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Date palm micropropagation: Advances and applications

Micropropagação de tamareira: Avanços e aplicações

ABSTRACT

Date palm (Phoenix dactylifera L.) is a fruit tree resilient to adverse climatic conditions predominating in hot arid regions of the Middle East and North Africa. The date fruit contains numerous chemical components that possess high nutritional and medicinal values. Traditional propagation by offshoots is inefficient to satisfy current demands for date palm trees. Alternatively, micropropagation provides an efficient means for large-scale propagation of date palm cultivars. Both somatic embryogenesis and organogenesis, either directly or indirectly though the callus phase, have been demonstrated in date palm in vitro regeneration. Culture initiation commonly utilizes shoot-tip explants isolated from young offshoots. Recently, the immature inflorescences of adult trees were utilized as an alternative nondestructive source of explants. In addition to the nature of the explant used, successful plant regeneration depends on the cultivar, composition of the culture medium and physical status. Challenges of date palm micropropagation include long in vitro cycle, latent contamination, browning, somaclonal variation as well as ex vitro acclimatization and transplanting. A remarkable amount of research investigating these factors has led to optimized protocols for the micropropagation of numerous commercially important cultivars. This has encouraged the development of several international commercial tissue culture laboratories. Molecular characterization provides an assurance of genetic conformity of regenerated plantlets, a key feature for commercial production. This article describes date palm micropropagation protocols and also discusses recent achievements with respect to somaclonal variation, molecular markers, cryopreservation and future prospects.

Index terms:
Cryopreservation; somatic embryogenesis; somaclonal variation; organogenesis; molecular marker.

RESUMO

A tamareira (Phoenix dactylifera L.) é uma arvore frutífera adaptada à condições climáticas adversas predominantemente em regiões áridas do Oriente Médio e Norte Africano. As tâmaras possuem vários componentes químicos com alto valor medicinal e nutricional. A propagação tradicional por estacas não é suficiente para satisfazer a demanda por mudas e assim, a micropropagação apresenta-se como uma alternativa eficiente para a produção de mudas em larga escala. Embriogênese somática e organogênese, tanto direta quanto indireta via calos, tem sido usada para obter a regeneração in vitro de tamareira. O inicio do cultivo in vitro normalmente utiliza meristemas excisados de brotações jovens. Recentemente, inflorescências imaturas de árvores adultas são usadas como fonte alternativa de explantes não destrutiva. Além da origem do explante, o sucesso da regenerção depende do cultivar, da composição do meio de cultura e de condições físicas. Desafios na micropropagação de tamareira incluem um longo ciclo in vitro, contaminação, escurecimento do tecido, variação somaclonal além do enraizamento e aclimatização ex vitro. Diversos estudos investigando esses fatores tem conduzido à otimização de protocolos de micropropagação de inúmeros cultivares comerciais proporcionando o estabelecimento de vários laboratórios de cultura de tecidos de plantas. A caracterização molecular permite uma segura conformidade genética do material regenerado, considerado uma característica chave na produção comercial. Essa revisão descreve protocolos de micropropagação de tamareira e aborda as mais recentes conquistas relacionadas à variação somaclonal, marcadores moleculatres, criopreservação e perspectivas futuras.

Termos para indexação:
Criopreservação; embriogênese somática; variação somaclonal; organogênese; marcador molecular.

INTRODUCTION

Date palm (Phoenix dactylifera L.) is a ‘tree of life’ which belongs to the family Arecaceae; it is distributed mainly in the Middle East and North Africa. Over the last century, date palm agriculture has spread to Australia, Southern Africa and the Americas. The distribution of date palm is very peculiar because of the inherent requirement for hot climate which is necessary for successful pollination and fruit setting (Chao; Krueger, 2007CHAO, C. C. T.; KRUEGER, R. R. The date palm (Phoenix dactylifera L.): Overview of biology, uses, and cultivation. Horticultural Science, 42(5):1077-1082, 2007.). Date palm cultivation has been recorded for thousands of years and over that time selection procedures have developed more than 3,000 known varieties of date palm around the world (Johnson, 2011JOHNSON, D. V. Introduction: Date palm biotechnology from theory to practice. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology , Dordrecht: Springer , 2011. p.1-11.). Date fruits possess important nutritional and medicinal values and as a part of religious practice, dates are consumed by people all over the world (Vayalil, 2012VAYALIL, P. K. Date fruits (Phoenix dactylifera Linn): An emerging medicinal food. Critical Reviews in Food Science and Nutrition, 52(3):249-271, 2012.). Dates are a good source of essential minerals, which enhance their nutritional value (Al-Shahib; Marshall, 2002AL-SHAHIB, W.; MARSHALL, R. J. Dietary fibre content of dates from 13 varieties of date palm Phoenix dactylifera L. International Journal of Food Science and Technology, 37:719-721, 2002., 2003AL-SHAHIB, W.; MARSHALL, R. J. The fruit of the date palm: Its possible use as the best food for the future? International Journal of Food Science and Nutrition, 54:247-259, 2003.; Elleuch et al., 2008ELLEUCH, M. et al. Date flesh: Chemical composition and characteristics of the dietary fibre. Food Chemistry , 111:67-82, 2008.). The aqueous extracts of dates possess antioxidant activity (Al-Farsi et al., 2007AL-FARSI, M. et al. Compositional characteristics of dates, syrups, and their by-products. Food Chemistry, 104:943-947, 2007.; Biglari; AlKarkhi; Easa, 2008BIGLARI, F.; ALKARKHI, A. F. M.; EASA, A. M. Antioxidant activity and phenolic content of various date palm (Phoenix dactylifera) fruits from Iran. Food Chemistry , 107:1636-1641, 2008.; Saafi et al., 2009SAAFI, E. B. et al. Phenolic content and antioxidant activity of four date palm (Phoenix dactylifera L.) fruit varieties grown in Tunisia. International Journal of Food Science and Technology , 44:2314-2319, 2009.). Phenolic compounds present in dates, have beneficial effects on human health and act against cancer and cardiovascular diseases (Vayalil, 2012VAYALIL, P. K. Date fruits (Phoenix dactylifera Linn): An emerging medicinal food. Critical Reviews in Food Science and Nutrition, 52(3):249-271, 2012.). Dates are a high energy source consisting of carbohydrates 44-88% in addition to proteins 2.3-5.6% and fats 0.2-9.3% (El Hadrami; El Hadrami, 2009EL HADRAMI, I.; EL HADRAMI, A. Breeding date palm. In: JAIN, S. M.; PRIYADARSHAN, P. M. Breeding Plantation Tree Crops, New York: Springer, 2009. p.191-216.). Palm syrup, palm sugar, vinegar, wine and honey are made from the date fruits (Chao; Krueger, 2007CHAO, C. C. T.; KRUEGER, R. R. The date palm (Phoenix dactylifera L.): Overview of biology, uses, and cultivation. Horticultural Science, 42(5):1077-1082, 2007.). The annual international market value (including import and export) of the date crops reached nearly 1.9 billion USD (FAOSTAT, 2013FAOSTAT. Food and Agricultural Organization of the United Nations. 2013. Available in: <Available in: http://www.fao.org/faostat/en/; >. Access in: June 20, 2017.
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), from which a country can build a strong economic platform. The vegetative part of the date tree is used as a raw material for roof coverings for houses, wooden boats, timber for wood industry and making numerous handicrafts (Al-Khayri; Jain; Johnson, 2015aAL-KHAYRI, J. M.; JAIN, S. M.; JOHNSON, D. V. Date palm genetic resources and utilization, Vol. 1: Africa and the Americas, Dordrecht: Springer, 2015a. 546p. , 2015bAL-KHAYRI, J. M.; JAIN, S. M.; JOHNSON, D. V. Date palm genetic resources and utilization, Vol. 2: Asia and Europe, Dordrecht: Springer , 2015b. 566p.).

In recent years, because of overexploitation, the diversity of the date palm groves has declined. The production and utilization of the date fruits also varies from country to country due to the influence of current environmental conditions. There are a number of elements which hinder the production of date palm, such as major pests and diseases, salinity and drought, poor harvest and postharvest practices (Al-Khayri; Jain; Johnson, 2015aAL-KHAYRI, J. M.; JAIN, S. M.; JOHNSON, D. V. Date palm genetic resources and utilization, Vol. 1: Africa and the Americas, Dordrecht: Springer, 2015a. 546p. , 2015bAL-KHAYRI, J. M.; JAIN, S. M.; JOHNSON, D. V. Date palm genetic resources and utilization, Vol. 2: Asia and Europe, Dordrecht: Springer , 2015b. 566p.). Date palm is conventionally propagated vegetatively by offshoots. Due to their heterozygous nature, seeds cannot be used for the propagation of commercially elite cultivars because they produce off-type propagules (Abahmane, 2011ABAHMANE, L. Date palm micropropagation via organogenesis. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology, Dordrecht: Springer, 2011. p.69-90.). The number of offshoots produced by a date palm tree during their life span is only about 20-30 (Zaid; El-Korchi; Visser, 2011ZAID, A.; EL-KORCHI, B.; VISSER, H. J. Commercial date palm tissue culture procedures and facility establishment. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology , Dordrecht: Springer , 2011. p.137-180.). The survival rate of offshoots in the field is low and chances of disease transmission are high (Abahmane, 2011ABAHMANE, L. Date palm micropropagation via organogenesis. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology, Dordrecht: Springer, 2011. p.69-90.). These factors hinder meeting the increasing agro-industrial demand for propagules. The use of micropropagation techniques eliminates these restrictions and allows for large-scale production of healthy, disease-free, true-to-type plants.

