*3.1.1 Callogenesis*

*Coffee - Production and Research*

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

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

*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

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].

the ability to form calluses or embryos although they appear to be senescent.

impairs nutrient absorption from the culture medium.

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

**6**

**Figure 3.**

**Figure 2.**

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

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

#### **Figure 4.**

*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 transferred to* ex vitro *environment.*

of high concentration of soluble sugars in the calogenesis phase while it was lower in the formation and maturation of somatic embryos [71].

In the indirect pathway, *C. arabica* leaf explants initiate callus formation from procambium cells around the seventh day after the beginning of cultivation [72]. In general, from the 14th day of cultivation, it is possible to see the occurrence of small callus on the edges of explants that reach up to 3 mm in size [45]. These calluses are usually formed on only one or two sides of the explant and later reach sizes that can occupy the four edges of the explant. In early development, calluses are hyaline, but gradually they tend to oxidize. In a set of explants of the same origin subjected to indirect pathways, it is possible to find calluses that do not develop. They remain small, up to 5 mm in size. However, some of these calluses have the capacity to form somatic embryos, which indicates that they appear to have been "latent" during this time. Furthermore, it is further noted that not all *C. arabica* explants form calli indicating that the somatic embryogenic capacity may vary between explants from the same plant or from the same leaf.

Callus of *C. arabica* can reach sizes up to 30 mm around 90–120 days from the beginning of cultivation. During this period, these calluses may also become oxidized. Oxidation of explants and callus is associated with the high content of phenols present in tissues of this species. It is also observed that normally oxidized calli have the capacity to form somatic embryos although they have an appearance of senescence.

#### *3.1.2 Embryogenesis, embryo induction*

For the second phase of the indirect route, MS/2 medium is used, with NAA and kinetin added and 20 g/L sucrose, according to the protocol of Sondhal and Sharp [34]. For this purpose, calli are transferred from the calogenesis induction medium to the embryogenesis medium. In this phase, there is the initiation and development of somatic embryos from certain cells, located in some sectors of the callus, which correspond to embryogenic centers [45, 55, 57]. Somatic embryos are usually formed on the bottom of the callus, which is in contact with the culture medium and also on the top surface of the callus. On the other hand, not all callus form somatic embryos.

The induction of embryogenic cells occurs during a precise moment of callus life [19]. This induction window may vary for identical explants of the same genotype depending on the cultivation conditions used and particularly the hormonal balance [20, 73]. In the same explant, it is possible to find competent or non-competent callus sections, which indicates that genetically identical cells may respond differently to and from a particular stimulus. Embryogenic cells rapidly lose their ability to divide due to the occurrence of the differentiation event, so the moment of callus transfer to embryogenesis is crucial. As the callus gets older, embryogenic cells lose their specificity in forming embryos. Somatic embryo formation is a continuous process and several embryonic stages can occur at the same time, in the same explant, in the same culture [74].

#### **3.2 Direct somatic embryogenesis in** *Coffea*

The process of direct somatic embryogenesis in *C. arabica* occurs in a single phase, which is the main feature of this pathway [75] (**Figure 5**). In this case, explant tissue cells are already determined and competent [76] for embryogenic development, before being extracted from the explant donor leaf [77] and may differentiate into somatic embryos soon after the start of cultivation. In this pathway, embryo formation occurs from leaf explant mesophyll cells [27]. The phenomenon

**9**

**Figure 5.**

*days; (E) Plantlets at 320 days; (F) adult plants [75].*

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

since somatic embryos are formed in a single phase.

of direct formation of embryogenic tissue in the explant is described as cloning of certain cells [78]. This cloning is a kind of large-scale reproduction of pro-embryos. Thus, pre-embryogenic cells present in explant tissues are cloned and require less epigenetic reprogramming compared to determined embryogenic cells in the indirect process. In the direct route, the need for cellular reprogramming is differ-

To respond to the direct pathway, *Coffea* explant cells only need contact with the cytokine-like plant growth regulator and the auxin normally inhibits its occurrence [79]. Explant edge cells differentiate into somatic embryos in response to cytokine in the culture medium [62, 80–83]. The efficiency of the direct pathway also seems to be related to explants from young leaves. On the other hand, in direct somatic embryogenesis, the occurrence of somaclonal variation tends to be minor or absent,

In the direct pathway, after inoculation of the explants in the culture medium, the formation of embryogenic structures occurs, which can be visualized around the 15th day of culture [75] (**Figure 5A**). Embryogenic structures are formed,

*Direct somatic embryogenesis on explants of* C. arabica *Catuaí cultivar maintained in the dark, at 30°C, for 320 days from the beginning of the experiment. (A) Embryogenic structure (arrow), up to 15 days; (B) oxidized embryogenic structure (arrow), up to 70 days; (C) embryos (lower arrow) formed from the embryogenic structure (top arrow), up to 100 days; (D) embryos formed from the border of the explant (arrow), up to 100* 

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

ent from the indirect route.

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

*Coffee - Production and Research*

the same plant or from the same leaf.

