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Pesquisa Agropecuária Brasileira

Print version ISSN 0100-204XOn-line version ISSN 1678-3921

Pesq. agropec. bras. vol.39 no.2 Brasília Feb. 2004 



Rescue of a non-viable accession and rapd analysis of recovered plants of Arachis retusa


Resgate de um acesso inviável de Arachis retusa e análise das plantas recuperadas por RAPD



Rachel Fatima GagliardiI; Georgia Pereira PachecoI; Carlos Alberto OliveiraII; Leonardo Alves CarneiroI; José Francisco Montenegro VallsIII; Maria Lucia Carneiro VieiraII; Elisabeth MansurI

IUniversidade do Estado do Rio de Janeiro, Laboratório de Micropropagação e Transformação de Plantas, Rua São Francisco Xavier, 524-PHLC, sala 505, Maracanã, CEP 20550-013 Rio de Janeiro, RJ, Brazil. E-mail:,,,
IIEscola Superior de Agricultura Luiz de Queiroz, Dep. de Genética, Caixa Postal 83, CEP 13418-900 Piracicaba, SP, Brazil. E-mail:,
IIIEmbrapa Recursos Genéticos e Biotecnologia, Caixa Postal 02372, CEP 70770-900 Brasília, DF, Brazil. E-mail:




In vitro regeneration of Arachis retusa was examined for the purpose of germplasm renewal and conservation. Random amplified polymorphic DNA (RAPD) fingerprinting was used to evaluate the genetic stability of plants derived from embryo axes and apical segments. Ten arbitrary decamer primers were screened and five of them were selected. Ninety genomic regions were evaluated, with an average of 18 loci per clone. All amplified segments were monomorphic. The results indicate that recovered plants are genetically stable at the assessed genomic regions and that both regeneration processes are suitable for in vitro germplasm preservation of Arachis species.

Index terms: germplasm conservation, micropropagation, molecular characterization, genetic stability.


A regeneração in vitro de Arachis retusa foi avaliada visando à renovação e conservação de germoplasma. A estabilidade genética de plantas derivadas de eixos embrionários e segmentos apicais foi avaliada por RAPD. Foram analisados dez oligonucleotídeos decâmeros arbitrários, dos quais cinco foram selecionados. Noventa regiões genômicas foram avaliadas, com uma média de 18 loci por clone. Todos os segmentos amplificados foram monomórficos. Estes resultados indicam que as plantas são geneticamente estáveis nas regiões genômicas examinadas e que ambos os processos são adequados para a conservação in vitro do germoplasma de Arachis.

Termos para indexação: conservação de germoplasma, micropropagação, caracterização molecular, estabilidade genética.



Some wild species of Arachis are endemic to Brazil and occur in areas degraded by human actions. This situation could lead to the loss of germplasm potencially useful for breeding programs and also to the reduction of genetic variability within populations. Therefore, it is highly desirable to develop appropriate in vitro conservation techniques, which may offer an alternative to seed banks for germplasm multiplication, conservation and distribution.

In vitro germplasm conservation relies on micropropagation methods. However, phenotypic and genetic variation are reported to occur during in vitro regeneration processes, originating somaclonal variants (Kaeppler et al., 2000). Thus, the risks of genetic changes induced by tissue culture and the importance of assessing the genetic stability of the biological material along all phases of storage must be considered in the context of conservation.

Random amplified polymorphic DNA (RAPD) fingerprinting has been considered a very suitable method for detecting molecular alterations in plants regenerated in vitro by different processes (Goto et al., 1998; Plader et al., 1998; Rout et al., 1998). In Arachis, this technique has also been used to evaluate genetic diversity (He & Prakash, 1997; Subramanian et al., 2000; Dwivedi et al., 2001), phylogenetic relationships within wild species, interspecific polymorphisms (Galgaro et al., 1998) and introgression analysis of interspecific hybrids (Garcia et al., 1995).

In this work, non-viable seeds of A. retusa Krapov., W.C. Gregory & Valls were rescued and multiplied by tissue culture and the genetic stability of plants originated was evaluated by RAPD analysis.

Seeds of A. retusa (accession V9950, BRA-022969, originally collected in Goiás State, Central Brazil) were provided by the seed bank of Embrapa Recursos Genéticos e Biotecnologia. Primary cultures were obtained from embryo axes as previously reported (Gagliardi et al., 2000). Primary plants derived from those explants were multiplied by culturing apical segments on MS medium with 2.7 mM NAA (Gagliardi et al., 2002).

Total DNA was extracted from sets of 4-7 plants derived from both explant types, using the CTAB procedure (Doyle & Doyle, 1987). Ten primers were assayed and five of them were selected (Table 1). RAPD reactions were performed in a PTC-100 MJ thermocycler at a final volume of 25 mL containing 10–20 ng of genomic DNA; 10 mM Tris-HCl pH 8.8; 50 mM KCl; 200 mM dNTP; 50 mM each primer; 4 mM MgCl2 and 2U Taq polymerase. After denaturing the DNA at 94°C for 5 min, 45 cycles of 94°C for 1 min, 40°C for 1 min, and 72°C for 2 min were performed, followed by a final extension step at 72°C for 6 min (Vieira et al., 1997). RAPD products were analyzed by electrophoresis (3V/cm) on 1.4% agarose gels and visualized after ethidium bromide staining. Gels were photographed under UV light and data were scored for the presence or absence of amplification products.



The culture of embryo axes in presence of 4.4 mM BAP resulted in multishoot formation and indirect organogenesis after the development of preexisting meristems, confirming previous results (Gagliardi et al., 2000). Shoots were rooted on MS devoid of growth regulators and used for RAPD analysis. The ten primers used in this work were previously employed in order to establish phylogenetic relationships in Arachis and Stylosanthes (Vieira et al., 1997; Subramanian et al., 2000; Dwivedi et al., 2001). Five of those primers were selected on the basis of the number of RAPD loci and good reproducibility.