Since the early reports of date palm in vitro regeneration (Tisserat, 1979TISSERAT, B. Propagation of date palm (Phoenix dactylifera L.) in vitro. Journal of Experimental Botany, 30:1275-1283, 1979., 1982TISSERAT, B. Factors involved in the production of plantlets from date palm callus cultures. Euphytica , 31:201-214, 1982.) numerous researchers have described different approaches to achieve date palm micropropagation: somatic embryogenesis (Al-Khayri, 2003AL-KHAYRI, J. M. In vitro germination of somatic embryos in date palm: Effect of auxin concentration and strength of MS salts. Current Science, 84:680-683, 2003., 2005AL-KHAYRI, J. M. Date palm (Phoenix dactylifera L.). In: JAIN, S. M.; GUPTA, P. K. Protocols of somatic embryogenesis in woody plants. Berlin: Springer, 2005. p.309-319.; Fki et al., 2003FKI, L. et al. An optimised protocol for plant regeneration from embryogenic suspension cultures of date palm, (Phoenix dactylifera L.), cv. Deglet Nour. Plant Cell Reports, 21:517-524, 2003., 2011aFKI, L. et al. Date palm micropropagation via somatic embryogenesis. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology , Dordrecht: Springer , 2011a. p.47-68.; Mazri et al., 2017MAZRI, M. A. et al. Somatic embryogenesis from bud and leaf explants of date palm (Phoenix dactylifera L.) cv. Najda. 3 Biotech , 7:58, 2017.; Naik; Al-Khayri, 2016NAIK, P. M.; AL-KHAYRI, J. M. Somatic embryogenesis of date palm (Phoenix dactylifera L.) through cell suspension culture. In: JAIN, S. M. Protocols for in vitro cultures and secondary metabolite analysis of aromatic and medicinal plants, 2nd ed. Methods in molecular biology. New York: Springer , 1391:357-366, 2016.; Othmani et al., 2009aOTHMANI, A. et al. Somatic embryogenesis and plant regeneration in date palm Phoenix dactylifera L., cv. Boufeggous is significantly improved by fine chopping and partial desiccation of embryogenic callus. Plant Cell, Tissue and Organ Culture , 97:71-79, 2009a.; Roshanfekrrad et al., 2017ROSHANFEKRRAD, M. et al. Effect of AgNO3 and BAP on root as a novel explant in date palm (Phoenix dactylifera cv. Medjool) somatic embryogenesis. Pakistan Journal of Biological sciences, 20(1):20-27, 2017.) and organogenesis (Bekheet, 2013BEKHEET, S. A. Direct organogenesis of date palm (Phoenix dactylifera L.) for propagation of true-to-type plants. Scientia Agriculturae, 4:85-92, 2013.; Jazinizadeh et al., 2015JAZINIZADEH, E. et al. In vitro production of date palm (Phoenix dactylifera L.) cv. ‘Barhee’ plantlets through direct organogenesis. Biological Forum - An International Journal, 7(2):566-572, 2015.; Khan; Bi, 2012KHAN, S.; BI, T. B. Direct shoot regeneration system for date palm (Phoenix dactylifera L.) cv. Dhakki as a means of micropropagation. Pakistan Journal of Botany, 44(6):1965-1971, 2012.; Khierallah; Bader, 2007KHIERALLAH, H. S.; BADER, M. S. M. Micropropagation of date palm (Phoenix dactylifera L.) var. Maktoom through organogenesis. Acta Horticulturae , 736:213-223, 2007.; Meziani et al., 2015MEZIANI, R. et al. Effects of plant growth regulators and light intensity on the micropropagation of date palm (Phoenix dactylifera L) cv. Mejhoul. Journal of Crop Science and Biotechnology , 18:325-331, 2015., 2016MEZIANI, R. et al. Organogenesis of Phoenix dactylifera L. cv. Mejhoul: Influences of natural and synthetic compounds on tissue browning, and analysis of protein concentrations and peroxidase activity in explants. Scientia Horticulturae , 204:145-152, 2016.). In addition to providing a means for rapid clonal propagation of date palm (Khierallah; Bader, 2007KHIERALLAH, H. S.; BADER, M. S. M. Micropropagation of date palm (Phoenix dactylifera L.) var. Maktoom through organogenesis. Acta Horticulturae , 736:213-223, 2007.), tissue culture techniques can be utilized for the production of synthetic seeds (Bekheet et al., 2002BEKHEET, S. A. et al. A synthetic seed system of date palm through somatic embryogenesis encapsulation. Annals of Agricultural Sciences, 47:325-337, 2002.), cell suspension culture (Othmani et al., 2009bOTHMANI, A. et al. In vitro cloning of date palm Phoenix dactylifera L., cv. Deglet Bey by using embryogenic suspension and temporary immersion bioreactor (TIB). Biotechnology and Biotechnological Equipment, 23(2):1181-1188, 2009b.), cryopreservation (Fki et al., 2013FKI, L. et al. Cold hardening and sucrose treatment improve cryopreservation of date palm meristems. Biologia Plantarum , 57(2):375-379, 2013. ), somaclonal variation introduces stress tolerant, disease resistance and with high quality fruits (El Hadrami; El Hadrami, 2009EL HADRAMI, I.; EL HADRAMI, A. Breeding date palm. In: JAIN, S. M.; PRIYADARSHAN, P. M. Breeding Plantation Tree Crops, New York: Springer, 2009. p.191-216.; Jain, 2001JAIN, S. M. Tissue culture-derived variation in crop improvement. Euphytica, 118:153-166, 2001.), the production of secondary metabolites (El-Sharabasy, 2004EL-SHARABASY, S. F. Effects of different precursors on characters and production of some secondary products from date palm (Phoenix dactylifera L.) cv. Sewi tissues during embryogenesis stage. Arab Journal of Biotechnology , 7:91-98, 2004.) and commercial production of date palm gives considerable profit to both public-sector and private agencies (Zaid; El-Korchi; Visser, 2011ZAID, A.; EL-KORCHI, B.; VISSER, H. J. Commercial date palm tissue culture procedures and facility establishment. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology , Dordrecht: Springer , 2011. p.137-180.).

The present article gives an update of the current approaches of date palm micropropagation with emphasis on the plant regeneration through somatic embryogenesis and organogenesis and use of a bioreactor. It highlights key factors that influence in vitro differentiation and culture growth including tissue browning, hyperhydricity, explant type, light and composition of culture medium. Also addressed are concerns related to potential somaclonal variation, molecular detection techniques, utilization of tissue culture in germplasm conservation and commercial production.

MICROPROPAGATION

The decades of efforts on date palm micropropagation by scientists achieved the goal of using different parameters which affect the in vitro development of date palm such as explant source (Figure 1A), age of explant, size of explant, intensity and quality of light, temperature, pH of the medium, plant hormones, culture medium and age of culture (Al-Khayri, 2013AL-KHAYRI, J. M. Factors affecting somatic embryogenesis in date palm (Phoenix dactylifera L.) In: ASLAM, J.; SRIVASTAVA, P. S.; SHARMA, M. P. Somatic embryogenesis and genetic transformation in plants, New Delhi: Narosa Publishing House, 2013. p.5-38.; Mazri, 2015MAZRI, M. A.; MEZIANI, R. Micropropagation of Date Palm: A Review. Cell and Developmental Biology, 4:160, 2015. ; Mazri; Meziani, 2015MEZIANI, R. et al. Effects of plant growth regulators and light intensity on the micropropagation of date palm (Phoenix dactylifera L) cv. Mejhoul. Journal of Crop Science and Biotechnology , 18:325-331, 2015.; Mazri et al., 2016MAZRI, M. A. et al. Optimization of medium composition for in vitro shoot proliferation and growth of date palm cv. Mejhoul. 3 Biotech, 6:111, 2016.). This is an efficient alternative to the conventional means of propagation. Somatic embryogenesis and organogenesis are the two main modes of micropropagation of date palm, which are widely accepted throughout the tissue culture arena in the world (Bhansali, 2010BHANSALI, R. R. Date palm cultivation in the changing scenario of Indian arid zones: Challenges and prospects. Desert plants, Springer, 2010. p.423-459.). The utilization of bioreactors is also making a significant contribution toward micropropagation of different plant species (Frómeta et al., 2017FRÓMETA, O. M. et al. In vitro propagation of Gerbera jamesonii Bolus ex Hooker f. in a temporary immersion bioreactor. Plant Cell, Tissue and Organ Culture, 129:543-551, 2017.; Gomes et al., 2016GOMES, H. T. et al. Regeneration of somatic embryos of oil palm (Elaeis guineensis) using temporary immersion bioreactors. Industrial Crops and Products, 89:244-249, 2016. ; Othmani et al., 2011OTHMANI, A. et al. Bioreactors and automation in date palm micropropagation. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology , Dordrecht: Springer , 2011. p.119-136.).

