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1 Background Information

Bananas and plantains, collectively known as bananas, belong to the genus Musa (Family: Musaceae); these monocotyledonous giant herbs are among the most important horticultural crops widely distributed throughout the humid tropical and sub-tropical regions of the world where they are cultivated. Both dessert and cooking bananas are important perennial fruit crops on which millions of people depend for their livelihood and as their main staple foods. The present-day cultivated bananas are diploid and triploid hybrids largely originated form natural inter-(sub) specific- and inter-specific crosses between diploid (2n = 2x = 22 chromosomes) wild subspecies, mainly of Musa acuminata Colla (A genome) and Musa balbisiana Colla (B genome) bananas (D’Hont et al. 2000; Husin et al. 2014), both belonging to section Musa (Häkkinen 2013).

Traditional breeding in bananas is limited due to polyploidy and vegetative parthenocarpy. Vegetative propagation through the conventional procedure of using banana lateral shoots that develop from the rhizome, so-called suckers, cannot always match the demand of elite genotypes for cultivation. Consequently, propagation through biotechnological approaches, viz., organogenesis and SE, has made a great contribution to large-scale production of demanded plants. In particular, ECS cultures are powerful tools for genetic improvement and clonal propagation of bananas, given the constrain production in many countries due to pests, diseases, and abiotic stresses and the troublesome of its conventional breeding. These procedures are of use for the conservation of the genetic diversity of the wild bananas ancestors of current edible plants (Asif et al. 2001), they also support breeding strategies by embryo rescue of seeds from open-pollinated seedy bananas and controlled new hybrids (Afele and de Langhe 1991; Bakry 2008; Uma et al. 2012). Propagation of edible clones for synthetic seed technology (Ganapathi et al. 2001b), and the use of somatic embryogenic cultures for in vitro selection of somaclonal variants (Ghag et al. 2014b) or genetic transformation for example edible vaccine against hepatitis B (Kumar et al. 2005) and resistance to biotic stress (Ghag et al. 2014a) are examples of the active topics of research in Musa.

2 Somatic Embryogenesis

Somatic embryogenesis is a process that illustrates the expression of plant developmental plasticity and of cellular totipotency, with tremendous potential for fundamental research and commercial application. This is a biological process in which under inductive conditions somatic cells reach embryogenic competence and after a series of biochemical and morphological changes precisely organized embryos are produced (Quiroz-Figueroa et al. 2006). The somatic embryos have a bipolar structure with apical and radical meristems and showed morphological characteristics resembling those found in zygotic embryos. Plant regeneration in banana through SE is important because of the recognized unicellular origin of embryos (Strosse et al. 2004; Roux et al. 2004; Escobedo-GraciaMedrano et al. 2014) and the risk of generating plants with chimeric tissues after genetic transformation is minimized. SE can be initiated directly from the explant of donor plant without callus formation (direct SE), or indirectly by means of a callus stage (indirect SE) (Quiroz-Figueroa et al. 2006). In the latter case, SE usually follows a similar path, such as: (i) initiation of calli followed by selection of the embryogenic callus, (ii) proliferation of the embryogenic cells of the callus (undifferentiated phase), (iii) regeneration of numerous embryos from these phase (embryogenic phase), and (iv) conversion of somatic embryos into mature embryos with the ability to regenerate healthy plants (Khalil et al. 2002; Gaj 2004; Ramírez-Villalobos and de García 2009). Either on semi-solid medium (calli) or in liquid medium (cell suspensions), the multiplication of embryogenic cells is a key step because it greatly and rapidly scales up the number of potential embryos that may be produced. Indirect SE with formation of a callus stage is a common experiential method use in bananas, while secondary somatic embryos/embryogenesis (SE2) stimulated directly from the primary somatic embryo or through formation of a callus stage have been both reported (Khalil et al. 2002). Whereas, direct SE from split shoot tips have been recently reported (Remakanthan et al. 2014).

