The control of somatic embryogenesis formation in coffee has not yet been fully defined. Knowing the factors that control the occurrence of somatic embryogenesis in C. arabica allows to optimize its application, and especially the direct pathway. The high or low capacity of somatic embryogenesis of a species is related to the presence or absence of sufficient cells in the explant, which is inherent in their totipotency. The maintenance of somatic embryogenesis capacity requires the use of conditions that are determined and maintain the proliferation of competent cells.
Somatic embryogenesis regeneration capacity is related to the explant donor plant development stage, physiological conditions of the explant donor plant and the explant position relative to the plant. However, it is also associated with other factors such as in vitro culture conditions and mainly plant growth regulators. Seasons influence the indirect somatic embryogenesis response of plant explants to eight C. Arabica genotypes in the field. Explants form more somatic embryos in the autumn-winter season than in the spring-summer season.
In Vitro Culture Status Factors
Somatic embryogenesis contributes to coffee growing programs. This is an asexual reproductive process based on the cellular totipotent concept. Every haploid or somatic cell of plant tissue has the genetic information necessary to create a complete and functional plant. Somatic embryogenesis can occur directly or indirectly. Coffee arabica genotypes may respond directly, indirectly, or both. In this species, indirect somatic embryogenesis consists of two stages: callogenesis and embryogenesis, while the direct pathway takes place in a single stage without callogenesis. In coffee, the indirect pathway in general is induced by auxin and cytokinin, and only by the direct pathway with cytokinin. Genotypes of this coffee strain respond readily, usually indirectly, with high somatic embryo production. However, these are directly inefficient because they offer low embryo production and the process takes a long time.
In the indirect somatic embryogenesis of C. arabica, calli is induced and initiated in the absence or presence of light. However, it reaches a larger size only if it is kept in the absence of light. These coffee beans gradually increase in size each month and can reach sizes up to 30 mm. In the direct pathway, C. arabica explants also have difficulty responding in the presence of light. Thus, small structures called embryogenic structures are formed at the edge of the explants, which remain without changes in size or shape in the presence or absence of light.
Rearing temperature is another factor that can influence the somatic embryogenesis response. Leaf explants of Catuaí variety and two hybrids show higher somatic embryo formation at 30 ° C compared to 25 ° C.
Plant Growth Regulation
Various studies show that plant regulators play a decisive role in controlling somatic embryo formation in Coffee leaf explants, the most researched aspect in this regard. The already well established combination of arabica, auxin or cytokines for this species is usually used. In this way, auxin 2,4 D has been most used to induce callogenesis in C. arabica leaf explants. This auxin is considered potent and is also used for induction of anthers and roots.
Most studies for induction of the direct pathway use the auxin-free cytokine because it tends to inhibit its formation. However, the effectiveness of the direct pathway response may vary depending on the type and concentration of cytokine used. A pioneering study reported that 6-BA at a 5-M dose matched direct pathway induction in C. arabica explants.
The formation of somatic embryos was also obtained from C. canephora explants inoculated in MS medium with the addition of different cytokines, 2-iP, ZE, Ki and 6-BA, all at 5-M concentration. Explants generate somatic embryos in the presence of all cytokines, but responses vary by cytokine type and are more efficient than 2-iP, ZE, Kin and 6-BA. In a study, it was found that auxins used in different concentrations also directly inhibit somatic embryogenesis of these genotypes. In another study, Zeatin induced a direct pathway response in C. canephora explants. In another study, 2-iP concentrations of 7.5 and 10 µM were found to support more somatic embryos than 2.5 and 5 µM doses.
The synthetic cytokine 6-benzylaminopurine also has the ability to directly induce the pathway in C. arabica explants. 6-BA, used at 30-M concentration, resulted in higher somatic embryo production than 10 and 20-M doses in leaf explants of Mundo Novo de C. arabica variety. However, although the 6-BA concentration was high, embryo production decreased and the process continued for a long time. However, this result is interesting because 6-BA is a cheaper and obtainable synthetic cytokine than zeatin and 2-iP, which helps to reduce the cost of clonal seedling generation. Cytokine TDZ has also been used to induce direct regeneration of C. arabica somatic embryos. Leaf explants of cultivar IAPAR 59 and C. arabica hybrid Sachimor showed a direct somatic embryogenesis response in the presence of TDZ at concentrations of 2.27.
The literature shows that the Coffee direct pathway is well proven to occur only in the presence of cytokines. However, it is also possible to find studies in which explants of this species form somatic embryos in the presence of auxin. When explants of the cultivar Acaia Cerrado were grown in a single culture medium with the addition of kinetin, GA 3 and NAA, somatic embryos were formed. In another study, direct pathway leaf explants of the Mundo Novo variety were compared with 2-iP alone. In this comparison, it was found that it showed high somatic embryo production in response to brassinosteroid-associated 2-iP treatment. On the other hand, explants treated with brassinosteroid alone without cytokines formed only embryogenic structures without any somatic embryo formation.
Stress factors are associated with promoting the acquisition of different types of embryogenic competence. Stressful conditions can also affect the acquisition of different types of embryogenic competence. Studies show that somatic embryogenesis formation is strongly associated with exposure of explants to some high-density stressors or high-concentration plant growth regulators. Osmotic stress treatments change the environment of the explant. This change in tissue and organ growth conditions may represent the estrium that causes cells to undergo changes in their developmental processes and empower them for inductive signals for somatic embryogenesis. Thus, the stress induction system consists of two stages: the acquisition of embryogenic competence and the formation of the somatic embryo.
It has been shown from the responses found in different species that the stress induction system can cause more somatic embryo formation. However, their control and mode of action are unknown, and this aspect has been little studied in the culture of coffee plants. It was confirmed that C. canephora explants sent directly for somatic embryogenesis produced more embryos in a medium with 3 g of agar than in a medium containing 6 g. This result is indirect evidence that altering the osmotic potential of the culture tends to support somatic embryogenesis capability. In another study, it was found that the change in the osmotic concentration of the culture medium affects the embryogenesis response. The use of 7% PEG 6000 resulted in greater somatic embryo formation in leaf explants of C. arabica genotypes AC1 and the cultivar Mundo Novo than using 5% PEG 6000. This reagent has a high molecular weight and is ineffective. Ionic, toxic It is non-water soluble, not absorbed by vegetable cells, and changes the osmotic potential when added to a culture medium.
Somatic embryogenesis contributes to the coffee crop in relation to both breeding programs and the production chain. Little is known about the factors that control somatic embryogenesis in coffee genotypes. However, it is known that plant hormones control the formation of this process. In addition, research has shown that environmental and mainly stress factors applied during the cultivation condition in Coffee play a role in the control of somatic embryogenesis.
Author: Ozlem Guvenc Agaoglu