the life of the trees themselves. Among the various maladies affecting date palm, two are of contemporary prominence. Bayoud disease, a fatal vascular wilt, is caused by a soil-borne fungus disease ( Fusarium oxysporum). This disease first appeared in Morocco in the late nineteenth century and has spread widely in that country and into neighboring Algeria. In particular it attacks the prized Moroccan Medjool cultivar; it is dispersed chiefly by the transfer of offshoots. From the onset of symptoms of leaf withering, death of the palm follows in several months. No chemical or biological control is known; the only present solution appears to be the selection of cultivars which are resistant to the disease (Zaid 2002). A second major threat to date palms is the red palm weevil (Rhychophorus ferrugineus). Native to tropical Asia, in recent decades the insect has spread westward steadily into South Asia, the Middle East, North Africa and southern Europe. It attacks a number of commonly-cultivated palms. The larvae burrow into the palm trunk, but infestation is not obvious until serious damage has been done and leaf wilt occurs. At that point the palm is beyond recovery and dies when the terminal bud is attacked. Following good plantation practices to avoid plant stress and attention to sanitation appear to lessen the incidence of infestation. A variety of chemical and biological controls are being studied to control this insect but a protocol for early detection and effective treatment of infested trees has not been achieved.

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Increasing fruit yield is the key objective of date improvement efforts. Current yield figures can provide an approximate benchmark against which to measure future success, but average fruit yields per tree are very difficult to determine. For example, according to FAOSTAT, world date fruit production in 2008 reached 7,048,089  mt, from a harvested area of 1,264,611  ha. Calculated data gives an average yield of 5.57 mt/ha. The basic problem with such a calculation is that the number of trees planted per hectare varies widely depending upon whether the plantings in a country are traditional and without fixed spacing, or modern plantations with fixed spacing, or, a combination of the two. To demonstrate the reported range: in Morocco 50 trees/ha is typical, whereas in Somalia it reportedly can be to 577 palms/ha. Another difficulty is that yields measured by weight vary considerable among cultivars because of fruit size and weight. The most reliable yield figures come from two popular high-value cultivars. Medjool and Barhee, grown under modern plantation conditions in the USA and Israel, achieve yields of 80–120 and 200 kg per tree, respectively (Zaid 2002).

1.3 Early Date Palm Technology Date fruits, along with grapes and cereals such as barley, played a key role in Mesopotamia and Ancient Egypt to make fermented alcoholic beverages. In fact, the word alcohol is derived from Arabic. The development of fermentation technology, therefore, is directly related to date palm. At some later time, the technique of tapping date palm inflorescences and trunks for sap was perfected; sap also ferments naturally into a mild alcoholic beverage. The consumption of laqmi, fermented from either sap or fruit, at certain locations in North Africa, represents a contemporary practice of an ancient technology. At least seven ancient technologies, directly or indirectly related to date palm, were developed in Mesopotamia and are summarized below (Dowson 1921, 1923, 1982; Popenoe 1973; Pruessner 1920): 1 . Plant sexuality recognized; male and female date palms distinguished. 2. Date groves organized using fixed tree spacing and with catch cropping of annual crops in initial years of establishment. 3. Date palms under cultivation segregated by gender. 4. Free flow irrigation and other water management practices of date palm groves. 5. Artificial pollination of date palms. 6. Offshoot separation propagation devised. 7. Stages of fruit development were recognized and given specific designations, as follows: From pollination to final date-fruit ripening takes about 200  days. Date palm flowering and fruiting were recognized to have five distinct stages over the ripening period. Hababauk, female flowers and immediate post-pollination period when the very young fruits are creamy white in color; kimri, green fruit undergoing rapid growth; khalal, fruit grows slowly to full size, sugar content increasing while moisture

