Anomalous somatic embryos in Acca sellowiana (O. Berg) Burret (Myrtaceae)
01 Mar 2008-Brazilian Journal of Botany (Sociedade Botânica de São Paulo)-Vol. 31, Iss: 1, pp 155-164
TL;DR: A formacao de embrioes somaticos anomalos, bem como a baixa taxa de seu desenvolvimento em plântulas (" emblings" ), refletiria a ocorrencia de disturbios fisiologicos e/ou geneticos protagonizada pela presenca de 2,4-D no meio of cultura.
Abstract: A embriogenese somatica representa uma ferramenta importante para estudos basicos do desenvolvimento das plantas. Atualmente este processo e utilizado como uma tecnica com potencial para a micropropagacao de plantas em grande escala, embora ainda com aplicacao restrita a poucas especies. Entretanto, quando sao formados embrioes somaticos anomalos a aplicacao economica e inviabilizada. Em Acca sellowiana (O. Berg) Burret, uma importante frutifera nativa, uma elevada quantidade de embrioes somaticos com fenotipos alterados (76,3%) foi encontrada ja no 40o dia de cultura na presenca de 2,4-D. Entre esses, 12,2% consistiam de embrioes fundidos, 40,4% apresentavam cotiledones concrescidos, 13% possuiam mais de dois cotiledones e 10,7% nao tinham cotiledones ou eles eram pouco desenvolvidos, incluindo os embrioes sem meristema apical caulinar. As analises histologicas mostraram que os embrioes malformados originavam-se tanto diretamente dos cotiledones, hipocotilo e radicula dos embrioes zigoticos utilizados como explantes, como indiretamente de calos formados a partir dessas estruturas. Sugere-se que a formacao de embrioes somaticos anomalos, bem como a baixa taxa de seu desenvolvimento em plântulas (" emblings" ), refletiria a ocorrencia de disturbios fisiologicos e/ou geneticos protagonizada pela presenca de 2,4-D no meio de cultura. Sao indicadas algumas abordagens experimentais alternativas visando reduzir a ocorrencia de embrioes somaticos com malformacoes.
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TL;DR: The results suggest that DNA methylation dramatically influences SE in Acca sellowiana, and globalDNA methylation dynamics are related to morphogenetic response.
Abstract: DNA methylation is an epigenetic regulatory mechanism of gene expression which can be associated with developmental phases and in vitro morphogenetic competence in plants. The present work evaluated the effects of 5-azacytidine (AzaC) and 2,4-dichlorophenoxyacetic acid (2,4-D) on Acca sellowiana somatic embryogenesis (SE) and global DNA methylation levels by high-performance liquid chromatography mass spectrometry (HPLC/MS/MS). 2,4-D-free treatments revealed no somatic embryo formation in both accessions tested. Treatments supplemented with 2,4-D pulse plus AzaC in the culture medium resulted in increased embryo formation. In AzaC-free treatment, HPLC/MS/MS analysis showed a gradual increase in methylation levels in cultures of both accessions tested during SE induction. Treatment with AzaC and 2,4-D-free resulted in a marked decrease in methylation for both accessions, ranging from 37.6 to 20.8 %. In treatment with 2,4-D and AzaC combined, the 85 accession showed increasing global methylation levels. Otherwise, the 101X458 accession, in the same treatment, showed a decrease between 10 and 20 days, followed by an increase after 30 days (39.5, 36.2 and 41.6 %). These results indicate that 2,4-D pulse combined with AzaC improves SE induction. However, the conversion phase showed that although positively influencing SE induction, AzaC had a dysregulatory effect on the stage of autotrophic plant formation, resulting in significantly lower conversion rates. The results suggest that DNA methylation dramatically influences SE in Acca sellowiana, and global DNA methylation dynamics are related to morphogenetic response. Key message 5-Azacytidine combined with 2,4-D increases the number of Acca sellowiana somatic embryos. Global DNA methylation is directly affected by these compounds.
72 citations
Cites background or methods from "Anomalous somatic embryos in Acca s..."
...Pescador et al. (2008) reported 10 % conversion rates, attributed to the high rates of anomalous somatic embryos....
