**3. Somatic embryogenesis in** *Coffea*

Somatic embryogenesis is applied to *C. arabica* and *C. canephora* species for the purpose of vegetative multiplication of elite cultivars, large-scale clonal cultivar, Arabica F1 hybrid [4], to obtain transgenic plants and also as plant differentiation study model [31]. In addition, cloning allows the *in vitro* stock of germplasm cultivars for exchange between research institutions and the preservation of materials since seeds of this species have some degree of recalcitrance [32, 33].

In *C. arabica* the formation of somatic embryos can be obtained either by indirect [34], direct [35] or both somatic embryogenesis (**Figure 2**). Indirect somatic embryogenesis occurs in two phases, callogenesis followed by embryogenesis. On the other hand, direct somatic embryogenesis occurs in one phase without callogenesis. Comparison between these two forms of embryogenesis shows that the direct pathway is more advantageous than the indirect pathway. The direct pathway that occurs in a single phase ends up reducing inputs, labor and cultivation time while the indirect pathway occurs in two phases with higher costs of producing somatic embryos [6, 36, 37]. But although the direct pathway is more advantageous, most studies show that vegetative multiplication of *C. arabica* genotypes is mainly achieved by indirect somatic embryogenesis [6]. In the indirect pathway, explants of this species produce embryos more easily and in high quantity while in the direct pathway the number of formed embryos is smaller and this process occurs in a long time. Studies have indicated that the difficulty of direct pathway occurrence in *C. arabica* genotypes seems to be related to the presence of substances produced by the explant tissue itself [38]. This was demonstrated in explants of *C. canephora* and *Daucos carota* that showed inhibition of direct route when they were cultivated in culture medium with addition of substances secreted by explants of *C. arabica*. This result is possibly related to the difficulty of direct pathway occurrence in *C. arabica* genotypes.

*Coffee* regeneration via somatic embryogenesis can be obtained from different types of explants (**Figure 3**), such as anthers [39, 40], leaves [34, 41–46] and roots [47, 48]. However, leaf explant is the most used type for the application of direct or indirect somatic embryogenesis in *C. arabica* genotypes, this has been occurring since the pioneering work of Sondhal and Sharp [34]. Normally, these explants are used in rectangular shape with dimensions close to 1.5 × 2 cm2 .

#### **Figure 2.**

*Details of the occurrence of indirect and direct somatic embryogenesis in* Coffea*.*

**Figure 3.**

*Types of explants used for the application of indirect and direct somatic embryogenesis in* Coffea *genotypes.*

There are indications that *Coffea* somatic embryogenesis is more efficient if applied to leaf explants from *in vitro* seedlings than from leaves collected from plants in the natural environment [49–51], and this response must be related to vegetable hormones endogenous. This aspect may also be related to morphology since *in vitro* seedling leaves have a thicker cuticular layer than those from environmental plants. This characteristic tends to favor greater absorption of culture media components leading to the efficiency of somatic embryogenesis response [51, 52]. In addition, *Coffea* leaf explants from environmental plants can become curled which impairs nutrient absorption from the culture medium.

*C. arabica* explants remain green until about 60 days after inoculation in the culture medium and after this period they oxidize. This response is verified in explants submitted to direct or indirect route [45, 46]. In general, oxidized explants also have the ability to form calluses or embryos although they appear to be senescent.

In the indirect pathway, somatic embryos of *C. arabica* are formed after about 210 days of explant inoculation in the culture medium [45] while in the direct pathway they can be observed after about 90 days, but in small numbers [46]. On the other hand, there are also genotypes that do not form somatic embryos when submitted to these two embryogenesis pathways and are called recalcitrant [53].

**7**

**Figure 4.**

*transferred to* ex vitro *environment.*

*Observations on Somatic Embryogenesis in* Coffea arabica *L.*

embryos via the indirect route than by the direct one [54].

**3.1 Indirect somatic embryogenesis in** *Coffea*

high concentrations of 2,4 D.

*3.1.1 Callogenesis*

Leaf explants of *C. arabica* cultivar Mundo Novo form somatic embryos in both pathways [45, 46]. But the same genotype may have different capacity for somatic embryogenesis in each of these pathways. Explants of cultivar Mundo Novo and decaffeinated genotypes AC1, AC2 and AC3 formed a greater number of somatic

In *Coffea*, indirect somatic embryogenesis occurs in two phases, the first is callogenesis followed by embryogenesis that corresponds to the formation of somatic embryos [34, 45, 55–57] (**Figure 4**). Another characteristic of the indirect somatic embryogenesis in this species is the occurrence of somaclonal variation in cloned plants, due to the long time that callus remains *in vitro* [58]. Somaclonal variation is undesirable because it leads to the formation of mutants, which can compromise plant growth and development. On the other hand, its occurrence is desirable to obtain genotypes with genetic variability that can be incorporated into the coffee breeding program [59, 60]. For the induction of explant mutations are cultivated at

Most studies use the Sondhal and Sharp protocol [34] for the application of indirect somatic embryogenesis in *C. arabica* genotypes and these usually respond with some somatic embryo production even when they have low regenerative capacity.

For the induction of callogenesis in *Coffea* genotypes in general it is used the protocol with MS medium [61] and the addition of phytoregulators 2,4 D and kinetin and 30 g/L sucrose [34]. At this stage, auxin 2,4 D is used at high concentration [26, 62–65] which causes disturbance of endogenous auxin metabolism of explant tissue leading to cell division [8]. Auxin stress is required to obtain calogenesis in most species [12, 66]. Thus, in the induction of callogenesis, differentiated somatic cells of the explant tissue undergo re-determination, with the occurrence of cell division and proliferation events that form a non-functional cell mass, the callus. But it is also found that the induction of calogenesis in *Coffea* can be obtained in response to

Sucrose is used in high concentration to provide energy for the induction of calogenesis [34]. Biochemical analyzes in *C. arabica* explants indicated the occurrence

*Indirect somatic embryogenesis in the cultivar Mundo Novo of* Coffea Arabica*. (A) Leaf explant with callus presence 15 days after the beginning of cultivation. (B) Calogenesis at 40 days of cultivation. (C) Calogenesis at 90 days of cultivation. (D) Callus with somatic embryos. (E) Embryonic axis. (F) Seedlings. (G) Plant* 

the use of other auxin types such as NAA [67, 68] and Picloram [69, 70].

*DOI: http://dx.doi.org/10.5772/intechopen.90853*

## *Observations on Somatic Embryogenesis in* Coffea arabica *L. DOI: http://dx.doi.org/10.5772/intechopen.90853*

Leaf explants of *C. arabica* cultivar Mundo Novo form somatic embryos in both pathways [45, 46]. But the same genotype may have different capacity for somatic embryogenesis in each of these pathways. Explants of cultivar Mundo Novo and decaffeinated genotypes AC1, AC2 and AC3 formed a greater number of somatic embryos via the indirect route than by the direct one [54].
