Vegetos
(An International Journal of Plant Research & Biotechnology)
Cryopreservation and photomixotrophic cultivation of Pfaffia glomerata (Spreng.) pedersen genotypes enable the conservation of a valuable medicinal resource
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Caproni Duanny Thais Rodrigues, Torres-Silva Gabriela, Batista Diego Silva, Cruz Ana Cláudia Ferreira, Felipe Sérgio Heitor Sousa, Chagas Kristhiano, Pinheiro Marcos Vinícius Marques, Otoni Wagner Campos, Torres-Silva Gabriela, Torres-Silva Gabriela
Research Articles | Published: 07 March, 2025
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Keywords: Long-term conservation, Cryopreservation, Genetic resources, Ecdysone, Brazilian-ginseng
Abstract
Pfaffia glomerata, commonly known as Brazilian-ginseng, attracts significant pharmaceutical interest due to its β-ecdysone content. However, the extraction of phytotherapeutic agents can threaten the existence of medicinal plants and contribute to their genetic erosion. Cryopreservation is the most promising technique for the long-term maintenance of plant genetic resources. This study aimed to establish a cryopreservation method for different accessions of P. glomerata, analyzing the impact of pre-cultivation, saturation, and vitrification solutions. Optimal cryopreservation involved pre-cultivation with 0.75 M sucrose at 4 °C under constant agitation for 24 h, followed by incubation in 2.0 M glycerol and 0.4 M sucrose for 20 min, and immersion in a plant vitrification solution (comprising 30.0% glycerol, 15.0% ethylene glycol, 15.0% dimethyl sulfoxide, and 13.6% sucrose) for 1 h. This method enabled the survival and regeneration of explants after exposure to liquid nitrogen. Next, the impact of photoautotrophic and photomixotrophic cultivation on the hardening of explants at ultra-low temperatures was assessed. Photoautotrophic conditions increased the proportion of vacuoles and limited nuclear development; whereas photomixotrophic cultivation of four P. glomerata accessions supplemented with 4.0 or 40.0 μM abscisic acid and 10.0 or 100.0 μM salicylic acid was more successful. Pre-acclimatization was found to be more efficient with 40 μM abscisic acid, and survival was greater in accessions with a higher accumulation of β-ecdysone.
References
Bettoni JC, Bonnart R, Shepherd AN, Kretzschmar AA, Volk GM (2019) Successful cryopreservation of Vitis vinifera Cv. ‘Chardonnay’ from both in vitro and growth chamber source plants. Acta Hortic 1234:211–218
Bharuth V, Naidoo C (2020) Responses to cryopreservation of recalcitrant seeds of Ekebergia capensis from different provenances. S Afr J Bot 132:1–14
Bissati S, Boujenah S, Morisset C, Chenchouni H (2020) Does pre-culture in sugar-rich media affect carbohydrate content and post-thawing recovery rate of cryopreserved potato (Solanum phureja) shoot tips? J King Saud Univ Sci 32:1917–1924
Bradaï F, Bastante JA, Romero CS (2023) Effect of sucrose preculture and culture inoculum density on cryopreservation of Olive somatic embryos. Sci Hortic 322:112–123
Coelho MAN, Moraes MA (2012) Pfaffia glomerata (AMARANTHACEAE). Lista Vermelha da Flora Brasileira: Centro Nacional de Conservação da Flora/ Instituto de Pesquisas Jardim Botânico do Rio de Janeiro. Available in https://proflora.jbrj.gov.br/html/Pfaffia glomerata_2012.html. Access in: 27 nov. 2024
Corrêa JPO, Vital CE, Pinheiro MVM, Batista DS, Azevedo JFL, Saldanha CW, Cruz ACF, DaMatta FM, Otoni WC (2015) In vitro photoautotrophic potential and ex vitro photosynthetic competence of Pfaffia glomerata (Spreng.) pedersen accessions. Plant Cell Tiss Org Cult 121:289–230
Corrêa JPO, Vital CE, Pinheiro MVM, Batista DS, Saldanha CW, Cruz ACF, Notini MM, Freitas DMS, DaMatta FM, Otoni WC (2016) Induced polyploidization increase 20-hydroxyecdysone content, in vitro photoautotrophic growth and ex vitro biomass accumulation in Pfaffia glomerata (Spreng.) Pedersen. In Vitro Cell Dev Biol - Plant 52:45–55
Cruz CD (2013) GENES - a software package for analysis in experimental statistics and quantitative genetics. Acta Sci Agron 35:271–276
