Elicitor-Induced Production of Biomass and Pharmaceutical Phenolic Compounds in Cell Suspension Culture of Date Palm (Phoenix dactylifera L.).

Plants that synthesize bioactive compounds that have high antioxidant value and elicitation offer a reliable in vitro technique to produce important nutraceutical compounds. The objective of this study is to promote the biosynthesis of these phenolic compounds on a large scale using elicitors in date palm cell suspension culture. Elicitors such as pectin, yeast extract (YE), salicylic acid (SA), cadmium chloride (CdCl2), and silver nitrate (AgNO3) at 50, 100, and 200 mg/L concentrations are used. The effects of elicitors on cell culture were determined in terms of biomass [packed cell volume (PCV), fresh and dry weight], antioxidant activity, and phenolic compounds (catechin, caffeic acid, kaempferol, apigenin) were determined using high-performance liquid chromatography (HPLC). Results revealed that enhanced PCV (12.3%), total phenolic content [317.9 ± 28.7 mg gallic acid equivalents (GAE)/100 g of dry weight (DW)], and radical scavenging activity (86.0 ± 4.5%) were obtained in the 50 mg/L SA treated cell culture of Murashige and Skoog (MS) medium. The accumulation of optimum catechin (26.6 ± 1.3 µg/g DW), caffeic acid (31.4 ± 3.8 µg/g DW), and kaempferol (13.6 ± 1.6 µg/g DW) was found in the 50 mg/L SA-treated culture when compared to the control. These outcomes could be of great importance in the nutraceutical and agronomic industries.

NAA-Induced Direct Organogenesis from Female Immature Inflorescence Explants of Date Palm.

Micropropagation has great potential for the multiplication of female and male date palms of commercially grown cultivars by using inflorescences. This approach is simple, convenient, and much faster than the conventional method of using shoot-tip explants. We describe here a stepwise micropropagation procedure using inflorescence explants of Iraqi date palm cultivar Maktoom. Cultured explants were derived from 0.5-cm-long spike segments excised from 8 to 10-cm-long spathes. About 70% formed adventitious buds on Murashige and Skoog (MS) medium supplemented with 2 mg/L naphthalene acetic acid (NAA), 4 mg/L benzylaminopurine (BAP), and 40 g/L sucrose and maintained in the dark for 16 weeks before transferring to normal light conditions. The best multiplication rate was achieved with 3 mg/L 2ip and 2 mg/L; for shoot elongation, the best medium is MS containing 0.5 mg/L BAP, 0.5 mg/L 2ip, and 1 mg/L GA3. Well-developed shoots were cultured for rooting in half MS medium amended with 1 mg/L NAA and 45 g/L sucrose. Plantlets with well-developed roots were successfully hardened in the greenhouse. Inflorescence explants proved to be a promising alternative explant source for micropropagation of date palm cultivars.

Direct Organogenesis from Immature Female Inflorescence of Date Palm by Gradual Reduction of 2,4-D Concentration.

Inflorescences represent an alternative explant source for superior date palm trees, especially those that do not produce offshoots. They provide large numbers of explants free of fungal and bacterial contamination for successful tissue culture initiation. Furthermore, they are characterized by the capacity of plant regeneration within a short time as compared to other explant types. This chapter focuses on the procedures employed for plant regeneration by direct organogenesis using immature female inflorescence explants, including initiation of adventitious buds, differentiation, multiplication, shoot elongation, rooting, and acclimatization. Adding 5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) into the initiation medium and gradually reducing it to 1 and then to 0.5 mg/L in the subsequent 2 subcultures, respectively, are determining factors in direct adventitious bud formation from the inflorescence. Bud differentiation is obtained on MS medium containing 0.25 mg/L kinetin (Kin), 0.25 mg/L benzyladenine (BA), 0.25 mg/L abscisic acid (ABA), 0.1 mg/L naphthaleneacetic acid (NAA), and 0.2 g/L activated charcoal (AC). Regenerated shoots exhibit sufficient root formation on MS medium supplemented with 2 mg/L indole butyric acid (IBA) and 1 mg/L NAA and subsequent survival in the greenhouse.

Optimized Direct Organogenesis from Shoot Tip Explants of Date Palm.

