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The embryogenic response of mango is based upon the morphogenic potential of the nucellus. Mango cultivars are either polyembryonic or monoembryonic, depending on their ecogeographic origin (see Section 1.1 Botany and History). The adventitious embryos within polyembryonic mango seeds are derived from the nucellus, a maternal tissue that surrounds the embryo sac; polyembryony in mango is under the control of a single dominant gene (Aron et al., 1998). A summary of those mango cultivars that have been regenerated via somatic embryogenesis is provided in Table 2.2.2.

Table 2.2.2. Somatic embryogenesis from nucellar cultures of mango (Litz and Lavi, 1998; Ara et al., 2000b).

There are no convenient markers enabling the prediction of the embryogenic response of the nucellus of various mango cultivars. Litz and Yurgalevitch (1997) reported that the induction of embryogenic competence in the cultured nucellus of monoembryonic ‘Tommy Atkins’ was inhibited by the ethylene antagonist aminoethoxyvinylglycine (AVG) and by dicyclohexylammonium sulfate (DCHA), an inhibitor of spermidine synthesis, in contrast to polyembryonic ‘Tuehau’ in which regeneration was not affected. This confirmed an earlier study that demonstrated that somatic embryogenesis in mango was partially mediated by spermidine (Litz and Schaffer, 1987). Therefore, the biosynthesis of ethylene and/or the sensitivity of nucellar tissue to ethylene may be an important determinant for induction of embryogenic cultures from this tissue.

INDUCTION. Induction of embryogenic competence is related to the developmental stage of the nucellus at the time of explanting and also can be influenced by the physiological condition of the tree (Litz, 1987). Fruits that are c.30–40 days after pollination contain seeds in which the nucellus is at the ideal stage for explanting. Mango fruit at the appropriate stage of development are surface-sterilized with 20–30% (v/v) domestic bleach containing Tween 20 for 30 min, rinsed with three changes of sterile deionized water, and each fruit is bisected along its longitudinal axis without damaging the seed under sterile conditions. The immature seed is removed and is also bisected carefully along its longitudinal axis. Manzanilla Ramiriez et al. (2000) obtained optimum results with ‘Ataulfo’ (polyembryonic), ‘Tommy Atkins’ (monoembryonic) and ‘Haden’ (monoembryonic) when the embryo (mass) to immature seed ratio was 1:3. The embryo mass is removed and discarded. The nucellus can be removed by carefully peeling it away from the interior of the seed coat using a sterile, flat spatula. After transferring the nucellus onto induction medium in sterile petri dishes, the cultures are incubated in darkness at 25°C. Thereafter, it is necessary to subculture the explants onto fresh medium at least daily until the oxidation of the explant ceases.

Induction of embryogenic mango cultures from the excised nucellus of immature mango seeds of polyembryonic and monoembryonic cultivars was first reported by Litz et al. (1983) and Litz (1984), respectively. These results were confirmed by Jana et al. (1994) and Ara et al. (2000b). The efficiency of induction is cultivar dependent. Litz et al. (1998) compared the induction responses of four cultivars and found that ‘Hindi’ (polyembryonic) has the highest embryogenic response, followed by ‘Lippens’ (monoembryonic), ‘Tommy Atkins’ (monoembryonic) and ‘Nam doc Mai’ (polyembryonic) in that order. Manzanilla Ramirez et al. (2000) compared the induction responses of three cultivars and observed that ‘Ataulfo’ (polyembryonic) was more embryogenic than either ‘Tommy Atkins’ (monoembryonic) or ‘Haden’ (monoembryonic) in that order. Optimization of conditions for induction of embryogenic mango cultures was reported by DeWald et al. (1989a) using polyembryonic ‘James Saigon’ and ‘Parris’ as models. The standard procedure has been only slightly modified since then and utilizes a basal medium consisting of B5 (Gamborg et al., 1968) major salts without (NH₄)₂SO₄, Murashige and Skoog (1962) (MS) minor salts and organic components, 60 g/l sucrose, 400 mg/l glutamine, 4.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 g/l gellan gum.

Lad et al. (1997) defined the temporal effect of 2,4-D for induction of embryogenic competence in explanted nucellus of ‘Carabao’ (polyembryonic). Culture initiation required a minimum of 7–14 days exposure to 2,4-D, and a maximum exposure of 56 days. Embryogenic competence of the cultures was optimum after a minimum 28 day exposure to 2,4-D. Nurse cultures consisting of highly embryogenic mango cultures can stimulate the induction of embryogenic competence from the nucellus of cultivars that are relatively difficult to induce (Litz et al., 1998). Use of a nurse culture involves explanting the nucellus onto a sterile filter paper which has been dampened with induction medium and which overlays the highly embryogenic mango culture growing on semi-sterile induction medium.

Litz (1987) demonstrated that somatic embryos can develop directly from the nucellus without an intermediate callus. Very shortly after the appearance of nucellar cultures, they are completely organized and consist of proembryos, embryogenic cells, cell aggregates and proembryonic masses (PEMs) (Litz et al., 1993, 1995; Litz and Lavi, 1998).

MAINTENANCE. Embryogenic mango cultures usually appear c.30 days after explanting and are friable and white-to-cream. The cultures rapidly darken on semisolid medium and must be subcultured at 3–4 weeks intervals. PEMs develop from globular somatic embryos in the presence of the primary induction agent, 2,4-D; somatic embryo development is suppressed by 2,4-D. In suspension culture and to a lesser extent on semisolid medium, the PEMs increase in size, and their protoderm dedifferentiates thereby becoming embryogenic. Secondary globular somatic embryos develop from embryogenic cells in the protoderm. This highly repetitive or secondary somatic embryogenesis in the presence of 2,4-D is the basis for maintenance of embryogenic cultures.

