Optimized somatic embryogenesis and plant regeneration in elite Argentinian sugarcane (Saccharum spp.) cultivars

Background Biotechnological breeding of elite sugarcane cultivars is currently limited because of the difficulty of regenerating plants by tissue culture. Here, we report that commercially elite sugarcane genotypes, which are adapted to Argentinian agro-ecological conditions, are capable of being regenerated via indirect somatic embryogenesis. Leaf rolls of five elite genotypes were cultured following two callus induction protocols using different concentrations of 2,4-D as the growth regulator. Embryogenic calluses were regenerated under light conditions. Regenerated plants were subsequently acclimatized in the greenhouse under two acclimatization procedures before being transplanted to the field. Results Four of the five genotypes were able to form somatic embryos following the two induction protocols. The variables related to embryogenic callus production were influenced by the interaction between genotype and culture conditions. For plant regeneration, the embryogenic calluses were further cultured on an IBA-supplemented medium, where we observed a high genotype dependence. Calluses from the four cultivars regenerated a good number of plants. With the procedures described here, we obtained more than 90% of well-acclimatized plants both in the greenhouse and in the field. Conclusions This protocol provides a simple way to regenerate sugarcane plants through indirect somatic embryogenesis. Also, the results confirm that tissue culture ability is highly genotype-dependent in sugarcane. Our findings suggest that these elite cultivars could be good candidates for biotechnological breeding.

Genomic Methylated Cytosine Level during the Dedifferentiation and Cellular Competence in Coffea arabica Lines: Insights about the Different In Vitro Responses

Coffea arabica genotypes present distinct responses in vitro, and somaclonal variation occurrence has been reported. Global cytosine methylation is one of the epigenetic mechanisms that influences the Coffea in vitro responses. We aimed to establish the indirect somatic embryogenesis in C. arabica ‘Catuaí Vermelho’, ‘Caturra’ and ‘Oeiras’, associate the distinct responses to the methylated cytosine genomic level, and check the ploidy stability. Leaf explants were cultured in callus induction and proliferation medium. The resulted calli were transferred to the regeneration medium, and the mature cotyledonary somatic embryos were transferred to the seedling medium. ‘Oeiras’ exhibited the highest number of responsive leaf explants, followed by ‘Caturra’ and ‘Catuaí Vermelho’. Global methylated cytosine level increased over time in the ‘Catuaí Vermelho’ and ‘Caturra’ friable calli, remaining constant in ‘Oeiras’. ‘Oeiras’ did not regenerate somatic embryos, while ‘Catuaí Vermelho’ exhibited the highest number. Somatic embryo regeneration was associated with the increase of the methylated cytosine level. However, the ‘Catuaí Vermelho’ embryogenic calli showed a lower methylated cytosine level than ‘Caturra’. Recovered plantlets exhibited the same 2C value and chromosome number to the explant donors. Therefore, cytosine hypermethylation occurred during C. arabica indirect somatic embryogenesis, influencing cell competence and somatic embryos regeneration.

Direct and Indirect Somatic Embryogenesis Induction in Camellia oleifera Abel

Camellia oleifera Abel. is an important woody oil species; however, the shortage of rapid and industrialized seedling culture is a large constraint on the development of the tea oil industry. Somatic embryogenesis (SE) is one of the main powerful biotechnological tools for plant mass regeneration, but the largely unknown SE in C. oleifera limits the scale production of clonal plants. In this study, we described a high-efficiency SE system via direct and indirect pathways in C. oleifera and investigated the effect of genotype, explant age and phytohormones on SE. In the direct pathway, somatic embryos were highly induced from immature seeds 220 days after full blossom, and the development of embryoids was achieved with a combination of 0.19 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg/L thidiazuron (TDZ). In the indirect pathway, embryogenic calli were induced from the same explants in medium containing 1.5 mg/L 2,4-D, while 0.75 mg/L 2,4-D treatment led to high proliferation rates for embryogenic calli. The addition of 0.19 mg/L 2,4-D alone stimulated the production of globular embryos while causing a 75% loss of the induction rate in the heart embryo stage. Upon transfer of the globular embryos to phytohormone-free medium, an optimal induction rate of 62.37% from globular embryos to cotyledonary embryos was obtained. These data suggest that the subsequent differentiation process after the globular embryo stage in ISE is more similar to an endogenous phytohormones-driven process. Mature embryos germinated to produce intact plantlets on half-strength MS basal medium with a regeneration rate of 63.67%. Histological analysis confirmed the vascular bundle isolation of embryoids from the mother tissue. We further studied the different varieties and found that there were no significant genotype differences for SE induction efficiency in C. oleifera. Thus, we established a high-efficiency induction system for direct and indirect somatic embryogenesis (ISE) in C. oleifera and regenerated intact plantlets via SE, not organogenesis. ISE has a more complicated induction and regulatory mechanism than direct somatic embryogenesis. The improved protocol of SE would benefit mass propagation and genetic manipulation in C. oleifera.

Indirect Somatic Embryogenesis and Cryopreservation of Agave tequilana Weber Cultivar ‘Chato’

Agave tequilana Weber cultivar ‘Chato’ represents an important genetic supply of wild severely in decline populations of ‘Chato’ for breeding and transformation programs. In this work, the indirect somatic embryogenesis and cryopreservation of Somatic Embryos (SEs) were investigated using the ‘Chato’ cultivar as a study case. Methods: Embryogenic calli were induced by the cultivation of 1 cm of young leaves from in vitro plants on MS semisolid medium supplemented with 24.84, 33.13, 41.41, 49.69, and 57.98 μM 4-amino-3,5,6-trichloro-2- pyridinecarboxylic acid (picloram) in combination with 2.21, 3.32, and 4.43 μM 6-benzylaminopurine (BAP). The origin and structure of formed SEs were verified by histological analysis. Cryopreservation studies of SEs were performed following the V-cryoplate technique and using for dehydration two vitrification solutions (PVS2 and PVS3). Results: The highest average (52.43 ± 5.74) of produced SEs and the Embryo Forming Capacity (estimated index 52.43) were obtained using 49.69 µM picloram and 3.32 µM BAP in the culture medium. The highest post-cryopreservation regrowth (83%) and plant conversion rate (around 70%) were achieved with PVS2 at 0 °C for 15 min. Conclusion: Our work provides new advances about somatic embryogenesis in Agave and reports the first results on cryopreservation of SEs of this species.