Present study reports successful in vitro clonal propagation of a potential medicinal plant, Cassia alata using mature nodal explants. Murashige and Skoog (MS) medium supplemented with different concentrations (0.5, 2.5, 5.0, 7.5, 10.0 and 12.5 μM) of 6-benzyladenine (BA), kinetin and 2-isopentenyl adenine (2-iP) singly as well as in combination with different auxins, α-naphthalene acetic acid (NAA), Indole-3-butyric acid (IBA) or Indole-3-acetic acid (IAA) (0.1, 0.5 and 1.0 μM) were used. MS medium enriched with 7.5 μM BA and 0.5 μM NAA yielded the highest regeneration frequency (92 %) with maximum multiple shoots (12.3 ± 0.6) and shoot length (4.7 ± 0.1 cm) after 12 weeks of culture. Shoots were rooted best on full MS containing 0.5 μM IBA. Ex vitro rooting of in vitro derived microshoots was also achieved in 150 μM IBA treatment for 20 min followed by transfer to thermocol cups containing sterile Soilrite™. About 85 % plantlets survived acclimatization procedure to the field. The genetic fidelity of in vitro regenerated plants was analyzed using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Of the 20 RAPD primers, 18 primers produced clear, reproducible and scorable bands while out of 13 ISSR primers screened, only ten generated well-defined and scorable bands in all the tested plants. A total of 69 and 71 bands were scored with an average of 3.8 and 7.1 bands per primer for RAPD and ISSR primers respectively. All banding profiles from micropropagated plants were monomorphic and similar to of the mother plant, thus confirming the true-to-type nature of the in vitro-raised clones.

In vitro clonal propagation and evaluation of genetic fidelity using RAPD and ISSR marker in micropropagated plants of Cassia alata L.: a potential medicinal plant

The first objective of this study was to assess and optimize somatic embryo production in a genetically diverse range of cacao genotypes. The primary and secondary somatic embryogenesis response of eight promising cacao clones and a positive control was evaluated using modified versions of standard protocols. The second objective was to optimize the efficiency of primary somatic embryogenesis for a commercially important cacao clone, CCN 51, which has proven to be quite recalcitrant to standard protocols, relative to CCN 10, a clone also included in our analysis. The efficiency of the overall process was assessed by determining the number of somatic embryos produced per starting somatic tissue explant, as well as the quality of embryos (normal vs. abnormal) produced. Donor floral explants were subjected to five tissue culture steps, each 15-25 days in duration. Although all studied genotypes produced primary somatic embryos, most of them originated only from brown or brown-white callus. Overall, flower petals performed better than staminodes, and our best performing genotype yielded an average of 7-10 embryos produced in brown callus explants with embryogenic response during primary somatic embryogenesis procedures. In conclusions our analysis from a pilot in a small-scale are: 1) it is possible to achieve a high production of plants by somatic embryogenesis, although the efficiency is highly genotype-dependent; it is therefore necessary to optimize hormone balance and hormone type, as well as the explant type for each genotype, 2) through the use of secondary somatic embryogenesis, it is possible to increase somatic embryogenesis production at least ten-fold, and 3) the observed response variation between genotypes may reflect differences in endogenous and exogenously-supplied hormones. The importance of adapting the tissue culture protocol to the genotype is discussed.

	Key words: Somatic embryogenesis, 2,4-D, cellular competence, propagation, abnormalities, ethylene, cacao.

/pdf/optimization-of-somatic-embryogenesis-procedure-for-4465utt7df.pdf

Optimization of somatic embryogenesis procedure for commercial clones of Theobroma cacao L.

Several biochemical components associated with different stages of somatic embryogenesis in coffee (Coffea arabica L.) are investigated using foliar explants. Soluble sugar, starch, free amino acids and total proteins were extracted and quantified at different stages of somatic embryogenesis, such as foliar segments (initial explants), primary calluses, embryogenic calluses, globular embryos, torpedoes, cotyledonary embryos and mature fruit zygotic embryos. Total soluble sugar levels increased sixfold at the initial stages of somatic embryogenesis induction. During this period, total soluble sugar in the cultures contained approximately 99.3% glucose and fructose. At 67.4 μg/mg MS, no significant changes were observed in total sugar content during the embryo’s somatic maturation and regeneration. During this stage, total soluble sugar was composed of 60% sucrose. After primary callus formation, starch contents increased gradually until the culture’s conclusion. Total free amino acids, particularly arginine, lysine, methionine, asparagine, glutamine and histidine, revealed a higher synthesis until the formation of the primary callus, after which they remain statistically constant up to the end of the process. During the induction of calluses, a gradual increase of total proteins occurred, which, in the differentiating and maturing of somatic embryos, did not differ statistically till the formation of a cotyledonary embryo, when rates decreased 21.8%.