Figure 1:
Micropropagation of date palm. (A) Shoot tip culture, (B) Embryogenic callus induction, (C) Embryogenic callus proliferation, (D) Embryo formation, (E) Rooting and (F) Transplanted plant.

Somatic embryogenesis

Somatic embryogenesis can be defined as the development of somatic embryos from somatic cells, which undergo a series of morphological and biochemical changes (Quiroz-Figueroa et al., 2006QUIROZ-FIGUEROA, F. R. et al. Embryo production through somatic embryogenesis can be used to study cell differentiation in plants. Plant Cell, Tissue and Organ Culture , 86:285-301, 2006.). Somatic embryogenesis includes a series of stages viz. embryogenic callus initiation, formation of somatic embryo, development of somatic embryo, maturation and plantlet formation. In date palm micropropagation somatic embryogenesis is considered as the most potent tool to achieve large-scale production.

Embryogenic callus initiation and embryo formation

The embryogenic calli initiation process in the date palm is very slow; this is mainly due to the tree nature of the plant. The embryogenic callus initiation and proliferation (Figure 1B and 1C) process depends on various parameters like the type of genotype, explant source, plant hormones, and culture condition. To induce embryogenic callus in most of the cases for either shoot tip or inflorescence, a high concentration of auxins is used. Most researchers suggest 2,4-dichlorophenoxyacetic acid (2,4-D) as the most efficient auxin to induce embryogenic callus used at 100 mg L-1 concentration (Al-Khayri, 2005AL-KHAYRI, J. M. Date palm (Phoenix dactylifera L.). In: JAIN, S. M.; GUPTA, P. K. Protocols of somatic embryogenesis in woody plants. Berlin: Springer, 2005. p.309-319.; Al-Khayri, 2010AL-KHAYRI, J. M. Somatic embryogenesis of date palm (Phoenix dactylifera L.) improved by coconut water. Biotechnology , 9:477-484, 2010.; Eshraghi; Zaghami; Mirabdulbaghi, 2005ESHRAGHI, P.; ZAGHAMI, R.; MIRABDULBAGHI, M. Somatic embryogenesis in two Iranian date palm cultivars. African Journal of Biotechnology , 4:1309-1312, 2005.; Naik; Al-Khayri, 2016NAIK, P. M.; AL-KHAYRI, J. M. Somatic embryogenesis of date palm (Phoenix dactylifera L.) through cell suspension culture. In: JAIN, S. M. Protocols for in vitro cultures and secondary metabolite analysis of aromatic and medicinal plants, 2nd ed. Methods in molecular biology. New York: Springer , 1391:357-366, 2016.). Fki et al. (2011aFKI, L. et al. Date palm micropropagation via somatic embryogenesis. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology , Dordrecht: Springer , 2011a. p.47-68.) reported that high concentrations of 2,4-D causes somaclonal variations. Many researchers applied low concentration of 2,4-D such as 1.5, 5 and 10 mg L-1 to induce somatic embryo (Aslam et al., 2011ASLAM, J. et al. Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars. Saudi Journal of Biological Sciences, 18:369-380, 2011.; El Hadrami; Cheikh; Baaziz, 1995EL HADRAMI, I.; CHEIKH, R.; BAAZIZ, M. Somatic embryogenesis and plant regeneration from shoot-tip explants in Phoenix dactylifera L. Biologia Plantarum , 37:205-211, 1995.; Othmani et al., 2009aOTHMANI, A. et al. Somatic embryogenesis and plant regeneration in date palm Phoenix dactylifera L., cv. Boufeggous is significantly improved by fine chopping and partial desiccation of embryogenic callus. Plant Cell, Tissue and Organ Culture , 97:71-79, 2009a.). Depending on the genotype the somatic embryo formation (Figure 1D) period also varies from a few to several months (Eshraghi; Zaghami; Mirabdulbaghi, 2005ESHRAGHI, P.; ZAGHAMI, R.; MIRABDULBAGHI, M. Somatic embryogenesis in two Iranian date palm cultivars. African Journal of Biotechnology , 4:1309-1312, 2005.; Hassan; Taha, 2012HASSAN, M. H.; TAHA, R. A. Callogenesis, somatic embryogenesis and regeneration of date palm Phoenix dactylifera L. cultivars affected by carbohydrate sources. International Journal of Agricultural Research, 7:231-242, 2012.; Othmani et al., 2009aOTHMANI, A. et al. Somatic embryogenesis and plant regeneration in date palm Phoenix dactylifera L., cv. Boufeggous is significantly improved by fine chopping and partial desiccation of embryogenic callus. Plant Cell, Tissue and Organ Culture , 97:71-79, 2009a.). Recently Mazri et al. (2017MAZRI, M. A. et al. Somatic embryogenesis from bud and leaf explants of date palm (Phoenix dactylifera L.) cv. Najda. 3 Biotech , 7:58, 2017.) reported somatic embryogenesis from the adventitious bud of date cv. Najda, where the MS medium was supplemented with 2,4-D, kinetin (KN) or 6-dimethylallylamino purine (2iP). Proximal leaf segment showed embryogenesis only in the medium supplemented with 2,4-D or picloram.

Development of somatic embryo and maturation

Various factors are responsible for the development of somatic embryo and maturation. Date palm has the capacity to develop as a mature embryo in both semisolid and liquid medium. The authors reported in the date palm cv. Deglet Nour, 1-month old suspension culture produces 200 embryos from 100 mg fresh weight callus inoculum, on the other hand it produces 10 embryos on semisolid medium (Fki et al., 2003FKI, L. et al. An optimised protocol for plant regeneration from embryogenic suspension cultures of date palm, (Phoenix dactylifera L.), cv. Deglet Nour. Plant Cell Reports, 21:517-524, 2003.). In the date cv. Khalas, application of thiamine and biotin showed an increased number of embryos and also helps in the elongation process (Al-Khayri, 2001AL-KHAYRI, J. M. Optimization of biotin and thiamine requirements for somatic embryogenesis of date palm (Phoenix dactylifera L.). In Vitro Cellular & Developmental Biology-Plant, 37:453-456, 2001.). The embryogenic calli with fine chopping and partial desiccation significantly improves the embryo maturation in cv. Boufeggous as reported by Othmani et al. (2009aOTHMANI, A. et al. Somatic embryogenesis and plant regeneration in date palm Phoenix dactylifera L., cv. Boufeggous is significantly improved by fine chopping and partial desiccation of embryogenic callus. Plant Cell, Tissue and Organ Culture , 97:71-79, 2009a.).

Plantlet formation

Somatic embryo germination or plantlet formation depends on different factors. The size of the somatic embryos also varies the germination percentage in date palm (Al-Khayri; Al-Bahrany, 2012AL-KHAYRI, J. M.; AL-BAHRANY, A. M. Effect of abscisic acid and polyethylene glycol on the synchronization of somatic embryo development in date palm (Phoenix dactylifera L.). Biotechnology , 11:318-325, 2012.). Al-Khayri (2003AL-KHAYRI, J. M. In vitro germination of somatic embryos in date palm: Effect of auxin concentration and strength of MS salts. Current Science, 84:680-683, 2003.) also reported that the somatic embryo germination is influenced by the strength of the medium and concentration of the 1-naphthalenacetic acid (NAA) and indole-3-butyric acid (IBA) used. Somatic embryo germination with 81% plantlet conversion was achieved with 1 mg L-1 NAA concentration (Othmani et al., 2009bOTHMANI, A. et al. In vitro cloning of date palm Phoenix dactylifera L., cv. Deglet Bey by using embryogenic suspension and temporary immersion bioreactor (TIB). Biotechnology and Biotechnological Equipment, 23(2):1181-1188, 2009b.). The hormone combination of 6-benzylamino purine (BAP), IBA and NAA also influences the plantlet formation or rooting (Figure 1E) in dates (Zouine; El Hadrami, 2007ZOUINE, J.; EL HADRAMI, I. Effect of 2,4-D, glutamine and BAP on embryogenic suspension culture of date palm (Phoenix dactylifera L.). Scientia Horticulturae , 112:221-226, 2007.). However, Mazri et al. (2017MAZRI, M. A. et al. Somatic embryogenesis from bud and leaf explants of date palm (Phoenix dactylifera L.) cv. Najda. 3 Biotech , 7:58, 2017.) achieved somatic embryo germination in hormone-free MS medium.

