ABSTRACT
Indirect somatic embryogenesis (ISE) establishment for Coffea species started in the 1970s. Since then, intraspecific variations in the morphogenic pathway have been reported, even in the common environmental condition in vitro. Several authors have suggested that these variations are the result of genetic, epigenetic, and/or physiological events, highlighting the need for investigations to know the causes. Along these lines, this study aimed to investigate and describe, for the first time, the global 5-methylcytosine and physiological changes that occur in the cells of the aggregate suspensions of Coffea canephora during proliferation and somatic embryo regeneration steps. The cell proliferation step was characterized by increase in cell mass in all subcultures; relatively low mean values of global 5-methylcytosine (5-mC%), abscisic acid (ABA), and indole-3-acetic acid (IAA); high mean value of 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor); and increase followed by decrease in spermidine (Spd, a polyamine) level. Therefore, these epigenetic and physiologic aspects promoted the cell proliferation, which is fundamental for ISE. In turn, the somatic embryo regeneration was correlated with global 5-mC% and physiological changes. The competence acquisition, determination, and cell differentiation steps were marked by increases in mean values of 5-mC%, IAA and ABA, and decreases in ACC and Spd, evincing that these changes are the triggers for regeneration and maturation of somatic embryos. Therefore, dynamic and coordinated epigenetic and physiologic changes occur in the cells of the aggregate suspensions during the C. canephora ISE in liquid system.
Subject(s)
5-Methylcytosine/metabolism , Coffea/metabolism , Plant Proteins/metabolism , Plant Somatic Embryogenesis Techniques/methodsABSTRACT
Polyploidy is more than two chromosomal sets per nucleus, as the allotetraploid Coffea arabica. Due to allotetraploidy, C. arabica shows different phenotypes compare to diploid Coffea species, highlighting by beverage quality produced from its grains. Looking for the possibility of new phenotypes coupled with economic feature, considerable progress since 60's was reached for synthetic chromosome set doubling (CSD) in vitro, involving especially the antitubulin compounds, biological material, and used tissue culture pathway as the indirect somatic embryogenesis (ISE). Here, we aimed to regenerate autotetraploid and auto-alloctaploid plantlets of Coffea canephora and C. arabica, respectively, from a novel in vitro CSD procedure for Coffea. Exploring the ISE pathway, we treated the cellular aggregate suspensions (CAS) with 0.0 (control), 0.5, 1.5, or 2.5 mM of colchicine solution for 48, 72, or 96 h and maintained in liquid medium under constant orbital shaking. After transferring the CAS to semisolid media for somatic embryo regeneration, we considered it as cellular mass. Mature cotyledonary somatic embryos were only regenerated from cellular masses treated with 2.5 mM/48 h and 2.5 mM/72 h for C. canephora and with 0.5 mM/48 h for C. arabica. Evaluating the DNA ploidy level and the chromosome counting revealed that 36 (34.9%) plantlets of C. canephora were autotetraploids (4C = 2.86 pg, 2n = 4x = 44) and 61 (21.1%) of C. arabica were auto-alloctaploids (4C = 5.24 pg, 2n = 8x = 88). The CSD procedure, exploring the CAS proliferation and ISE pathway, promoted whole-genome duplication and resulted in a relatively high number of solid polyploids of both Coffea species. Due to distinct responses, DNA sequence fidelity (genetic) and global level of 5-methylcytosine (epigenetic) were evaluated. We observed that the increase of 5-methylcytosine levels was associated with somatic embryo regeneration from cells showing DNA sequence fidelity for the tested SSR primers. In conclusion, the adopted procedure for in vitro CSD is reproducible for induction, regeneration and propagation of Coffea polyploids and potentially other shrubbery and woody species. In view of the novelty of this procedure to generate new germplasm, we show the key issues and the steps of the CSD procedure.
ABSTRACT
Cytogenetic studies in Primulaceae are mostly available for herbaceous species, and are focused on the chromosome number determination. An accurate karyotype characterization represents a starting point to know the morphometry and class of the chromosomes. Comparison among species within Myrsine, associating these data with the nuclear 2C value, can show changes that led the karyotype evolution. Here, we studied three Myrsine species [Myrsine coriacea (Swartz, 1788) Brown ex Roemer et Schultes, 1819, Myrsine umbellata Martius, 1841 and Myrsine parvifolia Candolle, 1841] that show different abilities to occupy the varied types of vegetation within the Brazilian Atlantic Forest. Cytogenetic characterization showed some individuals with 2n = 45 chromosomes for Myrsine parvifolia and Myrsine coriacea, with most individuals of the three species having 2n = 46. The first karyograms for Myrsine were assembled and presented morphologically identical and distinct chromosome pairs. In addition, differences in the mean 2C nuclear value and chromosome morphometry were found. Therefore, the first description of the Myrsine karyotype has been presented, as well as the nuclear 2C value. The procedures can be applied to other Myrsine species for future investigations in order to better understand its effects on the differential spatial occupation abilities shown by the species in Brazilian Atlantic Forest.
ABSTRACT
Chromosome morphometry and nuclear DNA content are useful data for cytotaxonomy and for understanding the evolutionary history of different taxa. However, the chromosome number is the only karyotype aspect reported for the species of Dorstenia so far. In this study, the nuclear genome size of Dorstenia arifolia (Lamarck, 1786), Dorstenia bonijesu (Carauta & C. Valente, 1983) and Dorstenia elata (Hooker, 1840) was evaluated and their karyotype morphometry accomplished, with the aim of verifying the potential of those parameters to understand evolutionary issues. Mean nuclear 2C value ranged from 2C = 3.49 picograms (pg) for Dorstenia elata to 2C = 5.47 pg for Dorstenia arifolia, a variation of ± 1.98 pg. Even though showing a marked difference in 2C value, the three species exhibited the same 2n = 32. Corroborating the flow cytometry data, differences in chromosome morphology were found among the karyotypes of the species investigated. Based on this and the only phylogeny proposed for Dorstenia thus far, structural rearrangements are related to the karyotype variations among the three species. Besides, the karyological analysis suggests a polyploid origin of the Dorstenia species studied here.