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...This morphogenetic route has been studied in this species, as shown by the works of Guerra et al. (1997, 2001); Dal Vesco and Guerra (2001); Stefanello et al. (2005); Cangahuala-Inocente et al. (2004, 2007, 2009) and Pescador et al. (2008, 2012)....
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TL;DR: The identification of the main factors that can cause abnormal SE development in different plant species are identified, how SE abnormalities are related to somaclonal variations and which genes may be involved with embryo abnormalities are suggested.
Abstract: Somatic embryogenesis is a morphogenetic event where somatic cells have the ability to produce embryos without gamete fusion. It is used as a technique for plant mass propagation. It is a process that has six well defined steps such as induction, expression, development, maturation, germination and plant conversion. These steps are characterized by distinct physiological, morphological and molecular events. Although somatic embryogenesis has been established in several plant species, there remains many problems to be solved. The main problem in somatic embryogenesis is the large number of abnormal embryos produced which cannot germinate nor convert into normal plants. Abnormalities in somatic embryos (SE) can be generated by genetic or epigenetic changes in the DNA. These changes in the DNA can be influenced by external factors such as the use of plant growth regulators and mutagenic substances or stress factors applied to the plant tissue such as high and low temperatures, drought, salinity, and heavy metals. Abnormalities generated by genetic changes in the DNA are hardly reversible; however, abnormalities generated by epigenetic changes may be reversible and the abnormal embryos are able to produce normal plants in most cases. This review focuses on the identification of the main factors that can cause abnormal SE development in different plant species, suggest how SE abnormalities are related to somaclonal variations and identify which genes may be involved with embryo abnormalities. Zygotic embryo abnormalities in Arabidopsis thaliana mutants are listed with the aim to understand the main genetic mechanisms involved in embryo aberrations. The abnormalities in somatic embryos are related to the use of 2,4-D in most of the published protocols, this sintetic auxin disrupts the endogenous auxin balance and the auxin polar transportation interfering with the embryo apical-basal polarity.
70 citations
01 Jan 2012
TL;DR: In this paper, the effects of 5-azacytidine (AzaC) and 2,4-dichlorophenoxyacetic acid (2-4-D) on Acca sellowiana somatic embryogenesis and global DNA methylation levels were evaluated.
Abstract: DNA methylation is an epigenetic regulatory mechanism of gene expression which can be associated with developmental phases and in vitro morphogenetic compe- tence in plants. The present work evaluated the effects of 5-azacytidine (AzaC) and 2,4-dichlorophenoxyacetic acid (2,4-D) on Acca sellowiana somatic embryogenesis (SE) and global DNA methylation levels by high-performance liquid chromatography mass spectrometry (HPLC/MS/MS). 2,4-D-free treatments revealed no somatic embryo formation in both accessions tested. Treatments supplemented with 2,4-D pulse plus AzaC in the culture medium resulted in increased embryo formation. In AzaC-free treatment, HPLC/MS/MS analysis showed a gradual increase in methylation levels in cultures of both accessions tested during SE induction. Treatment with AzaC and 2,4-D-free resulted in a marked decrease in methylation for both accessions, ranging from 37.6 to 20.8 %. In treatment with 2,4-D and AzaC combined, the 85 accession showed increasing global methylation levels. Otherwise, the 101X458 accession, in the same treatment, showed a decrease between 10 and 20 days, followed by an increase after 30 days (39.5, 36.2 and 41.6 %). These results indicate that 2,4-D pulse combined with AzaC improves SE induc- tion. However, the conversion phase showed that although positively influencing SE induction, AzaC had a dysregu- latory effect on the stage of autotrophic plant formation, resulting in significantly lower conversion rates. The results suggest that DNA methylation dramatically influences SE in Acca sellowiana, and global DNA methylation dynamics are related to morphogenetic response. Key message 5-Azacytidine combined with 2,4-D increases the number of Acca sellowiana somatic embryos. Global DNA methylation is directly affected by these compounds.
