1 Boletim de Pesquisa 245 e Desenvolvimento ISSN Fevereiro, 2009 Encapsulation of Cassava nodal segments for germoplasm storage
2 ISSN Fevereiro, 2009 Empresa Brasileira de Pesquisa Agropecuária Embrapa Recursos Genéticos e Biotecnologia Ministério da Agricultura, Pecuária e Abastecimento Boletim de Pesquisa e Desenvolvimento 245 Encapsulation of Cassava nodal segments for germoplasm storage L. Pedro Barrueto Cid Andréa R. Ramos Cruz Luiz Joaquin Castelo Branco Carvalho Embrapa Recursos Genéticos e Biotecnologia Brasília, DF 2009
3 Exemplares desta edição podem ser adquiridos na Embrapa Recursos Genéticos e Biotecnologia Serviço de Atendimento ao Cidadão Parque Estação Biológica, Av. W/5 Norte (Final) Brasília, DF CEP Caixa Postal PABX: (61) Fax: (61) Comitê de Publicações Presidente: Miguel Borges Secretária-Executiva: Maria da Graça Simões Pires Negrão Membros: Diva Maria de Alencar Dusi Luiz Adriano Maia Cordeiro José Roberto de Alencar Moreira Regina Maria Dechechi G. Carneiro Samuel Rezende Paiva Suplentes: João Batista Tavares da Silva Margot Alves Nunes Dode Supervisor editorial: Maria da Graça Simões Pires Negrão Normalização Bibliográfica: Rosamares Rocha galvão Editoração eletrônica: Maria da Graça Simões Pires Negrão Figura da Capa: Bead after 30 days at 15 C, seen under simple microscope. 1ª edição 1ª impressão (2009): Todos os direitos reservados A reprodução não autorizada desta publicação, no todo ou em parte, constitui violação dos direitos autorais (Lei nº 9.610). Dados Internacionais de Catalogação na Publicação (CIP) Embrapa Recursos Genéticos e Biotecnologia Embrapa 2009
4 Sumário Abstract... 5 Resumo... 6 Introduction... 7 Materials and methods... 7 Results and discussion... 9 Literature... 11
5 Encapsulation of Cassava nodal segments for germoplasm storage L. Pedro Barrueto Cid 1 Andréa R. Ramos Cruz 2 Luiz Joaquin Castelo Branco Carvalho 1 Abstract Caulinary nodes (axillary buds) from in vitro plants of three cassava genotypes were: (A) encapsulated with Ca-alginate and placed on SP medium; (B) encapsulated and placed on wet filter paper, (C) non-encapsulated and placed on SP medium; (D) non encapsulated but, placed on wet filter paper. All treated nodes were kept during 30 days at 15 C under dark and finally transferred to 27 C ± 2 and 16h light for 30 days more. High percentages of plant recovers (> 60 %) were observed in A and C treatments without signs of plantlet damage. Conversely, plant recovers from wet filter paper, B and D, were absent. This opens up another possibility for cassava long-term storage using in vitro culture techniques. Additional index words: Manihot esculenta; Ca-alginate; clonal storage; and SP medium. 1 Researchers. EMBRAPA Genetic Resources and Biotechnology, C.P , CEP , Brasília-DF, Brazil. 2 Under-graduate student
6 Encapsulamento de segmentos nodais de mandioca para armazenamento de germoplasma. Resumo Nós caulinares de mandioca oriundos de três genótipos cultivados in vitro foram: (A) encapsulados com alginato de cálcio e colocados em meio básico SP; (B) encapsulados e colocados em papel filtro umedecido, (C) não encapsulados, porém, colocados em meio básico SP; (D) não encapsulados e colocados em papel filtro. Todos os tratamentos foram mantidos por 30 dias no escuro e 15 C e logo depois, transferidos para luz, 16h, e temperatura de 27 ± 2 C. Nestas últimas condições, alta percentagem de conversão de plantas (> 60 %) foi observada nos tratamentos A e C, inclusive, sem sinais de dano no material clonal. Em contraste, a conversão em plantas, nos tratamentos B e D, não foi observada. Estes resultados abrem promissoras possibilidades para o armazenamento de germoplasma de mandioca por longo tempo, usando a cultura de tecido in vitro. Palavras chaves: Manihot esculenta, alginato de cálcio, armazenamento e meio SP básico.
