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Journal of Forest Research

ISSN: 1341-6979 (Print) 1610-7403 (Online) Journal homepage:

Designing ex-situ conservation strategies for seedsstorage of Piper aduncum and P. hispidinervumthrough cryopreservation and low-temperaturetechniques

Tatiane Loureiro Da Silva, Hugo Teixeira Gomes & Jonny EversonScherwinski-Pereira

To cite this article: Tatiane Loureiro Da Silva, Hugo Teixeira Gomes & Jonny EversonScherwinski-Pereira (2017) Designing ex-situ conservation strategies for seeds storage of Piperaduncum and P. hispidinervum through cryopreservation and low-temperature techniques, Journalof Forest Research, 22:6, 380-385, DOI: 10.1080/13416979.2017.1381494

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Published online: 28 Sep 2017.

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Designing ex-situ conservation strategies for seeds storage of Piper aduncum andP. hispidinervum through cryopreservation and low-temperature techniquesTatiane Loureiro Da Silvaa, Hugo Teixeira Gomesb and Jonny Everson Scherwinski-Pereira c

aInstituto Federal de Educao, Cincia e Tecnologia do Acre (IFAC), Xapuri, AC, Brazil; bDepartamento de Botnica, PPGBot, Universidade deBraslia, Braslia, DF, Brazil; cEmbrapa Recursos Genticos e Biotecnologia, Braslia, DF, Brazil

ABSTRACTPiper aduncum and Piper hispidinervum are species found primarily in the Amazon region, and havebeen highlighted by the fact that they contain, in their essential oils, significant amounts of dillapioland safrole. The aim of this work was to assess the seed behavior of P. aduncum and P. hispidi-nervum and to develop strategies for mediumto long-term storage of the species at sub-zero andcryogenic temperatures. In the first experiment, seeds were desiccated and their water content wasdetermined. Then, the seeds were kept at 20C for up to 90 days. In the second experiment, seedswere stored for up to 24 h in liquid nitrogen (LN; 196C), with and without the use of cryopro-tectants. Finally, for assessing the efficiency of these techniques, seeds were stored at 20C and inLN for up to 360 days. The germination rate was more than 90% for both species when stored at20C or LN, without compromising their viability and development. The results suggest that P.aduncum and P. hispidinervum have seeds with orthodox behavior, which can be stored for themedium to long term at sub-zero and cryogenic temperatures without the need for use ofcryoprotectants.

ARTICLE HISTORYReceived 5 September 2017Accepted 14 September2017

KEYWORDSPiperaceae; germplasmconservation;cryopreservation;desiccation; vitrification


The high rate of erosion of genetic resources, combinedwith the loss of components of biodiversity, has led severalcountries to conduct research geared toward conservationof their biological resources (Khoury et al. 2010). In Brazil,there is an urgent need to take initiatives to enable rapidadvancement of scientific knowledge on the compositionand conservation of Amazon species, before they begin todisappear, or even before they have even been cataloged andstudied. Among these species, Piper aduncum and Piperhispidinervum, popularly known in the Amazon region aspimenta-de-macaco and pimenta-longa, respectively,stand out due to the secondary compounds that areextracted from the essential oil present in their leaves andbranches in stages. These species also stand out becausethey can be commercially exploited without requiring theircomplete destruction (Souza Filho et al. 2010; Abreu et al.2015).

The essential oil of P. aduncum contains significantquantities (up to 80%) of dillapiol, a phenylpropanoid com-pound responsible for fungicidal, molluscicidal, larvicidal,and insecticidal activity (Brazo et al. 2014). Moreover,several studies also report the effect of this compound inthe control of fungi and insects that attack species of eco-nomic importance (Araujo et al. 2012; Guerrini et al. 2009;Piton et al. 2014), as well as parasites that cause diseases tohumans, such as Trypanosoma cruzi (Batista et al. 2008),Plasmodium falciparum (Cline et al. 2009), and Aedesaegypti (Santana et al. 2015), the etiological agents ofChagas disease, malaria and dengue, and zika, respectively.

The essential oil of P. hispidinervum contains high levels(up to 90%) of a phenyl ether known as safrole, which

currently presents a serious risk of not being able to meetthe growing international demand in the medium- to long-term period (Sauter et al. 2012). Heliotropine is obtainedfrom this compound, which is widely used as a fragrance,and piperonyl butoxide, an essential ingredient for the pro-duction of pyrethrum-based biodegradable insecticides(Barbosa et al. 2012). In view of the economic potentialthat these species have, coupled with the fact that as thereare yet no preservation programs to assure these species willbe maintained, it is essential to develop conservation stra-tegies to prevent them from becoming endangered species.According to Silva and Scherwinski-Pereira (2011), P. adun-cum and P. hispidinervum are included in the list of priorityspecies for conservation. According to the authors, in theseAmazon species, seed preservation is a preventive measurethat should be taken to assure the conservation of theirgenetic resources.

Conservation in seed banks is a methodology aimed atthe medium- to long-term preservation of plant geneticresources. But for the success of this technique, it is neces-sary to know the physiological behavior of the seed to bestored (Hay and Probert 2013). According to Walters(2015), orthodox seeds, which have tolerance to desiccation,have good resistance to sub-zero temperatures. On the otherhand, recalcitrant seedssensitive to decreased water con-tentdo not survive when exposed to low temperatures.

In light of the foregoing, the classification of the physio-logical behavior of seeds of a particular species as orthodoxor recalcitrant is of the utmost importance to define theconservation strategy to be used, given that only orthodoxseeds can be maintained in the medium to long periods incold-storage facilities (Engelmann 2011). Additionally,

CONTACT Jonny Everson Scherwinski-Pereira Embrapa Recursos Genticos e Biotecnologia, Av. W5 Norte (final),70770-917, Braslia, Brazil


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cryopreservation has also emerged as a rather promisingalternative for seeds preservation (Walters et al. 2013).Cryopreservation is the storage of biological material inliquid nitrogen (LN) at a temperature around 196C.Under these conditions, the plant material can be storedfor long periods, because the cell metabolism becomespractically paralyzed, thereby avoiding genetic variabilityand deterioration of the stored propagule (Kaviani 2011).

Given the potential of P. aduncum and P. hispidinervumfor the production of dillapiol and safrole, together with thelack of studies on the physiological behavior and preserva-tion of their seeds, this study aimed at analyzing the toler-ance of these propagules when submitted to a decrease inmoisture content and assessing strategies to promote theirmedium- to long-term conservation using sub-zero andcryogenic temperatures.

Material and methods

The seed lots used for the experiments were obtained fromthe germplasm bank of P. aduncum and P. hispidinervumbelonging to Embrapa Acre, located in the city of RioBranco, in the state of Acre, Brazil.

Morphological characterization, determination ofmoisture content, and tolerance to desiccation

For the morphological characterization, 50 seeds of eachspecies were evaluated for length, width, and thickness; thelength was measured from the base to the apex, and the widthand thickness were measured at the central portion of theseeds. In both cases, the measurements were taken with theaid of a digital caliper (Mitutoyo). In this phase, we alsoevaluated the weight of 10 sub-samples with 100 seeds each,to obtain a weight of 1,000 seeds of each species.

As for the determination of the desiccation curve, the P.aduncum and P. hispidinervum seeds were divided intothree lots of 400 seeds each, which were maintained for 0(control


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