64 citations
TL;DR: A high degree of similarity between protein profiles of the assayed somatic embryos was observed and proteins involved in the synthesis of phenylalanine ammonia-lyase, a conspicuous polyphenol present in the induction of feijoa embryogenic cultures were identified.
Abstract: Feijoa (Acca sellowiana, Myrtaceae), a native fruit species from southern Brazil and northern Uruguay, is considered to constitute a reference system for somatic embryogenesis in woody dicots. This in vitro regenerative pathway is an efficient micropropagation method, and a suitable model system for studies in plant developmental physiology. This study attempts to detect and identify proteins that are expressed during the different developmental stages of somatic embryos of A. sellowiana. Using high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE), a high degree of similarity between protein profiles of the assayed somatic embryos was observed. Of the 74 different protein spots extracted for analysis, 60 were identified by means of 2-DE/MALDI-TOF/MS. Twelve proteins were expressed in all the assayed stages. Ten proteins were expressed in the initial stages and 22 proteins were expressed in the mature developmental stages of somatic embryos. Only one protein was expressed exclusively in the torpedo stage, whereas four were expressed in the pre-cotyledonary, and none in the cotyledonary stage. The proteins identified were involved in the synthesis of phenylalanine ammonia-lyase, a conspicuous polyphenol present in the induction of feijoa embryogenic cultures. The presence of essential proteins of nitrogen metabolism, such as the cytosolic glutamine synthetase protein, was also observed. The physiological implications of these findings are discussed.
27 citations
Cites background from "Anomalous somatic embryos in Acca s..."
...…with its non-expression in the other developmental stages, could suggest an anomalous gene expression in mature somatic embryos showing morphologic abnormalities, as revealed by Pescador et al. (2008), or a low rate of embryo conversion to plantlets as shown by Cangahuala-Inocente et al. (2007)....
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TL;DR: The results outlined the hormonal variations occurring during zygotic and somatic embryogenesis regarding the timing, nature and hormonal status involved in both processes and could be related to the previously observed high frequency formation of anomalous somatic embryos formed in A. sellowiana, as well as to their low germination ability.
Abstract: Endogenous levels of IAA, ABA and four types of CKs were analyzed in zygotic and indirect (ISE) and direct somatic embryogenesis of Acca sellowiana. Zygotic and somatic embryos at different developmental stages were sampled for morphological and hormonal analysis. Both embryo types showed substantial asymmetry in hormone levels. Zygotic embryos displayed a conspicuous peak of IAA in early developmental stages. The results outlined the hormonal variations occurring during zygotic and somatic embryogenesis regarding the timing, nature and hormonal status involved in both processes. The short transient pulse of IAA observed on the 3rd day in culture was suggested to be involved with the signaling for the induction of somatic embryogenesis. Fertilized ovule development was associated with increased IAA levels 21–24 days after pollination, followed by a sharp decrease in the cotyledonary stage, both in zygotic and somatic embryos. There was a prominent increase in ABA levels in cultures which generated ISE 24–30 days after pollination, a period that corresponds to the heart and torpedo stages. The levels of total CKs (Z, [9R]Z, iP and [9R]iP) were also always higher in zygotic than in somatic embryogenesis. While zygotic embryogenesis was dominated by the presence of zeatin, the somatic process, contrarily, was characterized by a large variation of the other cytokinin forms and amounts studied. The above results, when taken together, could be related to the previously observed high frequency formation of anomalous somatic embryos formed in A. sellowiana, as well as to their low germination ability.
18 citations
Cites background or result from "Anomalous somatic embryos in Acca s..."
...In view of this, it is also plausible to believe that the relatively high frequency of anomalous somatic embryos connected to the very low percentage of germination observed in A. sellowiana (Pescador et al. 2008), could be seen as a consequence of these disturbances....
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...sellowiana with cotyledon fusion, malformed apical meristems, or even their complete absence (Pescador et al. 2008), could involve disturbances in IAA distribution...
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...The development of somatic embryos of A. sellowiana with cotyledon fusion, malformed apical meristems, or even their complete absence (Pescador et al. 2008), could involve disturbances in IAA distribution within the embryo or lower levels of IAA found in embryos derived from ISE and DSE compared to…...
