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Somaclonal Variation in Date Palm

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The exploration of somaclonal variation is an approach that could provide date palm breeding programs with new genotypes. Naturally occurring or induced variants may have superior agronomic quality and/or enhanced performance but could also harbor
  183S.M. Jain et al. (eds.),  Date Palm Biotechnology ,   DOI 10.1007/978-94-007-1318-5_9, © Springer Science+Business Media B.V. 2011 Abstract  The exploration of somaclonal variation is an approach that could provide date palm breeding programs with new genotypes. Naturally occurring or induced variants may have superior agronomic quality and/or enhanced performance but could also harbor new traits such as tolerance to drought and salinity or resistance to major diseases i.e. bayoud. This chapter summarizes recent progress in terms of studying and exploring date palm somaclonal variation, and provides an outlook about future applications of this biotechnology in this socioeconomically important crop. Keywords  Biotic and abiotic stress • Bayoud • Conventional breeding • Date palm • Drought •  In vitro -selection • Phoenix dactylifera  L. • Salt • Somaclonal variation A. El Hadrami ( * ) OMEX Agriculture Inc. Canada, P.O. Box 301, 290 Agri Park Road, Oak Bluff, Manitoba, R0G 1N0, Canada  e-mail: abdele@omexcanada.comF. Daayf  Department of Plant Science, University of Manitoba, 222, Agriculture Building, Winnipeg, MB R3T 2N2, CanadaS. Elshibli • S.M. JainDepartment of Agricultural Sciences, University of Helsinki, FI-00014 Helsinki, Finland  e-mail:; mohan.jain@helsinki.fiI. El Hadrami Department of Biology, Faculty of Sciences Semlalia, Laboratory of Biotechnologies, Cadi Ayyad University, B.P. 2390, 40 000 Marrakech, Morocco Chapter 9 Somaclonal Variation in Date Palm A. El Hadrami, F. Daayf, S. Elshibli, S.M. Jain, and I. El Hadrami  184A. El Hadrami et al. 9.1 Introduction Genetic variation is essential to fulll the needs of conventional and non-conventional date palm-breeding programs. Sufficient genetic diversity and variation can be found among cultivated germplasm, and in wild relatives such as species of Phoenix   and Sabal  palms. Once a trait is bred-in or introgressed into a new cultivar, several years of selection follow in the field to assess its stability and affect on agronomic perfor-mance and yield. Date palm can be propagated sexually and recombination becomes the main genetic event that allows for the incorporation of genetic variability into segregating populations. However, this is a lengthy process that may take decades before introgressing the new desirable traits. To accelerate the process, the species can be vegetatively propagated, which allows the introgression of the new traits through mechanisms other than the recombination. Induced mutagenesis and in vitro  techniques represent some of the alternatives to alter desired genetic traits, at the same time as preserving the integrity of the genome and the clone characteristics.Somaclonal variation and in vitro -selection represent useful biotechnology tools in date-palm breeding for tolerance to biotic and abiotic stresses i.e. drought, salinity, diseases and pests. These techniques also offer an improvement of the value-added of the new genotypes with traits such as an increase in the number and/or size of fruits or improved texture or taste, or modification in flower structure (Ahloowalia and Maluszynski 2001 ; Pedrieri  2001 ; Witjaksono 2003). Somaclonal variation represents a real advantage in widening the genetic basis of this species, relying more or less solely on vegetative propagation. By applying specific selective agents or providing particular conditions to in vitro- propagated tissues, soma-clones with desired traits can be produced at a high frequency (Karp 1995). The causes of somaclonal variation during multiplication are diverse and tightly dependent upon the genotype, its level of ploidy, the growth conditions and dura-tion of selection (Maluszynski and Kasha 2002). Studies of the determinants of such variation have revealed that it can be due to changes at the gene level through genetic events such as duplication, translocation, mutation by insertion or deletion of transposable elements, or methylation. It can also occur at the chromosome level through instability, inversion and transient or permanent ploidy changes (Dennis 2004; Kumar and Marthur 2004 ; George and Sherington  1984 ; Phillips et al.  1990). These phenomena often lead to irreversible pleiotropic and epigenetic events and the production of variants called chimera. Commonly used mutagens include micro -bial synthetic toxins i.e. crude fungal-culture filtrates; fusaric acid (El Hadrami et al. 2005); chemicals such as ethylene scimine (ES), diethyl sulphonate (DES), ethyl methane-sulphonate (EMS), and the azida group (i.e. NaN3) or physical mutagens such as  c  - and g -rays (Co60), fast and thermal neutrons (nf and Nth). Depending on the selective agent, in vitro -selection could be conducted using regenerative and embryogenic calli, cell suspensions, zygotic and somatic rescued embryos, fused protoplasts and cybrids, but also at later stages during the regenera-tion of shoot and root meristems. The method of choice often depends on the advanced control of the micropropagation technique as well as the ease of applica-tion and the efficiency of the selective agent in inducing high levels of variation.  