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content decreases, hard, glossy, red or yellow in color; rutab, fruits ripening to a soft stage, brown in color; tamar, fruits fully ripened, wrinkled, brown or black in color. In Arabic, tamar is also a generic term for dates. These designations, especially the last four, are basic technical terms in use today. The terms appear in the literature with variable English spellings. Apparently, the naming of date palm cultivars also originated in Mesopotamia to distinguish different fruit types. Popenoe (1973), writing about the ancient Zahidi cultivar of Iraq, reports on a comment to him by V.H.W. Dowson in the 1920s to the effect that Zahidi was a Basrah variety that, according to local tradition, was the first female variety known and that all other varieties were derived from it. Pruessner (1920) made calculations of fruit productivity of date palms in Mesopotamia through deciphering stone tablets from about 4,000  years ago, and estimated that annual yields per tree were in a range of 105–180 kg. These could be considered moderate as compared to contemporary yields. Two issues that early date palm technology could not deal with effectively were rapid cultivar propagation and an efficient method of breeding improved cultivars. Seed propagation, intentional or spontaneous, has a major drawback of producing mixed populations of female and male palms, and those progeny, because of crosspollination, did not fully replicate the desirable fruit characteristics sought. Offshoot propagation was able to solve the true-to-typeness problem, but with a major limitation because relatively small numbers of offshoots are available, far short of the numbers needed for rapid expansion of date palm cultivation. Since domestication, sexually propagated date palms, past and present, have produced many new forms and some of those with desirable fruit or other characteristics have become named cultivars, in turn being perpetuated and propagated by offshoots. But this did not contribute much to achieving positive changes in certain cultivars that only plant breeding could accomplish. The Department of Agriculture was a major promoter of date cultivation in the USA beginning in the early twentieth century with the importation of some 20,000 offshoots, representing about 150 cultivars, from Iraq, Pakistan, Egypt, Algeria, Tunisia and Morocco. Once the industry was well established, a breeding program was initiated at a date palm research station in Indio, California, which ran from 1948 to 1978. The primary goal of the breeding program, using backcrossing, was to obtain female Deglet Noor cultivars with fruit adapted to mechanical harvesting and processing, but without any loss of fruit quality. Deglet Noor has always been the most important cultivar in California and even today accounts for 70% of total production (Fig. 1.1). The program ended before that goal could be achieved. Some promising progeny from those efforts are preserved in the National Date Palm Germplasm Repository, Thermal, California. Genetic improvement through date palm breeding is not feasible because of the length of time for a palm to reach sexual maturity (5–7 years). Moreover, offshoot propagation cannot produce large numbers of progeny. Traditional date cultivation typically carried out on a small scale, faces special difficulties, often because of inaccessibility to modern technologies developed for and utilized by large commercial orchards. Cost is most often the main obstacle.

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Fig. 1.1  Mature stand of Deglet Noor cultivar in Indio, California. Note the metal ladders permanently affixed to the upper portion of the trees to facilitate access to the crown for date culture and management

However, traditional producers can achieve enhanced productivity by adopting inexpensive manual practices relative to watering, drainage, fertilizer, weed control, tree spacing, etc.

1.4 Tissue Culture and Molecular Biology The last half century has been characterized by scientific efforts to advance agricultural production through new methods of propagation and to gain insights into the molecular structure of plants to select progeny with enhanced productivity and resistance to specific pests and diseases. Both subjects treated in this section are being studied intensively as evidenced by the numerous recently-published references cited in the chapters which follow.

1.4.1 Tissue Culture Research on date-palm propagation via tissue culture (also called in vitro propagation or micropropagation) began in about the 1970s. At about the same time, palm tissue culture research also was undertaken with coconut (Cocos nucifera) and the

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African oil palm (Elaeis guineensis). Research progress made since then in date palm micropropagation has been reviewed by Al-Khayri (2005, 2007). Zaid (2002) provides a comprehensive discussion of all aspects of date palm propagation. In it he summarized the general advantages of tissue culture techniques. In slightly modified form they are as follows: • Production of healthy disease- and pest-free cultivars of female or male palms with desirable qualities, e.g. bayoud resistance in females; superior pollen in males; • Large-scale multiplication of plantlets at any season of the year; • Production of genetically-uniform progeny; • Ability to propagate elite cultivars which lack offshoots and seed-only derived plants; • Facilitate exchange of plant materials among laboratories for research purposes without risk of spreading diseases or pests and avoiding often cumbersome plant quarantine regulations; • Reliable source of a large quantity of plantlets, if required. Two tissue culture techniques are used. The first is somatic embryogenesis which involves plantlet production by generating embryos from cells not originating from reproductive organs. Tisserat (1981) is credited with developing the technique for date palm. In this technique, derived cells are cultured in a medium of growth regulators to produce a mass of disorganized cells called callus. Subsequently, the culture medium is modified to induce embryos from the callus. Key advantages of this method are that it produces bipolar structures ostensibly able to germinate, avoiding distinct phases of shoot and root induction. Continuous production is feasible to produce large numbers of plantlets at relatively low cost per unit. The main disadvantage is that high hormone levels in the medium make the plantlets prone to mutation which yields off-types, which are only manifested a few years later after field planting. Off-types produced by somatic embryogenesis, however, do represent induced genetic variability which may have desirable genetic traits of value in molecular breeding. The second technique, direct organogenesis, utilizes meristematic cells and low concentrations of plant growth regulators in the medium. Axillary bud, root tip or floral bud cells can also be used. The advantage of this technique is that it avoids callus formation and produces plantlets directly, which are true-to-type and not susceptible to mutation. On the other hand, a comparatively small number of plantlets are produced because many do not survive the rooting stage. Organogenesis is slow and expensive as compared to somatic embryogenesis. Research related to tissue culture is also investigating cell and protoplasm culture to produce callus and radiation to induce mutations in vitro to create promising genetic variants which then can be multiplied. In several countries, commercial plantings have been and are being carried out using tissue-cultured plants derived by both techniques. Private companies and government-supported facilities are engaged in plantlet production. Tissue culture laboratories are expensive to establish and run, and need to produce large quantities of plantlets to achieve an economy of scale. The future potential of tissue culture appears to be greatest in countries with large areas in production needing rehabilitation

