Gilberto Barbante Kerbauy
Bio: Gilberto Barbante Kerbauy is an academic researcher from University of São Paulo. The author has contributed to research in topics: Explant culture & Somatic embryogenesis. The author has an hindex of 4, co-authored 4 publications receiving 104 citations.
TL;DR: Histological analysis indicated that buds formed at the leaf base at the 65th day of culture, and the endogenous level of IAA and the IAA/cytokinin ratio decreased after the 65rd day of Culture, while the level of ABA was reduced from the time of inoculation up to the 75th dayof culture.
Abstract: Shoot apices of Musa subgroup AAA `Grande Naine' were used for in vitro culture establishment. The endogenous hormone levels and their effects on bud formation were evaluated during a 75-day period. Cytokinins, IAA and ABA were separated by HPLC and quantified by means of ELISA. Enzymatic degradation of IAA was determined by the colorimetric method. Explants were maintained on establishment medium for 60 days. The endogenous cytokinins were higher in the basal portion of the explant. Subculture to proliferation medium (65 to 75 days) resulted in a substantial increase of cytokinins in the basal portion and in a decline in the apical portion. 2iP was the predominant cytokinin in the tissue. The endogenous level of IAA and the IAA/cytokinin ratio decreased after the 65th day of culture. The level of ABA was reduced from the time of inoculation up to the 75th day of culture. Histological analysis indicated that buds formed at the leaf base at the 65th day of culture.
TL;DR: Zygotic embryo formation occurred under a situation represented by high sucrose amounts, simultaneously with low fructose and glucose levels, while in contrast, somatic embryo formation took place under an endogenous sugar status characterized by a substantial fructose enhancement.
Abstract: Comparative analysis of zygotic and somatic embryogenesis of Acca sellowiana showed higher amounts of sucrose, fructose, raffinose, and myo-inositol in zygotic embryos at different developmental stages than in corresponding somatic ones. These differences were mostly constant. In general, glucose levels were significantly lower than the other soluble carbohydrates analyzed, showing minor variation in each embryo stage. Despite the presence of sucrose in the culture medium, its levels conspicuously diminished in somatic embryos compared with the zygotic ones. Raffinose enhanced parallel to embryo development, regardless of its zygotic or somatic origin. Analysis of the soluble carbohydrate composition of mature zygotic cotyledon used as explant pointed out fructose, glucose, myo-inositol, sucrose, and raffinose as the most important. Similar composition was also found in the corresponding somatic cotyledon. Total soluble carbohydrates varied inversely, decreasing in zygotic embryos and increasing in somatic embryos until the 24th d, at which time they increased rapidly about sixfold in zygotic embryos until the 27th d, a period coinciding with the zygotic proembryos formation. Such condition seems to reflect directly the variation of endogenous sucrose level, mainly because glucose and fructose diminished continuously during this time period. This means that, in terms of soluble sugars, zygotic embryo formation occurred under a situation represented by high sucrose amounts, simultaneously with low fructose and glucose levels, while in contrast, somatic embryo formation took place under an endogenous sugar status characterized by a substantial fructose enhancement. Starch levels increased continuously in zygotic embryos and decreased in somatic ones, the reverse to what was found in fructose variation. Starch accumulation was significantly higher in somatic torpedo and cotyledonary embryos than in the corresponding zygotic ones.
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.
25 Jun 1992
TL;DR: Os efeitos de diferentes tipos de meios de cultura, nutrientes orgânicos, carvao ativado, estado fisico do meio e tamanho dos explantes foram estudados em relacao a conversao de apices radiculares de C. pileatum em protocormoides (PLB).