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...Another difference is the continuous growth of somatic embryos resulting from a lack of developmental arrest, often leading to the formation of abnormal structures with very low frequency of plant development (Faure et al. 1998; Pescador et al. 2008)....
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...sellowiana (Pescador et al. 2008), could be seen as a consequence of these disturbances....
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References
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Journal Article•
8,559 citations
"Anomalous somatic embryos in Acca s..." refers methods in this paper
...For histological analyses, samples were fixed in Karnovsky’s solution (Karnovsky 1965), modified by using 1% glutaraldehyde in 0....
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...For histological analyses, samples were fixed in Karnovsky’s solution (Karnovsky 1965), modified by using 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), 4% formaldehyde, and 0.2 M phosphate buffer (pH 7.2), 5:3:2 (v/v), for 4 hours at room temperature....
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TL;DR: It is argued that this variation in plant cell culture itself generates genetic variability (somaclonal variation) that may be employed to enhance the exchange required in sexual hybrids for the introgression of desirable alien genes into a crop species.
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3,113 citations
"Anomalous somatic embryos in Acca s..." refers methods in this paper
...…used as a technique to propagate plants in large scale (Gray & Purohit 1991) but at the same time it is a prominent source of somaclonal variation (Larkin & Scowcroft 1981) in a representative number of plants (Lazzeri et al. 1987, Fourré et al. 1997, Rodriguez & Wetzstein 1998, Dedicová et al.…...
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The effects of various common fixatives and the effects of the pH of the staining solution are evaluated.
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...Longitudinal and transverse sections (5-7 µm thick) were obtained with a rotary microtome, mounted on glass slides and stained with 0.05% toluidine blue O (Color Index 52040) in 0.1 M phosphate buffer (pH 6.8) (O’Brien et al. 1965) for five minutes....
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TL;DR: The results indicate how the establishment of cell polarity, polar auxin efflux and local auxin response result in apical–basal axis formation of the embryo, and thus determine the axiality of the adult plant.
Abstract: Axis formation occurs in plants, as in animals, during early embryogenesis. However, the underlying mechanism is not known. Here we show that the first manifestation of the apical-basal axis in plants, the asymmetric division of the zygote, produces a basal cell that transports and an apical cell that responds to the signalling molecule auxin. This apical-basal auxin activity gradient triggers the specification of apical embryo structures and is actively maintained by a novel component of auxin efflux, PIN7, which is located apically in the basal cell. Later, the developmentally regulated reversal of PIN7 and onset of PIN1 polar localization reorganize the auxin gradient for specification of the basal root pole. An analysis of pin quadruple mutants identifies PIN-dependent transport as an essential part of the mechanism for embryo axis formation. Our results indicate how the establishment of cell polarity, polar auxin efflux and local auxin response result in apical-basal axis formation of the embryo, and thus determine the axiality of the adult plant.
1,767 citations
"Anomalous somatic embryos in Acca s..." refers background in this paper
...D can change the conformation of the PIN protein, negatively interfering with the efflux of IAA, and altering the normal establishment of the apical-basal somatic embryos axis (Friml et al. 2003)....
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TL;DR: The purpose of this paper is to show the growth of freely suspended cells from certain dicotyledonous plants may be carried forward into the formation of roots and shoots and, in fact, the development of whole plants.
Abstract: THE FIRST of this group of papers (Steward et al., 1958) described the various ways in which freely suspended cells from certain dicotyledonous plants grow and multiply to form a relatively unorganized nmulticellular mnass. The growth in question occurs under prescribed and controlled nutritional and environmental conditions. The purpose of this paper is to show hoAv the growth may be carried forward into the formation of roots and shoots and, in fact, the development of whole plants.
748 citations
"Anomalous somatic embryos in Acca s..." refers background in this paper
...Haccius work preceded the classic work of Steward et al. (1958) on carrot somatic embryos induction without the use of 2,4-D. Halperin & Wetherell (1969) observed the abnormal development of apical meristems and further inhibited growth on carrot embryos when they were treated with relative high…...
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