7 7 Introduction Cassava (Manihot esculenta Crantz) is a very important staple food because of its starchy root. Brazil produces 31% of the global production (MATTOS e DANTAS, 1981). Novel mutants with potential agronomic traits have been found with high levels of free sugar, β- carotene, lutein, and lycopene content (CARVALHO et al., 2000; CARVALHO et al., 2004). Nearly of 56% of the 97 wild species of Manihot are found in Latin America. Unfortunately for diverse reasons some of these landraces are being lost and urgently need to be saved (GROS et al., 2000). The maintenance of part or all of this germplasm in experimental or nursery fields is laborious and expensive. Plant tissue culture provides a useful tool to maintain this material with low cost under laboratory conditions (BARRUETO CID e CARVALHO, 2008). Synthetic seeds production by encapsulation with calcium alginate of axillary buds or somatic embryos and low temperature storage has been reported for various agronomic crops (BAPAT et al., 1987; REDENBAUGH, 1990; GHOSH e SEN, 1994). It has been shown that storage of synthetic seeds can alleviate frequent subculture practice in germplasm collection maintenance, facilitate germplasm transport in activities related to germplasm exchange between partners (countries or laboratories), in addition to it offers easy handling material for the scale up of elite clones (PICCIONI e STANDARDI, 1995; GANAPATHI et al., 2001; LISEK e ORLIKOWSKA, 2004). The present study describes a method for the encapsulation of axillary buds specifically for cassava to accesses its potential for in vitro long-term germplasm conservation. Materials and methods a) Cuttings. Cassava cuttings (Manihot esculenta Crantz) (200 mm mm) from two landraces: CAS 36.18; F and from cultivars IAC each having several dormant axillary buds, were collected at the experimental Embrapa/Cenargen field in Brasilia DF. Cuttings were transferred to the greenhouse and grown in black polyethylene bags containing vermiculite (1 kg capacity). Cuttings were kept moist under environment temperature of 23 ± 3 C. After 30 days they started to sprout. When the shoots reached ca. 100 mm (long) by 5mm (width), they were removed and taken to laboratory. b) Surface sterilization and inoculation Shoots were washed with tap water and detergent and cut into segments of 10 to mm 15 mm each with one axillary bud (mini-cutting). In a laminar air-flow cabinet the mini-cuttings were placed in commercial bleach (sodium hypocloride 2.5 % (w/v) for 10 minutes and washed with distilled de-ionized water (DDW).
8 8 After this procedure, the mini-cuttings were transferred to a glass beaker having a sterile solution of Benlate 500 (Dupont) and Claforan (Hoechs/Roussel) 0.1 % (w/v) respectively, and stirred for 30 min. The mini-cuttings were inoculated in assay tubes (22 mm x 150 mm) having 15 ml of SP medium (BARRUETO CID e CARVALHO, 2008) and kept under standard conditions of light and temperature (27 ± 2 C and 16 h photoperiod with a photosynthetic photon flux of 50 µmol m -2 s -1 emitted from cool-white fluorescent tubes). After days, the shoots obtained from axillary buds now 10 mm-20 mm long were removed and transferred to assay tubes containing 15 ml fresh SP medium. After 45 days under standard conditions, each shoot now ca. 50 mm long, produced 4 or 5 nodes with one axillary bud per node and two or three roots per shoot (ca. 20 mm-30 mm long). c) Alginate treatment Plantlets were withdrawn from the assay tubes and their leaves and roots were removed. The caulinary node was cut into 5-6 mm segments each having one axillary bud per node (Fig.1 A). The segments were transferred to a 50 ml glass beaker with 3 % (w/v) sodium alginate (500 cps, Nacalai Tesque Inc. Kyoto-Japan) and 2 % (w/v) sucrose. Using a Pasteur pipette with tip cut off, the nodes were then placed into 80 ml of CaCl2.2H20 (1 % w/v) for the preparation of drops each having one node. The CaCl2 solutions were kept on a magnetic stirrer (ca. 80 rpm) for 30 minutes (Fig. 1B). The beads that were formed in the CaCl2 were sieved (plastic sieve of 50 mm of diameter) and placed into an open Petri dish and were not rinsed, Fig. 1 C. The beads containing the nodes, Fig. 1C, were submitted to different treatments. Fig. 1. A: Visual aspect of caulinary nodes with axillary buds (~ 6,0 mm of length) CAS B: encapsulated node induction, beads, in Ca-Cl2 solution kept on a magnetic stirrer. C: beads isolated and sieved from CaCl2 solutions. D: Visual aspect under simple microscope of a bead (~ 7 mm of length) after 30 days at 15 C. E-F: axillary bud sprouting and growing from bead respectively (A treatment).
