Use of Electron Microscopy for the Detection of Contaminant Endophytic Bacteria In Vitro.

The success of in vitro cultivation, particularly for micropropagation purposes, depends on the efficient control of contaminants. In this context, the sterilization of plant material constitutes a fundamental step in initiating cultures. Microbial contaminants can be found either on the surface (epiphyte) or inside plant explants (endophyte). However, the latter is generally challenging to detect and may not always be eradicated through surface sterilization alone. Endophyte contaminants, such as bacteria, can persist within plant material over several cultivation cycles, potentially interfering with or inhibiting in vitro establishment, growth, or recovery of cryopreserved materials. Therefore, microscopy techniques, such as electron microscopy, can yield valuable insights into bacterial endophytes' localization, tissue colonization patterns, and functions in in vitro plant culture. This information is essential for adopting effective strategies for eliminating, preventing, or harmonious coexistence with contaminants.

Cellular responses of oil palm genotypes during somatic embryogenesis involve participation of procambial cells, DNA demethylation, and auxin accumulation.

KEY MESSAGE: Cell markers of somatic embryogenesis initiation from leaf tissues in oil palm involve the participation of procambial cells, DNA demethylation, and auxin accumulation. Low callogenesis and genotype-dependent response have been mentioned in the development of somatic embryogenesis protocols of Elaeis oleifera × E. guineensis elite hybrids, which requires more detailed investigations of the process. Thus, the initial cellular responses of immature leaves of adult genotypes of this hybrid were investigated for the first time, emphasizing histological, epigenetic, and endogenous auxin changes. Leaf segments from two genotypes, one responsive to somatic embryogenesis (B351733) and another non-responsive (B352933), were inoculated in Murashige and Skoog medium with 450 µM of 4-amino-3, 5, 6-trichloropicolinic acid. For anatomical analysis, samples of both genotypes were collected at 0, 20, 90, and 105 days of cultivation. Samples of both genotypes were also taken at different cultivation periods to analyze DNA methylation status (% 5-mC-5 methylcytosine) via ELISA test. Immunolocalization assays were performed with anti-indole-3-acetic acid and anti-5-methyl-deoxycytosine antibodies from samples of hybrid B351733. We distinguished two groups of cells reactive to the induction of embryogenic callogenesis, parenchymatous sheath cells, and procambial cells; however, only the latter are directly involved with the formation of calluses. The data obtained indicate that the formation of calluses in hybrid B351733 is related to DNA hypomethylation, while the non-responsiveness of leaf explants in hybrid B352932 is related to DNA hypermethylation. The in situ immunolocalization enabled the identification of initial markers of the callogenic process, such as IAA accumulation and hypomethylation. Identifying these events brings the possibility of establishing strategies for efficient manipulation of somatic embryogenesis protocols in palm trees.

Indirect somatic embryogenesis of Piper hispidinervum L. and evaluation of the regenerated plants by flow cytometry.

BACKGROUND: Piper hispidinervum is a species native from the Amazon region with great economic potential, given its scientifically proven insecticidal properties. In this study, an efficient protocol of plant regeneration via indirect somatic embryogenesis has been established for the first time. In a first experiment, for the induction of calluses, foliar explants of non-discriminated accesses of P. hispidinervum were inoculated in MS medium supplemented with α-naphtalenacetic acid (NAA) and 6-benzylaminopurine (BAP), in different combinations. For a second experiment, foliar explants from five different accesses of P. hispidinervum (PH17, PH21, PH28, PH37, and PH39) were analyzed regarding the formation of calluses when cultivated in MS medium with 5 mg L-1 NAA + 2.5 mg L-1 BAP. To obtain somatic embryos-like structures, calluses were cultivated in MS medium with 10 mg L-1 NAA + 2.5 mg L-1 of BAP. The somatic embryos-like structures obtained were inoculated in MS medium devoid of growth regulators and the plantlets were subjected to acclimatization. Calluses and somatic embryos-like structures were subjected to anatomical analysis and genetic stability of regenerated plants was analyzed by flow cytometry. RESULTS: The treatments 2.5 mg L-1 BAP and 5 mg L-1 NAA + 2.5 mg L-1 BAP, after 60 days of cultivation, provided each 32% of primary callus, not being verified the formation of calluses in medium devoid of BAP. It was found that accesses differed among them with respect to the formation of primary calluses, with emphasis on accesses PH28, PH37, and PH39, with mean percentage of 95.3%. Regarding the percentage of embryogenic calluses and formation of somatic embryos-like structures, there were no statistical differences between accesses, with mean values of 90.6% and 77.3%, respectively. The somatic embryos-like structures of P. hispidinervum have conspicuous morphoanatomical similarities with the zygotic embryo, and flow cytometry analysis showed no significant variation in nuclear DNA size among plants regenerated in vitro and plants coming from seed germination, which indicates ploidy level stability. CONCLUSION: This protocol is the first cited in the literature that demonstrates an efficient micropropagation process by somatic embryogenesis of P. hispidinervum. It can be used either to enable large-scale vegetative production or to subsidize germplasm conservation or genetic engineering of P. hispidinervum.