Abstracts
The objective of this work was to evaluate the in vitro maintenance of oil palm (Elaeis guineensis and E. oleifera) accessions under slow-growth conditions. Plants produced by embryo rescue were subject to 1/2MS culture medium supplemented with the carbohydrates sucrose, mannitol, and sorbitol at 1, 2, and 3% under 20 and 25±2ºC. After 12 months, the temperature of 20°C reduced plant growth. Sucrose is the most appropriate carbohydrate for maintaining the quality of the plants, whereas mannitol and sorbitol result in a reduced plant survival.
Elaeis guineensis; Elaeis oleifera; Arecaceae; plant genetic resources.
O objetivo deste trabalho foi avaliar a manutenção in vitro de acessos de óleo de palma (Elaeis guineenses e E. oleifera) sob condições de crescimento lento. Plantas produzidas pelo resgate de embriões foram submetidas ao meio de cultura 1/2MS, acrescido dos carboidratos sacarose, manitol e sorbitol nas concentrações de 1, 2 e 3%, em 20 e 25±2ºC. Após 12 meses, a temperatura de 20°C retardou o crescimento das plantas. A sacarose é o carboidrato mais adequado para a manutenção da qualidade das plantas, enquanto o manitol e o sorbitol resultam em menor sobrevivência das plantas.
Elaeis guineenses; Elaeis oleífera; Arecaceae; recursos genéticos vegetais.
Plant species of the genus Elaeis produce seeds with intermediary
characteristics, i.e., that can withstand partial dehydration, but are cold-sensitive and
cannot support prolonged exposure under conventional genebank conditions (Ellis et al., 1990ELLIS, R.H.; HONG, T.D.; ROBERTS, E.H. An intermediate category of seed
storage behaviour? I. Coffee. Journal of Experimental Botany, v.41, p.1167-1174,
1990. DOI: 10.1093/jxb/41.9.1167.
https://doi.org/10.1093/jxb/41.9.1167....
, 1991ELLIS, R.H.; HONG, T.D.; ROBERTS, E.H.; SOETISNA, U. Seed storage
behaviour in Elaeis guineensis. Seed Science Research, v.1, p.99-104, 1991. DOI:
10.1017/S0960258500000726.
https://doi.org/10.1017/S096025850000072...
). Therefore, these plants present serious limitations for ex situ germplasm
conservation in the long term. The routine form of conservation is maintaining the
collections in the field, which generally requires high costs, specialized labor, and
extensive areas. Added to this, is the fact that collections maintained in the field are
under constant threat of losses or depredation through exposure to biotic and abiotic
factors that compromise the safety of accessions conserved in this way (Engelmann, 2011ENGELMANN, F. Use of biotechnologies for the conservation of plant
biodiversity. In vitro Cellular and Developmental Biology, v.47, p.5-16, 2011. DOI:
10.1007/s11627-010-9327-2.
https://doi.org/10.1007/s11627-010-9327-...
).
As an alternative and auxiliary measure for plant germplasm conservation in the field, biotechnological strategies based on concepts of in vitro culture of plant cells, tissues, and organs have been developed and improved. These techniques, besides acting as auxiliary measures for the conservation of germplasm in the field, have the potential to overcome some limitations associated with conventional methods of conservation ex situ and to facilitate the exchange of pest-free germplasm (Scherwinski-Pereira et al., 2010SCHERWINSKI-PEREIRA, J.E.; COSTA, F.H.S.; CAMILLO, J.; SILVA, D.B.; ALVES, R.B.N.; VIEIRA, R.F.Tissue culture storage of Brazilian medicinal plants germplasm. Acta Horticulturae, v.860, p.211-214, 2010.). In vitro conservation protocols should promote maximum survival, genetic stability, as well as enable the subculture frequencies of the plant material to be extended.
In general, in vitro germplasm banks are maintained under minimal growth conditions,
obtained by slowing down the plant metabolism, which is usually achieved by reducing
temperature and light intensity, or by modifying the formulations of the culture media,
including dilution of mineral elements, reduction of sugar concentrations, changes in the
use of growth regulators, or the addition of osmotically active compounds (Lédo et al., 2007LÉDO, A. da S.; CUNHA, A.O.; ARAGÃO, W.M.; TUPINAMBÁ, E.A. Efeito da
sacarose e do manitol na conservação in vitro por crescimento lento de coqueiro anão.
