DATE PALM

Date palm, Phoenix dactylifera L., a monocotyledonous angiosperm belonging to the Palmaceae (Arecacea) family, is native to the tropical or subtropical regions of Africa or Southern Asia.  Thousands of years of selection have produced the 3,000 varieties currently cultivated around the world in regions where hot arid climatic conditions favored by date palm prevail.   Date palm is mainly a diploid, 2n = 2x = 36, dioecious tree species with separate male and female plants.  For fruit setting, fertilization of the female flowers is required which often involves manual or mechanical pollination.  The date fruit is a single, oblong, one-seeded berry, consisting of pericarp or fruit skin, fleshy mesocarp, and membranous endocarp around the seed.  It is highly nutritious and rich in source of sugar, minerals, and vitamins.

Date palm may reach an age of over 100 years and a height of over 20 m.  Only about 20 offshoots are produced during the first 10 to 15 years of the tree life.  This puts limitations on the efficacy of the traditional propagation method involving separating and planting offshoots.  Propagation by seeds is not applicable for cultivation of known cultivars because genetic traits including fruit characteristics are not maintained.  Alternatively, micropropagation is gaining increased interest as it provides a rapid mass clonal propagation means.

In vitro propagation
Clonal propagation of date palm (Phoenix dactylifera L.) elite cultivars with known performance is highly desired in date palm growing countries. This long-lived dioecious monocotyledon has traditionally been vegetatively propagated from offshoots produced by individual trees.  The slowness of this conventional method, which, moreover, yields only a limited number of offshoots from a parent tree, has made in vitro tissue culture an attractive alternative method for mass propagation of date palm.  A satisfactory process for the regeneration of date palm elite cultivars from tissue culture should result in individuals phenotypically and genetically identical to the explant from which they are derived.  However, due to somaclonal variation generated during in vitro culture, it cannot be certified that the genetic organization of tissue culture derived material is identical to the explant from which it originates, and that it is inherently homogeneous.  The assessment of the genetic stability of in vitro derived clones is then an essential step in the application of biotechnology for micropropagation of true-to-type clones, for in vitro germplasm conservation, and for planting of high quality materials.

Date palm micropropagation began a quarter of a century ago and has progressed relatively slowly due to its inherent slow growth nature and limited research resources available to the developing countries where date palm is mostly grown.  Reports demonstrating the ability to regenerate date palm through direct regeneration, without callus stage, are also available. Micropropagation through direct regeneration is thought to reduce the potential for undesirable somaclonal variants among regenerants while somatic embryogenesis mediated by callus stage is highly efficient and more popular in research laboratories.  In recent years, several studies have examined various components of the culture medium including sucrose, silver nitrate, biotin and thiamine, auxins and salt strength, and other tissue culture factors.

Cell suspension cultures have been employed to study aspects related to physiology, and somatic embryogenesis.  Expected to revolutionize date palm propagation, development of synthetic seeds is gaining research interest. In addition to offering an effective propagation means, tissue culture proved applicable in genetic improvement of date palm through induced mutations and in vitro selection. Zygotic embryo and shoot tip explants of Phoenix canariensis were used for plantlet regeneration via somatic embryogenesis. In date palm as for many monocotyledons, callogenesis is a prerequisite for the initiation of somatic embryogenesis, and requires the presence of auxin in the medium.  Plant regeneration through somatic embryogenesis from young leaf explants (5–10 mm long) adjacent to the apex of 5–6 year old offshoots of Tunisian date palm. Factors affecting embryogenic callus initiation, including plant growth regulators and explant size, were investigated.  The highest induction frequencies of embryogenic calli occurred after 6–7 months on MS medium supplemented with 10 mg l-1 2,4-D and 0.3 mg l-1 activated charcoal.

The subculture of these calli onto maintenance medium resulted in the formation of proembryos.  Fine chopping and partial desiccation (6 and 12 h) of embryogenic calli with proembryos  prior to transfer to MS medium supplemented with 1 mg l-1 ABA stimulated the rapid maturation of somatic embryos.  Subsequent substitution of ABA in MS medium with 1 mg l-1 NAA resulted in the germination and conversion of 81% of the somatic embryos into plantlets with normal roots and shoots.  The growth of regenerated somatic plants was also monitored in the field.  This was the first report of a protocol for somatic embryo maturation in date palm following fine chopping and partial desiccation of embryogenic callus. The reason for the improvement with fine chopping embryogenic calli has not been investigated at the physiological or molecular level and may be attributed to an inhibitory substance(s) released by surrounding embryogenic cells.   Also, it is possible that the physical stress caused by fine chopping of callus stimulates de novo protein synthesis allowing a rapid division and differentiation of embryogenic cells.  In addition, chopping of embryogenic calli into small pieces minimizes the intercellular connections between cells and contributes to a partial isolation of cells.  The desiccation treatment might trigger rapid biochemical changes in the calli and under water stress specific enzymes or polypeptides probably appear in callus culture and stimulates the accumulation of storage reserves and triglycerides that have a positive effect on somatic embryo maturation.  It was suggested that the improvement of the maturation process of somatic embryos via partial desiccation and especially via fine chopping embryogenic callus is feasible, reproducible and offers a tremendous potential for an inexpensive method for large scale propagation of superior date palm cultivars.