Dias FCR, Gomes MLM, Melo FCSA, Menezes TP, Martins AL, Cupertino MC, Otoni WC, DaMatta SLP (2020) Pfaffia glomerata hydroalcoholic extract stimulates penile tissue in adult Swiss mice. J Ethnopharmacol 261:113–132
Dias FCR, Matta SLP, Lima GDA, Souza ACF, Menezes TP, Melo FCSA, Otoni WC, Neves MM, Gomes LM (2023) Pfaffia glomerata polyploid accession compromises male fertility and fetal development. J Ethnopharmacol 314:116–129
Engelmann F (2011) Use of biotechnologies for the conservation of plant biodiversity. Vitro Cell Dev Biol - Plant 47:5–16
Engelmann F, Ramanatha R (2012) Major research challenges and directions for future research. In: Normah MN, Chin HF, Reed BM (eds) Conservation of tropical plant species. Springer, Berlin, pp 511–526
Fang X, Szoltysik R, Tang J, Bajkacz S (2022) Efficient extraction and sensitive HPLC-MS/MS quantification of selected ecdysteroids in plants. J Food Compost Anal 110:104–118
Felipe SHS, Batista DS, Vital CE, Chagas K, Silva PO, Silva TD, Fortini EA, Correia LNF, Ávila RT, Maldaner J, Festucci-Busellif RA, DaMatta FM, Otoni WC (2019) Salinity-induced modifications on growth, physiology and 20-hydroxyecdysone levels in Brazilian-ginseng [Pfaffia glomerata (Spreng.) Pedersen]. Plant Physiol Biochem 140:43–54
Festucci-Buselli RA, Contim LAS, Barbosa LCA, Stuart JJ, Otoni WC (2008) Biosynthesis and potential functions of the ecdysteroid 20-hydroxyecdysone – a review. Botany 86:978–987
Fraga HPF, Vieira LN, Puttkammer CC, Silva JM, Anjos KG, Oliveira EM, Guerra MP (2016) High-efficiency cryopreservation of Araucaria angustifolia (Bertol.) Kuntze embryogenic cultures: ultrastructural characterization and morpho-physiological features. Plant Cell Tiss Org Cult 124:307–318
Franco RR, Franco RM, Justino AB, Borges ALS, Bittar VP, Saito N, Saraiva AL, Nicolau N Jr., Otoni WC, Espíndola FS (2024) Phytochemical composition of aerial parts and roots of Pfaffia glomerata (Spreng.) Pedersen and anticholinesterase, antioxidant, and antiglycation activities. Protoplasma 261:609–624. https://doi.org/10.1007/s00709-023-01916-9
Gomes SSL, Saldanha CW, Neves CS, Trevizani M, Raposo NRB, Notini MM, Santos MO, Campos JMS, Otoni WC, Viccini LF (2014) Karyotype, genome size, and in vitro chromosome doubling of Pfaffia glomerata (Spreng.) pedersen. Plant Cell Tiss Org Cult 118:45–56
Gonzalez-Arnao MT, Panta A, Roc AWM, Roosevelt H, Engelmann F (2008) Development and large scale application of cryopreservation techniques for shoot and somatic embryo cultures of tropical crops. Plant Cell Tiss Org Cult 92:1–13
Heringer AS, Steinmacher DA, Schimidt EC, Bouzon ZL, Guerra MP (2013) Survival and ultrastructural features of Peach palm (Bactris Gasipes Kunth) somatic embryos submitted to cryopreservation through vitrification. Protoplasma 250:1185–1193
Htwe CSS, Rajkumar S, Pathania P, Agrawal A (2023) Transcriptome profiling during sequential stages of cryopreservation in banana (Musa AAA Cv Borjahaji) shoot meristem. Plants 12:116–125
Iarema L, Cruz ACF, Saldanha CW, Dias LLC, Vieira RF, Oliveira EJ, Otoni WC (2012) Photoautotrophic propagation of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]. Plant Cell Tiss Org Cult 110:227–238
Kamada T (2006) Avaliação da diversidade genética de populações de fáfia (Pfaffia glomerata (Spreng.) Pedersen) por RAPD, caracteres morfológicos e teor de beta-ecdisona. Tese de Doutorado, Universidade Federal de Viçosa, Viçosa, MG, Brazil. Available online: <http://locus.ufv.br/handle/123456789/1298. Access in: 27 nov. 2024
Karnovsky MJA (1965) Formaldehyde–glutaraldehyde fixative of high osmolarity for use in electron microscopy. J Cell Biol 27:127–128
Khoddamzadeh AA, Sinniah UR, Lynch P, Kadir MA, Kadzimin SB, Mahmood M (2011) Cryopreservation of protocorm-like bodies (PLBs) of Phalaenopsis Bellina (Rchb.f.) christenson by encapsulation-dehydration. Plant Cell Tiss Org Cult 107:471–481
Matsumoto T (2017) Cryopreservation of plant genetic resources: conventional and new methods. J Agric Sci 5:13–20
Ming-Hua Y, Sen-Rong A (2010) Simple cryopreservation protocol of Dioscorea bulbifera L. embryogenic calli by encapsulation-vitrification. Plant Cell Tiss Org Cult 101:349–358
