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Somatic embryogenesis of coconut was first reported by Eeuwens and Blake (1977) and later by Pannetier and Buffard-Morel (1982). These studies used a range of somatic tissues as explants: young leaves, stem slices from young seedlings and sections of rachillae of young inflorescences (Branton and Blake, 1983; Gupta et al., 1984). Somatic tissues, including immature inflorescences and ovaries, and the easier to manipulate embryonic tissues (immature or mature embryos and embryo-derived plumules) have been used. Immature embryos were initially considered to be the most responsive tissue type; however, the response of mature embryos can be significantly increased by longitudinal slicing (Adkins et al., 1998; Samosir, 1999) and the use of isolated plumular tissue (Chan et al., 1998; Lopez-Villalobos, 2002; Pérez-Núñez et al., 2006). Focus has now shifted to other explants from mature phases, including young inflorescences (Antonova, 2009; Sandoval-Cancino et al., 2016). The in vitro regeneration pathway is primarily by somatic embryogenesis, although organogenesis cannot be ruled out.

The Y3 (Eeuwens, 1976) and BM72 (Karunaratne and Periyapperuma, 1989) plant growth media are generally used, whereas MS (Murashige and Skoog, 1962) and B5 (Gamborg et al., 1968) formulations are less effective (Branton and Blake, 1983; Bhallasarin et al., 1986). The addition of 3–4% sucrose is critical, and activated charcoal (0.1–0.3%) prevents explant and culture browning, which is related to the release of phenols and ethylene (Samosir, 1999). Activated charcoal interferes with the effectiveness of exogenously applied plant growth regulators and other media components, resulting in uncertainty as to the precise functional concentration of these additives (Pan and van Staden, 1998). Variations in particle size and the potency of activated charcoal can influence the induction of embryogenic cultures (Sáenz et al., 2009). Polyvinylpyrrolidone (PVP) does not appear to have a significant effect in reducing tissue browning (Basu et al., 1988). By contrast, polyvinylpolypyrrolidone (PVPP) does have a positive effect on embryogenic culture induction (Samosir, 1999). Frequent subculturing of explants and embryogenic cultures also decreases exposure to accumulated phenols in the plant growth medium (Fernando and Gamage, 2000; Pérez-Núñez et al., 2006).

Somatic embryogenesis of coconut consists of: (i) callus initiation and maintenance; (ii) somatic embryo development/maturation; and (iii) germination/plant recovery (Fig. 4.1.2). Embryogenic cultures are induced in the presence of a high concentration of auxin (usually 2,4-dichlorophenoxyacetic acid (2,4-D)). The 2,4-D concentration differs depending on genotype and explant, and if activated charcoal is included in the medium, a low 2,4-D (24 μM) concentration is optimal for induction from zygotic embryos of Sri Lanka Tall (Fernando and Gamage, 2000), whereas a much higher concentration (125 μM) is required for Malayan Yellow Dwarf and Buta Layar Tall (Adkins et al., 1998; Samosir, 1999). Embryogenic culture induction from immature inflorescences and plumules necessitates a higher concentration of 2,4-D (up to 600 μM) (Verdeil et al., 1994). High concentrations of 2,4-D can cause chromosomal aberrations after long-term exposure (Blake and Hornung, 1995). It is thought that coconut tissues can rapidly metabolize 2,4-D into triacylglycerol derivatives (López-Villalobos et al., 2004). These derivatives represent a stable and stored form of 2,4-D that can inhibit somatic embryo development even when 2,4-D is removed from the medium. Other auxins, i.e. naphthaleneacetic acid (NAA) (27 μM), have been successfully used in combination with 2,4-D (452 μM) to promote embryogenic culture induction from rachillae explants (Gupta et al., 1984). Ultrastructural changes that occur during induction suggests that gametophytic-like conditions produced by 2,4-D are essential for transition from the vegetative to embryogenic state (Verdeil et al., 2001).

Fig. 4.1.2. Clonal propagation of coconut via somatic embryogenesis.

A cytokinin, i.e. benzylaminopurine (BA), thidiazuron (TDZ), kinetin or 2-isopentyl adenine (2iP), is essential for the proliferation of embryogenic cultures and for maturation. Induction of embryogenic cultures is best achieved at 28°C in darkness for 1–3 months after explanting (Adkins et al., 1998; Pérez-Núñez et al., 2006), although Chan et al. (1998) described optimum conditions as a 12/12 h day/night photoperiod (45–60 μmol/m²/s). Polyamines, particularly putrescine or spermine, appear to enhance the embryogenic response, possibly by protecting explants from ethylene damage (Adkins et al., 1998). Inhibitors of ethylene biosynthesis, e.g. aminoethoxyvinylglycine (AVG) and silver thiosulfate, can stimulate culture proliferation and somatic embryo development (Adkins et al., 1998). The transition from undifferentiated callus to embryogenic cultures is promoted by the reduction or removal of 2,4-D from the culture medium. Supplementing the culture medium with 650–300 μM BA promotes recovery of viable plantlets (Chan et al., 1998; Pérez-Núñez et al., 2006). The development and maturation of somatic embryos is enhanced by 5 μM abscisic acid (ABA) (Samosir et al., 1999; Fernando and Gamage, 2000; Fernando et al., 2003). Osmotically active agents such as polyethyleneglycol (3%) in combination with 45 μM ABA can enhance somatic embryo production, maturation and germination (Samosir et al., 1998). Antonova (2009) demonstrated that 30 μM ancymidol, a growth retardant, improves somatic embryo germination by up to 56%.

To improve the recovery of somatic embryos and their conversion, suspension cultures have been useful for oil palm (Teixeira et al., 1995). Temporary immersion has been used to improve plantlet recovery of date palm (Tisserat and Vandercook, 1985) and peach palm Bactris gasipaes (Steinmacher et al., 2011). The acclimatization of somatic embryo-derived plantlets still requires further improvement; typical rates of conversion are c.50% (Fuentes et al., 2005). Progress towards improved acclimatization of plantlets could also be achieved with the photoautotrophic culture system (Fig. 4.1.3; Samosir and Adkins, 2014) and enhanced through the use of fatty acids, particularly lauric acid in the culture medium (López-Villalobos et al., 2001, 2011).

Fig. 4.1.3. A CO₂ enrichment system for improved acclimatization of tissue-cultured plantlets.