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or replacement, or to establish extensive new areas. As research reveals more about the date palm genome, tissue culture represents the best technique for selecting and propagating new improved cultivars. Tissue culture also has a large potential role to play in date palm germplasm conservation. Long-term storage of tissue cultured material can be achieved by in vitro cryopreservation. Storage requires maintaining in vitro material at temperatures approaching −196°C, the temperature of liquid nitrogen. At that temperature all biological processes cease and the material remains genetically stable. Cryopreservation requires expensive refrigeration equipment and a reliable source of electrical power, but the actual storage space needed is minimal. The conservation of date palm germplasm would be well served if a website were created and maintained to provide a means for researchers and others to post and retrieve data and information on the conservation status of cultivars. Such a website could function as a clearinghouse to identify cultivars in particular need of cryopreservation. It could also provide valuable information on the existence and status of cultivars maintained as in vivo germplasm collections. As research on the molecular biology of date palms progresses, in vivo palms will provide a means to compare the DNA, for example, of a Medjool cultivar infected with bayoud with one that is healthy.

1.4.2 Molecular Biology Complementary to advances in tissue culture techniques are the development of new biotechnologies which can accelerate plant breeding efforts. DNA, or genetic, fingerprinting provides a genetic profile to reveal molecular markers linked to ancestry, cultivar and relationship to related plants, morphology, disease and pest resistance, drought tolerance, soil adaptability and so on. There are four major fingerprinting techniques: • • • •

Restriction Fragment Length Polymorphisms (RFLPs); Randomly Amplified Polymorphic DNAs (RAPDs); Amplified Fragment Length Polymorphisms (AFLP); Simple Sequence Repeats (SSR), often referred to as microsatellites.

These techniques are discussed in detail in the chapters which follow. An important achievement in understanding the taxonomy of genus Phoenix palms was realized through a study of microsatellite markers which affirmed the validity of 14 species within the genus, and that P. dactylifera was initially domesticated from wild populations of the same species, and not derived from other species of Phoenix (Pintaud et al. 2010). Previously, there was uncertainty about the validity of certain taxonomic names, which was complicated by the fact that Phoenix species readily hybridize naturally in the wild where species distributions overlap or in cultivation when grown near each other. DNA fingerprinting research is being carried out to confirm the identity of date palm cultivars in various date-growing countries including Algeria (Benaceur et al. 1991);

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Egypt (El-Assar et  al. 2005); Iraq (Khierallah et  al. 2010); Morocco (Sedra et  al. 1998); Oman (Al-Ruqaishi et al. 2008); Qatar (Ahmed and Al-Qaradawi 2010); Saudi Arabia (Al-Khalifah and Askari 2003); Sudan (Elshibli and Korpelainen 2008); Tunisia (Zehdi et  al. 2004); UAE (Al Kaabi et  al. 2007); and USA-California (Johnson et al. 2009). These selected references show the breadth of research activities under way, which are using the various techniques available. Genetic analysis can also reveal more about individual cultivars, such as the work on the Medjool cultivar which confirmed its status as a landrace variety (Elhoumaizi et al. 2006). A milestone in molecular research on date palm was achieved with the sequen­ cing of the draft genome of the Khalas cultivar. (http://qatar-weill.cornell.edu/ research/datepalmGenome/download.html). This resource is freely-available to researchers on the internet and should bring about an increase in the use of mole­ cular markers. Reflective of the rise of genomic studies of the date palm, there were four papers on the subject presented at the March 2010 Fourth International Date Palm Conference in Abu Dhabi. A current key research issue is to determine date palm gender at a very early stage of development through genetic analysis. Some progress along these lines reveals the potential of molecular biology to differentiate gender. However, thus far molecular markers linked to gender have not been identified and this represents a key impediment to improvement of date palm. When this particular problem is solved it will represent a significant breakthrough and speed crop improvement. Future DNA studies also may reveal the genetic markers for fruit flesh being soft, semidry or dry, and for early, midseason and late ripening.

