In vitro rescue of interspecific embryos from Elaeis guineensis x E. oleifera (Arecaceae)
Paula Cristina da Silva Angelo, Larissa Alexandra Cardoso Moraes, Ricardo Lopes, Nelcimar Reis Sousa, Raimundo Nonato Vieira da Cunha & Regina Caetano Quisen
The African oil palm (Elaeis guineensis) is the most effective oil producer in tons per hectare. Nevertheless, its increasing cultivation in Latin America is harmed by the “lethal yellowing”. Genetic resistance to this anomaly can be found in the germplasm of American ]]>
E. oleifera), a native species from the Amazon rainforest. However, the procedures adopted to induce seeds of E. guineensis to germination frequently result mild for interespecific hybrids. Embryo in vitro cultivation can be a viable option. This work was aimed initially to test liquid MS medium supplemented with different glucose or sucrose concentrations for the in vitro ]]>
E. guineensis x E. oleifera controlled pollinations. Additionally we investigated different compost mixtures to acclimatize the regenerated hybrid plantlets. Concentrations of 10, 20 and 30g/L of both sugars were tested on flasks containing five mature zygotic embryos, with 15 repetitions per treatment in a total of 450 explants. The number of embryos displaying shoots and radicles at least 2mm in length per experimental unit was evaluated during phase one of in vitro cultivation. Plantlets ]]>
medium supplemented with 20g/L (110mM) of glucose. This sugar in ]]>
media with glucose above 110mM. Acclimatization could benefit from an extended period of in ]]>
development. Rev. Biol. Trop. 59 (3): 1081-1088. Epub 2011 September 01.
Elaeis guineensis es el productor de aceite más eficaz en toneladas por hectárea, su cultivo, cada vez mayor en América Latina, se ha visto perjudicado por el “amarilleamiento ]]>
E. oleifera), una especie nativa de la selva amazónica. Sin embargo, los procedimientos adoptados para inducir la germinación de las semillas de E. guineensis frecuentemente produce resultados modestos para híbridos interespecíficos. El cultivo de embrionesin vitro puede ser una ]]>
E. guineensis x E. oleifera originados de polinización controlada. Además se investigaron diferentes mezclas de compost para aclimatar los híbridos regenerados. Las concentraciones de 10, 20 y 30 g/L de ambos azúcares se probaron en frascos que contenían cinco embriones cigóticos maduros, con 15 ]]>
Palabras clave: ]]>
Currently, breeding programs in Latin America explore the interspecific hybridization between the African (Elaeis ]]>
Jacq.) and the American oil palm or caiaué [E. oleifera (Kunth) Cortés]. African oil palm is the cultivated oil plant that presents the highest yields of palm oil, used for cosmetics (Fiorese 2008, Natura 2010), food and biofuel and has social (Homma et al. 2000, Fiorese 2008) and ecologic relevance (Veiga et al. 2000). American oil palm has been introduced into breeding programs as a source of resistance to the ]]>
et al. 2000, Lopes et al. 2008).
However, interspecific hybrid seeds ]]>
et al. 1998) only gave rise to approximately 30% plantlets on average (R.N.V. Cunha & R. Lopes 2008, pers. comm.), far below the 80% obtained for African oil palm seeds (Nunes et al. 1998), and slightly below the 50% observed for seeds of American oil palm.
In vitro cultivation can be useful in determining which would be the appropriate conditions to facilitate the development of somatic embryos in the future and in other applications too. This technology was used to optimize germination of avocado genotypes inflicted by precocious fruit abscission (Sánchez-Romero et al. 2007). Besides, immature embryos from Hybiscus interspecific crosses that did not produce normal fruits were recently rescued in ]]>
(van Laere et al. 2007).
Specifically concerning species that have oil as the principal reserve present in the seeds, plantlets of Arabidopsis thaliana were established in vitro independently of the glyoxalate cycle as long as ]]>
et al. 2000). Sugar concentrations from 30 up to 90mM were sufficient to promote the germination of A. thaliana seeds in the presence of exogenous ABA, but root elongation occurred only under higher sugar concentrations (Finkelstein & Lynch 2000, Finkelstein & Gibson 2001). In vitro culture of zygotic embryos was used to rescue hybrids from Brassica ]]>
x B. juncea and to improve the efficacy of interspecific breeding, thus overcoming the abortion of hybrid embryos caused by the differences in the number of chromosomes between the two species (Zhang et al. 2003). In vitro, B. napus isolated embryos absorbed hexoses faster than sucrose. The activities of hexokinases and sucrose synthase increased in vitro during embryo development and sucrose synthase was ]]>
et al. 2003). In vitro cultivation of tenera immature embryos produced by crossing E. guineensis types dura and pisifera was reported by Aberlenc-Bertossi et al. (2003), although the rescue of interspecific hybrids from intercrosses of E. guineensis ]]>
E. oleifera has not been evaluated.
The objectives of the present work were to evaluate sucrose and glucose as carbon sources to rescue mature hybrid embryos produced by controlled pollination of E. guineensis x E. oleifera and compost ]]>
in vitro.