Magistra, v.19, p.346-351, 2007.; Engelmann, 2011ENGELMANN, F. Use of biotechnologies for the conservation of plant
biodiversity. In vitro Cellular and Developmental Biology, v.47, p.5-16, 2011. DOI:
10.1007/s11627-010-9327-2.
https://doi.org/10.1007/s11627-010-9327-...
; Yun-peng et al., 2012YUN-PENG, D.; WEN-YUAN, L.; MING-FANG, Z.; HENG-BIN, H.; GUI-XIA, J. The
establishment of a slow-growth conservation system in vitro for two wild lily
species. African Journal of Biotechnology, v.11, p.1981-1990, 2012. DOI:
10.5897/AJB11.2868.
https://doi.org/10.5897/AJB11.2868....
;
Lédo et al., 2014LÉDO, A. da S.; MOURA, C.R.F.; MACHADO, C. de A.; RAMOS, S.R.R.; SILVA,
A.V.C. da; LÉDO, C.A. da S.Mannitol for coconut ex situ conservation by minimum
growth. Pesquisa Agropecuária Brasileira, v.49, p.148-151, 2014. DOI:
10.1590/S0100-204X2014000200010.
https://doi.org/10.1590/S0100-204X201400...
; Nogueira et al., 2014NOGUEIRA, G.F.; PIO, L.A.S.; PASQUAL, M.; AMARAL, A.;
SCHERWINSKI-PEREIRA, J.E.An approach on the in vitro maintenance of sugarcane with
views for conservation and monitoring of plant nuclear DNA contents via flow
cytometry. In Vitro Cellular and Developmental Biology - Plant, 2014. DOI:
10.1007/s11627-014-9660-y.
https://doi.org/10.1007/s11627-014-9660-...
).
Currently, in vitro technology for the conservation of oil palm germplasm is focused almost
exclusively on the technique of cryopreservation of zygotic embryos (Villa et al., 2007VILLA, A.L.; JIMÉNEZ, P.E.; VALBUENA, R.I.; BASTIDAS, S.; NÚÑEZ, V.M.
Estudio preliminar para el establecimiento de un protocolo de crioconservación para
palma de aceite (Elaeis guineensis Jacq.). Agronomia Colombiana, v.25, p.215-223,
2007.), somatic embryos (Konan et al., 2010KONAN, K.E.; DURAND-GASSELIN, T.; KOUADIO, Y.J.; FLORI, A.; RIVAL, A.;
DUVAL, Y.; PANNETIER, C. In vitro conservation of oil palm somatic embryos for 20
years on a hormone-free culture medium: characteristics of the embryogenic cultures,
derived plantlets and adult palms. Plant Cell Reports, v.29, p.1-13, 2010. DOI:
10.1007/s00299-009-0787-y.
https://doi.org/10.1007/s00299-009-0787-...
), and pollen (Tandon et al.,
2007TANDON, R.; CHAUDHURY, R.; SHIVANNA, K.R. Cryopreservation of oil palm
pollen. Current Science, v.92, p.182-183, 2007.). However, despite its relative effectiveness, cryopreservation requires a
highly-specialized structure and labor, and it is a technique aimed at long-term
conservation (Sánchez-Chiang & Jiménez, 2010SÁNCHEZ-CHIANG, N.; JIMÉNEZ, V.M. Técnicas de conservación in vitro para
el establecimiento de bancos de germoplasma en cultivos tropicales. Agronomía
Mesoamericana, v.21, p.193-205, 2010. DOI: 10.15517/am.v21i1.11836.
https://doi.org/10.15517/am.v21i1.11836....
). A
complementary alternative is in vitro maintenance of whole plants, for the purpose of
making the germplasm available at short and medium terms. However, for this technique to be
truly effective, it is necessary to adapt the used methodologies, by determining the best
formulation of the culture media, the number of samples to be conserved, the frequency of
the subcultures, and the optimal temperature and light conditions for the maintenance of in
vitro collections (Sánchez-Chiang & Jiménez,
2010SÁNCHEZ-CHIANG, N.; JIMÉNEZ, V.M. Técnicas de conservación in vitro para
el establecimiento de bancos de germoplasma en cultivos tropicales. Agronomía
Mesoamericana, v.21, p.193-205, 2010. DOI: 10.15517/am.v21i1.11836.
https://doi.org/10.15517/am.v21i1.11836....
; Scherwinski-Pereira et al., 2010SCHERWINSKI-PEREIRA, J.E.; COSTA, F.H.S.; CAMILLO, J.; SILVA, D.B.;
ALVES, R.B.N.; VIEIRA, R.F.Tissue culture storage of Brazilian medicinal plants
germplasm. Acta Horticulturae, v.860, p.211-214, 2010.).