3 Initiation of Somatic Embryogenesis in Banana

The initiation of SE response is influenced by the developmental stage of the initial plant material, the genetic background of the stating banana plant material (Escalant et al. 1994; Youssef et al. 2010) and on the appropriate balance of plant growth regulators (PGR) in the medium perceived by the cells in culture. Indirect SE is induced by explant inoculation onto semi-solid or liquid medium in presence of exogenous PGR, predominantly auxin and sometimes cytokinin well-defined by the SE method employed (Krikorian and Scott 1995; Table 38.1). During indirect SE, dedifferentiation of the cells in the initial explant supports the formation of a mass of cells with embryogenic potential. From the earlier reports in the late 1980s, and thereafter, five main procedures for establishment of embryogenic callus and proliferation of ESC in banana are recognized. They rely on the type of explant in which SE is initiated for a large range of Musa genotypes tested, as follows: (i) immature zygotic embryos (IZEs), (Cronauer-Mitra and Krikorian 1988; Escalant and Teisson 1989; Navarro et al. 1997; Escobedo-GraciaMedrano et al. 2014; Maldonado-Borges et al. 2013; Krikorian and Scott 1995), and mature zygotic embryos (MZEs) (Uma et al. 2012), with modifications by the use of 2,4-dichlorophenoxyacetic acid (2,4-D), 3,6-dichloro-2-meth-oxybenzoic acid (dicamba) or 4-amino-3,5,6-trichloropyridine-2-carboxylic acid (picloram) as synthetic auxin (Novak et al. 1989; Escobedo-GraciaMedrano et al. 2014; Remakanthan et al. 2014). (ii) Rhizome slices and leaf sheaths (Novak et al. 1989), cultures of proliferating meristem called “scalps” (Dhed’a et al. 1991; Schoofs et al. 1998; Strosse et al. 2003), immature male (Ma 1991; Escalant et al. 1994; Côte et al. 1996; Grapin et al. 1996; Navarro et al. 1997), and female flowers (Grapin et al. 2000). More recently, proliferative male flower buds so-called “curds” (Pérez-Hernández and Rosell-García 2008) and male flower bract (Divakaran and Nair 2011) cultures, obtained in media supplemented with TDZ (thidiazuron), have been also used for initiation of SE. While, direct somatic embryos developed from split shoot tips under a combination of the PGR picloram and 6-benzyladenine (BA), (Remakanthan et al. 2014). On the other hand, as shown by their higher SE response, immature zygotic embryos have greater competence for embryogenic callus formation compared to differentiated tissue that need to undergo dedifferentiation (Marroquin et al. 1993; Escobedo-GraciaMedrano et al. 2014), yet, immature embryos responded better than mature embryos (Uma et al. 2012). From the inflorescence male bud (Fig. 21.1a), immature male flowers (Fig. 21.1b) are used for induction of SE callus (Fig. 21.1c). This method has been successfully employed on a large range of Musa cultivars (Strosse et al. 2003); however, this kind of explant is only suitable for genotypes having a male bud, the alternative for banana cultivars lacking male buds is to either use female flowers or the scalp methods. The frequency of embryogenic responsive male flower buds varied between 0.5 and 37 %, a response which is dependent on the genetic constitution of the donor plant, its physiological status, and some seasonal factors which influence the response (Escalant et al. 1994). Among the reported genotypes, the cv Enano Gigante (AAA, Cavendish subgroup) showed lower, from 0.7 to 10 %, responses (Navarro et al. 1997; Youssef et al. 2010). An average of 8 % embryogenic callus response has been reported for the scalp method for a range of genotypes (Strosse et al. 2003). Whereas, 12.5 and 25 % of embryogenic response was found using the method of proliferative male flower buds”, under semisolid and liquid inductive medium, respectively, (Pérez-Hernández and Rosell-García 2008). Thus, the mentioned SE protocols suitable for different purposes i.e., banana germplasm conservation, and massive propagation, needs assessment on a larger pool of samples in order to adjust them for each banana genotype. In general, for indirect SE the embryogenic reaction of the diverse banana explants to the different media (Table 21.1) follow three or four main patterns of callus development (Strosse et al. 2004; Youssef et al. 2010). Variation in callus formation response to the components of the induction media depend on the genotype (Strosse et al. 2006). Formation of fast/growing yellowish withe callus is undesirable because the callus oxidized rapidly and shows no embryogenic structures. In the scalps method, a positive embryogenic response occurs on 3–8 months old induced explants (Strosse et al. 2004), while the same response can take nearly 2–3 months in the male inflorescence, zygotic embryo (Escalant et al. 1994; Navarro et al. 1997) and curds (Pérez-Hernández and Rosell-García 2008) methods. The appearance of individual embryos under the different embryogenic method used is a promising indication of the embryogenic competence and plasticity of the starting material. In particular, it seems that direct SE is possible, though the plant conversion rates need to be improved (Remakanthan et al. 2014). As for indirect SE, the presence of a friable white callus, consisting of only early stage globular somatic embryos (Fig. 21.1d) and non-organized cell clusters, is the most wanted callus for the initiation of embryogenic cell suspension (ECS) cultures (Fig. 21.1d), from either of the cited methods (Ma 1991; Escalant et al. 1994; Côte et al. 1996; Grapin et al. 1996; Navarro et al. 1997; Marroquin et al. 1993; Escobedo-GraciaMedrano et al. 2014).

