372 AJCS 13(03):372-379 (2019) ISSN:1835-2707 doi: 10.21475/ajcs.19.13.03.p1209 Conservation of Hibiscus acetosella germplasm by seed cryopreservation Rodrigo Miranda Moraes 1 , Fernanda Carlota Nery 2 , Mayara Caroline Carvalho Pinto 1 , Renato Paiva 1 , Diogo Pedrosa Corrêa da Silva 1 , Patrícia Duarte de Oliveira Paiva 1 , Sandro Barbosa* 3 1 Universidade Federal de Lavras – UFLA, Setor de Fisiologia Vegetal, Campus universitário, CEP 37200-000, Lavras- MG, Brazil 2 Universidade Federal de São João del-Rei – UFSJ, Departamento de Engenharia de Biossistemas, Praça Dom Helvécio, 74, CEP 36301-160, São João del-Rei – MG, Brazil 3 Universidade Federal de Alfenas, Instituto de Ciências da Natureza, Rua Gabriel Monteiro da Silva, 700, CEP 37130- 000, Alfenas – MG, Brasil *Corresponding author: sandrobiogen@gmail.com; sandro.barbosa@unifal-mg.edu.br Abstract Hibiscus acetosella (Malvaceae) is a shrub of great importance for landscaping, food and medicinal purposes. The objective of this study was to preserve H. acetosella germplasm by seed cryopreservation. Half of the seed batch was scarified and the other half was kept intact. Cryopreservation occurred by immersion in liquid nitrogen for 1 hour. Moisture content (MC%), germination percentage (G%), germination speed index (GSI), normal seedling formation (NS%), shoot length (SL), dry matter (DM), biometry and plant survival were evaluated after treatment. MC% ranged between 7.7% and 6.65% in intact and scarified seeds, respectively. Scarification raised G% and GSI compared to intact seeds. Intact and scarified seeds had 100% and 70% NS%, respectively, when not cryopreserved. Cryopreservation reduced NS% to 62% and 12.75%, respectively. The highest SL was observed in intact and non- cryopreserved seeds, with an average of 10.21 cm in height. However, the cryopreservation of intact seeds reduced SL by about 50%, and scarification led to a further reduction, either with (3.32 cm) or without (2.47 cm) cryopreservation. Seedlings from intact and non-cryopreserved seeds showed higher DM in relation to seedlings from cryopreserved seeds. The association of cryopreservation and scarification further reduced DM. The cryopreservation of intact seeds yielded 100% survival at the end of the acclimatization process. However, cryopreservation of scarified seeds reduced the survival percentage to 15%. Changes in color were observed for seeds scarified and subjected to cryopreservation. Thus, cryopreservation is considered an efficient technique for the conservation of intact H. acetosella seeds in the long term. Keywords: In vitro conservation; Medicinal plants; Mechanical scarification; Vinagreira roxa. Abbreviations: MC%_moisture content; G%_germination percentage; GSI_germination speed index; NS%_percentage of normal seedling formation; SL_shoot length; DM dry matter. Introduction The genus Hibiscus L. is the largest in the family Malvaceae, and consists of about 300 species (Rocha and Neves, 2000), which are appreciated for the beauty of their flowers and also for their medicinal properties (Ojeda et al., 2010; Tsumbu et al., 2012). Among the species of the genus Hibiscus L., Hibiscus acetosella Welw., a shrub native to Africa, popularly known in Brazil as vinagreira roxa, groselheira or quiabo-roxo (Sakhanokho and Kelley, 2009), stands out. This species, which has botanical similarities with the medicinal plant Hibiscus sabdariffa, is mainly exploited for food purposes, and it is consumed fresh, as salads, teas and jellies (Lorenzi, 2008). According to Tsumbu et al. (2011) and (2012), H. acetosella has aroused special interest in its medicinal properties due to its great content of polyphenols, coumarins and flavonoids, which act as antioxidants and anti-inflammatory substances, as well as caffeic acid. This secondary metabolite is also involved in antioxidant (Özyurt et al., 2004), anti- inflammatory (Toyoda et al., 2009) and anticancer activities (Borrelli et al., 2002; Wang et al., 2005), besides the modulation of heart rate and blood pressure (Tan et al., 2005). Due to its extensive exploitation for its medicinal potential, efforts are justified for the conservation of H. acetosella. In addition, preserving the genetic diversity of this species, through appropriate germplasm conservation techniques, is necessary for the maintenance of biological material and management of this resource in the future (Lopes et al., 2013; Pandey et al., 2015). According to Goldfarb et al. (2011), seed germplasm conservation is the most viable form of storage, since it is a more organized system and requires small spaces. Among the ex situ germplasm conservation techniques, cryopreservation stands out as a long-term conservation method. This technique is efficient in preserving biological