**4.2 Plant growth regulation**

Several studies indicate that phytoregulators play a decisive role in controlling the formation of somatic embryos in *Coffea* leaf explants, which is the most explored aspect on this subject. For the induction of the indirect pathway in *C*. *arabica*, the auxin/cytokine combination, which is already well established for this species, is generally used. In this pathway, auxin 2,4 D has been the most used to induce callogenesis in *C. arabica* leaf explants. This auxin is considered strong and is also used for the induction of anthers [39, 40] and roots [47, 48].

For induction of the direct pathway, most studies use cytokine without auxin because it tends to inhibit its occurrence. However, the efficiency of the direct pathway response may vary depending on the type and concentration of cytokine employed. A pioneer study showed that 6-BA at 5 μM dose matched direct pathway induction in *C. arabica* explants [91].

The formation of somatic embryos was also obtained from *C. canephora* explants inoculated in MS medium only with addition of different cytokines being 2-iP, ZE, Ki and 6-BA, all at a concentration of 5 μM [92]. Explants formed somatic embryos in the presence of all cytokines, but responses varied according to cytokine type. The 2-iP was more efficient than the ZE, Kin and 6-BA. In this study, it was also found that auxins used at different concentrations inhibited the direct somatic embryogenesis of these genotypes. In another study, Zeatin caused the direct pathway response in *C. canephora* explants [87]. Cytokine 2-iP also caused direct pathway induction in *Coffea* [93, 94]. In another study, it was found that 2-iP concentrations of 7.5 and 10 μM favored a greater number of somatic embryos than the 2.5 and 5 μM doses [95].

Synthetic cytokine 6-benzylaminopurine also has the ability to induce the direct pathway in *C. arabica* explants [69, 91, 96–98]. The 6-BA used at 30 μM concentration caused higher production of somatic embryos than the 10 and 20 μM doses in leaf explants of the cultivar Mundo Novo de *C. arabica* [46]. But although the 6-BA concentration was high, embryo production was reduced and the process also took place over a long time. However, this result is interesting because it favors the cost reduction of clonal seedling formation since 6-BA is a cheaper and available synthetic cytokine than zeatin and 2-iP. Cytokine TDZ

#### **Figure 6.**

*Direct somatic embryogenesis from leaf explants of* C. arabica*. (A) Macroscopic view of coffee leaf explants after 60, 90 and 130 days of culture on induction medium supplemented with 10 μM 2-iP + 1.0 μM 24-epiBR. (B) Number and (C) size of somatic embryos obtained by treatment during the somatic embryogenesis process. (D) Somatic embryo germination. (E) Elongating regenerated plantlets after 90 days on ½MS medium. Bars = 0.5 and 2 cm [99].*

has also been used to induce regeneration of *C. arabica* somatic embryos via the direct pathway [47, 48]. Leaf explants of cultivar IAPAR 59 and *C. arabica* hybrid Sachimor showed direct somatic embryogenesis response in the presence of TDZ at concentrations of 2.27; 4.54; 6.81; 9.08; 13.62 μM, but with low embryo production [96].

The literature shows that it is well established that the *Coffea* direct pathway only occurs in the presence of cytokine, but it is also possible to find studies in which explants of this species formed somatic embryos in the presence of auxin. Explants of cultivar Acaia Cerrado formed somatic embryos when grown in a single culture medium with addition of kinetin, GA3 and NAA [67]. In another study, it was found that leaf explants of cultivar Mundo Novo submitted to direct pathway showed high production of somatic embryos in response to 2-iP treatment associated with brassinosteroid compared to 2-iP alone control [99]. On the other hand, explants treated with brassinosteroid alone without cytokine formed only embryogenic structures without any occurrence of somatic embryos (**Figure 6**).

#### **4.3 Stress factor**

The stress factors have been related to promoting the acquisition of embryogenic competence in different species [20, 66]. Stressful conditions can also influence the acquisition of embryogenic competence in different species [19, 20, 30, 66, 100, 101].

**13**

**5. Conclusions**

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

Studies indicate that the occurrence of somatic embryogenesis is strongly related to the exposure of explants to some high intensity stress factor and or the high concen-

*Effect of 3 and 6 g/L agar on the direct somatic embryogenesis in explants of* C. canephora *cultivar "Robusta 2264 Mar" maintained at 25°C in the dark [106]. (A) 6 g/L of agar: explants with curvature without contact with the medium. (B) Explant with few somatic embryos in the presence of 6 g/L agar. (C) 3 g/L of agar: the medium is in contact with the border of the explant. (D) Explant with many somatic embryos in the presence of 3 g/L agar.*

Osmotic stress treatments alter the explant's environment. This change in tissue/ organ growth conditions may represent the estrum that enables cells to undergo changes in developmental processes and make them competent for inductive signals for somatic embryogenesis. Thus, the stress-induction system is composed of two phases: the acquisition of embryogenic competence and the formation of the