Biochemical events during somatic embryogenesis in Coffea arabica L.

The objective of this study was to characterize the histodifferentiation of somatic embryogenesis obtained from leaf explants of C. arabica. Therefore, we histologically analyzed the respective stages of the process: leaf segments at 0, 4, 7, 15 and 30 days of cultivation, Type 1 primary calli (primary calli with embryogenic competence) and 2 (primary calli with no embryogenic competence), embryogenic calli, globular, torpedo and cotyledonary embryos, and mature zygotic embryos. Callus formation occurred after seven days of culture, with successive divisions of procambium cell. In this cultivation phase, it was found that Type 1 primary calli are basically formed by parenchymal cells with reduced intercellular spacing, whereas Type 2 primary calli are predominantly composed of parenchymal cells with ample intercellular spaces and embryogenic calli composed entirely of meristematic cells. After 330 days, it was evident from the differentiation of somatic embryogenesis that there was formation of globular somatic embryos, consisting of a characteristic protoderm surrounding the fundamental meristem. With the maturation of these propagules after 360 days, torpedo-stage somatic embryos arose, in which tissue polarization and early differentiation of procambial strands were verified. After 390 days, cotyledonary somatic embryos were obtained, where the onset of vessel elements differentiation was verified, a characteristic also observed in mature zygotic embryos. We concluded that somatic embryogenesis obtained from C. arabica leaves initiates from procambium cell divisions that, in the course of cultivation, produce mature somatic embryos suitable for regenerating whole plants.

Histology of somatic embryogenesis in Coffea arabica L.

Coffea arabica L. belongs to the Rubiaceae family, and the genus Coffea is believed to have a primary center of origin and genetic variability in the highlands of southwestern Ethiopia. It is a vital beverage commodity across the world and a valuable export product, ranking second in international trade after petroleum. Ethiopia is among the top five major coffee-producing countries and is Africa’s leading producer. However, its full production capacity has not yet been exploited, and research efforts to reduce biotic and abiotic factors through reproduction have been extremely limited. Hence, improvement through different breeding methods is essential to overcome the constraints in its production. Thus, the objective of this study is to review the different breeding methods applied for different traits in Ethiopia. Breeding methods depend on the type and the source of traits and the final breeding objectives. The main breeding objectives are production, resistance/tolerance to diseases, and cup quality. The commonly applied breeding methods are selected and intra-specific hybridization, germplasm enhancement, and the development of improved varieties with wider adaptability. There is also a practice of crossing parental lines selected for certain desirable traits for the development of hybrid varieties. Accordingly, some promising success has been obtained. Forty-one coffee varieties have been released so far. Because conventional breeding methods are time-consuming, integrating conventional breeding methods with biotechnological techniques could have an instrumental role in the rapid development of suitable varieties for the changing climate.

Coffee (Coffea arabica L.): Methods, Objectives, and Future Strategies of Breeding in Ethiopia—Review

Summary Coffea arabica is one of the most traded agricultural commodities in the World, and the major export commodity of Ethiopia. Improvement of this crop requires the development of hybrid varieties that possess desirable traits. The production and distribution of hybrid coffee is difficult due to the high costs required for manual crossing and maintenance. The use of somatic embryogenesis is an effective means of propagation. A protocol for somatic embryogenesis of hybrid C. arabica from leaf explants is described here. Leaf explants collected from healthy 1-year-old seedlings were used. The highest percentage of callus induction was observed from explants cultured on MS medium containing 1.0 mg l–1 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 2.0 mg l–1 6-benzylaminopurine (BAP). Embryogenic calli were obtained from leaf explants cultured on MS medium supplemented with 0.05 mg l–1 kinetin in combination with 0.1 mg l–1 indole-3-butyric acid (IBA). The highest number of embryos that germ...

Somatic embryogenesis of a coffee (Coffea arabica L.) hybrid using leaf explants

https://link.springer.com/content/pdf/10.1186/s43141-021-00168-5.pdf

Workia Ahmed

Papers

Somatic embryogenesis of a coffee (Coffea arabica L.) hybrid using leaf explants

Genetic diversity and population structure of date palms (Phoenix dactylifera L.) in Ethiopia using microsatellite markers.