Material and methods
Plant material: ]]>
Elaeis guineensis plants with pollen from selected plants of E. oleifera, in the Rio Urubu Experimental Station, Rio Preto da Eva, Amazonas, Brazil, 2°35’ S - 59°28’ W, at 200m in elevation. Fruits from progenies labeled as CI1488 and CI1603 were collect 150 days after pollination and stored at 20°C for 156 and 143 days, respectively. After removal of fruit tissues, seeds were washed with ]]>
media.
In vitro ]]>
Embryos from progenies CI1603 and CI1488 were cultivated over cellulose acetate membranes on liquid MS basal salts and vitamins (Murashige & Skoog 1962) supplemented with 10, 20 or 30g/L sucrose or 10, 20 or 30g/L glucose, under 26±2°C and irradiated with 24µmol/m2s of photosynthetically active photons for 16h photoperiods. The emergence of shoots and radicles with at least 2mm in length was scored up to 50 days. Experiments were conducted in a completely randomized design, with each experimental ]]>
in vitro. Raw data -consisting of the average number of embryos exhibiting emerged shoots and radicles per experimental unit- were transformed to (x+0.5)1/2 for ANOVA analyzes and the subsequent Tukey tests. These procedures comprised phase one of in vitro cultivation (P1VC). Following P1VC, plantlets that exhibited shoots and radicles were transferred to half ]]>
medium for 60 days, under the conditions of temperature and irradiation described previously, until acclimatization. These procedures constituted phase two of in vitro cultivation (P2VC).
Acclimatization: ]]>
ex vitro transfer, plantlets were evaluated and randomly distributed in 200cm3 plastic tubes with drainage holes fitted in square cages and filled with commercial horticultural compost (constituted principally of milled wood) pure or mixed 3:1 or 1:1 with sand. Plantlets in tubes were maintained under greenhouse conditions with approximately 70% shading and manual water supplying and evaluated 43, 100 and 125 days after transfer. At transfer and in the final evaluation, roots and leaves were counted along with the measurement of ]]>
et al. 1995).
Results
]]>
Elaeis guineensis x E. oleifera hybrid embryos displayed no significant differences in average values observed for shoot emergence per experimental unit under distinct glucose treatments (n=45, F=2.337, p=0.115) but significantly higher averages (n=45, F=13.742, p<0.001 and Tukey, p<0.05) were observed for radicle emergence under glucose 20 (110mM) and 30g/L (165mM) (Table 1).
For cultivation in sucrose-containing media the average values observed for radicle (n=45, F=5.441, p=0.010) and shoot (n=45, F=21.899, p<0.001) emergence were highest for the highest concentration tested. There was no significant difference between 30g/L (87mM) and 10g/L (29mM) for shoot emergence (Tukey, p≥0.05), but radicle emergence was significantly higher in 30g/L sucrose (
Table 1).
It became evident that germinating oil palm interspecific embryos exhibited higher shoot emergence frequencies in comparison to radicle emergence, independently of the sugar source in the cultivation media (Table 1). Although ]]>
media, it was noticed that alleviation of the impairment in root elongation was achieved in 20g/L (110mM) or highest concentration of this sugar. Shoot emergence was similarly frequent for plantlets maintained in medium containing 10 (55mM), 20 (110mM) or ]]>
media containing 20g/L (110mM) glucose or higher (Table 1). Accordingly, among sucrose treatments, increase in the emergence of radicles with subsequent development was observed almost exclusively for the highest concentration (30g/L or 89mM, Fig. 1), despite it ]]>
Most plantlets ]]>
From P1VC, 130 plantlets were transferred to the greenhouse. Data ]]>
ex vitro transfer revealed the absence of significant influence of culture media used during P1VC (20 or 30g/L glucose or 30g/L sucrose) on the number of leaves per plant (n=130, H=0.0361, p=0.982), on the lengths of the longest roots (n=118, H=0.719, p=0.698) or on the longest leaves (n=130, H=1.171, p=0.425).
At transfer, a ]]>
Table 1).