*3.1.2 Embryogenesis, embryo induction*

same explant, in the same culture [74].

**3.2 Direct somatic embryogenesis in** *Coffea*

of senescence.

somatic embryos.

of high concentration of soluble sugars in the calogenesis phase while it was lower in

In the indirect pathway, *C. arabica* leaf explants initiate callus formation from procambium cells around the seventh day after the beginning of cultivation [72]. In general, from the 14th day of cultivation, it is possible to see the occurrence of small callus on the edges of explants that reach up to 3 mm in size [45]. These calluses are usually formed on only one or two sides of the explant and later reach sizes that can occupy the four edges of the explant. In early development, calluses are hyaline, but gradually they tend to oxidize. In a set of explants of the same origin subjected to indirect pathways, it is possible to find calluses that do not develop. They remain small, up to 5 mm in size. However, some of these calluses have the capacity to form somatic embryos, which indicates that they appear to have been "latent" during this time. Furthermore, it is further noted that not all *C. arabica* explants form calli indicating that the somatic embryogenic capacity may vary between explants from

Callus of *C. arabica* can reach sizes up to 30 mm around 90–120 days from the beginning of cultivation. During this period, these calluses may also become oxidized. Oxidation of explants and callus is associated with the high content of phenols present in tissues of this species. It is also observed that normally oxidized calli have the capacity to form somatic embryos although they have an appearance

For the second phase of the indirect route, MS/2 medium is used, with NAA and kinetin added and 20 g/L sucrose, according to the protocol of Sondhal and Sharp [34]. For this purpose, calli are transferred from the calogenesis induction medium to the embryogenesis medium. In this phase, there is the initiation and development of somatic embryos from certain cells, located in some sectors of the callus, which correspond to embryogenic centers [45, 55, 57]. Somatic embryos are usually formed on the bottom of the callus, which is in contact with the culture medium and also on the top surface of the callus. On the other hand, not all callus form

The induction of embryogenic cells occurs during a precise moment of callus life [19]. This induction window may vary for identical explants of the same genotype depending on the cultivation conditions used and particularly the hormonal balance [20, 73]. In the same explant, it is possible to find competent or non-competent callus sections, which indicates that genetically identical cells may respond differently to and from a particular stimulus. Embryogenic cells rapidly lose their ability to divide due to the occurrence of the differentiation event, so the moment of callus transfer to embryogenesis is crucial. As the callus gets older, embryogenic cells lose their specificity in forming embryos. Somatic embryo formation is a continuous process and several embryonic stages can occur at the same time, in the

The process of direct somatic embryogenesis in *C. arabica* occurs in a single phase, which is the main feature of this pathway [75] (**Figure 5**). In this case, explant tissue cells are already determined and competent [76] for embryogenic development, before being extracted from the explant donor leaf [77] and may differentiate into somatic embryos soon after the start of cultivation. In this pathway, embryo formation occurs from leaf explant mesophyll cells [27]. The phenomenon

the formation and maturation of somatic embryos [71].

**8**

of direct formation of embryogenic tissue in the explant is described as cloning of certain cells [78]. This cloning is a kind of large-scale reproduction of pro-embryos. Thus, pre-embryogenic cells present in explant tissues are cloned and require less epigenetic reprogramming compared to determined embryogenic cells in the indirect process. In the direct route, the need for cellular reprogramming is different from the indirect route.

To respond to the direct pathway, *Coffea* explant cells only need contact with the cytokine-like plant growth regulator and the auxin normally inhibits its occurrence [79]. Explant edge cells differentiate into somatic embryos in response to cytokine in the culture medium [62, 80–83]. The efficiency of the direct pathway also seems to be related to explants from young leaves. On the other hand, in direct somatic embryogenesis, the occurrence of somaclonal variation tends to be minor or absent, since somatic embryos are formed in a single phase.

In the direct pathway, after inoculation of the explants in the culture medium, the formation of embryogenic structures occurs, which can be visualized around the 15th day of culture [75] (**Figure 5A**). Embryogenic structures are formed,

#### **Figure 5.**

*Direct somatic embryogenesis on explants of* C. arabica *Catuaí cultivar maintained in the dark, at 30°C, for 320 days from the beginning of the experiment. (A) Embryogenic structure (arrow), up to 15 days; (B) oxidized embryogenic structure (arrow), up to 70 days; (C) embryos (lower arrow) formed from the embryogenic structure (top arrow), up to 100 days; (D) embryos formed from the border of the explant (arrow), up to 100 days; (E) Plantlets at 320 days; (F) adult plants [75].*

on average, on one or two sides of the rectangular leaf explant. Normally, these structures range in size from 2 to 4 mm (**Figure 5B**) and remain this way until the end of cultivation. About 50 days after the start of cultivation the structures start to oxidize (**Figure 5C**) and by 150 they are completely oxidized [75]. The formation of somatic embryos usually starts from 90 days of cultivation but in low quantity and around 120 days this number tends to increase. Somatic embryos in addition to being formed at the edges of explants (**Figure 5D**) also develop over the surface of embryogenic structures (**Figure 5C**).