For that reason, studies on in vitro conservation of whole oil palm plants are still
incipient.
The objective of this work was to evaluate the in vitro maintenance of oil palm (Elaeis guineensis and Elaeis oleifera) accessions under slow-growth conditions.
The materials used in the experiments were obtained from the oil palm germplasm bank, of Embrapa Amazônia Ocidental, located in the municipality of Rio Preto da Eva, in the state of Amazonas, Brazil. To determine slow-growth conditions, the effects both of temperature and of carbohydrate types and concentrations were tested. To study the effects of temperature on in vitro conservation, the following genotypes were tested: three of E. oleifera, EOD08, EOD09, and EOD15; three of E. guineensis var. dura, EGD01, EGD05, and EGD11; and three of E. guineensis var. tenera, EGT03, EGT05, and EGT11 (Table 1).
Survival rate, length of the aerial part (LAP), growth rate, and appearance (color) of the leaves of oil palm (Elaeis sp.) accessions after 12 months of in vitro storage at 20 and 25ºC temperature(1).
The plants used in the experiments were regenerated from zigotic embryos, isolated from
mature fruits and germinated on half-strength Murashige and Skoog (1/2MS) medium,
supplemented with biotin, pantothenic acid, 3% sucrose, and 0.25% Phytagel (Sigma, St.
Louis, MO, USA) (Luis et al., 2010LUIS, Z.G.; BEZERRA, K.M.G.; SCHERWINSKI-PEREIRA, J.E. Adaptability and
leaf anatomical features in oil palm seedlings produced by embryo rescue and
pre-germinated seeds. Brazilian Journal of Plant Physiology, v.22, p.209-215, 2010.
DOI: 10.1590/S1677-04202010000300008.
https://doi.org/10.1590/S1677-0420201000...
). The in vitro
plants were selected, giving preference to those of similar size (10±1 cm) and with
equal numbers of leaves and roots (1 to 2 roots and 2 to 3 expanded leaves). After
selection, the roots and leaves of the plants were cut to initial average height of
approximately 4±1 cm. Then, the plants were inoculated in 1/2MS medium, supplemented
with 3% sucrose and 0.25% Phytagel (Sigma, St. Louis, MO, USA). The experiment was
maintained simultaneously in growth rooms with temperatures of 20 and 25±2ºC, light
intensity of 30 μmol m-2 s-1, and photoperiod of 12 hours.
To test the effect of different carbohydrates and concentrations, the EOD08 and EGT11 genotypes of the E. oleifera and E. guineensis oil palms, respectively, were used. Plants were selected as described in the previous experiment. The plants were inoculated in 1/2MS medium, supplemented with 0.25% Phytagel and with the carbohydrates sucrose, mannitol, and sorbitol (Sigma, St. Louis, MO, USA) at the concentrations of 1, 2, and 3%. The experiment was maintained in a growth room at 25±2ºC, with 30 μmol m-2 s-1 light intensity and a 12-hour photoperiod.
For both experiments (temperature and carbohydrates), the plants were inoculated in 25x250-mm test tubes containing 15 mL of culture media. The pH was adjusted to 5.7±0.1, and sterilization was performed by autoclaving at 121ºC for 20 min, under 1.2 atm of pressure. The experiment was conducted in a completely randomized design, with ten replicates per treatment; each treatment consisted of a test tube with one plant. The evaluations were performed over 12 months for the following leaf traits: survival rate, length of the aerial part, growth rate, and color (appearance). For the variable appearance of leaves, the following scale of scores was attributed: 1, dark-green leaves; 2, light-green leaves; and 3, yellowish leaves with dark spots or completely necrosed leaves. The data were subjected to analysis of variance, followed by the Scott & Knott post hoc test, at 5% probability.
Plants maintained at a temperature of 20°C showed a significant reduction in height and
growth rate, although their normal development was not affected (Table 1). The obtained results are in accordance with those reported
for the conservation of the germplasm of tropical species, which suggest that the most
favorable temperature for the maintenance of these species under minimal growth conditions
is between 15 and 20°C, providing a reduction in metabolic activity and a decrease in
growth (Engelmann, 2011ENGELMANN, F. Use of biotechnologies for the conservation of plant
biodiversity. In vitro Cellular and Developmental Biology, v.47, p.5-16, 2011. DOI:
10.1007/s11627-010-9327-2.
https://doi.org/10.1007/s11627-010-9327-...
).