Of the responses found in different species, it was shown that the stress-induction system could cause a greater formation of somatic embryos [101–107] although its forms of control and action are unknown and this aspect has been little studied in the culture of coffee plants. It was verified that *C. canephora* explants submitted to direct somatic embryogenesis formed a greater number of embryos in a medium to which 3 g of agar had been added, than in one containing 6 g [106] (**Figure 7**). This result was indirect evidence that altering the osmotic potential of the culture tends to favor the ability of somatic embryogenesis. In another study, it was found that the alteration of the osmotic concentration of the culture medium influenced the embryogenesis response [108]. The use of 7% PEG 6000 caused a greater formation of somatic embryos in foliar explants of the *C. arabica* genotypes AC1 and cultivar Mundo Novo than the use of 5% PEG 6000. This reagent has a high molecular weight and is inert, non-ionic, non-toxic, water soluble [109], not absorbed by vegetable cells and alters

Somatic embryogenesis contributes to coffee crop both in relation to breeding programs and its production chain. Little is known about the factors controlling

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

tration of plant growth regulator [22].

the osmotic potential when added to a culture medium.

somatic embryo [102].

**Figure 7.**

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

#### **Figure 7.**

*Coffee - Production and Research*

has also been used to induce regeneration of *C. arabica* somatic embryos via the direct pathway [47, 48]. Leaf explants of cultivar IAPAR 59 and *C. arabica* hybrid Sachimor showed direct somatic embryogenesis response in the presence of TDZ at concentrations of 2.27; 4.54; 6.81; 9.08; 13.62 μM, but with low embryo

*Direct somatic embryogenesis from leaf explants of* C. arabica*. (A) Macroscopic view of coffee leaf explants after 60, 90 and 130 days of culture on induction medium supplemented with 10 μM 2-iP + 1.0 μM 24-epiBR. (B) Number and (C) size of somatic embryos obtained by treatment during the somatic embryogenesis process. (D) Somatic embryo germination. (E) Elongating regenerated plantlets after 90 days on ½MS medium. Bars =* 

The literature shows that it is well established that the *Coffea* direct pathway only

The stress factors have been related to promoting the acquisition of embryogenic competence in different species [20, 66]. Stressful conditions can also influence the acquisition of embryogenic competence in different species [19, 20, 30, 66, 100, 101].

occurs in the presence of cytokine, but it is also possible to find studies in which explants of this species formed somatic embryos in the presence of auxin. Explants of cultivar Acaia Cerrado formed somatic embryos when grown in a single culture medium with addition of kinetin, GA3 and NAA [67]. In another study, it was found that leaf explants of cultivar Mundo Novo submitted to direct pathway showed high production of somatic embryos in response to 2-iP treatment associated with brassinosteroid compared to 2-iP alone control [99]. On the other hand, explants treated with brassinosteroid alone without cytokine formed only embryogenic structures

without any occurrence of somatic embryos (**Figure 6**).

**12**

production [96].

**Figure 6.**

*0.5 and 2 cm [99].*

**4.3 Stress factor**

*Effect of 3 and 6 g/L agar on the direct somatic embryogenesis in explants of* C. canephora *cultivar "Robusta 2264 Mar" maintained at 25°C in the dark [106]. (A) 6 g/L of agar: explants with curvature without contact with the medium. (B) Explant with few somatic embryos in the presence of 6 g/L agar. (C) 3 g/L of agar: the medium is in contact with the border of the explant. (D) Explant with many somatic embryos in the presence of 3 g/L agar.*

Studies indicate that the occurrence of somatic embryogenesis is strongly related to the exposure of explants to some high intensity stress factor and or the high concentration of plant growth regulator [22].

Osmotic stress treatments alter the explant's environment. This change in tissue/ organ growth conditions may represent the estrum that enables cells to undergo changes in developmental processes and make them competent for inductive signals for somatic embryogenesis. Thus, the stress-induction system is composed of two phases: the acquisition of embryogenic competence and the formation of the somatic embryo [102].

Of the responses found in different species, it was shown that the stress-induction system could cause a greater formation of somatic embryos [101–107] although its forms of control and action are unknown and this aspect has been little studied in the culture of coffee plants. It was verified that *C. canephora* explants submitted to direct somatic embryogenesis formed a greater number of embryos in a medium to which 3 g of agar had been added, than in one containing 6 g [106] (**Figure 7**). This result was indirect evidence that altering the osmotic potential of the culture tends to favor the ability of somatic embryogenesis. In another study, it was found that the alteration of the osmotic concentration of the culture medium influenced the embryogenesis response [108]. The use of 7% PEG 6000 caused a greater formation of somatic embryos in foliar explants of the *C. arabica* genotypes AC1 and cultivar Mundo Novo than the use of 5% PEG 6000. This reagent has a high molecular weight and is inert, non-ionic, non-toxic, water soluble [109], not absorbed by vegetable cells and alters the osmotic potential when added to a culture medium.

## **5. Conclusions**

Somatic embryogenesis contributes to coffee crop both in relation to breeding programs and its production chain. Little is known about the factors controlling

somatic embryogenesis in *Coffea* genotypes. But it is known that plant hormones act in controlling the occurrence of this process. In addition, studies have shown that environmental and mainly stress factors applied during the cultivation condition are involved in the control of somatic embryogenesis in *Coffea*.