Fig. 21.1
figure 1

Plant regeneration of Musa acuminata (AAA) cv. Grand Naine (subgroup Cavendish) via indirect somatic embryogenesis. a Inflorescence shown new opened female flowers and the distal male bud, b immature male inflorescence used to induce callus, c yellow friable callus obtained from immature inflorescence cultured on M1 medium for 3 months. d Homogeneous embryogenic callus with expression of globular somatic embryos, e homogenous cell suspension obtained after filtration of initial suspension (f) and subculture for 3 months in M2 medium. g Somatic embryos after 45 days in maturation medium, showing evidence of initiation of starch accumulation. h Germinated embryo after 8 days of culture on PGR-free medium, emergence of shoot is shown. i Emblings after 15 days of acclimation at the glasshouse

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Table 21.1 Composition of culture media frequently use to initiate indirect somatic embryogenesis in Musa spp.
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4 Proliferation of Embryogenic Callus and Initiation of Cell Suspension Cultures

After the formation of the embryogenic callus (3–5 months) in an auxin-supplemented semisolid medium, the callus is multiplied by a monthly refreshment of the medium (MA1, for immature male flower methods and MI medium for the immature zygotic embryo method), except that the concentration of the sole auxin 2,4-D is reduced to 4.5 µM. Whereas for the scalp method, both auxin and cytokinin plant growth regulators (PGR) are use and maintained in the culture medium (Table 21.1). The existing protocols for somatic embryogenesis using embryogenic cell suspension (ESC) cultures have the potential to produce non-chimeric plants. Thus, establishment and maintenance of embryogenic cultures with high capacity to generate somatic embryos is an important and desired step in any protocol for plant regeneration at a massive level and transformation success. Banana ECS cultures are established by transferring embryogenic callus (Fig. 21.1d) into M2, MA2 liquid medium (M1 and MA1, without gelling agent) for male flower method, or the MI (Marroquin et al. 1993) or S1 and S2 liquid media for the immature-zygotic-embryo method (Table 21.2); most of these media are added with l-glutamine and malt extract, respectively, (Côte et al. 1996; Strosse et al. 2003; Escobedo-GraciaMedrano et al. 2014; Jafari et al. 2015). Under such media, the ECS cultures multiply (Fig. 21.1e, f), with refreshment of the medium every one to two weeks. It is important to note that one of the main difference with the scalp method is that the concentration of exogenous PGR (2,4-D and zeatin) is kept the same throughout the induction and multiplication phases (ZZss and ZZl) and neither glutamine nor malt extract are added to the medium(Strosse et al. 2003). Whereas, for the flower method, with the onset of embryogenic callus formation at high auxin concentration, reduction of auxin concentration is essential for proliferation of somatic embryogenic callus and expression of somatic embryos (Ma 1991; Côte et al. 1996; Jafari et al. 2015). In spite of extensive advances in SE, low embryo germination and a loss of ECS morphogenetic competence are still the bottlenecks of SE procedures in various banana cultivar and embryogenic systems (Schoofs et al. 1999).

Table 21.2 Composition of culture media frequently used for the establishment of embryogenic cell suspension cultures in Musa spp.
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5 Development and Maturation of Somatic Embryo