In vitro propagation is an available alternative to produce uniform and good-quality planting material to establish large-scale date palm cultivation in a short time. This study was carried out to achieve organogenesis and multiplication directly from shoot tips without callus formation, thus avoiding any possibility of undesirable genetic variability among the regenerated plants. The shoot tips explants are cultured on Murashige and Skoog (MS) medium supplemented with 1 mg/L naphthaleneacetic acid (NAA), 1 mg/L naphthoxyacetic acid (NOA), 2.5 mg/L benzyladenine (BA), and 2.5 mg/L isopentenyladenine (2iP). Numerous adventitious buds appeared from the shoot tip explants in darkness after six subcultures at 4-week intervals. Vegetative buds pass through three stages: initiation bud formation, vegetative bud differentiation, and shoot bud proliferation. Shoots are transferred onto medium containing low concentrations of growth regulators for shoot multiplication. The organogenesis protocol described herein consists of six steps: initiation of meristematic buds, multiplication, elongation, rooting, pre-acclimatization, and finally plant acclimatization.

Direct Organogenesis and Indirect Somatic Embryogenesis by In Vitro Reversion of Mature Female Floral Buds to a Vegetative State.

This protocol describes in vitro plant regeneration from mature female inflorescence explants of date palm (Phoenix dactylifera L.) by reversion of floral state (reproductive phase) to the vegetative state. The mature female inflorescence (fully developed) is cultured on MS induction medium containing 10 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 3 mg/L 2-isopentenyladenine (2iP), and 2 mg/L paclobutrazol (PBZ) or 2 mg/L abscisic acid (ABA). The basal part of the petals has meristematic cells, which can be induced to initiate callus or direct shoot formation depending on the plant growth regulator amendments. Callus forms on the induction medium supplemented with PBZ after 12 weeks, whereas it differentiates into somatic embryos on a medium containing 0.1 mg/L naphthaleneacetic acid (NAA). Direct shoots are regenerated on the induction medium amended with ABA after 24 weeks. Procedures for plant regeneration from mature female inflorescence explants are described, and histological changes which occur during the reversion process are presented.

Indirect Somatic Embryogenesis from Mature Inflorescence Explants of Date Palm.

Due to the limitations associated with shoot tip explants in the micropropagation of date palm, inflorescence explants are an ideal alternative. This chapter focuses on the protocol for the induction of callus from inflorescence tissue, establishment for proliferation of somatic embryos, germination, elongation, rooting, and acclimatization. Female inflorescences, 30-40 cm in length, cv. Shaishee, were used for culture initiation. After disinfection, the outer inflorescence cover (spathe) is cut open, and the spikelet explants, 1 cm long, are cultured on modified Murashige and Skoog (MS) medium containing 100 mg/L 2,4-D, 3 mg/L kinetin, and 3 mg/L 2ip and incubated at 25 ± 2 °C in the dark. Callus obtained after 6-8 months of culturing is transferred to the culture medium to induce somatic embryogenesis and plant regeneration. Well-developed regenerated shoots are cultured on MS medium containing 0.2 mg/L NAA for root induction and plantlets acclimatized in the greenhouse before transfer to the field.

Histological Evidence of Indirect Somatic Embryogenesis from Immature Female Date Palm Inflorescences.

Rapid production of somatic embryogenesis and date palm regeneration is achieved by culturing immature female inflorescence explants. Inflorescence explants are soft, creamy in color, average 6-7 cm in length, and cultured on Murashige and Skoog (MS) medium containing 1 mg/L thidiazuron (TDZ). Callus induction occurs after 4-5 weeks of culture on the callus induction medium. Subsequently, callus develops embryogenic calli on MS medium supplemented with 0.1 mg/L naphthalene acetic acid (NAA). Histological samples were collected successively at the culturing time and during morphogenetic changes throughout the developmental stages of somatic embryos. Initiation of callus and different successive developmental stages for somatic embryos including two-celled, four-celled, globular, bipolar, and fully developed cotyledonary somatic embryos were observed. Mature somatic embryos develop within 10-12 weeks after culture establishment.

Histological Analysis of the Developmental Stages of Direct Somatic Embryogenesis Induced from In Vitro Leaf Explants of Date Palm.

