Abstract
The medicinal plant Piper crassinervium is a source of bioactive compounds with potential use in agrochemical and pharmaceutical industries. However, its propagation is slow and influenced by the composition of the culture medium and growth conditions. In the genus Piper, tissue oxidation limits the biomass and recovery of bioactive compounds such as phenols. We evaluated the effect of medium formulations, sucrose, growth regulators, medium consistency, explant type, gas exchange and irradiance levels on growth and phenols and total flavonoids content in P. crassinervium in vitro plants. Murashige and Skoog (MS) and DKW/Juglans (DKW/J) media induced the highest production of chlorophyll and phenolic compounds; however, the regeneration rate of the explants in the DKW/J medium was the lowest (~75%) in comparison with the other media (90%). The maximum biomass production was achieved when half-strength MS (½MS) liquid medium was used; while the optimum sucrose level depended on the medium salt concentration used. Notably, the irradiance altered the accumulation of biomass and phenolic compounds. The presence of one remaining leaf in the nodal segments, used as explants, produced taller plants in ½MS liquid medium, combined or not with 2-iP. The optimal growth conditions for biomass and phenolic compound production in P. crassinervium in vitro plants established were: nodal explant with a preexisting leaf cultured on ½MS stationary liquid medium, 15 g L−1 sucrose, using lids with one PTFE 0.45-µm-pore size membrane and an irradiance of 100 µmol m−2 s−1.
Key message
Medium formulation, sucrose concentration, irradiance levels, gas exchange and explants with preexisting leaf improved the in vitro propagation and phenolic compounds production in Piper crassinervium.
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Acknowledgements
The authors are thankful to Prof. Oscar Marino Mosquera and MSc. Yeny Juliana Cortes of the Grupo de Biotecnología—Productos Naturales (GB-PN) [Escuela de Tecnología Química, Universidad Tecnológica de Pereira (UTP), Pereira, Risaralda, Colombia] for their guidance in the establishment of chemical analysis protocols; to Dr. Adriano Valentin-Silva (Graduate Program of Biological Sciences (Botany), Institute of Biosciences, UNESP—São Paulo State University, Botucatu, SP, Brazil) for his assistance in plant identification and collection of seeds of P. crassinervium and to Dr. Manuel Alejandro Ix Balam for the schematic drawing of the Fig. 1. The financial support was provided by Fundacão de Amparo à Pesquisa do Estado de Minas Gerais—Brazil (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico—Brazil (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES—Finance Code 001). WCO and SAF are supported by Research Fellowships from CNPq.
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AMRR, JVSS and JVMF raised the in vitro plants for the experiments and performed the experiments. AMRR, JVSS, JVMF, TDS, KC and DVF performed growth and photosynthetic pigments analysis. AMRR, JVSS, JVMF and DVF performed extractions and phytochemical analysis. AMRR, DSB and MVMP performed statistical analyses. AMRR, DSB, MVMP, DVF, WCO and SAF designed the research, interpreted the data and wrote the paper.
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Communicated by Silvia Moreno.
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Online Resource 2 In vitro plant growth variables of Piper crassinervium after 35 days of culture under different salt medium composition (a – f), culture medium consistency and sealing systems (g – l) and salt concentration of MS medium (m – r). (MS: Murashige and Skoog; DKW/J: DKW/Junglans; QL: Quoirin and Lepoivre; WPM: McCown’s Woody Plant; JADSm: JADS without the antioxidant polyvinylpyrrolidone (PVP); SL: Stirring liquid medium; StL: Stationary liquid medium; S: Semi-solid medium; 0M and 1M: Autoclavable rigid polypropylene lid without membrane and cover with 1 membrane; ½MS: MS medium with half the concentration of macro and micronutrients. Means followed by the same lowercase letter did not differ significantly among treatments (Tukey’s test at 5%). Error bars indicate the standard error among replicates). (JPG 5045 KB)
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Online Resource 3 In vitro plant growth variables of Piper crassinervium after 35 days of culture under irradiance of 45, 70, 100 and 150 µmol m-2 s-1 (a – f) and from stem segments without, with half and one preexisting leaf, respectively (0L, ½L and 1L) (g – l). (Means followed by the same lowercase letter do not differ significantly among treatments (Tukey’s test at 5%). Error bars indicate the standard error among the replicates). (JPG 3678 KB)
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Online Resource 4 Growth variables of Piper crassinervium plantlet grown in vitro under different photoperiods. (DOC 40 KB)
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Online Resource 5 Growth variables of Piper crassinervium plantlet grown in vitro with different growth regulators. (DOC 41 KB)
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Online Resource 6 In vitro plant growth variables of Piper crassinervium after 35 days of in vitro culture in ½MS liquid medium supplemented with 2-iP (2, 5 and 10 μM) (a - i) and ½OM and ½MS medium in combination with sucrose (7.5, 15 and 30 g L-1) (j - o). (2-iP: N6-(2-isopentenyl) adenine; ½OM and ½MS: Rugini Olive medium (OM) and Murashige and Skoog (MS) medium with half the concentration of macro and micronutrients. Means followed by the same lowercase letter do not differ significantly between 2-iP and sucrose concentrations; Averages followed by the same capital letter do not differ when comparing means ½OM and ½MS (Tukey’s test at 5%). Error bars indicate the standard error among the replicates). (JPG 4897 KB)
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Ríos-Ríos, A.M., da Silva, J.V.S., Fernandes, J.V.M. et al. Micropropagation of Piper crassinervium: an improved protocol for faster growth and augmented production of phenolic compounds. Plant Cell Tiss Organ Cult 137, 495–509 (2019). https://doi.org/10.1007/s11240-019-01585-8
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DOI: https://doi.org/10.1007/s11240-019-01585-8