The gain brought to the morphogenetic aptitude of female date palm inflorescences through in vitro hermaphrodism induction. They investigated the main factors involved in the process of sex modification through hormonal induction, such as the floral developmental stage and hormone combination and concentration. They demonstrated that the vestigial stamens (staminodes) of female date palm flowers display a new and high capacity to proliferate under particular in vitro conditions, without blocking carpel’s development, leading to morphologically typical hermaphrodite flowers. This de novo activation of repressed stamens was found to occur rapidly. The isolated pollen mother cells appear in the obtained anther’s locules and undergo an ordinary microsporogenesis process. The data showed that hermaphrodism induction depended heavily on both the growth regulators applied and the flower’s developmental stage. This report also confirms the early theory that suggests that dioecious plants come from a hermaphrodite ancestor. Such hermaphrodism control can provide new prospects and opportunities for the investigation of the in vitro self-fertilization process. It can also be useful in improving the understanding of the genetic mechanism involved in sex organ development in date palm. The mechanical or manual pollination procedure is an ancestral practice that is undeniably useful and indispensable in date palm.

Molecular markers
An RFLP analysis of five date palm (Phoenix dactylifera L.) elite cultivars (cvs. Barhee, Deglet Nour, Khalassa, Khadrawy, and Medjool) has been performed on offshoot leaves surrounding the shoot tips used to initiate tissue culture. With the availability of probes such as cDNA 1, the use of RFLP for rapid and reliable cultivar identification, and screening of cultivated populations with economically important traits in date palm growing countries is now conceivable.

The potential of biochemical and molecular markers in sex identification of in vivo grown and in vitro differentiated cultures of date palm. In vitro zygotic lines were proliferated from mature and immature zygotic embryos of date palm. The mature embryos were more potential in the in vitro differentiation responses compared with immature embryos.  Incorporation of tissue culture medium by 3 mg/L 2ip + 5 mg/L 2,4-D gave the highest percentages of embryo germination and growth. Isozymes were used for identification of sex type of date palm cultures. High level of peroxidase activity has been observed in adult female and offshoot female. Acid phosphatase and glutamate oxaloacetate enzymes gave a strong difference between male and female date palm. Early estimation of sex type of in vitro differentiated lines has been realized via the activity levels of the two enzymes. Random amplified polymorphic DNA (RAPD) technique was used to compare genetic material from male, female and unknown lines of date palm. RAPD analysis showed a relatively closely relation between the two females (adult and offshoot) cultures, since they have large number of homologous bands. Although, there is low relationship between male and female, results of similarity could not confirm link to sex or estimate the sex type of unknown clones.

Random amplified microsatellite polymorphisms (RAMPOs) to assess genetic diversity among 30 date-palm cultivars and10 male trees. Using 18 primers combinations, 197 bands were scored and 186 were polymorphic suggesting the high level of polymorphism among studied cultivars. Moreover, taking into account the high percentage of polymorphic bands (ppb), the resolving power (Rp) together with the polymorphism information content (PIC) scored values, all the tested primer sets contribute strongly in the discrimination of date-palm genotypes. In addition, the topology of the derived UPGMA dendrogram exhibited cultivars’ clustering made independently both from the geographical origin and/or from the sex of trees. The present data support the Mesopotamian origin of the date-palm domestication.

The availability of reproducible regeneration systems for date palm has paved the way to several biotechnological applications during the current decade including somaclonal variation selection, cryopreservation, synthetic seeds, as well as cell and protoplast cultures. Furthermore, molecular approaches have been utilized in cultivar identification and assessment of genetic fidelity of regenerants. At the present, researchers are striving to develop a genetic transformation system to enable improvement of essential characteristics. The future appears promising for date palm biotechnology particularly in commercial production.

Source: Dr.V.Ponnuswami, PhD, PDF (Taiwan), Former Dean & Professor (Horticulture), Horticultural College & Research Institute, Tamil Nadu Agricultural University, Coimbatore