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Brazilian ginseng [ glomerata (Spreng.) Pedersen] germplasm conservation

Alves, R. B. N1.; Mendes, R. A.1; Mendes, M. A.1; Carneiro, R. M. D. G.1; Silva, D. B.1; Cardoso, L. D.1; Salomão, A. N.1; Vieira, R. F.1 1Embrapa Recursos Genéticos e Biotecnologia, Caixa Postal 02372, Brasília, D.F., . E-mail: [email protected]

ABSTRACT: Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen] germplasm conservation. The main goal of this project was to establish a Brazilian ginseng (Pfaffia glomerata) germplasm collection. In 2001, Embrapa Genetic Resources and Biotechnology, in collaboration with the Emater Paraná (Paraná State Rural Assistance Coorporation), and São Paulo State University (Unesp), Botucatu, has launched a field expedition to the borders of the rivers Paraná, Ivaí, and Paranapanema, including areas in the States of Mato Grosso do Sul, Paraná and São Paulo. The total germplasm collection has 15 accessions, including 145 individuals, which are maintained at field, in vitro and greenhouse conditions.

Key words: fáfia, , Pfaffia, genetic resources, ex situ conservation.

INTRODUCTION high risk to this species survival as well as to raw material supply (Magalhães, 2000). Pfaffia glomerata (Amaranthaceae) is a large, The main goal of this project was to establish shrubby ground vine, which has a deep root system. a Brazilian ginseng germplasm collection. It grows mainly at the borders of Paraná river, and predatory collection has greatly reduced its natural MATERIAL AND METHOD populations. Pfaffia is known as “brazilian ginseng”, since it is widely used as an adaptogen for many material ailments and to promote weakless, much like American and Asian ginseng (Panax spp.). In May, 2001, Embrapa Genetic Resources This action is attributed to the anabolic agent, and Biotechnology, in collaboration with the Emater beta-ecdysterone, as well as three new ecdysteroid Paraná (Paraná State Rural Assistance Coorporation), glycosides, which are found in high amounts in P. and São Paulo State University (Unesp), Botucatu, glomerata roots. The root of Pfaffia also contains has launched a field expedition (Fig. 1) to the borders about 11% saponins. These saponins include a group of the rivers Paraná, Ivaí, and Paranapanema, of novel chemicals called pfaffosides as well as pfaffic including areas of the States of Mato Grosso do acids, glycosides, and nortriperpenes. These saponins Sul, Paraná and São Paulo (Table 1). Fifteen have clinically demonstrated the ability to inhibit accessions of Brazilian ginseng [Pfaffia glomerata cultured tumor cell melanomas and to help regulating (Spreng.) Pedersen], Amaranthaceae, including 145 blood sugar levels. individuals, were collected by cuttings, labeled, Brazilian ginseng exploitation is mostly made enrolled in a wet toilet paper and placed in a plastic by small growers and local communities (Corrêa bag. Cuttings of each individual were planted and Júnior, 2003). This type of collection represents an maintained at greenhouse conditions until complete additional family income, and is the only activity they rooting. This material has been transferred to both do outside their properties. field and in vitro germplasm collections. Voucher Although most of the pharmacological studies specimens were identified by Dr. Josafá Carlos on Brazilian ginseng have been done in Japan, the Siqueira (taxonomist of Amaranthaceae) and stored raw material from this species is still collected from in Embrapa Genetic Resources and Biotechnology wild populations in Brazil (Magalhães, 2000). It is herbarium (CEN). estimated that around 720 ton of P. glomerata roots and 190 ton of powder of Brazilian ginseng have been Conservation strategies traded onto the national and external market in 2002. Field and seed conservation Most of raw material from wild populations is collected at the margins of Paraná river (Fig.1), leading to a A sample of each individual was transplanted to field conditions, although the original collection was Recebido para publicação em agosto/2004 kept at greenhouse conditions. During October/2001 Aceito para publicação em julho/2006

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TABLE 1. Germplasm collection of the Brazilian Ginseng (Pfaffia glomerata) from the States of Paraná, São Paulo and Mato Grosso do Sul, 2001.

a F=Field collection; I=In vitro collection bPN = National Park the samples were planted in the Experimental Field Germination tests were performed on seeds of Embrapa Genetic Resources and Biotechnology, from accessions 2209, 2211, 2217, 2219 and 2221 at Brasília, DF. The field is located at 15o46’ south latitude 25ºC incubation temperature in gerbox with four and 47o55’ west longitude, at an altitude of 1079 replications of 100 seeds. Seed moisture content was meters, and the soil in this area is oxysoil with clay determined with three replicates of 20 seeds by oven texture. Each individual was planted in a 0,5x2m drying at 105ºC for 24 h. The results were expressed spaces that were irrigated throughout the year by as an average of percentages on fresh weight basis. sprinkler system irrigation.