Acclimatization of plantlet

The success of acclimatization is hidden in the nature of the substrate which should be high in organic matter, possess optimal water-holding capacity and proper aeration attributes (Hegazy, 2008HEGAZY, A. E. Micropropagation of Egyptian date palm cv. Selmy through floral buds culture. Journal of Agricultural Sciences, Mansoura University, 33(4):2803-2815, 2008.). Plantlets derived from date palm somatic embryos have been successfully acclimatized in many cultivars. Kurup et al. (2014KURUP, S. S. et al. Rapid in vitro regeneration of date palm (Phoenix dactylifera L.) cv. Kheneizi using tender leaf explant. Emirates Journal of Food and Agriculture , 26(6):539-544, 2014.) found nearly 60% survival rate of cv. Kheneizi when transferred to pots with a peat/vermiculite mixture of 2:1. Othmani et al. (2009aOTHMANI, A. et al. Somatic embryogenesis and plant regeneration in date palm Phoenix dactylifera L., cv. Boufeggous is significantly improved by fine chopping and partial desiccation of embryogenic callus. Plant Cell, Tissue and Organ Culture , 97:71-79, 2009a., 2009bOTHMANI, A. et al. In vitro cloning of date palm Phoenix dactylifera L., cv. Deglet Bey by using embryogenic suspension and temporary immersion bioreactor (TIB). Biotechnology and Biotechnological Equipment, 23(2):1181-1188, 2009b.) reported 60 and 80% survival rates in date palm cvs. Boufeggous and Deglet Nour, respectively. Al-Khayri (2010)AL-KHAYRI, J. M. Somatic embryogenesis of date palm (Phoenix dactylifera L.) improved by coconut water. Biotechnology , 9:477-484, 2010. observed 72-84% survival rate in cvs. Khasab and Nabout Saif plantlets after ex vitro transfer (Figure 1F).

Organogenesis

Organogenesis refers to the development of organ tissue with a vascular connection and finally which influences the plant formation from an explant with or without the intermediate callus stage. Date palm micropropagation favors the direct organogenesis of an explant without callus formation stage. To produce elite cultivars from rapid clonal propagation, direct organogenesis techniques have been widely adopted (Khierallah; Bader, 2007KHIERALLAH, H. S.; BADER, M. S. M. Micropropagation of date palm (Phoenix dactylifera L.) var. Maktoom through organogenesis. Acta Horticulturae , 736:213-223, 2007.). There are various steps involved in date palm organogenesis: adventitious bud formations, multiplication of shoot bud, shoot elongation, and rooting (Abahmane, 2011ABAHMANE, L. Date palm micropropagation via organogenesis. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology, Dordrecht: Springer, 2011. p.69-90.; Bekheet, 2013BEKHEET, S. A.; TAHA H. S. Complementary strategy for conservation of date palm germplasm. Global Journal of Biodiversity Science and Management, 3(1):96-107, 2013.; Mazri; Meziani, 2013MAZRI, M. A.; MEZIANI, R. An improved method for micropropagation and regeneration of date palm (Phœnix dactylifera L). Journal of Plant Biochemistry and Biotechnology , 22:176-184, 2013., 2015).

Adventitious bud formations

The formation of adventitious buds from date palm explants depends on several factors. Based on the findings of Al-Khateeb (2006)AL-KHATEEB, A. A. Role of cytokinin and auxin on the multiplication stage of date palm (Phoenix dactylifera L.) cv. Sukry. Biotechnology, 5:349-352, 2006. a high concentration of plant hormones induces abnormal growth of the tissue without bud formation, and low concentrations influence the formation of adventitious buds. Many researchers have found that different auxin and cytokinin combinations promote bud formation in different date palm cultivars. Cultivar Maktoom induced buds in the MS medium (Murashige; Skoog, 1962MURASHIGE, T.; SKOOG, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15:473-497, 1962.) supplemented with 1 mg L-1 BAP, 2 mg L-1 2iP, 1 mg L-1 NAA and 1 mg L-1 2-naphthoxyacetic acid (NOA) (Khierallah; Bader, 2007KHIERALLAH, H. S.; BADER, M. S. M. Micropropagation of date palm (Phoenix dactylifera L.) var. Maktoom through organogenesis. Acta Horticulturae , 736:213-223, 2007.). Bekheet (2013BEKHEET, S. A.; TAHA H. S. Complementary strategy for conservation of date palm germplasm. Global Journal of Biodiversity Science and Management, 3(1):96-107, 2013.) reported that MS medium fortified with 2 mg L-1 2iP and 1 mg L-1 NAA promotes buds in cv. Zaghlool, while Al-Mayahi (2014)AL-MAYAHI, A. M. W. Thidiazuron-induced in vitro bud organogenesis of the date palm (Phoenix dactylifera L.) cv. Hillawi. African Journal of Biotechnology , 13:3581-3590, 2014. used cv. Hillawi which induced buds in the MS medium supplemented with 1 mg L-1 BAP and 0.5 mg L-1 thidiazuron (TDZ).

Multiplication of shoot buds

Shoot bud multiplication is influenced by various factors such as medium composition, genotype and plant hormones. Khierallah and Bader (2007KHIERALLAH, H. S.; BADER, M. S. M. Micropropagation of date palm (Phoenix dactylifera L.) var. Maktoom through organogenesis. Acta Horticulturae , 736:213-223, 2007.) mentioned that the date palm cv. Maktoom showed higher shoot-bud multiplication in MS medium with a hormone combination of 1 mg L-1 NAA, 1 mg L-1 NOA, 4 mg L-1 2iP and 2 mg L-1 BAP. Mazri and Meziani (2013MAZRI, M. A.; MEZIANI, R. An improved method for micropropagation and regeneration of date palm (Phœnix dactylifera L). Journal of Plant Biochemistry and Biotechnology , 22:176-184, 2013.) found that half-strength MS medium augmented with 0.5 mg L-1 NOA and 0.5 mg L-1 KN produced 23.5 shoot buds per explant after 3 months of multiplication in cv. Najda. Al-Mayahi (2014)AL-MAYAHI, A. M. W. Thidiazuron-induced in vitro bud organogenesis of the date palm (Phoenix dactylifera L.) cv. Hillawi. African Journal of Biotechnology , 13:3581-3590, 2014. reported production of an average of 18.2 buds per culture in cv. Hillawi, in the MS medium containing 1 mg L-1 BAP and 0.5 mg L-1 TDZ.

Shoot Elongation and rooting

The medium with or without hormone promotes shoot elongation and rooting in date palm. Mazri and Meziani (2013MAZRI, M. A.; MEZIANI, R. An improved method for micropropagation and regeneration of date palm (Phœnix dactylifera L). Journal of Plant Biochemistry and Biotechnology , 22:176-184, 2013.) found that in the cv. Najda shoot elongation was faster in the medium supplemented with hormones when compared to hormone free medium, which also adds high frequency of root formation, the hormone free medium showed wider and green leaves with optimum survival rates. Bekheet (2013BEKHEET, S. A.; TAHA H. S. Complementary strategy for conservation of date palm germplasm. Global Journal of Biodiversity Science and Management, 3(1):96-107, 2013.) suggested 1 mg L-1 NAA induces better and optimum rooting at the same concentration IAA or IBA. Meziani et al. (2015MEZIANI, R. et al. Effects of plant growth regulators and light intensity on the micropropagation of date palm (Phoenix dactylifera L) cv. Mejhoul. Journal of Crop Science and Biotechnology , 18:325-331, 2015.) reported cv. Mejhoul shoots grew an average of 13.4 cm with an average 4.6 roots number per shoot with wide and green leaves from 3 months old hormone-free half MS medium.

Acclimatization of plantlet

Acclimatization of plantlets is the final step of micropropagation. Mazri and Meziani (2013MAZRI, M. A.; MEZIANI, R. An improved method for micropropagation and regeneration of date palm (Phœnix dactylifera L). Journal of Plant Biochemistry and Biotechnology , 22:176-184, 2013.) achieved good survival frequency in cv. Najda when a peat-gravel mixture was used as the substrate. Meziani et al. (2015MEZIANI, R. et al. Effects of plant growth regulators and light intensity on the micropropagation of date palm (Phoenix dactylifera L) cv. Mejhoul. Journal of Crop Science and Biotechnology , 18:325-331, 2015.) reported after 3 months of observation in the greenhouse the cv. Mejhoul plantlets showed 88% survival rate. The cv. Boufeggous shoots from semisolid medium, found the highest survival rate up to 92.5% whereas the shoots from stationary liquid media culture showed a survival rate of 50% after 3 months in the greenhouse (Mazri, 2015MAZRI, M. A.; MEZIANI, R. Micropropagation of Date Palm: A Review. Cell and Developmental Biology, 4:160, 2015. ).