9 9 The following group treatment were made: A: encapsulated nodes were placed in a Petri dish, 100 mm x 10 mm with 25 ml SP medium. B: encapsulated nodes were inoculated into a Petri dish containing two filter paper layers and 5 ml of DDW. C: nodes were not encapsulated and placed into a Petri dish with 25 ml SP medium. D: nodes were not encapsulated and placed into Petri dish with two filter paper layers and 5 ml DDW. E (control) similar to A, but kept at standard conditions of light and temperature without prior exposure to 15 C. All sterile-disposable Petri dishes with nodes: A, B, C, D and E were sealed with plastic film. All except E were then kept in a dark incubator for 30 days at 15. Claforan was sterilized using a Millipore filter (0.2µm pore). The material inside the laminar flow hood (glassware, forceps, alginate and CaCl2 solutions, SP medium, etc.) was autoclaved at 120 C for 20 minutes. The experimental design consisted of 12 nodes per Petri dish. All treatments with a total of 36 nodes were replicated three times. Results and discussion Under standard conditions of temperature and light, nodes of the different clones inoculated into SP medium showed high percentages of conversion into plants (Table 1: A, C; Fig.1E). No oxidative browning was observed. After 30 days the plantlets in tubes were mm high and produced several roots (Fig. 1 F). For all nodes inoculated on filter paper plus DDW, encapsulated or not ( Table 1: B-D), the plant conversions after 30 days was zero. No stem or roots were formed because the axillary bud did not sprout or grow. Other authors reported similar results for plant conversions in nutritive medium and filter paper (GANAPATHI et al., 2001). Table1. Effect of different treatments on cassava axillary buds germination under standard conditions of light and temperature after treatment under 15 C and darkness (except E). Genotype A B C D E CAS ± ± ± 23 IAC 79± ± 0,0 0 79± 6 F ± ± ± 6 Average frequency (%) ± standart deviation The lack of plant conversion from axillary buds into plants (B-D treatments), was not attributed to the temperature at 15 C. The alternative treatments (A-C) was kept at the same temperature, but plants developed from the calcium alginate beads. At low temperature, axillary buds remained dormant during the 30 days, Fig. D. No signs of rooting or shooting were seen emerging from beads. Cassava being a tropical plant, we do
10 10 not know why the 15 C was sufficient to stop axillary bud growth during the short storage. This dormancy was not observed in the control (E), whose buds began to germinate after ten days. Plant development was not evident with filter paper plus DDW. The lack of conversion may be explained mainly by nutrient deficiency and not by the need for hormones. Hormones e.g., auxin or cytokinin, reported by Ganapathi et al. (1992), were not added to the SP medium. Even so, in A and C treatments plant conversion occurred. Sucrose deficiency was implicated for the lack of germination in present study, green nodes turned pale yellow or brown after 30 days at 15 C in the B-D treatments. The lack of plant conversion (Table 1) is a strong indication that the axillary buds were dead during this period. Piccioni e Standardi, (1995) considered the nodes to be alive if they could be maintained green. In our work, the presence of sugar and mineral elements were very important in the medium for explant survival during storage, e.g., A-C treatments and control (E). By contrast, DDW alone was not sufficient to keep the axillary buds alive for 30 days. In Santalum album tap water as sole source of nutrient in Petri dishes showed a low percentage of conversion to plants (BAPAT e RAO, 1988). Further research is needed to assess how sugars and/or mineral nutrition improve the quality of the conversion. The C treatment was included to compare plant conversion with and without Ca-alginate and to show that it was not toxic nor depressive (Table 1) The high levels of plant conversion in C, are in accordance with our routine results in SP medium (BARRUETO CID e CARVALHO, 2008). The E treatment was a good indicator for vigor and viability of the nodes. When they were placed in the SP medium at standard conditions, the percent plant conversion of nodes (axillary buds) after one week was similar to the A treatment. This again showed that the Caalginate did not reduce axillary buds growth in the three genotypes tested. In conclusion, the use of a Ca-alginate matrix to encapsulate three different cassava genotype nodes (A treatment) did not depress nor produce toxic effects during the conversion to plants. All recovered plantlets were without phenotypic variation. On the other hand, low temperature (15 C) was fundamental to repress the explant grow without to affect axillary bud survival. Our method enables a simple and practical protocol for the production of encapsulated nodes from cassava (axillary buds), however, more research are desirable to evaluate in vitro long-term germplasm conservation at 15 C. Literature BAPAT, V. A.; MHATRE, M.; RAO, P. S. Propagation of Morus indica L. (Mulberry) by encapsulated shoot buds. Plant Cell Reports, Berlin, v. 6, p , 1987.
