Somatic embryogenesis and plant regeneration in Piper aduncum L

Abstract

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 L1 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%.