185 9 Somaclonal Variation in Date Palm The regeneration method of tolerant cells is also important in order to preserve the inheritance of the desired trait or traits. 9.2 Somaclonal Variation in Date Palm Somaclonal variation is an essential component of date-palm breeding in which variation regenerated from somatic cells can be used for the introduction of new agronomic, tolerance or quality traits (El Hadrami and El Hadrami 2009; Jain 2001). Variation in the somaclones has often been associated with changes in chromosome numbers and/or structure, punctual mutations or DNA methylation or other epige-netic events (Brown et al. 1993; Larkin and Scowcroft 1981). Somaclonal variation is undesirable from an industrial production stand point of view but may provide an enrichment of the gene pool. It also provides additional advantages such as the mass production of plants, opportunities for synthetic seeds, cryopreservation and direct delivery system for genetic variation. Its frequency depends, among other factors, on the genotype and the length of the proliferation process.Jain (2007) reported that rapid shoot proliferation can be achieved from various parts of the plant including shoot tips, stem cuttings, auxiliary buds and roots. He also pointed out that the selection of the genotype and the number of sub-culture cycles help limit the appearance of somaclones after the step of plant regeneration. Many off-type plants and abnormal dwarf phenotypes with low fruit sets may still be observed among the in vitro- propagated populations with high frequencies. These phenotypes are not always detectable at seedling stages and often become apparent a few years after planting. However, the technological advances and the development of molecular markers have made it possible, in recent years, to early and accurately detect these variants and eliminate them from the mass production (Baaziz et al. 1994 ; Corniquel and Mercier 1994 ; Cullis et al. 1999 ; Powell et al. 199 6 ; Saker et al. 2000; Salman et al. 1988). These off-types and somaclones can be further investigated to enrich the genetic pool.  9.2.1 Somaclonal Variation The concept of somaclonal variation was introduced in the early 1980s to describe any variation observed in tissue culture (Larkin and Scowcroft 1981). Somaclones spontaneously develop during tissue culture due to the plasticity of the genome and its ability to restructure in response to exo- or endogenous conditions encountered in vitro . Although this can lead to novel sources of genetic variation for breeding programs, it is a phenomenon that needs to be regulated to guarantee uniformity during multiplication.In comparison with the findings on African oil palm, it can be assumed that the use of higher concentrations of auxins during the redirection of calli to regenerate plant-lets has a dramatic effect on the rearrangement of the genome and often leads to epigenetic events and the formation of somaclones. Some auxins such as 2,4-D, more  186 A. El Hadrami et al. than others, have been shown to be highly effective in inducing this phenomenon. Date palm genotypes also differ in terms of their responses to these growth regulators. Some cultivars may exhibit a genotype-fidelity and produce a high rate of true-to-type plants while others are more prone to variations. It is noteworthy that most date palm cultivars remain to be tested for their in vitro  propagation abilities through tissue culture. Among those tested, a certain degree of recalcitrance has been observed, especially for those exhibiting agronomic, production, and quality or resistance traits (El Bellaj 2000; El Hadrami 1995 ; Gueye et al. 2009; Zouine et al. 2005). The body of knowledge regarding the potential use of somaclonal variation in date palm breeding remains to be fulfilled and most available data are either sporadic or lack conclusive thoroughness. On the other hand, it is usually described and acknowledged in many other systems that micropropagation via somatic embryogen-esis may lead, as compared to organogenesis, to higher percentages of somaclonal variation. Comparison between the two systems is lacking in date palm due to the lengthy process of achieving tissue cultures and producing vitroplants.  