11 11 BAPAT, V. A; RAO, P. S. Sandalwood plantlets from Synthetic seeds. Plant Cell Reports, Berlin, v. 7, p , BARRUETO CID, L. P.; CARVALHO, L. J. C. B. Importance of abscisic acid (ABA) in the in vitro conservation of cassava (Maniholt esculenta CRANTZ). Chilean Journal of Agricultural Research, Santiago de Chile, v. 68, n. 3, p , CARVALHO, L. J. C. B.; CABRAL, B. G.; CAMPOS, L. Raiz de reserva de mandioca: um sistema biológico de múltipla utilidade. Brasília, DF: Recursos Genéticos e Biotecnologia, p. (Embrapa Recursos Genéticos e Biotecnologia. Documentos, 44). CARVALHO, L. J. C. B; SOUZA, C. R. B. de; CASCARDO, J. C. M. de; BLOCH JUNIOR, C.; CAMPOS, L. Identification and characterization of a novel cassava (Manihot esculenta Crantz) clone with high free sugar content and novel starch. Plant Molecular Biology, Dordrecth, Holanda, v. 56, p , GANAPATHI, T. R.; SRINIVAS, L.; SUPRASANNA, P.; BAPAT, V. A. Regeneration of plants from alginate-encapsulated somatic embryos of banana cv. Rasthali (Musa spp. AAB group). In Vitro Cellular and Developmental Biology Plant, Washington, v. 37, p , GANAPATHI, T. R., SUPRASANNA, P.; BAPAT, V. A.; RAO, P. S. Propagation of banana encapsulated shoot tips. Plant Cell Reports, Berlin, v. 11, p , GHOSH, B.; SEN, S. Plant regeneration from alginate encapsulated somatic embryos of Asparagus cooperi baker. Plant Cell Reports, Berlin, v. 13, p , GROS, M. de; MENDES, R. A.; SOUZA SIQUEIRA, C. S.; LABUTO, L. B. Cultura de tecidos de três espécies silvestres de Manihot. In: INTERNATIONAL SCIENTIFIC MEETING ON CASSAVA BIOTECHNOLOGY, 4., 1998, Salvador. Proceedings... Brasília-DF: Embrapa Recursos Genéticos e Biotecnologia/CBN, p LISEK, A.; ORLIKOWSKA, T. In vitro storage of strawberry and raspberry in calcium-alginate beads at 4º C. Plant Cell Tissue and Organ Culture, Hague, Holanda, v. 78, p , MATTOS, P. L. P. de; DANTAS, J. L. L. Utilização do cultivo da mandioca consorciada com feijão. Cruz das Almas: Embrapa-CNPMF, p. (Embrapa-CNPMF. Circular Técnica, 2). PICCIONI, E.; STANDARDI, A. Encapsulation of micropropagated buds of six woody species. Plant Cell Tissue and Organ Culture, Hague, Holanda, v. 42, p , REDENBAUGH, K. Application of artificial seed to tropical crops. Hortscience, Alexandria, US, v. 25, p , 1990.