Bioreactor

In recent years micropropagation protocols have used liquid media instead of semisolid media. The production of somatic embryos in liquid culture systems was 17-fold higher than on semisolid medium (Othmani et al., 2011OTHMANI, A. et al. Bioreactors and automation in date palm micropropagation. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology , Dordrecht: Springer , 2011. p.119-136.). The Temporary Immersion Bioreactor (TIB) showed that shoot clusters were regenerated 5.5-fold faster as compared to semisolid medium (Othmani et al., 2011OTHMANI, A. et al. Bioreactors and automation in date palm micropropagation. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology , Dordrecht: Springer , 2011. p.119-136.). The bioreactor system may be used in the micropropagation optimization process, which needs less manipulation of medium, reduces the cost of gelling agents, experienced labor, laboratory space and induces the multiplication frequency of the particular plant species (Gomes et al., 2016GOMES, H. T. et al. Regeneration of somatic embryos of oil palm (Elaeis guineensis) using temporary immersion bioreactors. Industrial Crops and Products, 89:244-249, 2016. ). Recently, Frómeta et al. (2017FRÓMETA, O. M. et al. In vitro propagation of Gerbera jamesonii Bolus ex Hooker f. in a temporary immersion bioreactor. Plant Cell, Tissue and Organ Culture, 129:543-551, 2017.) successfully achieved the regeneration of the Barberton daisy (Gerbera jamesonii) using TIB system. Ramírez-Mosqueda and Iglesias-Andreu (2016RAMÍREZ-MOSQUEDA M. A.; IGLESIAS-ANDREU, L. G. Evaluation of different temporary immersion systems (BIT®, BIG, and RITA®) in the micropropagation of Vanilla planifolia Jacks. In Vitro Cellular & Developmental Biology-Plant , 52:154-160, 2016.) reported the micropropagation of the vanilla orchid (Vanilla planifolia) in different bioreactor systems such as TIB, Gravity Immersion Bioreactors (GIB) and Recipient for Automated Temporary Immersion (RITA). The high number of shoots observed in the TIB followed by RITA and GIB and optimum rooting was found in TIB compared to GIB and RITA. Gomes et al. (2016GOMES, H. T. et al. Regeneration of somatic embryos of oil palm (Elaeis guineensis) using temporary immersion bioreactors. Industrial Crops and Products, 89:244-249, 2016. ) evaluated the regeneration of somatic embryos of oil palm (Elaeis guineensis) by using semisolid, RITA and Temporary Immersion System (TIS) and found TIS was most suitable for regeneration. Whereas, the use of TIB system in date palm (cv. Deglet Bey) in vitro regeneration was reported by Othmani et al. (2009bOTHMANI, A. et al. In vitro cloning of date palm Phoenix dactylifera L., cv. Deglet Bey by using embryogenic suspension and temporary immersion bioreactor (TIB). Biotechnology and Biotechnological Equipment, 23(2):1181-1188, 2009b., 2011OTHMANI, A. et al. Bioreactors and automation in date palm micropropagation. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology , Dordrecht: Springer , 2011. p.119-136.). Improved embryogenic callus and proliferation of regenerated shoots was observed in TIB system compared to semi-solid medium.

MAJOR PROBLEMS AND FACTORS AFFECTING THE DATE PALM MICROPROPAGATION

In date palm micropropagation various problems are encountered like tissue browning, hyperhydricity and genetic variation. The major factors are as follows: nature of the explant, light and medium composition used. The problems and factors affecting micropropagation are briefly discussed in this section.

Tissue browning

The browning of the explant is the most frequent problem in date palm tissue culture which finally leads to the death of the tissue. During micropropagation both somatic embryogenesis (Abohatem; Zouine; El Hadrami, 2011ABOHATEM, M.; ZOUINE, J.; EL HADRAMI, I. Low concentrations of BAP and high rate of subcultures improve the establishment and multiplication of somatic embryos in date palm suspension cultures by limiting oxidative browning associated with high levels of total phenols and peroxidase activities. Scientia Horticulturae, 130:344-348, 2011.) and organogenesis (Mazri, 2015MAZRI, M. A.; MEZIANI, R. Micropropagation of Date Palm: A Review. Cell and Developmental Biology, 4:160, 2015. ) encounter this problem. The reason is that date tissue contain high levels of phenolic compounds which are toxic to the tissue and finally causes its death (Loutfi; El Hadrami, 2005LOUTFI, K.; EL HADRAMI, I. Phoenix dactylifera date palm. In: LITZ, R. E. Biotechnology of fruit and nut crops, Wallingford: CAB International, 2005. p.144-156.). During the surface sterilization procedure, ascorbic acid and citric acid are used to control browning in the explant tissue (Al-Khayri, 2010AL-KHAYRI, J. M. Somatic embryogenesis of date palm (Phoenix dactylifera L.) improved by coconut water. Biotechnology , 9:477-484, 2010.; Khierallah et al., 2015KHIERALLAH, H. S. M. et al. Influence of sucrose and pacloburtazol on callus growth and somatic embryogenesis in date palm cv. Bream. International Journal of Current Research and Academic Review, 1:270-276, 2015.; Naik; Al-Khayri, 2016NAIK, P. M.; AL-KHAYRI, J. M. Somatic embryogenesis of date palm (Phoenix dactylifera L.) through cell suspension culture. In: JAIN, S. M. Protocols for in vitro cultures and secondary metabolite analysis of aromatic and medicinal plants, 2nd ed. Methods in molecular biology. New York: Springer , 1391:357-366, 2016.). In order to prevent this browning many researchers add activated charcoal and polyvinylpyrrolidone (PVP) in the culture medium (Al-Khayri, 2010AL-KHAYRI, J. M. Somatic embryogenesis of date palm (Phoenix dactylifera L.) improved by coconut water. Biotechnology , 9:477-484, 2010.; Mazri; Meziani, 2013MAZRI, M. A.; MEZIANI, R. An improved method for micropropagation and regeneration of date palm (Phœnix dactylifera L). Journal of Plant Biochemistry and Biotechnology , 22:176-184, 2013.; Naik; Al-Khayri, 2016NAIK, P. M.; AL-KHAYRI, J. M. Somatic embryogenesis of date palm (Phoenix dactylifera L.) through cell suspension culture. In: JAIN, S. M. Protocols for in vitro cultures and secondary metabolite analysis of aromatic and medicinal plants, 2nd ed. Methods in molecular biology. New York: Springer , 1391:357-366, 2016.).

Hyperhydricity

Hyperhydricity refers to the accumulation of water content in the cultured tissue. This physiological disorder is common in somatic embryogenesis and organogenesis of date palm (Mazri, 2015MAZRI, M. A. Role of cytokinins and physical state of the culture medium to improve in vitro shoot multiplication, rooting and acclimatization of date palm (Phoenix dactylifera L.) cv. Boufeggous. Journal of Plant Biochemistry and Biotechnology , 24(3):268-275, 2015.; Mazri; Meziani, 2013MAZRI, M. A.; MEZIANI, R. An improved method for micropropagation and regeneration of date palm (Phœnix dactylifera L). Journal of Plant Biochemistry and Biotechnology , 22:176-184, 2013.; McCubbin; Zaid, 2007MCCUBBIN, M. J.; ZAID, A. Would a combination of organogenesis and embryogenesis techniques in date palm micropropagation be the answer? Acta Horticulturae , 736:255-259, 2007.). Kriaa et al. (2012KRIAA, W. et al. The date palm (Phoenix dactylifera L.) micropropagation using completely mature female flowers. Comptes Rendus Biologies, 335:194-204, 2012.) reported when hydrated calli were cultured on the low concentration of 2,4-D for a long period, calli will induce somatic embryos or shoots. The factors such as plant hormones, liquid media and concentration of ammonia used are responsible for hyperhydricity (Al-Khateeb, 2008AL-KHATEEB, A. A. The problems facing the use of tissue culture technique in date palm (Phoenix dactylifera L.). Scientific Journal of King Faisal University, 9:85-104, 2008.; Mazri; Meziani, 2015MAZRI, M. A.; MEZIANI, R. Micropropagation of Date Palm: A Review. Cell and Developmental Biology, 4:160, 2015. ). The use of high agar concentration in the semisolid media and low concentration of plant hormones and ammonium concentration will help in the reduction of formation of hyperhydricity in the cultured tissue (Al-Khateeb, 2008AL-KHATEEB, A. A. The problems facing the use of tissue culture technique in date palm (Phoenix dactylifera L.). Scientific Journal of King Faisal University, 9:85-104, 2008.).