Somatic embryogenesis is an ideal technique for the micropropagation of date palm using different explant tissue; however, histological studies describing the ontogenesis of plant regeneration are limited. This chapter provides a simple protocol for the histological analysis of the successive developmental stages of direct somatic embryogenesis induced from in vitro leaf explants. Direct somatic embryos are obtained from Murashige and Skoog (MS) medium containing 2 mg/L 6-benzylaminopurine. In order to observe the different developmental stages, histological analysis is carried out on samples at 15-day intervals for 60 days. Samples are fixed in formalin acetic alcohol and embedded in paraffin wax. Stain serial transverse and longitudinal sections, 8 µm thick, are stained with safranin-Fast Green. After 15 days on the induction medium, somatic embryos exhibit multicellular origin directly from the procambium cells, whereas the mesophyll and the epidermal cells are not involved in this process. After 2 months, several developmental stages (pre-globular, globular, early bipolar, bipolar, and cotyledonary-shaped) are observed. These embryos germinate after transferring to MS medium without plant growth regulators and rooting on 2 mg/L NAA-containing medium resulting in complete plantlets.

Plant Regeneration from Somatic Embryogenic Suspension Cultures of Date Palm.

Somatic embryogenesis is one of the most important technologies for plant regeneration of elite date palm cultivars. Recently, considerable progress has been made in the development and optimization of this technique from embryogenic cell suspension cultures. This chapter describes a procedure for the rapid development of a large number of somatic embryos from embryogenic cell suspension cultures. An efficient plant regeneration protocol via somatic embryogenesis from cell suspension cultures starting with shoot-tip explants to plantlet acclimatization also is fully described. Low concentrations of 6-benzylaminopurine (BAP) to 0.3 mg/L and high rate of subcultures each 7 days lead to improve the establishment and multiplication of somatic embryos in suspension cultures by limiting oxidative browning, associated with high total phenols and peroxidase activities. The detailed morphological observations have revealed the cells destined to become somatic embryos. Activated charcoal (AC) at 0.15 g/L has a positive effect on growth rate of somatic embryos by reducing tissue and medium browning, phenolics, and peroxidase activity.

Synchronization of Somatic Embryogenesis in Date Palm Suspension Culture Using Abscisic Acid.

Somatic embryogenesis is considered the most effective method for commercial propagation of date palm. However, the limitation of obtaining synchronized development of somatic embryos remains an impediment. The synchronization of somatic embryo development is ideal for the applications to produce artificial seeds. Abscisic acid (ABA) is associated with stress response and influences in vitro growth and development. This chapter describes an effective method to achieve synchronized development of somatic embryos in date palm cell suspension culture. Among the ABA concentrations tested (0, 1, 10, 50, 100 µM), the best synchronized growth was obtained in response to 50-100 µM. Here we provide a comprehensive protocol for in vitro plant regeneration of date palm starting with shoot-tip explant, callus initiation and growth, cell suspension establishment, embryogenesis synchronization with ABA treatment, somatic embryo germination, and rooting as well as acclimatized plantlet establishment.

Microcalli Induction in Protoplasts Isolated from Embryogenic Callus of Date Palm.

Date palm (Phoenix dactylifera L.) production is severely hampered due to several pests and diseases. Biotechnological tools such as protoplast fusion appear as an alternative to ensure rapid genetic improvement and multiplication of this species. However, establishment of an effective system of plant regeneration from protoplasts culture is a prerequisite for date palm somatic hybridization. In this chapter, we describe an effective protocol to induce microcalli in protoplasts isolated from nodular callus of important Algerian date palm cultivars. In this protocol, the main factors influencing the isolation (i.e., enzymatic solution, mannitol concentration, duration, and mode of maceration) of protoplasts from the calli of Algerian date palm cultivars were optimized. Purified protoplasts were cultured on a semisolid medium supplemented with a hormonal balance of auxin and cytokinin to obtain microcalli formation.

Temporary Immersion System for Date Palm Micropropagation.