At the different temperatures, the survival rate of the oil palm plants remained high (>
95%). Furthermore, no significant differences were observed in the appearance of the plants
after 12 months of conservation. Similar results were found for coconut (Cocos
nucifera L.) plants maintained in vitro at 27±1°C, for which the survival
rate was 75% after 18 months of cultivation (García et
al., 2008GARCÍA, M.B.; MALAURIE, B.; VILTRES, S.P.; CAMPOS, D.C. Efecto de las
distintas concentraciones de sacarosa en la conservación in vitro de coco (Cocos
nuciferas L.). Revista Colombiana de Biotecnologia, v.10, p.111-119,
2008.). Lédo et al.
(2007)LÉDO, A. da S.; CUNHA, A.O.; ARAGÃO, W.M.; TUPINAMBÁ, E.A. Efeito da
sacarose e do manitol na conservação in vitro por crescimento lento de coqueiro anão.
Magistra, v.19, p.346-351, 2007. observed, for this same species, the maintenance of 100% plant survival
after 365 days of cultivation, and considered in vitro cultivation to be a promising
alternative for the conservation of germplasm in palms. The use of low temperatures in the
in vitro maintenance of the germplasm of palm species was previously reported only for date
palm (Phoenix dactylifera L.) (Bekheet,
2011BEKHEET, S.A. In vitro conservation of date palm germplasm. In: JAIN,
S.M.; AL-KHAYRI, J.M.; JOHNSON, D.V. (Ed.). Date palm biotechnology. Dordrecht:
Springer, 2011. p.337-360. DOI: 10.1007/978-94-007-1318-5_17.
https://doi.org/10.1007/978-94-007-1318-...
).
In general, media containing sucrose at 3% favor the normal development of the conserved plants and result in plants with better appearance, with the predominance of uniform green leaves throughout the storage process (Table 2). The greatest delay in the growth of the oil palm plants was in the culture media containing mannitol. In spite of the positive result in growth reduction, this carbohydrate resulted in accentuated necrosis of the aerial part and in a consequent reduction in plant survival. Similar results were found in coconut (Lédo et al., 2007LÉDO, A. da S.; CUNHA, A.O.; ARAGÃO, W.M.; TUPINAMBÁ, E.A. Efeito da sacarose e do manitol na conservação in vitro por crescimento lento de coqueiro anão. Magistra, v.19, p.346-351, 2007.), in which the addition of mannitol resulted in lower growth of the aerial part and roots, significantly affecting plant survival. In the present study, the use of sorbitol produced intermediate results, but close to those obtained when the oil palm plants were cultivated in sucrose, with high explant survival, predominance of light-green leaves, and low incidence of necrosis.
Survival rate, length of the aerial part (LAP), growth rate, and appearance (color) of the leaves of oil palm (Elaeis sp.) accessions after 12 months of in vitro storage at 25ºC with different carbohydrates at concentrations of 1, 2, and 3%(1).
Therefore, the obtained data show that oil palm plants can be maintained under minimal growth conditions simply by reducing the temperature in the growth room from 25 to 20°C. This method, besides being effective, can provide stability to the conserved materials, given that the occurrence of stress during the conservation period is one of the factors that could induce somaclonal variation. Sucrose is the most appropriate carbohydrate for maintaining the quality of the plants, whereas mannitol and sorbitol result in a reduced plant survival.
Acknowledgments
To Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), to Financiadora de Estudos e Projetos (Finep), and to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), for financial support and fellowships; and to Dr. Ricardo Lopes and Dr. Raimundo Nonato from the Brazilian Oil Palm Breeding Program of Embrapa Amazônia Ocidental, Brazil, for providing the biological material for the experiments.
- BEKHEET, S.A. In vitro conservation of date palm germplasm. In: JAIN, S.M.; AL-KHAYRI, J.M.; JOHNSON, D.V. (Ed.). Date palm biotechnology. Dordrecht: Springer, 2011. p.337-360. DOI: 10.1007/978-94-007-1318-5_17.
» https://doi.org/10.1007/978-94-007-1318-5_17. - ELLIS, R.H.; HONG, T.D.; ROBERTS, E.H. An intermediate category of seed storage behaviour? I. Coffee. Journal of Experimental Botany, v.41, p.1167-1174, 1990. DOI: 10.1093/jxb/41.9.1167.
» https://doi.org/10.1093/jxb/41.9.1167. - ELLIS, R.H.; HONG, T.D.; ROBERTS, E.H.; SOETISNA, U. Seed storage behaviour in Elaeis guineensis. Seed Science Research, v.1, p.99-104, 1991. DOI: 10.1017/S0960258500000726.