References Ahmed TA, Al-Qaradawi AY (2010) Genetic diversity of date palm genotypes in Qatar as determined by SSR and ISSR markers. Paper presented at fourth international date palm conference, Abu Dhabi. Abstract Book p 80 Al Kaabi HH, Zaid A, Shephard H, Ainsworth C (2007) AFLP variation in tissue culture-derived date palm (Phoenix dactylifera L.) plants. Acta Hort 736:135–159 Al-Khalifah NS, Askari E (2003) Molecular phylogeny of date palm (Phoenix dactylifera L.) cultivars from Saudi Arabia by DNA fingerprinting. Theor Appl Genet 107:1266–1270 Al-Khayri JM (2005) Date palm Phoenix dactylifera L. In: Jain SM, Gupta PK (eds.) Protocols of somatic embryogenesis in woody plants. Springer, Netherlands, pp 309–319 Al-Khayri JM (2007) Date palm Phoenix dactylifera L. micropropagation. In: Jain SM, Häggman H (eds.) Protocols for micropropagation of woody trees and fruits. Springer, Netherlands, pp 509–526 Al-Ruqaishi IA, Davey M, Alderson P, Mayes S (2008) Genetic relationships and genotype tracing in date palms (Phoenix dactylifera L.) in Oman, based on microsatellite markers. Plant Gen Res Char Util 6:70–72 Benaceur M, Lanaud C, Chevallier M-H, Bounaga N (1991) Genetic diversity of the date palm (Phoenix dactylifera) from Algeria revealed by enzyme markers. Plant Breed 107:56–57 Dowson VHW (1921, 1923) Dates and date cultivation of the ‘Iraq. Parts I, II, III. Heffer & Sons, Cambridge Dowson VHW (1982) Date production and protection. FAO, Rome

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El-Assar AM, Krueger RR, Devanand PS, Chao CT (2005) Genetic analyses of Egyptian date (Phoenix dactylifera L.) accessions using AFLP markers. Genet Res Crop Evol 52:601–607 Elhoumaizi MA, Devanand PS, Fang J, Chao CT (2006) Confirmation of ‘Medjool’ date as a landrace variety through genetic analysis of ‘Medjool’ accessions in Morocco. J Am Soc Hort Sci 131:403–407 Elshibli S, Korpelainen H (2008) Microsatellite markers reveals high genetic diversity in date palm (Phoenix dactylifera L.) germplasm from Sudan. Genet 134:251–260 Johnson C, Cullis TA, Cullis MA, Cullis CA (2009) DNA markers for variety identification in date palm (Phoenix dactylifera L.). J Hort Sci Biotech 84:591–594 Khierallah HSM, Bader SM, Baum M, Hamwich A (2010) Assessment of genetic diversity for some Iraqi date palms (Phoenix dactylifera L.) using AFLP markers. Paper presented at fourth international date palm conference, Abu Dhabi. Abstract Book p 84 Pintaud J-C, Zehdi S, Couvreur T (2010) Species delimitation in the genus Phoenix (Arecaceae) based on SSR markers, with emphasis on the identity of the date palm (Phoenix dactylifera L.). In: Seberg A, Peterson G, Barfod A, Davis J (eds) Diversity, phylogeny, and evolution in the monocotyledons. Aarhus Univ Press, Denmark, pp 267–286 Popenoe PC (1973) The date palm. Field Research Projects, Coconut Grove, Miami Pruessner AH (1920) Date culture in ancient Babylonia. Am J Sem Lang Lit 36:213–232 Sedra MH, Lashermes P, Trouslot P, Combes M-C (1998) Identification and genetic diversity analysis of date palm (Phoenix dactylifera L.) varieties from Morocco using RAPD-markers. Euphy 103:75–82 Tisserat B (1981) Date palm tissue culture. Agricultural Research Service, Advances in Agricultural Technology, Western series, No. 17. Oakland, California UN (1993) The United Nations convention on biological diversity. Concluded at Rio de Janeiro 5 June 1992, into force 29 Dec 1993 Zaid A (ed.) (2002) Date palm cultivation. Rev. ed. FAO, Rome Zehdi S, Trifi M, Marrakchi M, Pintaud J-C (2004) Genetic diversity of Tunisian date palms (Phoenix dactylifera L.) revealed by nuclear microsatellite polymorphism. Hered 141:278–287 Zohary D, Hopf M (2000) Domestication of plants in the old world, 3rd edn. Oxford University Press, Oxford