Nature of explant

In the beginning era of date palm tissue culture, researchers used different kind of explants: embryos, immature fruits, roots, leaf petioles, lateral buds, shoot tips, pieces of stem and rachilla tissue (Sharma; Deepak; Chowdhury, 1986SHARMA, D. R.; DEEPAK, S.; CHOWDHURY, J. B. Regeneration of plantlets from somatic tissues of date palm (Phoenix dactylifera L.). Indian Journal of Experimental Biology, 24:763-766, 1986.; Tisserat, 1979TISSERAT, B. Propagation of date palm (Phoenix dactylifera L.) in vitro. Journal of Experimental Botany, 30:1275-1283, 1979.). After decades of research it has been proved that date palm micropropagation is most responsive with explants having meristematic origin and these includes apical shoot tips, lateral buds and leaf primordial isolated from shoot tip (Aslam et al., 2011ASLAM, J. et al. Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars. Saudi Journal of Biological Sciences, 18:369-380, 2011.; Khan; Bi, 2012KHAN, S.; BI, T. B. Direct shoot regeneration system for date palm (Phoenix dactylifera L.) cv. Dhakki as a means of micropropagation. Pakistan Journal of Botany, 44(6):1965-1971, 2012.). The meristematic cells or tissue respond quickly to the components of the culture medium. However, Khan and Bi (2012KHAN, S.; BI, T. B. Direct shoot regeneration system for date palm (Phoenix dactylifera L.) cv. Dhakki as a means of micropropagation. Pakistan Journal of Botany, 44(6):1965-1971, 2012.) reported the multiplication of date palm cv. Dhakki through direct shoot regeneration where different explants such as shoot tips, leaf primordia and apical meristem were used, among these shoot tips emerged as a most promising explant with highest capacity for direct shoot regeneration. In recent years use of the inflorescence as an explant has increased, where harvesting of the offshoots is not required. Micropropagation from inflorescence was achieved in date palm cvs. such as Gulistan, Aseel, Dedhi, Gajar, Kashoo-wari, Khar, Kharbline, Khormo, Deglet Noor, Blombek, Menakher and Barhee (Abahmane, 2013ABAHMANE, L. Recent achievements in date palm (Phoenix dactylifera L.) micropropagation from inflorescence tissues. Emirates Journal of Food and Agriculture, 25(11):863-874, 2013.; Abul-Soad, 2012ABUL-SOAD, A. A. Influence of inflorescence explant age and 2,4-D incubation period on somatic embryogenesis of date palm. Emirates Journal of Food and Agriculture , 24(5):434-443, 2012.; Abul-Soad; Mahdi, 2010ABUL-SOAD, A. A.; MAHDI, S. M. Commercial production of tissue culture date palm (Phoenix dactylifera L.) by inflorescence technique. Journal of Genetic Engineering and Biotechnology , 8:39-44, 2010.; Kriaa et al., 2012KRIAA, W. et al. The date palm (Phoenix dactylifera L.) micropropagation using completely mature female flowers. Comptes Rendus Biologies, 335:194-204, 2012.).

Effect of light

Light serves as an external factor to regulate the growth and development of in vitro plants. The broad spectrum of fluorescent lamps with a wavelength range from 380-750 nm are used as a light source for tissue culture (Kim et al., 2004KIM, H. H. et al. Green-light supplementation for enhanced lettuce growth under red and blue-light-emitting diodes. Horticultural Science , 39:1617-1622, 2004.). Light intensity and type of light affect date palm micropropagation (Al-Mayahi, 2016AL-MAYAHI, A. M. W. Effect of red and blue light emitting diodes ‘‘CRB-LED’’ on in vitro organogenesis of date palm (Phoenix dactylifera L.) cv. Alshakr. World Journal of Microbiology and Biotechnology , 32:160, 2016.; Meziani et al., 2015MEZIANI, R. et al. Effects of plant growth regulators and light intensity on the micropropagation of date palm (Phoenix dactylifera L) cv. Mejhoul. Journal of Crop Science and Biotechnology , 18:325-331, 2015.). Meziani et al. (2015MEZIANI, R. et al. Effects of plant growth regulators and light intensity on the micropropagation of date palm (Phoenix dactylifera L) cv. Mejhoul. Journal of Crop Science and Biotechnology , 18:325-331, 2015.) evaluated the organogenesis of date palm cv. Mejhoul using different levels of light intensities. The results explained that the 2000-3000 lux light intensity enhances shoot elongation and greening but reduction in the shoot bud proliferation was observed. Darkness and low light intensity (500 lux) significantly induced advanced rooting. The 1000 lux light intensity during the multiplication stage showed optimal growth of culture with respect to shoot buds per explant, greening an advanced rooting. Recently Al-Mayahi (2016AL-MAYAHI, A. M. W. Effect of red and blue light emitting diodes ‘‘CRB-LED’’ on in vitro organogenesis of date palm (Phoenix dactylifera L.) cv. Alshakr. World Journal of Microbiology and Biotechnology , 32:160, 2016.) conducted an experiment to test the effect of combinations of red + blue light emitting diode (18:2) (CRB-LED) and white fluorescent light on direct organogenesis by induction of adventitious buds from shoot tip and multiplication shoots of date palm cv. Alshakr. The results showed CRB-LED performed better in the production of shoot numbers than white fluorescent light. CRB-LED also significantly increased the total soluble carbohydrate, starch, free amino acids, peroxidase activity, potassium, magnesium and sodium contents of the in vitro shoots.

Medium composition

Tissue culture media is composed of a wide range of micro and macro elements, these compositions of basal media regulates the growth and development of tissue in the in vitro culture. The date palm tissue culture also showed different growth patterns with respect to the various media composition (Al-Khayri, 2011AL-KHAYRI, J. M. Basal salt requirements differ according to culture stage and cultivar in date palm somatic embryogenesis. American Journal of Biochemistry and Biotechnology, 7:32-42, 2011.). Al-Khayri (2011AL-KHAYRI, J. M. Basal salt requirements differ according to culture stage and cultivar in date palm somatic embryogenesis. American Journal of Biochemistry and Biotechnology, 7:32-42, 2011.) tested five different types of media such as MS medium, W medium (White, 1963WHITE, P. R. The Cultivation of animal and plant cells, 2nd ed., New York: Ronald Press Co., 1963. 228p.), NN medium (Nitsch; Nitsch, 1969NITSCH, J. P.; NITSCH, C. Haploid plants from pollen grains. Science, 163:85-87, 1969.), SH medium (Schenk; Hildebrandt, 1972SCHENK, R. U.; HILDEBRANDT, A. C. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Canadian Journal of Botany, 50:199-204, 1972.), Woody Plant Medium, WPM (Lloyd; McCown, 1981LLOYD, G.; MCCOWN, B. Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by shoot tip culture. International Plant Propagators Society Proceedings, 30:421-427, 1981.) to evaluate the callus growth and somatic embryogenesis. Optimal callus growth was attained in cv. Barhee using SH, W and MS media, cv. Berny using SH and NN medium, in cv. Khusab using W and WPM media. The best embryo production was seen in cv. Barhee on W and SH media, cv. Berny using WPM and MS media and cv. Khusab using W and SH media. The optimum regeneration percentage obtained in the cv. Barhee occurred on W and WPM media, cv. Berny on WPM medium and cv. Khusab using W medium. Mazri et al. (2016MAZRI, M. A. et al. Optimization of medium composition for in vitro shoot proliferation and growth of date palm cv. Mejhoul. 3 Biotech, 6:111, 2016.) evaluated the effect of various concentrations of major mineral salts including ammonium nitrate, potassium nitrate, calcium chloride dehydrate, potassium dihydrogen phosphate and magnesium sulfate heptahydrate on shoot bud proliferation of date palm cv. Mejhoul, they also tested the different concentration of L-glutamine and myo-inositol. The results showed that maximum number of shoot buds per explant obtained on the medium fortified with 825 mg L-1 NH4NO3, 1900 mg L-1 KNO3, 220 mg L-1 CaCl2.2H2O, 170 mg L-1 KH2PO4, 370 mg L-1 MgSO4.7H2O as well as 1 g L-1 L-glutamine, 2 g L-1 myo-inositol and 30 g L-1 sucrose. The media supplemented with 1650 mg L-1 NH4NO3 significantly influenced the frequency of hyperhydricity. The results also showed that the number of shoot buds per explant was significantly affected by the concentration of L-glutamine, and myo-inositol present in the medium.