» https://doi.org/10.1017/S0960258500000726. - ENGELMANN, F. Use of biotechnologies for the conservation of plant biodiversity. In vitro Cellular and Developmental Biology, v.47, p.5-16, 2011. DOI: 10.1007/s11627-010-9327-2.
» https://doi.org/10.1007/s11627-010-9327-2. - GARCÍA, M.B.; MALAURIE, B.; VILTRES, S.P.; CAMPOS, D.C. Efecto de las distintas concentraciones de sacarosa en la conservación in vitro de coco (Cocos nuciferas L.). Revista Colombiana de Biotecnologia, v.10, p.111-119, 2008.
- KONAN, K.E.; DURAND-GASSELIN, T.; KOUADIO, Y.J.; FLORI, A.; RIVAL, A.; DUVAL, Y.; PANNETIER, C. In vitro conservation of oil palm somatic embryos for 20 years on a hormone-free culture medium: characteristics of the embryogenic cultures, derived plantlets and adult palms. Plant Cell Reports, v.29, p.1-13, 2010. DOI: 10.1007/s00299-009-0787-y.
» https://doi.org/10.1007/s00299-009-0787-y. - LÉDO, A. da S.; CUNHA, A.O.; ARAGÃO, W.M.; TUPINAMBÁ, E.A. Efeito da sacarose e do manitol na conservação in vitro por crescimento lento de coqueiro anão. Magistra, v.19, p.346-351, 2007.
- LÉDO, A. da S.; MOURA, C.R.F.; MACHADO, C. de A.; RAMOS, S.R.R.; SILVA, A.V.C. da; LÉDO, C.A. da S.Mannitol for coconut ex situ conservation by minimum growth. Pesquisa Agropecuária Brasileira, v.49, p.148-151, 2014. DOI: 10.1590/S0100-204X2014000200010.
» https://doi.org/10.1590/S0100-204X2014000200010. - LUIS, Z.G.; BEZERRA, K.M.G.; SCHERWINSKI-PEREIRA, J.E. Adaptability and leaf anatomical features in oil palm seedlings produced by embryo rescue and pre-germinated seeds. Brazilian Journal of Plant Physiology, v.22, p.209-215, 2010. DOI: 10.1590/S1677-04202010000300008.
» https://doi.org/10.1590/S1677-04202010000300008. - NOGUEIRA, G.F.; PIO, L.A.S.; PASQUAL, M.; AMARAL, A.; SCHERWINSKI-PEREIRA, J.E.An approach on the in vitro maintenance of sugarcane with views for conservation and monitoring of plant nuclear DNA contents via flow cytometry. In Vitro Cellular and Developmental Biology - Plant, 2014. DOI: 10.1007/s11627-014-9660-y.
» https://doi.org/10.1007/s11627-014-9660-y. - SÁNCHEZ-CHIANG, N.; JIMÉNEZ, V.M. Técnicas de conservación in vitro para el establecimiento de bancos de germoplasma en cultivos tropicales. Agronomía Mesoamericana, v.21, p.193-205, 2010. DOI: 10.15517/am.v21i1.11836.
» https://doi.org/10.15517/am.v21i1.11836. - SCHERWINSKI-PEREIRA, J.E.; COSTA, F.H.S.; CAMILLO, J.; SILVA, D.B.; ALVES, R.B.N.; VIEIRA, R.F.Tissue culture storage of Brazilian medicinal plants germplasm. Acta Horticulturae, v.860, p.211-214, 2010.
- TANDON, R.; CHAUDHURY, R.; SHIVANNA, K.R. Cryopreservation of oil palm pollen. Current Science, v.92, p.182-183, 2007.
- VILLA, A.L.; JIMÉNEZ, P.E.; VALBUENA, R.I.; BASTIDAS, S.; NÚÑEZ, V.M. Estudio preliminar para el establecimiento de un protocolo de crioconservación para palma de aceite (Elaeis guineensis Jacq.). Agronomia Colombiana, v.25, p.215-223, 2007.
- YUN-PENG, D.; WEN-YUAN, L.; MING-FANG, Z.; HENG-BIN, H.; GUI-XIA, J. The establishment of a slow-growth conservation system in vitro for two wild lily species. African Journal of Biotechnology, v.11, p.1981-1990, 2012. DOI: 10.5897/AJB11.2868.
» https://doi.org/10.5897/AJB11.2868.
Publication Dates
-
Publication in this collection
May 2015
History
-
Received
12 Dec 2014 -
Accepted
20 Mar 2015