The development of the embryo (zygotic and non-zygotic) follows a sequence of organized events, which comprise active cell division that contribute to the formation of an undifferentiated globular shape embryo with a defined protodermis, followed by cellular differentiation and enlargement of the embryo. Maturation, the final stage of embryo development is distinguished by overall cell expansion and accumulation of reserve substances of the embryo. In general, at this stage, repression of germination and the acquisition of desiccation tolerance, is frequent in the zygotic embryo maturation, and often is lacking in somatic embryo terminal development (Merkle et al. 1995; de Moura Vale et al. 2014). In several edible bananas genotypes, the basis for the low quality and quantity of the somatic embryos, and the poor regeneration and conversion to vigorous plants is frequently the least studied aspects in banana (Krikorian and Scott 1995). In this regard, the quality and quantity of banana somatic embryos have shown to be influenced by medium pH, the so-called ECS acidogenic growth hypothesis (Chung et al. 2015). Five types of cell aggregates has been recognized in banana ECS cultures of Musa AAA cv. Grande naine, each type can have a significant effect on the quality of the embryo established during the development–maturation stage, and likewise can promote embryo germination (Domergue et al. 2000). The pH of buffered media during ECS cultures have an effect on the type of cell aggregates developed. Addition of l-glutamine and malt extract which is a mix of amino acids, carbohydrates and vitamins, in the culture medium during the initial step of embryogenesis and embryo development until the maturation step is a general practice in banana protocols (Tables 21.2 and 21.3). These compounds rapidly incorporated into the carbon skeletons for metabolism and protein synthesis. The addition of the amino acids l-glutamine, alanine or l-glutamic acid have shown to strongly stimulate the development of somatic embryos in carrot (Higashi et al. 1996). The effect of l-glutamine, and to a lesser extent proline, in somatic embryo development has proven to enhance the efficiency of banana (Musa acuminata cv. Berangan) regeneration (Husin et al. 2014). In the absence of exogenous PGR, histodifferentiation of developing embryos in M. a. ssp. malaccensis proceed well under limited water availability and reached the stage of maturity (coleoptilar stage) perceived by the typical white-opaque-color of the embryo and exhibit the cotydelonary slit (Maldonado-Borges et al. 2013; Escobedo-GraciaMedrano et al. 2014). One of the most important factors during somatic embryo maturation is the impact of water stress (Attree et al. 1991). During this stage with the gradual loss of water, the embryos initiate the process of desiccation tolerance (Bomal et al. 2002), which gives the embryo the capacity to survive under water-limiting conditions (Bewley et al. 2013; Dekkers et al. 2015). During this state of “dehydration,” the embryo is stimulated to accumulate reserve proteins, carbohydrates and lipid compounds (Klimaszewska et al. 2000; de Moura Vale et al. 2014). Available banana protocols control the water availability to the developing somatic embryos by using either higher concentration of gelling gum and/or filter paper or both (Table 21.3), under this circumstances accumulation of starch in during embryo maturity is indicated by the white-opaque color (Fig. 21.1g) around 45 days of culture (Côte et al. 1996; Chung et al. 2015), as is in other plant species (Márquez-Martín et al. 2011). Molecular evidences from transcribed expressed sequences have suggested that an early responsive to dehydration proteins (ERDs) are involved in embryo maturation. The ERD genes are defined as genes rapidly activated during drought stress and involved in Abscisic acid mediated developmental and stress responses (Maldonado-Borges et al. 2013).

Table 21.3 Comparison of the composition of six culture media used for the development and maturation of somatic embryos of Musa spp.
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6 Somatic Embryo Derived Plants (Emblings)

Plant recovery by germination of somatic embryo with emergence of normal root and shoot (Fig. 21.1h) is achieved on media germination with added or without PGR, which depends on the genotype and the previous culture procedure followed during embryo development (Table 21.4). Following the acclimatization and hardening of emblings (Fig. 21.1i), conversion rates are estimated and the values are compared with the percentages of embryo germination. The percentage of somatic embryo germination recorded for some genotypes fluctuates between 3 and 46 % in triploid Cavendish (AAA) bananas. These values can reach up to 91 % when somatic embryos differentiated from ECS cultures of type 2 cell aggregates (Domergue et al. 2000). Higher rates (90–95 %) found in the triploid (AAB) cv Dwarf Brasilian (AAB) and the seminiferous M. a. ssp. malaccensis (AA), have in common that embryo development passed through a differentiation–maturation phase. In the case of somatic embryos obtained by the direct SE obtained from split shoot tips (M. a. AAA, cv. Grand Naime), a 2–3 % of embryo conversion has been reported (Remakanthan et al. 2014). Although several SE protocols have been described for different bananas genotypes, the comparisons of results as to the percentage of somatic embryo germination and emblings conversion rates is sometimes difficult, because the presented data do not always make a distinction between both situation. The emblings conversion rates vary from 13 % in the edible (AA) Pisang mas banana, 13–25 % for Grand Nain of the Cavendish subgroup (AAA), 66.7 % in the highland African banana (AAA) (Namanya et al. 2004), and 100 % on the wild M. a. ssp. malaccensis (AA), respectively, (Table 21.4). With regard to non-conventional breeding (genetic transformation) for the pest and disease problems of banana and plantains worldwide, high germination and conversion rates underscore the fact that SE is essential in the development of in vitro regeneration systems which are a critical step for development of resistant varieties (Ghag et al. 2014a). So far, the data indicates that continuous work is needed for the development and optimization of SE protocols of many different cultivated clones.

