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… Show more
“…4 B). These results indicate high efficiency of the somatic embryogenesis process of P. hispidinervum induced by NAA and BAP, similar to the one mentioned for P. aduncum [ 24 ]. Fig.…”
Section: Resultssupporting
confidence: 83%
“…It is noteworthy, the apparently positive role of oxidation of primary calluses in the induction of somatic embryogenesis of P. hispidinervum , previously reported also in P. aduncum [ 24 ] . The formation of somatic embryos from oxidized calluses is not a feature limited to species of the genus Piper .…”
Section: Resultsmentioning
confidence: 74%
“…According to statistical analysis, the treatments 2.5 mg/l BAP and 5 mg/l NAA + 2.5 mg/l BAP provided each, after 60 days of cultivation, 32% of primary callus formation (Table 1 ). Significantly higher percentage of primary calluses, mean value of 87%, was obtained by [ 24 ] in leaf segments of P. aduncum also cultivated in medium with 5 mg/l NAA + 2.5 mg/l BAP. Fig.…”
Section: Resultsmentioning
confidence: 88%
“…Initially, the seeds of the different accesses were subjected to disinfestation, characterized by immersion for three minutes in 70% ethanol (v/v), followed by immersion for 20 min in sodium hypochlorite (NaOCl) (2.0–2.5% active chlorine) and four washings in distilled and autoclaved water. Then, the seeds were inoculated in vials containing 30 ml of MS culture medium [ 24 ] plus 30 g/l sucrose (Sigma, St. Louis, MO, USA). The pH of the medium was adjusted to 5.8 ± 0.1 prior to the addition of the gelling agent (2.5 g/l Phytagel–Sigma, St. Louis, MO, USA).…”
Section: Methodsmentioning
confidence: 99%
“…After the germination of somatic embryos-like structures and plant growths, the acclimatization process was carried out [ 24 ]. After washing in running water to eliminate the culture medium, the plants were inoculated in plastic cups of 500 mL containing commercial substrate Bioplant®, which were accommodated in trays, covered with plastic bags and taken for incubation for 10 days in incubator chamber (Percival, model I-30NL), with temperature of 25 °C and photoperiod of 16 h. After acclimatization, the surviving plants were transferred to greenhouse to complete growth.…”
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.
“…4 B). These results indicate high efficiency of the somatic embryogenesis process of P. hispidinervum induced by NAA and BAP, similar to the one mentioned for P. aduncum [ 24 ]. Fig.…”
Section: Resultssupporting
confidence: 83%
“…It is noteworthy, the apparently positive role of oxidation of primary calluses in the induction of somatic embryogenesis of P. hispidinervum , previously reported also in P. aduncum [ 24 ] . The formation of somatic embryos from oxidized calluses is not a feature limited to species of the genus Piper .…”
Section: Resultsmentioning
confidence: 74%
“…According to statistical analysis, the treatments 2.5 mg/l BAP and 5 mg/l NAA + 2.5 mg/l BAP provided each, after 60 days of cultivation, 32% of primary callus formation (Table 1 ). Significantly higher percentage of primary calluses, mean value of 87%, was obtained by [ 24 ] in leaf segments of P. aduncum also cultivated in medium with 5 mg/l NAA + 2.5 mg/l BAP. Fig.…”
Section: Resultsmentioning
confidence: 88%
“…Initially, the seeds of the different accesses were subjected to disinfestation, characterized by immersion for three minutes in 70% ethanol (v/v), followed by immersion for 20 min in sodium hypochlorite (NaOCl) (2.0–2.5% active chlorine) and four washings in distilled and autoclaved water. Then, the seeds were inoculated in vials containing 30 ml of MS culture medium [ 24 ] plus 30 g/l sucrose (Sigma, St. Louis, MO, USA). The pH of the medium was adjusted to 5.8 ± 0.1 prior to the addition of the gelling agent (2.5 g/l Phytagel–Sigma, St. Louis, MO, USA).…”
Section: Methodsmentioning
confidence: 99%
“…After the germination of somatic embryos-like structures and plant growths, the acclimatization process was carried out [ 24 ]. After washing in running water to eliminate the culture medium, the plants were inoculated in plastic cups of 500 mL containing commercial substrate Bioplant®, which were accommodated in trays, covered with plastic bags and taken for incubation for 10 days in incubator chamber (Percival, model I-30NL), with temperature of 25 °C and photoperiod of 16 h. After acclimatization, the surviving plants were transferred to greenhouse to complete growth.…”
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.
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