In addition to the mineral salts in tissue culture medium, the carbon source also plays an important role in the tissue growth. In the tissue culture of different plant species and also in the date palm the major carbon source used is sucrose at 30 g L-1 (Al-Khayri, 2013AL-KHAYRI, J. M. Factors affecting somatic embryogenesis in date palm (Phoenix dactylifera L.) In: ASLAM, J.; SRIVASTAVA, P. S.; SHARMA, M. P. Somatic embryogenesis and genetic transformation in plants, New Delhi: Narosa Publishing House, 2013. p.5-38.). In addition to the sucrose, other carbon source such as sorbitol, maltose, mannitol or commercial granulated sugar are also being used to improve the callus growth and shoot bud proliferation in date palm (Al-Khayri, 2013AL-KHAYRI, J. M. Factors affecting somatic embryogenesis in date palm (Phoenix dactylifera L.) In: ASLAM, J.; SRIVASTAVA, P. S.; SHARMA, M. P. Somatic embryogenesis and genetic transformation in plants, New Delhi: Narosa Publishing House, 2013. p.5-38.; Mazri et al., 2016MAZRI, M. A. et al. Optimization of medium composition for in vitro shoot proliferation and growth of date palm cv. Mejhoul. 3 Biotech, 6:111, 2016.). Mazri et al. (2016MAZRI, M. A. et al. Optimization of medium composition for in vitro shoot proliferation and growth of date palm cv. Mejhoul. 3 Biotech, 6:111, 2016.) evaluated the various concentrations of carbon sources to improve the shoot bud multiplication in date palm cv. Mejhoul. The experiments revealed the medium supplemented with 30 g L-1 sucrose obtained the optimal number of shoot buds per explant when compared to sorbitol, mannitol and commercial granulated sugar. Low and high concentrations of sucrose did not induce optimum number of shoot buds. The superior morphology of the shoot buds was generated from sucrose supplemented media when compared to buds obtained from the media augmented with sorbitol, mannitol and commercial granulated sugar.

VARIATION IN MICROPROPAGATED PLANTS

Somaclonal variation

The morphological and genotypical differences observed in micropropagated plants are called somaclonal variations. Sometimes micropropagated plants carry advantageous characters with respect to commercial and agronomical interest which finally leads to some new varieties (El Hadrami et al., 2011EL HADRAMI, A. et al. Somaclonal variation in date palm. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology , Dordrecht: Springer , 2011. p.183-204.). Somaclonal variation act as a raw material for the mass propagation, cryopreservation and synthetic seed production. Date palm micropropagation and somatic variation introduces new genotypes with stress tolerant, disease resistant and with high quality fruits (El Hadrami; El Hadrami, 2009EL HADRAMI, I.; EL HADRAMI, A. Breeding date palm. In: JAIN, S. M.; PRIYADARSHAN, P. M. Breeding Plantation Tree Crops, New York: Springer, 2009. p.191-216.; Jain, 2001JAIN, S. M. Tissue culture-derived variation in crop improvement. Euphytica, 118:153-166, 2001.). Research has showed that somaclonal variation frequency is depend on the age of the tissue cultured date palm (Saker et al., 2000SAKER, M. et al. Detection of seasonal variations in tissue culture derived date palm plants using isozyme analysis and RAPD fingerprints. Biologia Plantarum , 43:347-51, 2000.). The high concentration of 2,4-D induced 25% somaclonal variation and observed change in the leaf morphology and also cause poor flower pollination leads to low quality date fruit (Fki et al., 2011bFKI, L. et al. Palm cryobanking. CryoLetters, 32(6):451-462, 2011b.). To reduce the risk of somaclonal variation researchers suggest using juvenile explants, low auxin concentration, especially 2,4-D and minimum numbers of subcultures (El Hadrami et al., 2011EL HADRAMI, A. et al. Somaclonal variation in date palm. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology , Dordrecht: Springer , 2011. p.183-204.; Fki et al., 2011aFKI, L. et al. Date palm micropropagation via somatic embryogenesis. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology , Dordrecht: Springer , 2011a. p.47-68., 2011bFKI, L. et al. Palm cryobanking. CryoLetters, 32(6):451-462, 2011b.; Khan; Bi, 2012KHAN, S.; BI, T. B. Direct shoot regeneration system for date palm (Phoenix dactylifera L.) cv. Dhakki as a means of micropropagation. Pakistan Journal of Botany, 44(6):1965-1971, 2012.). The use of high concentration of plant growth regulators induces somaclonal variation in date palm (Al-Mazroui et al., 2007AL-MAZROUI, H. S.; ZAID, A.; BOUHOUCHE, N. Morphological abnormalities in tissue culture-derived date palm (Phoenix dactylifera L.). Acta Horticulturae , 736:329-335, 2007.).

Molecular characterization

The date palm is heterozygous and its out-breeding nature creates progeny with 50% male and 50% female trees that are not true-to-type (Othmani et al., 2010OTHMANI, A. et al. Regeneration and analysis of genetic stability of plantlets as revealed by RAPD and AFLP markers in date palm (Phoenix dactylifera L.) cv. Deglet Nour. International Research Journal and Plant Science , 1:48-55, 2010.). In commercial micropropagation it is very important to test the genetic conformity/true-to-type of the regenerated plants. Several researchers have used molecular markers (AFLP, ISSR and RAPD) to confirm the genetic conformity of the tissue-cultured plants (Kumar et al., 2010KUMAR, N. et al. Assessment of genetic fidelity of micropropagated date palm (Phoenix dactylifera L.) plants by RAPD and ISSR markers assay. Physiology and Molecular Biology of Plants, 16:207-213, 2010.; Othmani et al., 2009cOTHMANI, A. et al. Regeneration and molecular analysis of date palm (Phoenix dactylifera L.) plantlets using RAPD markers. African Journal of Biotechnology , 8:813-820, 2009c., 2010OTHMANI, A. et al. Regeneration and analysis of genetic stability of plantlets as revealed by RAPD and AFLP markers in date palm (Phoenix dactylifera L.) cv. Deglet Nour. International Research Journal and Plant Science , 1:48-55, 2010.). The use of RAPD primers showed micropropagated date cv. Ferhi with high level of polymorphism and 37.8% of variability (Moghaieb; Abdel-Hadi; Ahmed, 2011MOGHAIEB, R. E. A.; ABDEL-HADI, A. A.; AHMED, M. R. A. Genetic stability among date palm plantlets regenerated from petiole explants. African Journal of Biotechnology , 10:14311-14318, 2011.). The variation in tissue-cultured plants is also observed in cvs. Barhee and Khalas which display dwarfism and abnormal flower development (Al-Kaabi; Zaid; Ainsworth, 2007AL-KAABI, H. H.; ZAID, A.; AINSWORTH, C. Plant-off-types in tissue culture-derived date palm (Phoenix dactylifera L.) plants. Acta Horticulturae, 736:267-281, 2007.; Zaid; Al-Kaabi, 2003ZAID, A.; AL-KAABI, H. Plant-off types in tissue culture-derived date palm. (Phoenix dactylifera L.). Emirates Journal of Food and Agriculture , 15:17-35, 2003.). Kumar et al. (2010KUMAR, N. et al. Assessment of genetic fidelity of micropropagated date palm (Phoenix dactylifera L.) plants by RAPD and ISSR markers assay. Physiology and Molecular Biology of Plants, 16:207-213, 2010.) evaluated 27 micropropagated date plants with 160 RAPD and 21 ISSR primers, in which 30 RAPD and 12 ISSR primers produced a total of 347 reproducible monomorphic band; the results suggested that the micropropagated plants are true-to-type. Aslam, Khan and Naqvi (2015ASLAM, J.; KHAN, S. A.; NAQVI, S. H. Evaluation of genetic stability in somatic embryo derived plantlets of six date palm (Phoenix dactylifera L.) cultivars through RAPD based molecular marker. Science Technology and Development, 34(1):1-8, 2015.) established the somatic embryo derived regeneration protocol for the six cvs. Barhee, Zardai, Khalasah, Muzati, Shishi and Zart, in which the RAPD profile showed a similar banding pattern for micropropagated plants and mother plants and confirmed the genetic stability. Abass, Al-Utbi and Al-Samir (2017ABASS, M. H.; AL-UTBI, S. D.; AL-SAMIR, E. A. R. H. Genotoxicity assessment of high concentrations of 2,4-D, NAA and Dicamba on date palm callus (Phoenix dactylifera L.) using protein profile and RAPD markers. Journal of Genetic Engineering and Biotechnology, 15(1):287-295, 2017. ) screened the genetic toxicity of hormones on date palm callus using RAPD and protein profile. Low concentration of 2,4-D and Dicamba showed no polymorphism in RAPD primers and protein profile when compared to the control profile. The high concentration of hormones induced more polymorphism in both the tested markers compared to control.