Plants surviving up to the second evaluation under greenhouse conditions 43 days after transfer corresponded to 55% (71 plants). This result was similar to the 58.33% survival observed for coconut plantlets (Lédo et al. 2007). From the total, 24 plants were ]]>
Plantlets gained on average 0.04cm/day in length of the longest leaf during the first 43 days in the greenhouse. Subsequent progress was significantly better (t-tests, p<0.05) comparing data registered upon ex vitro transfer
versus subsequent evaluations. It was observed the increase in the number of leaves and the lengthening of the longest leaf (Table 2), with gains of 0.123cm/day between the second and third evaluations and 0.110cm/day from the third to the final evaluations were observed. A correlation between the length of the longest leaf and the number of leaves not observed at transfer was established by the second evaluation (Spearman, n=71, r=0.778, p<0.001) and persisted through the experiment. ]]>
Distinct compost mixtures had no influence on shoot development until the final evaluation, when the number of leaves was higher for pure commercial compost (n=66, H=11.23, p=0.004). Root length followed a similar pattern and was equally high in pure compost or 3:1 compost:sand mixture (n=66, H=7.66, p=0.022). ]]>
Discussion
The phase of reserve (including sucrose and proteins) accumulation ends approximately 110 days after pollination, when the acquisition of tolerance to desiccation is initiated, for seeds of
Elaeis guineensis type tenera. About 120 days after pollination, water loss stops, embryos preserve a water content slightly higher than the values commonly observed for orthodox species and the seed remains dormant until fruit abscission, which can last 60 additional days or for longer periods (Aberlenc-Bertossi et al. 2003). Besides sucrose accumulation, abscisic acid (ABA) and late embryogenesis abundant (LEA) proteins are possibly involved in this process and the acquired desiccation ]]>
et al. 2003). This process could be somewhat different for interspecific hybrids, since E. oleifera seeds have relatively lower viability and the combination of genomes from two species can produce novel phenotypes for a massive number of traits. For instance, different Arabidopsis landraces or ecotypes vary in their degree of seed dormancy ]]>
et al. 2006). Nevertheless, E. guineensis remains the most suitable reference for the study of interspecific hybrids, and since fruits were collect 150 days after pollination and stored, they were considered to be dormant.
The hormone ABA can accumulate in roots of dormant species just after ]]>
et al. 2002) and in the inhibition of invertase activity and root elongation, which are complex signal transduction processes orchestrated by sugars, water and light (Bewley 1997, Finkelstein & Gibson 2001). The impairment in root elongation is gradually supplanted while the radicle becomes a photosynthate sink (Finkelstein & Gibson 2001), and this physiological mechanism is possibly coordinated with the accumulation of sufficient sucrose in shoots. Embryos of E. ]]>
type tenera exposed to light display chlorophyll accumulation in the haustorium due to chloroplast development, and subsequent starch accumulation (Rabéchault & Cas 1974). In plants, haustorium functions to transfer reserves from the endosperm to the germinating embryo (Carvalho 2000) and is not exposed to light. In the present study, greening of haustoria was observed. Accumulation of starch would also agree with the availability of hexoses in the shoots ]]>
media, when compared to 10g/L of the same sugar, and in the highest concentration of sucrose compared to the lower concentrations tested (Table 1). The optimum absorption ]]>
et al. 2003) would compensate for a possible inhibition of root invertases following emergence (Finkelstein & Lynch 2000).
During the first 43 days under acclimatization process, hybrid plantlets dying were presumably those with a higher number of leaves or ]]>
Table 2). Plantlets bearing more leaves would lose higher contents of water due to the higher number of stomata and thinner superficial wax layer (Abdelouahhab & Hughes 1995), thus demanding more absorption from an insufficiently developed root system. ]]>
To attain better results in future studies, embryos at different maturation stages will be evaluated as reported for avocado (Sanchéz- Romero et al. 2007) and the effects of growth regulators on germination will be tested (Aberlenc-Bertossi et al. 2003, Lédo et al. ]]>
in vitro in comparison to the average number of interspecific hybrid seeds germinating from progeny pools, the loss of plantlets during acclimatization was of concern. This losses could be reduced by a longer P2VC, by pruning of the roots and by the use of an automated system to control the moisture in the greenhouse. Germination rates and subsequent plantlet development may, in addition, be influenced by embryo genotype, and therefore, different results may be found in future experiments.
In conclusion, interspecific zygotic embryos of E. guineensis x E. oleifera succeeded in metabolize the glucose provided in the media for in vitro culture, and displayed the highest frequencies of emerged and ]]>
]]>
Acknowledgments
To CNPq for financial support, as grant number 401078/04-3. To Rosimar F. de Souza, Jeferson C. da Cruz and Hilma A. R. do Couto at Embrapa Western Amazon Plant Biotechnology Laboratory for technical support. To ]]>
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