Table 21.4 Medium composition commonly used for somatic embryo germination in Musa spp.
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7 Somaclonal Variation in Somatic Embryo Regenerated Plants

The in vitro culture environment, viz., type and concentration of applied plant growth regulators (PGRs), genetic background of the explant, and total number and duration of subcultures, can affect the properties of plants regenerated by somatic embryos (Konieczny et al. 2012). All these factors can contribute to the generation of genetic and epigenetic variation (Bairu et al. 2011) revealed in the phenotype, a phenomenon so-called somaclonal variation (SV) (Larkin and Scowcroft 1981). SV might be a pre-existent genetic variation in the explant due to changes in chromosome numbers, i.e., polyploidy and aneuploidy, chromosome structure (D’Amato 1990), or induced during the in vitro culture (Evans et al. 1984), in addition, mutations and epigenetic changes can take place at the DNA sequence level (Roux et al. 2004; Bairu et al. 2006; De-la-Peña et al. 2015). Banana SV have been reported to be associated with long-term cultures or cultures that involve a callus phase or high rates of multiplication treatments (Roux et al. 2004; Bairu et al. 2006). The decline in regeneration capacity of ECS cultures have been associated with cytogenetic instabilities in triploid (AAA, genome) Cavendish bananas, off-type regenerants from long-term Bluggoe suspension cultures (ABB, cooking banana) and subsequent loss of regeneration potential (Schoofs et al. 1998; Roux et al. 2005). Cytogenetically, regenerants from short-term SE showed genetic stability as compare to higher DNA amount found in emblings derived from longer-term cultures (Escobedo-GraciaMedrano et al. 2014). The genetic stability/instability evaluated by morphological and agronomical parameters have shown that variation is around 0.3–3.6 %, and molecular marker recorded a low variation (1.4–1.6 %) within the natural variation found in the mother plant used as explant source (Table 21.5). On the other hand, somaclonal variants are currently been used as a source for the selection of Fusarium resistance clones (Ghag et al. 2014b).

Table 21.5 Somaclonal variation in banana plants regenerated by in vitro somatic embryogenesis
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8 Banana Genetic Transformation Using ECS Cultures

In vitro culture of a wide range of commercial banana cultivars through SE using ECS cultures has been essential in developing reliable procedures for gene transfer in banana. Several groups working with different biotechnological approaches have succeeded in developing genetic transformation protocols taking advances of the available SE systems for the improvement of bananas (Crouch et al. 1998). These works have evaluated reporter genes, and transformation methods, i.e., DNA delivery by Agrobacterium, or particle bombardment using biolistic gun device and introduction of DNA into regenerable, ECS-derived protoplasts by electroporation (Sági et al. 2000). These procedures depend on the efficient regeneration of emblings from cells for delivery of genes that may confer resistance to biotic or abiotic stresses (Table 21.3). Protocols for ECS transformation by particle bombardment (Becker et al. 2000; Sagi et al. 1995) and co-cultivation with Agrobacterium are available for both, dessert and cooking bananas (Ganapathi et al. 2001a). The Agrobacterium-mediated transformation method may be more widely applicable as it offers advantages over direct gene transfer methodologies like particle bombardment and electroporation (Arvanitoyannis et al. 2008). The currently technology for banana transformation uses ECS cultures initiated form either scalps or immature male flower as starting plant material (Table 21.6), which have been applied to a range of banana cultivars and genotypes in which new genes (trans or cis) are successfully introduced. These approaches that include the expression of genes encoding plant, fungal, or bacterial hydrolytic enzymes (Vishnevetsky et al. 2011), or the production of edible vaccine against hepatitis B (Kumar et al. 2005) are examples of topics in Musa research.

Table 21.6 Genetic transformation in different banana cultivars using embryogenic cell suspension (ECS) systems initiated from tissues of different origin
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9 Conclusions

With regard to nonconventional breeding (genetic transformation) for pest and disease problems of banana and plantains worldwide, high germination of somatic embryos and emblings conversion rates underscore the fact that SE is essential in the progress of in vitro regeneration systems which are a critical step for development of resistant varieties. So far, the data indicates that continuous efforts are needed for the optimization of SE protocols of many different clones cultivated, the assessment of SV of emblings derived from different SE procedure is an important aim to underpin basic knowledge of the physiological, biochemical and molecular process that underlie the SE process and SV.