An efficient protocol is reported for in vitro plant regeneration through somatic embryogenesis in Piper aduncum, a Brazilian Amazon species with high economic potential. The species is important due to a variety of components found in its essential oil, with emphasis on dillapiole. Leaf explants from five accessions identified for high oil yield and levels of dillapiole were evaluated for their embryogenic potential. To induce embryogenic calli, the explants were cultivated in MS medium supplemented with 5 mg L −1 of 1-naphthaleneacetic acid (NAA) and 2.5 mg L −1 of N6-benzylaminopurine (BAP) for 80 d. For somatic embryogenesis, the embryogenic calli were transferred to MS medium with 10 mg L −1 of NAA and 2.5 mg L 1 of BAP and incubated for 45 d. The obtained somatic embryos were germinated in MS medium without regulators by 45 d and the obtained plantlets were subjected to acclimatization. Somatic embryos and calli from this process were subjected to anatomical and histochemical analyses. Biochemical analyses (total soluble sugars, starch, total amino acids, and proteins) were also performed to identify markers for embryogenic competence acquisition. In addition, the germination of somatic embryos was evaluated in a semi-solid and liquid system (R.I.T.A.® temporary immersion bioreactors). The obtained plants were evaluated for genetic fidelity using ISSR markers. The present study indicate that the accessions did not differ in embryogenic potential, with a mean percentage of calli with somatic embryos of 82.4%. Anatomical analyses confirmed the occurrence of the embryogenic route and the histochemical analyses identified starch grains in somatic embryos at different developmental stages. The biochemical analyses showed high total soluble sugars and total amino acids in embryogenic calli, marks of the acquisition of the embryogenic competence of P. aducum. The R.I.T.A.® temporary immersion bioreactors were highly efficient in the regeneration of somatic plants, with 100% germination. The plants regenerated in the semi-solid and liquid systems showed high genetic homogeneity. The survival rate of the acclimatized plants was 100%.

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.