The temporary immersion system (TIS) is being used with tremendous success for automation of micropropagation of many plant species. TIS usually consists of a culture vessel comprising two compartments, an upper one with the plant material and a lower one with the liquid culture medium and an automated air pump. The latter enables contact between all parts of the explants and the liquid medium by setting overpressure to the lower part of the container. These systems are providing the most satisfactory conditions for date palm regeneration via shoot organogenesis and allow a significant increase of multiplication rate (5.5-fold in comparison with that regenerated on agar-solidified medium) and plant material quality, thereby reducing production cost.

Plantform Bioreactor for Mass Micropropagation of Date Palm.

A novel protocol for the commercial production of date palm through micropropagation is presented. This protocol includes the use of a semisolid medium alternation or in combination with a temporary immersion system (TIS, Plantform bioreactor) in date palm micropropagation. The use of the Plantform bioreactor for date palm results in an improved multiplication rate, reduced micropropagation time, and improved weaning success. It also reduces the cost of saleable units and thus improves economic return for commercial micropropagation. The use of the Plantform bioreactor successfully addresses other hindrances that can occur during the scale-up of date palm micropropagation, including asynchrony of somatic embryos, limited maturation of somatic embryos, and highly variable germination frequencies of embryos.

Extraction and Estimation of Secondary Metabolites from Date Palm Cell Suspension Cultures.

The health benefits of dates arise from their content of phytochemicals, known for having pharmacological properties, including flavonoids, carotenoids, phenolic acids, sterols, procyanidins, and anthocyanins. In vitro cell culture technology has become an attractive means for the production of biomass and bioactive compounds. This chapter describes step-by-step procedures for the induction and proliferation of callus from date palm offshoots on Murashige and Skoog (MS) medium supplemented with plant growth regulators. Subsequently cell suspension cultures are established for optimum biomass accumulation, based on the growth curve developed by packed cell volume as well as fresh and dry weights. The highest production of biomass occurs at the 11th week after culturing. Moreover, this chapter describes methodologies for the extraction and analysis of secondary metabolites of date palm cell suspension cultures using high-performance liquid chromatography (HPLC). The optimum level of catechin, caffeic acid, apigenin, and kaempferol from the cell suspension cultures establishes after the 11th and 12th weeks of culture. This protocol is useful for scale-up production of secondary metabolites from date palm cell suspension cultures.

In Vitro Assessment of Abiotic Stress in Date Palm: Salinity and Drought.

Date palm is one of the major crops growing in regions where abiotic stress conditions are extreme. Abiotic stress affects plant growth, development, physiology, and biochemical processes. This chapter describes a protocol to evaluate the response of date palm cultures to abiotic stresses. Tolerance to salinity stress is assessed using calcium chloride (CaCl2), potassium chloride (KCl), and sodium chloride (NaCl) at 11.96, 12.06, and 9.45 g/L, respectively (equivalent to 0.8 MPa osmotic potential), with different exposure durations (1-12 days). Polyethylene glycol (PEG 8000) is tested at 0-30% (w/v) to assess tolerance to drought stress. Techniques are described to define the effects of these stress agents on the growth of callus and cell suspension cultures, water content, proline accumulation, and Na+ and K+ content ratio, in addition to the technique used for determining the median lethal dose (LD50) for PEG (29.5%) and NaCl (11.54 g/L). This protocol will be useful for future studies of in vitro selection of tolerant cell lines.

Somatic Embryogenesis of Date Palm (Phoenix dactylifera L.) Through Cell Suspension Culture.

Date palm (Phoenix dactylifera L.) is the oldest and most economically important plant species distributed in the hot arid regions of the world. Propagation of date palm by seeds produces heterogeneous offspring with inferior field performance and poor fruit quality. Traditionally, date palm is propagated by offshoots, but this method is inefficient for mass propagation because of limited availability of offshoots. Plant regeneration through tissue culture is able to provide technologies for the large-scale propagation of healthy true-to-type plants. The most commonly used technology approach is somatic embryogenesis which presents a great potential for the rapid propagation and genetic resource preservation of this species. Significant progress has been made in the development and optimization of this regeneration pathway through the establishment of embryogenic suspension cultures. This chapter focuses on the methods employed for the induction of callus from shoot tip explants, establishment of cell suspension culture, and subsequent somatic embryogenesis and plant regeneration.