9.2.2 Sources of Variation Besides the microchanges involved in the variation in the expression of specific genes, macrochanges such as deletion/addition of chromosomes or chromosome breakage may occur during tissue culture. These modifications remain dependent upon the explant srcin, the concentrations of plant growth regulators used and the type of multiplication technique used to propagate the tissues. Undifferentiated cells induced under in vitro  culture conditions are often genetically variable and unstable, with variations in chromosome numbers and ploidy levels. The causes of such chro-mosomal variation include irregular cell events that can occur during the induction of calli from explants i.e. endoreduplication, amitosis and DNA amplification (D’Amato 1985). Other factors also include higher ploidy levels and polysomaty. In date palm, the high frequency of retroelements and the sensitivity to certain auxins often triggers variations during tissues culture (Jain 2007). The relationship between polysomaty and the level of diploidy remains unclear in date palm. Studies using tomato vitroplants, for instance, have shown that the frequency of polyploidy production is likely to be higher using hypocotyl segments as compared to leaf and cotyledon explants, which predominantly produce diploid seedlings (Van den Bulk et al. 1990). On the other hand, the use of older cotyledons as explants had led to either a poor regeneration of plants or to the regeneration of tetraploid or mixoploid seedlings with a high polysomaty status (Colijn-Hooymans et al. 1994). Chromosomal variation has been previously reported in date palm. Various studies showed a chromosome number varying from 26 to 36 (Al-Salih and Al-Jarrah 1987; Al-Salih and Al Rawi 1987; Al-Salih et al. 1987; Beal 1937; Ibrahim et al. 1998; Loutfi 1999; Nemec 1910). Such variation may be linked to phenomena such as polysomaty, chromosome breakage or nucleolar heterochromatin aggregation/disaggregation that occur in in vitro -propagated material. In addition, somaclonal variation can occur in micropropagated tissues as a result of a gameto-, proto- or soma-clonal srcin.  187 9 Somaclonal Variation in Date Palm Gametoclonal Variation Gametoclonal variation refers to variants derived from gametic and gametophytic cells (Evans et al. 1984). The value of gametoclonal variation in plant breeding comes from the development of double-haploids after anther culture. Gametes, as a product of meiosis, receive according to Mendel segregation laws half of the genetically segregating alleles as opposed to somatic cells, which divide their genetic material equally during mitosis. Gametoclones are able to express both recessive and dominant alleles while crossovers may occur during meiosis, creating new sources of variability. Once doubled for stability, gametoclones can be examined for residual heterozygosis (Evans and Sharp 198 6 ) and used in breeding programs. This strategy has been explored in date palm using double haploids derived from micro- and macrospores (Chaibi et al. 2002; Zouine and El Hadrami 2004). Anther and ovule culture recovery is difficult in date palm. Investigations in this regard have led to the achievement of cell divisions and to the formation of globular embryoids from uninucleate microspores. Successful attempts report the importance of cold treatment combined with the use of two auxins and one cytokinin to generate embryoids (Chaibi et al. 2002), that unfortunately were unable to develop further. Other studies, using various treatments and exogenous factors, did not provide any major improvements apart from production of weak and short-living calli from these propagules. It is also important to mention that one of the main difficulties in devel-oping these studies is related to the short duration of the flowering period in date palm, preventing the harvest of fresh anthers with uninucleate microspores. These anthers are also likely to turn brown and die a few weeks after their culture. Chaibi et al. (2002 ) reported that treating anthers with a thermal shock at 37–38°C is suitable only within a narrow window of time prior to their in vitro  culture. Combined with that the use of MS medium amended with 2,4-D and 2-isopentenylaminopurin (2-iP), as well as activated charcoal, often help prevent tissue browning, and increase the percentage of microspore division.Some haploid recovery attempts have also been conducted using unfertilized date-palm ovules. Due to the small size of these ovules, browning and necrosis were the main limits encountered by these cultures. Although, the carpel enlarged and became quite prominent when cultured, the use of activated charcoal was required to ensure a much longer survival, and root or callus formation. Until now, the best results obtained were from flowers taken from closed spaths in which the embryo sacs were formed and contained undifferentiated cells. Recently, Masmoudi-Allouche et al. (2009) also reported on the potential induction of hermaphrodism in date palm inflorescences.In spite of its current unsuccessful use in date palm, gametoclones offer a great opportunity for breeding of this plant. Clonal propagation generates identical copies of the selected genotype but applying specific stresses at given times may result in loosing-up the control mechanisms, guaranteeing the stability of the genome of the gametoclones. Long-term propagation of multiple shoots in vitro  and excessive sub-cultures of the same stock may expose the tissue to an environment where their genetic stability gets altered, creating internal repeats of genetic sequences or trans-position of retroelements. All these events may affect essential genes required for
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