Ninety genomic regions (loci) were evaluated, with an average of 18 loci per A. retusa clone (Table 1). All RAPD loci were monomorphic in the plantlets obtained both from embryo axes and apical segments (Figure 1). Among the several investigators that have addressed the issue of genetic stability of micropropagated plants using RAPD, many have also found monomorphic loci, showing no intraclonal variation (Isabel et al., 1993; Valles et al., 1993; Rout et al., 1998). RAPD analysis is able to detect variations in the whole genome as the loci are scattered in all chromosomes and both repetitive and non-repetitive sequences are amplified. From this perspective, the recovered shoots evaluated in this work seem to be genetically stable at the genomic regions tested, indicating that these regeneration processes can be used for in vitro conservation of Arachis germplasm. Nevertheless, considering that the absence of in vitro-induced polymorphism does not exclude the possibility of chromosomal alterations, additional analysis as cytogenetics and AFLP (Amplified Fragments Length Polymorphism) must be considered to further evaluation of the genetic fidelity of these in vitro clones.




This work was supported by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro and Conselho Nacional de Desenvolvimento Científico e Tecnológico.



DOYLE, J.J.; DOYLE, J.L. A rapid DNA isolation procedure to small amounts of fresh leaf tissue. Phytochemical Bulletin, v.19, p.11-15, 1987.        [ Links ]

DWIVEDI, S.L.; GURTU, S.; CHANDRA, S.; YUEJIN, W.; NIGAM, N. Assessment of genetic diversity among selected groundnut germplasm - I: RAPD analysis. Plant Breeding, v.120, p.345-349, 2001.        [ Links ]

GAGLIARDI, R.F.; PACHECO, G.P.; COCULILO, S.P.; VALLS, J.F.M.; MANSUR, E. In vitro plant regeneration from seed explants of wild groundnut species (Genus Arachis, Section Extranervosae). Biodiversity and Conservation, v.9, p.943-951, 2000.        [ Links ]

GAGLIARDI, R.F.; PACHECO, G.P.; VALLS, J.F.M.; MANSUR, E. Germplasm preservation of wild Arachis species through culture of shoot apices and axillary buds from in vitro plants. Biologia Plantarum, v.45, p.353-357, 2002.        [ Links ]

GALGARO, L.; LOPES, C.R.; GIMENES, M.; VALLS, J.F.M.; KOCHERT, G. Genetic variation between several species of sections Extranervosae, Caulorrhizae, Heteranthae and Triseminatae (genus Arachis) estimated by DNA polymorphism. Genome, v.41, p.445-454, 1998.        [ Links ]

GARCIA, G.M.; STALKER, H.T.; KOCHERT, G. Introgression analysis of an interspecific hybrid population in peanuts (Arachis hypogaea L.) using RFLP and RAPD markers. Genome, v.38, p.166-176, 1995.        [ Links ]

GOTO, S.; THAKUR, R.C.; ISHII, K. Determination of genetic stability in long-term micropropagated shoots of Pinus thunbergii Parl. using RAPD markers. Plant Cell Reports, v.18, p.193-197, 1998.        [ Links ]

HE, G.; PRAKASH, C.S. Identification of polymorphic DNA markers in cultivated peanut (A. hypogaea L.). Euphytica, v.97, p.143-149, 1997.        [ Links ]

ISABEL, N.; TREMBLAY, L.; MICHAUD, M.; TREMBLAY, F.M.; BUSQUET, J. RAPDs as an aid to evaluate the genetic integrity of somatic embryogenesis-derived populations of Picea mariana (Mill.) B.S.P. Theoretical and Applied Genetics, v.86, p.81-87, 1993.        [ Links ]

KAEPPLER, S.M.; KAEPPLER, H.F.; RHEE, Y. Epigenetic aspects of somaclonal variation in plants. Plant Molecular Biology, v.43, p.179-188, 2000.        [ Links ]

PLADER, W.; MALEPSZY, S.; BURZA, W.; RUSINOWSKI, Z. The relationship between the regeneration system and genetic variability in the cucumber (Cucumis sativus L.). Euphytica, v.103, p.9-15, 1998.        [ Links ]

ROUT, G.R.; DAS, P.; GOEL, S.; RAINA, S.N. Determination of genetic stability of micropropagated plants of ginger using random amplified polymorphic DNA (RAPD) markers. Botanical Bulletin of Academia Sinica, v.39, p.23-27, 1998.        [ Links ]

SUBRAMANIAN, V.; GURTU, S.; NAGESWARA, R.C.; NIGAM, S.N. Identification of DNA polymorphism in cultivated groundnut using random amplified polymorphic DNA (RAPD) assay. Genome, v.43, p.656-660, 2000.        [ Links ]

VALLES, M.P.; WANG, Z.Y.; MONTAVON, P.; PTRYCUS, I.; SPANGENBERG, G. Analysis of genetic stability of plants regenerated from suspension cultures and protoplasts of meadow fescue (Fetusca pratensis Huds.). Plant Cell Reports, v.12, p.101-106, 1993.        [ Links ]

VIEIRA, M.L.C.; FUNGARO, M.H.P.; JUBIER, M.F.; LEJEUNE, B. Determination of taxonomic relationships among Brazilian taxa of Stylosanthes Sw., Leguminosae, using RAPD markers. Pesquisa Agropecuária Brasileira, v.32, p.305-310, 1997.        [ Links ]



Received on April 10, 2003 and accepted on November 27, 2003

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