Browsing by Author "Oropeza, C."
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Item 16S rRNA interoperon sequence heterogeneity distinguishes strain populations of palm lethal yellowing phytoplasma in the Caribbean region(2002) Harrison, N.A.; Myrie, W.; Jones, P.; Carpio, M.L.; Castillo, M.; Doyle, M.M.; Oropeza, C.DNA of phytoplasmas in lethal yellowing (LY)-diseased palms was detected by a nested polymerase chain reaction (PCR) assay employing rRNA primer pair P1/P7 followed by primer pair LY16Sf/ LY16-23Sr. Polymorphisms revealed by Hinfl endonuclease digestion of rDNA products differentiated coconut-infecting phytoplasmas in Jamaica from those detected in palms in Florida, Honduras and Mexico. A three fragment profile was generated for rDNA from phytoplasmas infecting all 21 Jamaican palms whereas a five fragment profile was evident for phytoplasmas infecting the majority of Florida (20 of 21), Honduran (13 of 14) and Mexican (5 of 5) palms. The RFLP profile indicative of Florida LY phytoplasma was resolved by cloning into two patterns, one of three bands and the other of four bands, that together constituted the five fragment profile. The two patterns were attributed to presence of two sequence heterogeneous rRNA operons, rrnA and rrnB, in most phytoplasmas composing Florida, Honduran and Mexican LY strain populations. Unique three and four fragment RFLP profiles indicative of LY phytoplasmas infecting Howea forsteriana and coconut palm in Florida and Honduras, respectively, were also observed. By comparison, the Jamaican LY phytoplasma population uniformly contained one or possibly two identical rRNA operons. No correlation between rRNA interoperon heterogeneity and strain variation in virulence of the LY agent was evident from this study.Item Detection of a SERK-like gene in coconut and analysis of its expression during the formation of embryogenic callus and somatic embryos(2009) Perez-Nunez, M.T.; Souza, R.; Chan, J.L.; Saenz, L.; Zuniga-Aguilar, J.J.; Oropeza, C.Somatic embryogenesis involves different molecular events including differential gene expression and various signal transduction pathways. One of the genes identified in early somatic embryogenesis is SOMATIC EMBRYOGENESIS RECEPTOR-like KINASE (SERK). Cocos nucifera (L.) is one of the most recalcitrant species for in vitro regeneration, achieved so far only through somatic embryogenesis, although just a few embryos could be obtained from a single explant. In order to increase efficiency of this process we need to understand it better. Therefore, the purpose of the present work was to determine if an ortholog of the SERK gene is present in the coconut genome, isolate it and analyze its expression during somatic embryogenesis. The results showed the occurrence of a SERK ortholog referred to as CnSERK. Predicted sequence analysis showed that CnSERK encodes a SERK protein with the domains reported in the SERK proteins in other species. These domains consist of a signal peptide, a leucine zipper domain, five LRR, the Serine- Proline-Proline domain, which is a distinctive domain of the SERK proteins, a single transmembrane domain, the kinase domain with 11 subdomains and the C terminal region. Analysis of its expression showed that it could be detected in embryogenic tissues before embryo development could be observed. In contrast it was not detected or at lower levels in non-embryogenic tissues, thus suggesting that CnSERK expression is associated with induction of somatic embryogenesis and that it could be a potential marker of cells competent to form somatic embryos in coconut tissues cultured in vitro.Item Effect of 22(S),23(S)-homobrassinolide on somatic embryogenesis in plumule explants of Cocos nucifera (L.) cultured in vitro(2003) Azpeitia, A.; Chan, J.L.; Saenz, L.; Oropeza, C.Item The effect of gibberellic acid on the in vitro germination of coconut zygotic embryos and their conversion into plantlets(The Society for In Vitro Biology, 2007) Pech y Ake, A.; Maust, B.; Orozco-Segovia, A.; Oropeza, C.The effect of gibberellic acid (GA3) was tested on germination of coconut zygotic embryos, their conversion into plantlets and ex vitro survival. There were four treatments consisting of 5 wk of culture in semi-solid medium or liquid medium, with or without GA3. Embryos were then transferred to GA3 free-liquid medium for the rest of a 32-wk culture. Germination and conversion percentages were higher in semi-solid medium than in liquid medium, and with both media percentages increased with GA3 treatment (with the exception of the highest GA3 concentration). Embryos of two varieties (MGD and MYD) were used. The following are the results with MGD embryos. Optimum GA3 concentration in liquid medium was 0.46 μM, with 80% germination (62% in the control without GA3) and 4.6 μM in semi-solid medium with 98% germination (71% in the control). With GA3 treatment, germination was also faster. Conversion in semi-solid medium with GA3 was 87% (60% in the control), and 45% in liquid medium with GA3 (25% in the control). Once the plantlets had at least three bifid leaves and three primary roots at the time of transfer to ex vitro, they survived independently of the treatment. When MYD embryos were used, germination and conversion percentages were higher in semi-solid medium than in liquid medium, and they increased when GA3 was used, although percentages were lower than those obtained with MGD embryos. The results showed that the use of GA3 benefited coconut embryos in culture because it favored germination and conversion to plants on semi-solid medium, and hence improved previous protocols.Item Endogenous isoprenoid and aromatic cytokinins in different plant parts of Cocos nucifera (L.)