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FIGURE 2 - Germination percentages and moisture content of Pfaffia glomerata seeds from five accessions.

In vitro conservation molecular studies and subsequent chemical Ten accessions were introduced at in vitro characterization of β-ecdysone, aiming the conditions for conservation. Apexes were collected identification of the genotypes best adapted to the from growing at field conditions to begin the in local conditions. vitro culture. The apexes were disinfested and disinfected using a sodium hypoclorite solution at 2% RESULT AND DISCUSSION concentration for 20 minutes prior to being inoculated A field and in vitro germplasm collection has into tubes. The initial media used was composed of been established at Embrapa Genetic Resources and micro and macronutrients of MS (Murashige & Skoog, Biotechnology. A preliminary survey has shown the 1962) on half concentration plus vitamins (5g.L-1 myo- presence of pathogenic fungi and root-knot nematodes Inositol, 25mg.L-1 Nicotinic acid, 25mg.L-1 Pyridoxine, at field conditions. Septoria spot was detected in 50mg.L-1 Thiamine, 100mg.L-1 Glycine and 100mg.L- leaves of P. glomerata (Mendes et al. 2003) and 1 Calcium pantothenate) and 20mg.L-1 Giberelic acid Meloidogyne incognita, M. javanica and Meloidogyne 3, 100mg.L-1 Ascorbic acid, 100mg.L-1 Calcium nitrate, sp. has been detected in roots. Germplasm collection 20mg.L-1 Putrecin, 100mg.L-1 L-Arginine and 0,4mg.L- will be useful to find sources of pathogenic fungi and 1 Kinetin. The plantlets were multiplied into the same nematode resistance. The presence of pathogenic media before they were transferred to the conservation fungi and nematodes has created a challenge to media composed of MS complete concentration of maintain this collection at field condition. micro and macronutrients plus vitamins (10g.L-1 myo- Germination percentage varied from 12 to 67% Inositol, 50mg.L-1 Nicotinic acid, 50mg.L-1 Pyridoxine, and moisture content varied from 9,6 to 12,5% (Figure 2). 100mg.L-1 Thiamine, 200mg.L-1 Glycine and 200mg.L- Seeds from each accessions were desiccated to 8,0% 1 Calcium pantotenate). They were earlier cultivated moisture content and stored at -20oC. in a growth chamber at 25±2°C, photoperiod of 12 A protocol for in vitro conservation for Pfaffia hours and light intensity of 35ì M.m-2s-1 for about 10 glomerata accessions was successfully established, days before being transferred to the conservation and nine accessions containing 80 individuals have chamber at 20±2°C, photoperiod of 12 hours and light been deposited in our in vitro collection. intensity of 68ì M.m-2s-1. The Brazilian ginseng in vitro The challenge now is to promote the conservation activities started in the second semester chemical, agronomical, and genetic characterization of 2002. The time period between subcultures was of of these accessions in order to identify materials for 16 months. use in breeding programs, including nematodes and fungi resistance, and high content of beta-ecdysone. Germplasm exchange The domestication of this species will reduce the In 2004, cuttings of all the accessions were exploitation from wild population. sent to Viçosa Federal University for agronomic and

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ACKNOWLEDGEMENT Botucatu, 2003. MAGALHAES, P. M. . Agrotecnología para el cultivo de Authors wish to thank Mr. Aécio Amaral fáfia o ginseng brasilero. In: José Vicente Martínez A.; Santos, Dr. Lin C. Ming (Unesp), Mr. Mineiro (Emater- Henry Yesid Bernal; Armando Cáceres. (Org.). Fundamentos de Agrotecnología de Cultivo de PR) and the director of Ilha Grande National Park for Plantas Medicinales iberoamericanas. 1/1 ed. their support and help during our field expedition. Santafé de Bogotá, 2000, v. , p. 323-332. MURASHIGE, T; SKOOG, F. A revised medium for rapid REFERENCE growth and bio assays with tobacco tissue cultures, CORRÊA JUNIOR, C. Estudo Agronômico de [Pfaffia Physiol. Plant., v.15, p.473-497, 1962. glomerata (Spreng.) Pedersen]: Sazonalidade na MENDES, M. A. S., OLIVEIRA, A. S. Mancha de Septoria Produção de Raízes e Conteúdos de b-ecdisona em Pfaffia glomerata no Distrito Federal. In: em Diferentes Acessos de São Paulo, Paraná e Congresso Brasileiro de Fitopatologia, 36, 2003, Mato Grosso do Sul. 2003. 94f. Tese (Doutorado em Uberlândia, MG. Fitopatologia Brasileira. Brasília, Agronomia/Horticultura) – Faculdade de Ciências DF: Sociedade Brasileira de Fitopatologia, 2003. Agronômicas, Universidade Estadual Paulista, p.097.

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