GERMPLASM CONSERVATION

Micropropagation methods help in the germplasm conservation of many plant species. The disappearance of date palm farms due to urbanization, and which also leads in the declining of the genetic diversity of the date palm. Shortage of arable land, adverse climatic conditions, diseases and natural disasters are some major factors affecting the diversity of date palm (Bekheet; Taha, 2013BEKHEET, S. A.; TAHA H. S. Complementary strategy for conservation of date palm germplasm. Global Journal of Biodiversity Science and Management, 3(1):96-107, 2013.; Zaid et al., 1999ZAID, A. et al. Diseases and pests of date palm. In: ZAID A,; ARIAS-JIMENEZ, E. J. Date palm cultivation, Rome: Food and Agriculture Organization (FAO Plant Production and Protection Paper No. 156), 1999. p.223-278.). There is a crucial need for the conservation of date palm germplasm and better utilization of available genetic resources. Although farmers are playing an important role in the conservation of the date palm germplasm, through cultivating different varieties in their traditional groves (Bekheet; Taha, 2013BEKHEET, S. A.; TAHA H. S. Complementary strategy for conservation of date palm germplasm. Global Journal of Biodiversity Science and Management, 3(1):96-107, 2013.), they tend to propagate a limited number of commercially desired cultivars. The use of modern techniques are necessary to conserve date palm germplasm. The establishment of a germplasm bank based on available in vitro technologies for this important species is yet to be realized.

Cryopreservation

One of the modern conservation method is cryopreservation, where genetic materials like shoot tips, callus, cell suspension, microspore and somatic embryos are stored long-term under super-low temperatures (-196 ºC in liquid nitrogen). A genuine micropropagation method is the primary requirement of cryopreservation of germplasm and in vitro conservation. Over decades researchers have standardized date palm micropropagation and started the cryopreservation of date palm tissue (Bekheet et al., 2007BEKHEET, S. A. et al. Cryopreservation of date palm (Phoenix dactylifera L.) cultured in vitro. Acta Horticulturae , 736:283-291, 2007.). Embryogenic calli of date palm were treated with cryogen and stored for several months under ultra-low temperature (-196 ºC). Tisserat (1982TISSERAT, B. Factors involved in the production of plantlets from date palm callus cultures. Euphytica , 31:201-214, 1982.) reported date palm regeneration from latent calli revived after 4-8 weeks. The successful cryopreservation of date palm callus after 4 months at -25 ºC was achieved by Mater (1987MATER, A. A. Production and cryogenic freezing of date palm germplasm and regeneration of plantlets from frozen material. Iraqi Journal of Agricultural Science (Zanco), 5:35-49, 1987.) by treating with cryoprotectant mixture. The normal growth of date palm embryos was achieved by Mycock et al. (1997MYCOCK, D. J. et al. Cryopreservation of somatic embryoids of Phoenix dactylifera. In: ELLIS, R. H.; BLACK, M.; MURDOCH, A. J.; Hong, T. D. Basic and applied aspects of seed biology, Dordrecht: Kluwer, 1997. p.75-82.) by pre-treating the embryos with glycerol and sucrose and dried to a water content of 0.4-0.7 g g-1. Bekheet, Taha and Saker (2002bBEKHEET, S. A.; TAHA, H. S.; SAKER, M. M. In vitro long-term storage of date palm. Biologia Plantarum, 45:121-124, 2002.) reported a long-term preservation method for in vitro shoot bud and callus culture with optimum percent of viability after 12 months treatment at 5 ºC. Al-Bahrany and Al-Khayri (2012AL-BAHRANY, A. M.; AL-KHAYRI, J. M. In vitro responses of date palm cell suspensions under osmotic stress induced by sodium potassium and calcium salts at different exposure durations. American Journal of Plant Physiology, 7:120-134, 2012.) described the germplasm conservation by providing reliable cryoprotectant solution for date palm (cv. Khalas) cell suspension. Fki et al. (2011bFKI, L. et al. Palm cryobanking. CryoLetters, 32(6):451-462, 2011b.) reported the successful cryopreservation method for proembryogenic masses (PEMs) of date palm variety Barhee and the morphology of the regenerated plants confirmed the stability of the clonal material. Furthermore, Fki et al. (2013FKI, L. et al. Cold hardening and sucrose treatment improve cryopreservation of date palm meristems. Biologia Plantarum , 57(2):375-379, 2013. ) reported the regeneration of cryopreserved callogenic meristems of date palm cv. Khenizi. Cryopreserved date palm tissue with optimum regeneration capacity can boost the micropropagation process.

COMMERCIAL PRODUCTION

Date palm micropropagation is an economical means to achieve rural development (Rajmohan, 2011RAJMOHAN, K. Date palm tissue culture: A pathway to rural development. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology , Dordrecht: Springer , 2011. p.29-45.). It has been proven that date palm micropropagation protocols have been developed and achieved commercial production (Hoop, 2000HOOP, B. M. Date palm micropropagation in Saudi Arabia: Policies and technology transfer. International Journal of Biotechnology , 2:333-341, 2000.). Numbers of cultivars are being developed and multiplied throughout the world. It has been reported that United Arab Emirates University developed micropropagation protocol for about 50 date palm cultivars (Rajmohan, 2011RAJMOHAN, K. Date palm tissue culture: A pathway to rural development. In: JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology , Dordrecht: Springer , 2011. p.29-45.). In India Atul Rajasthan Date Palms Ltd, has mounted a project to grow tissue cultured plants in the desert area using appropriate cultivars. As commercial production of date palm involves considerable profit, a number of private and public-sector agencies are involved in developing protocols for micropropagation of date palm. Some of the countries and their leading agencies/companies are producing millions of micropropagated plants annually are as follows: Marionnet G.F.A. in France, Palmdat in France and Namibia, Atul Rajasthan Date Palms Ltd. in India, Rahan Meristem in Israel, Domaine Agricole El Bassatine in Morocco, Al Rajhi Tissue Culture Laboratory in Saudi Arabia, Sapad Tissue Culture Date Palm Co. in Saudi Arabia, United Arab Emirates University - Date Palm Development Research Unit in U.A.E. and Date Palm Developments in United Kingdom. Most of the institutions and laboratories are focused on cv. Medjool because of its high economic importance; recently cv. Barhee also has been a focus for micropropagation, selling for USD 24-26 per plant. The price of the plant depends on the growth stage, cultivar and quantity ordered.

CONCLUSIONS AND FUTURE PROSPECTS

Date palm is a traditional crop in the Arab world which has the capacity to withstand adverse climatic conditions. For thousands of years date pam was propagated through conventional breeding which is a time-consuming and tedious process. The application of the tissue culture techniques gave date palm an improved efficiency compared to other crops. Micropropagation of date palm through somatic embryogenesis and organogenesis reduces labor cost and time. Date palm micropropagation depends on various factors which include tissue browning, hyperhyricity, explant source, age of explant, size of explant, cultivars, intensity and quality of light, temperature, pH of the medium, plant hormones, culture medium composition and age of culture. The various researchers all over the world are working on the factors which control date palm micropropagation via somatic embryogenesis and organogenesis. Optimization of all these factors in a particular elite cultivar will enhance the chances of biotechnological application such as somaclonal variation selection, cryopreservation, synthetic seeds, cell culture, protoplast cultures, haploid production, mutation studies, secondary metabolite production, use of bioreactor, assessment of genetic fidelity and genetic transformation (Al-Khayri; Jain; Johnson, 2015aAL-KHAYRI, J. M.; JAIN, S. M.; JOHNSON, D. V. Date palm genetic resources and utilization, Vol. 1: Africa and the Americas, Dordrecht: Springer, 2015a. 546p. , 2015bAL-KHAYRI, J. M.; JAIN, S. M.; JOHNSON, D. V. Date palm genetic resources and utilization, Vol. 2: Asia and Europe, Dordrecht: Springer , 2015b. 566p.; Aslam; Khan; Azad, 2015ASLAM, J.; KHAN, S. A.; AZAD, M. A. K. Agrobacterium-mediated genetic transformation of date palm (Phoenix dactylifera L.) cultivar “Khalasah” via somatic embryogenesis. Plant Science Today, 2(3):93-101, 2015.; Jain; Al-Khayri; Johnson, 2011JAIN, S. M.; AL-KHAYRI, J. M.; JOHNSON, D. V. Date palm biotechnology , Dordrecht: Springer , 2011. 743p.). Thus, there is a need for the optimization of date palm micropropagation in elite cultivars to encourage the development of commercial production of date via tissue culture laboratories in various parts of the world.

ACKNOWLEDGEMENT

The authors are grateful to Prof. Dennis V. Johnson (Cincinnati, Ohio, USA) for critical reading and commenting on the manuscript.

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Publication Dates

  • Publication in this collection
    Jul-Aug 2017

History

  • Received
    15 June 2017
  • Accepted
    28 July 2017
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