Mosa KA, Ahmed AE, Hazem Y, Kanawati IS, Abdullah A, Hernandez-Sori L, Ali MA, Vendrame W (2023) Insights into cryopreservation, recovery and genetic stability of medicinal plant tissues. Fitoterapia 169:105–117
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497
Nascimento EX, Mota JH, Vieira MC, Zárate NAH (2007) Produção de Biomassa de Pfaffia glomerata (Spreng.) pedersen e Plantago major L. em cultivo Solteiro e consorciado. Cienc Agrotecnol 31:742–730
Nausch H, Buyel JF (2021) Cryopreservation of plant cell cultures – Diverse practices and protocols. New Biotechnol 62:86–95
Nguyen QT, Xiao Y, Kozai T (2016) Photoautotrophic Micropropagation. In Toyoki Kozai, Genhua Niu, Michiko Takagaki editors: Plant Factory, Burlington: Academic Press, pp. 271–283. ISBN: 978-0-12-801775-3
O’Brien TP, McCully M (1981) The study of plant structure principles and select methods. Termarcarphi Pty Ltd, Melbourne
Oliveira DV, Santos IRI, Martins IS, Salomão AN (2019) Cryopreservation of shoot tips of Brazilian ginseng (Pfaffia glomerata (Spreng.) Pedersen) by vitrification. J Agric Sci 11:146–155
Pathirana R, McLachlan A, Hedderley D, Panis B (2016) Pre-treatment with Salicylic acid improves plant regeneration after cryopreservation of grapevine (Vitis spp.) by droplet vitrification. Acta Physiol Plant 38:12–21
POWO (2024). Plants of the World Online Available online: Access in: 27 https://powo.science.kew.org/
Ren R, Ji Z, Guo J, Yang X (2023) Cryopreservation of herbaceous Asteraceae seeds: effects of seed reserves on seed germination and seedling growth. Cryobiology 112:104–122
Ribeiro STC, Silva TFOD, Castro JC, Cabral MRP, Abreu-Filho BA, Oliveira AJB, Gonçalves RAC (2022) Chemical characterization and bioactivities of Fructans from Pfaffia glomerata roots. Bioact Carbohydr Diet Fibre 27:100–113
Saldanha CW, Otoni CG, Azevedo JL, Dias LLC, Rêgo MM, Otoni WC (2012) A low-cost alternative membrane system that promotes growth in nodal cultures of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]. Plant Cell Tiss Org Cult 110:413–422
Saldanha CW, Otoni CG, Rocha DI, Cavette PC, Detmann KSC, Tanaka FAO, Dias LLC, DaMatta FM, Otoni WC (2014) CO2-enriched atmosphere and supporting material impact the growth, morphophysiology and ultrastructure of in vitro Brazilian-ginseng [Pfaffia glomerata (Spreng.) Pedersen] plantlets. Plant Cell Tiss Org Cult 118:87–99
Savchenko RG, Veskina NA, Odinokov VN, Benkovskaya GV, Parfenova LV (2022) Ecdysteroids: isolation, chemical transformations, and biological activity. Phytochem Rev 21:1445–1486
Senna LR (2024) Pfaffia in Flora e Funga do Brasil. Jardim Botânico do Rio de Janeiro. Available in: <https://floradobrasil.jbrj.gov.br/FB4330. Access in: 27 nov. 2024
Sisunandar S, Rival A, Turquay P, Samosir Y, Adkins S (2010) Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos does not induce morphological, cytological or molecular changes in recovered seedlings. Planta 232:435–447
Vendrame WA, Faria RT (2011) Phloroglucinol enhances recovery and survival of cryopreserved Dendobrium nobile protocorms. Sci Hortic 128:131–135
Villalobos-Oliveira A, Ferreira CF, Yanes-Paz E, Lorente GY, Souza FV, Engelmann F, Martínez-Montero ME, Lorenzo JC (2022) Inter simple sequence repeat (ISSR) markers reveal DNA stability in pineapple plantlets after shoot tip cryopreservation. Vegetos 35:360–366. https://doi.org/10.1007/s42535-021-00327-6
Whelehan LM, Funnekotter B, Bunn E, Mancera RL (2022a) Review: the case for studying mitochondrial function during plant cryopreservation. Plant Sci 315:111–134
Whelehan LM, Dalziell EL, Bunn E, Mancera RL, Funnekotter B (2022b) How does metabolic rate in plant shoot tips change after cryopreservation? Cryobiology 109:1–9
Zhou XI, Ren X, Luo H, Huang L, Liu N, Chen W, Lei Y, Liao B, Jiang H (2022) Safe conservation and utilization of peanut germplasm resources in the oil crops Middle-term genebank of China. Oil Crop Sci 7:9–13
Author Information
Plant Biology Department, Laboratory of Plant Tissue Culture II – BIOAGRO, Federal University of Viçosa (UFV), Viçosa, Brazil