(2003) Saenz, L.; Jones, L.H.; Oropeza, C.; Vlacil, D.; Strnad, M.The present study reports the analyses of both isoprenoid and aromatic cytokinins in the coconut palm by combined high performance liquid chromatography and group specific enzyme immunoassays (HPLC-ELISA). The results showed that the isoprenoid cytokinins were several fold more abundant than the aromatic cytokinins in each of the plant parts analysed: immature inflorescence, shoot apical meristem (SAM), spear leaf and embryo. Within the isoprenoid cytokinins, the most abundant ones by type were the zeatin- (Z-), the isopentenyladenine- (iP-) and the dihydrozeatin- (DHZ-) type in decreasing order for most plant parts studied, and individually, zeatin riboside (ZR) or zeatin riboside-5 -monophosphate (ZR5 P) depending on the part. In the case of the iP-type cytokinins, the results showed that its 9-glucoside was the most abundant one in most parts. The isoprenoid cytokinin profiles in coconut showed a predominant pattern of 9-conjugation as a major metabolism route for these cytokinins. Analyses also showed the occurrence of the aromatic cytokinin 6-benzylaminopurine (BAP) and its riboside (BAPR), 9-glucoside (BAP9G), and nucleotide (BAPR5 P). Their presence in coconut palm was unequivocally identified after permethylation by gas chromatography-mass spectrometry. They were more concentrated in the embryo and in the immature inflorescence than in the other two parts studied, however their concentration in each part was several times lower than that of isoprenoid cytokinins. All four were detected in each of the parts studied. The most abundant ones were BAPR and BAP9G in immature inflorescence; and BAPR in all of the other parts. When all cytokinins analysed are considered, differences between the plant parts studied were found. The zygotic embryos showed the highest content, double that in immature inflorescence, and five times more that in spear leaf and SAM. These differences are even greater when individual cytokinins are compared.Item First report of a phytoplasma-associated leaf yellowing syndrom of Palma Jipi plants in Southern Mexico(2000-07) Cordova, I.; Oropeza, C.Item Improved somatic embryogenesis from cocos nucifera (l.) Plumule explants(Society for In Vitro Biology, 2006-02) Perez-Nunez, M.T.; Chan, J.L.; Saenz, L.; Gonzalez, T.; Verdeil, J.L.; Oropeza, C.Coconut is one of the most recalcitrant species to regenerate in vitro. Although previous research efforts using plumule explants have resulted in reproducible somatic embryogenesis, efficiency is only 4 or 10 somatic embryos per plumule without or with a brassinolide treatment, respectively. In order to increase the efficiency of somatic embryogenesis in coconut, two different approaches were evaluated and reported here: secondary somatic embryogenesis and multiplication of embryogenic callus. Primary somatic embryos obtained from plumule explants were used as explants and formed both embryogenic callus and secondary somatic embryos. The embryogenic calluses obtained after three multiplication cycles were capable of producing somatic embryos. The efficiency of the system was evaluated in a stepwise process beginning with an initial step for inducing primary somatic embryogenesis followed by three steps for inducing secondary somatic embryogenesis followed by three steps for embryogenic callus multiplication, and finally production of somatic embryos from callus. The total calculated yield from one plumule was 98 000 somatic embryos. Comparing this to the yield obtained from primary somatic embryogenesis results in about a 50 000-fold increase. When compared to the yield previously reported in the literature with the use of a brassinolide treatment, it is about a 10 000-fold increase in yield. The present protocol represents important progress in improvement in the efficiency of coconut somatic embryo production.Item Morphological and Histological Changes During Somatic Embryo Formation from Coconut Plumule Explants(Society for In Vitro Biology, 2006-02) Saenz, L.; Azpeitia, A.; Chuc-Armendariz, B.; Chan, J.L.; Verdeil, J.L.; Hocher, V.; Oropeza, C.Studies on the development of protocols for the clonal propagation, through somatic embryogenesis, of coconut have been reported for the past three decades, mostly using inflorescence explants, but with low reproducibility and efficiency. Recent improvements in these respects have been achieved using plumular explants. Here, we report a developmental study of embryogenesis in plumule explants using histological techniques in order to extend our understanding of this process. Coconut