Abstract: Os efeitos de diferentes tipos de meios de cultura, nutrientes orgânicos, carvao ativado, estado fisico do meio e tamanho dos explantes foram estudados em relacao a conversao de apices radiculares de C. pileatum em protocormoides (PLB). O meio MS modificado foi o que mais favoreceu a inducao de protocormoides quando comparado aos meios mais diluidos. A manutencao do crescimento do explante radicular e a formacao de protocormoides foi dependente da presenca de uma fonte de carbono no meio: sacarose, glicose e frutose mostraram ser as fontes mais efetivas. Bacto-peptona estimulou a regeneracao de protocormoides. Em meio liquido os explantes mostraram uma tendencia para a regeneracao de PLB do tipo multiplo. O carvao ativado mostrou ser eficaz na transformacao dos apices radiculares em protocormoides. Verificou-se uma relacao inversa entre o tamanho do explante e a capacidade de formacao de protocormoides.
TL;DR: The effect of the type and concentration of plant growth regulators and sub-culturing on somaclonal variation were studied in Cavendish banana cv.
Abstract: The effect of the type and concentration of plant growth regulators and sub-culturing on somaclonal variation were studied in Cavendish banana cv. ‘ Zelig ’ obtained from African Biotechnologies Ltd., South Africa. In vitro grown plants at the fourth multiplication cycle were used for the investigation. Auxins (IAA, IBA and NAA) and cytokinins (BA and TDZ) were used to multiply shoots for 10 generations. Bands generated through RAPD-PCR were scored according to whether they were present (1) or absent (0) to determine the extent of somaclonal variation. Results were then analyzed using cluster analysis. The relationship between multiplication rate and somaclonal variation was assessed using correlation analysis. Results indicated that treatments with higher multiplication rates produced more variants; sometimes as high as 72%. Dwarf off-types accounted for 88% of the variation. A dwarf-specific band, about 1500 kb in size, was amplified by the primer OPC-15. The band appeared consistently in normal plants but was absent in all dwarf plants.
TL;DR: Using a protocol for high rate shoot multiplication, certain morphological abnormalities observed during proliferation of shoot buds in vitro were not observed during acclimatization ex vitro, and the genetic stability of plantlets was assessed using RAPD and ISSR markers.
Abstract: Use of high levels of growth regulators during micropropagation results in undesirable clonal variability in important commercial crops such as banana. The present study investigated the effects of high levels of cytokinins on micropropagation in banana (genotype AAB), and the genetic stability of plantlets was assessed using RAPD and ISSR markers. Cytokinins, such as BA and kinetin were added to the routine shoot multiplication medium at concentrations up to 10 mg l−1. After 12 weeks of culture involving three subcultures, the maximum number of shoot buds were produced in cultures receiving either 5 mg l−1 BA (80 shoot buds) or 4 mg l−1 kinetin (62 shoot buds). Certain morphological abnormalities observed during proliferation of shoot buds in vitro were not observed during acclimatization ex vitro. To check the genetic stability, RAPD and ISSR profiles of micropropagated plantlets obtained from different cytokinin-treatments were compared with control microplants maintained on MS medium as well as the field-grown mother plant. A total of 50 RAPD and 12 ISSR primers resulted in 625 distinct and reproducible bands. Thus a total of 17,400 bands were generated showing homogeneous RAPD and ISSR patterns. Band intensity histogram of each gel confirmed their monomorphic nature with no genetic variation in all the plantlets analysed. Based on these results a protocol for high rate shoot multiplication was worked out leading to uniform shoot production.
TL;DR: MS (Murashige and Skoog) media supplemented with benzylaminopurine showed that the number of bud formation in shoot cultures of Musa acuminata cv.
Abstract: MS (Murashige and Skoog) media supplemented with benzylaminopurine (BAP) showed that the number of bud formation in shoot cultures of Musa acuminata cv.Berangan during the initiation stage increased proportionately with the concentrations used (11, 22 and 33 µM). However, the highest concentration of BAP (33 µM) simultaneously increased the formation of abnormal shoots. After the first apical bud appeared, explants were transferred to MS medium with lower concentrations of BAP either with or without indole acetic acid (IAA). Proliferation media supplemented with IAA showed enhanced shoot multiplication and elongation but did not help to reduce the abnormality index that occurred.
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.
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.