Embryogenic mango cultures can be maintained on semisolid medium, although proliferation of embryogenic cultures of many cultivars can be optimized in liquid medium supplemented with 4.8 μM 2,4-D (Litz et al., 1984; DeWald et al., 1989a); rapid proliferation of embryogenic cultures in suspension is cultivar dependent (Litz et al., 1993). Embryogenic suspension cultures are initiated by inoculating c.300 mg of PEMs into sterile 80 ml maintenance medium in a 250 ml Erlenmeyer flask. The flasks are maintained on a rotary shaker at 100 rpm in semi-darkness at 25°C with regular transfers of PEMs into fresh medium at 10–14-day intervals. Regular and frequent subculture is essential to avoid loss of morphogenic potential and darkening of the tissue. A typical suspension culture consists of PEMs, embryogenic cells and multicellular complexes.

MATURATION. Development of somatic embryos from embryogenic cultures maintained on semisolid maintenance medium can occur sporadically and without synchronization, due to the polarity within the tissue culture and lack of direct contact of parts of a culture with the medium containing 2,4-D. Transfer of embryogenic cultures from maintenance medium formulation to medium without 2,4-D is essential to initiate large-scale somatic embryo development. For embryogenic cultures that have been maintained in suspension, the cultures are decanted through filtration fabric with a 1000 μm opening size. The larger fraction is reinoculated into liquid maintenance medium for continued proliferation and the smaller fraction is transferred either into a liquid medium or onto a semisolid medium without 2,4-D to arrest repetitive somatic embryogenesis and to initiate somatic embryo development.

The plant growth media and conditions for stimulating somatic embryo development and maturation were described by DeWald et al. (1989b). The initial maturation medium consists of B5 major salts, MS minor salts and organic components, 60 g/l sucrose and 400 mg/l glutamine with or without 2.0 g/l gellan gum. Litz et al. (1993) reported that addition of either 4.65 μM kinetin or 4.44 μM BA to the maturation medium can stimulate the development of the shoot apex and cotyledons and reduce the maturation period. Cultures are incubated in darkness at 25°C.

When highly embryogenic cultivars are optimized as suspension cultures, the early cotyledonary somatic embryos that develop are hyperhydric and cannot develop to maturity (Monsalud et al., 1995) and become necrotic. Hyperhydricity of early mango somatic embryos can be reversed by partially desiccating heart-stage embryos (2–3 mm length) under high relative humidity for 24 h or by plating them onto maturation medium solidified with 6.0 g/l gellan gum (Monsalud et al., 1995). The reversion of hyperhydricity can induce precocious germination of mango somatic embryos, but this can be inhibited by 500 μM abscisic acid (ABA) in the maturation medium.

Mango embryos generally require 4–5 months to develop to maturity in vivo, and mature embryos can exceed 6–8 cm length (Fig. 2.2.1). Therefore, the plant growth medium formulations that have been adopted for stimulating growth and development of mango somatic embryos from the heart stage to maturity reflect the differing requirements of these enlarging somatic embryos. The maturation medium consists of B5 major salts, MS minor salt and organic components, 400 mg/l glutamine, 20% (v/v) filter-sterilized coconut water, 40 g/l sucrose and 2.0 g/l gellan gum (DeWald et al., 1989b). The sucrose concentration is gradually reduced to 10 g/l during sequential subculture to fresh media. The cultures are maintained in darkness at 25°C.

Fig. 2.2.1. Mango somatic embryos developing on maturation medium.

GERMINATION. Germinating somatic embryos are transferred to light conditions. The hypocotyl elongates, followed by growth of the tap root. The shoot apex remains underdeveloped, and c.2 weeks after germination, the shoot emerges (Fig. 2.2.2). Although many mango somatic embryos germinate under these conditions, their survival rate or conversion is low due to apical shoot necrosis, a physiological disorder that is associated with Ca⁺⁺ deficiency. Different strategies have been proposed to improve the rate of survival. (i) Litz and Lavi (1998) suggested that the period for embryogenic cultures in/on maintenance medium should be minimal. (ii) Ara et al. (1998) described the in vitro rooting of in vitro shoots obtained from germinated somatic embryos by pulsing them for 24 h with 24.6 μM IBA in liquid medium followed by transfer to auxin-free medium in darkness. (iii) Enhanced recovery of mango plantlets was described by Litz et al. (1993) following the induction of photoautotropism by transferring small plantlets onto minimal plant growth medium, containing <5% sucrose and 1% (w/v) activated charcoal. A filter-sterilized air mixture consisting of 20,000 ppm CO₂ in a nitrogen gas carrier was introduced into the growing containers, and the cultures were exposed to a 16 h photoperiod at 180 μmol/s/m² provided by cool white fluorescent tubes. (iv) Early heart-stage somatic embryos were encapsulated in calcium alginate containing modified standard mango medium with half strength major salts and supplemented with 2.9 μM GA₃ (Ara et al., 1999). (v) Shoot tips of germinating somatic embryos can be micrografted onto seedling rootstocks (Fig. 2.2.3).

Fig. 2.2.2. Mango plantlet derived from somatic embryo.

Fig. 2.2.3.Ex vitro graft of ‘Keitt’ shoot derived from a somatic embryo on ‘Turpentine’ seedling rootstock.