plumule explants consisted of the shoot meristem including leaf primordia. At day 15 of culture, the explants did not show any apparent growth; however, a transverse section showed noticeable growth of the plumular leaves forming a ring around the inner leaves and the shoot meristem, which did not show any apparent growth. At day 30, the shoot meristem started to grow and the plumular leaves continued growing. At day 45, the explants were still compact and white in color, but showed partial dedifferentiation and meristematic cell proliferation leading to the development of callus structures with a translucent appearance. After 60 d, these meristematic cells evolved into nodular structures. At day 75, the nodular structures became pearly globular structures on the surface of translucent structures, from which somatic embryos eventually formed and presented well-developed root and caulinar meristems. These results allow better insights and an integrated view into the somatic embryogenesis process in coconut plumule explants, which could be helpful for future studies that eventually could lead us to improved control of the process and greater efficiency of somatic embryo and plantlet formation.Item Phytoplasma distribution in coconut palms affected by lethal yellowing disease(2011) Oropeza, C.; Cordova, I.; Chumba, A.; Narvaez, M.; Saenz, L.; Ashburner, R.; Harrison, N.Lethal yellowing (LY), the most devastating disease affecting the coconut palm correspondence in America, is caused by phytoplasmas known to be distributed in different Dr C. Oropeza, Biotechnology Unit, Centro de pans of infected plants. However, no comprehensive reports exist on the phytoInvestigaci6n Cientlfica de Yucatan, Calle 43 plasma distribution. This study refers to the detection of LY phytoplasma DNA No. 130. Colonia Chuburna de Hidalgo, 97200 using PCR in different coconut plant pans, throughout the development of the Merida, Yucatan, Mexico. disease. Sample analysis of positive palms taken at different stages of disease Email: cos@cicy.mx development (either symptomatic or symptomless) showed differences in the Received: 19 November 2010; revised version percentage of LY detection between plant parts. Some parts showed a very high accepted: 1 April 2011. level of LY DNA (stem, young leaves, inflorescences, stem apex and root apex), low levels were found in the intermediate leaves and roots without apex, dOi:10.11111j.1744-7348.2011.00480.x whereas no LYphytoplasma DNA was detected in mature leaves. The detection percentage of LY phytoplasma DNA was lowest in symptomless-infected palms for all parts, except the stem, where phytoplasma accumulations were consistently detected. This pattern of detection among parts is consistent with the hypothesis that phytoplasmas move from photosynthate source tissues to sink tissues via the phloem mass flow process. The accumulations in the (lower) . stem, prior to the appearance of symntoms, suggest that this pan of the palm is where phytoplasmas first move from leaves after foliar feeding by vectors and in which they probably multiply and distribute to other palm parts, including roots. Embryos from infected palms were analysed by nested-PCR and 28% of 394 embryos were positive. Phytoplasma DNA was detected in embryos from fruit on any of the fruiting bunches regardless the age, but no pattern of quantitative distribution throughout the bunch developmental stages was observed. Germination of seeds from LY·positive symptomatic palms was 58% and from LY-negative symptomless palms were 71 %. No phytoplasma was detected in seedlings tested from both symptomatic and non-symptomatic palms. Seedlings tested after 2 years did not develop LY symptoms or eventually died.Item Regeneration of coconut (Cocos nucifera L.) from plumule explants through somatic embryogenesis(1998) Chan, J.L.; Saenz, L.; Talavera, C.; Hornung, R.; Robert, M.; Oropeza, C.A protocol was developed for coconut regeneration using plumules from mature zygotic embryos as explants, and media with the synthetic growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Evidence for the regeneration process from these tissues occurring through somatic embryogenesis is presented. The somatic embryos were capable of germination, subsequent development into plantlets and successful transfer to the nursery. The yields were larger, nearly twofold for calli and over tenfold for calli bearing somatic embryos, than those previously reported with inflorescence explants. The present protocol thus represents an improvement in time and yield over previous protocols. Even though plumule explants are not the ideal tissue source due to possible genetic heterogeneity, the improvements made here may be applicable to tissues from mature plants. In addition, micropropagation of coconut using plumules is potentially useful when they are obtained from fruit produced from selected parents of outstanding performance, such as those resistant to diseases.Item What are the possible applications for coconut (Cocos nucifera L.) micropropagation(1998) Verdeil, J.L.; Baudouin, L.; Hocher, V.; Bourdeix, R.; N' cho, Y.P.; Sangare, A.; Rillo, E.; Hamon, S.; Oropeza, C.