Neftalí Ochoa-alejo - Academia.edu (original) (raw)

Papers by Neftalí Ochoa-alejo

Methods in molecular biology (Clifton, N.J.), 2006

Fruits of chili peppers (Capsicum spp.) specifically synthesize and accumulate a group of analogs... more Fruits of chili peppers (Capsicum spp.) specifically synthesize and accumulate a group of analogs known as capsaicinoids in the placenta tissues. These secondary metabolites are responsible for the hot taste of chili pepper fruits. Capsaicinoids are of economic importance because of their use in the food, cosmetic, military, and pharmaceutical industry. Several efforts have been focused to investigate the biosynthetic capacity of in vitro chili pepper cells and tissue cultures in order to determine the production feasibility of these compounds at the industrial level under controlled conditions. A description of techniques for the establishment of in vitro cultures of chili pepper, the addition of precursors and intermediates to the culture medium, and the selection of cell lines as a means to increase the production of capsaicinoids as well as the extraction, separation, and quantification of capsaicinoids from chili pepper cell cultures is reported in this chapter.

Methods in Molecular Biology, 2012

Plant cell, tissue, and organ culture (PTC) techniques were developed and established as an exper... more Plant cell, tissue, and organ culture (PTC) techniques were developed and established as an experimental necessity for solving important fundamental questions in plant biology, but they currently represent very useful biotechnological tools for a series of important applications such as commercial micropropagation of different plant species, generation of disease-free plant materials, production of haploid and doublehaploid plants, induction of epigenetic or genetic variation for the isolation of variant plants, obtention of novel hybrid plants through the rescue of hybrid embryos or somatic cell fusion from intra- or intergeneric sources, conservation of valuable plant germplasm, and is the keystone for genetic engineering of plants to produce disease and pest resistant varieties, to engineer metabolic pathways with the aim of producing specific secondary metabolites or as an alternative for biopharming. Some other miscellaneous applications involve the utilization of in vitro cultures to test toxic compounds and the possibilities of removing them (bioremediation), interaction of root cultures with nematodes or mycorrhiza, or the use of shoot cultures to maintain plant viruses. With the increased worldwide demand for biofuels, it seems that PTC will certainly be fundamental for engineering different plants species in order to increase the diversity of biofuel options, lower the price marketing, and enhance the production efficiency. Several aspects and applications of PTC such as those mentioned above are the focus of this edition.

Plant Science, 1994

ABSTRACT Cell cultures were established from the drought sensitive chili pepper (Capsicum annuum ... more ABSTRACT Cell cultures were established from the drought sensitive chili pepper (Capsicum annuum L.) and from Larrea tridentata, a plant species highly tolerant to drought. The cell cultures were exposed to water stress imposed by the presence of polyethylene glycol (PEG) in the culture medium. Cell cultures of L. tridentata showed higher resistance to PEG (6.6 times higher) than those of C. annuum. More negative ψs values were found to occur in cell cultures of L. tridentata in response to PEG as compared to cell suspensions from chili pepper. Analysis of solutes revealed an important contribution of K+ to ψs. K+ levels were higher in chili pepper cells than in L. tridentata cultures. The Na+ and Ca2+ contents in the cell suspensions of the two species did not contribute significantly to ψs. A positive correlation was observed between proline accumulation and the capacity of cell cultures to grow in conditions of water stress. Glycine betaine levels did not show a positive correlation with drought resistance.

Plant Cell Reports, 1998

Transgenic Mexican lime [Citrus aurantifolia (Christm.) Swing] plants were regenerated from tissu... more Transgenic Mexican lime [Citrus aurantifolia (Christm.) Swing] plants were regenerated from tissues transformed by Agrobacterium rhizogenes strain A4, containing the wild-type plasmid pRiA4 and the binary vector pESC4 with nos-npt II and cab-gus genes. Transgenic shoots were generated by two different approaches. The first approach used internodal stem segments cocultured with A. rhizogenes. These were placed onto regeneration medium containing Murashige and Skoog salts and B5 organic compounds supplemented with 8 g · l -1 agar, 7.5 mg · l -1 6-benzylaminopurine, 1.0 mg · l -1 -naphthaleneacetic acid, 300 mg · l -1 cefotaxime and 80 mg · l -1 kanamycin as a selective agent, and incubated under continuous light at 25°C. Under these conditions, 76% of the explants produced shoots directly with no hairy root phase, with a mean of 1.3 shoots per explant, and 88% of these shoots were genetically transformed as determined by β-glucuronidase (GUS) assays. In the second approach, segments of transformed roots (15 mm long) obtained from internodal stem segments cocultured with A. rhizogenes were cultured on the above regeneration medium under similar conditions. Forty-one percent of these transformed root segments produced adventitious shoots, with a mean of 2.2 shoots per explant and with 90% of shoots transformed. GUS activity was evident in the transformed roots and in all parts of both transformed shoots and regenerated plants. The presence of the npt II and rolB genes in the regenerated plants was confirmed by PCR analysis. The presence of the npt II gene in the regenerated plants was also confirmed by Southern blot. Using these transformation systems, more than 300 Mexican lime transgenic plants were obtained, 60 of which were adapted to growing in soil.

BMC Genomics, 2014

The set of all mRNA molecules present in a cell constitute the transcriptome. The transcriptome v... more The set of all mRNA molecules present in a cell constitute the transcriptome. The transcriptome varies depending on cell type as well as in response to internal and external stimuli during development. Here we present a study of the changes that occur in the transcriptome of chili pepper fruit during development and ripening. Results: RNA-Seq was used to obtain transcriptomes of whole Serrano-type chili pepper fruits (Capsicum annuum L.; 'Tampiqueño 74') collected at 10, 20, 40 and 60 days after anthesis (DAA). 15,550,468 Illumina MiSeq reads were assembled de novo into 34,066 chili genes. We classified the expression patterns of individual genes as well as genes grouped into Biological Process ontologies and Metabolic Pathway categories using statistical criteria. For the analyses of gene groups we added the weighted expression of individual genes. This method was effective in interpreting general patterns of expression changes and increased the statistical power of the analyses. We also estimated the variation in diversity and specialization of the transcriptome during chili pepper development. Approximately 17% of genes exhibited a significant change of expression in at least one of the intervals sampled. In contrast, significant differences in approximately 63% of the Biological Processes and 80% of the Metabolic Pathways studied were detected in at least one interval. Confirming previous reports, genes related to capsaicinoid and ascorbic acid biosynthesis were significantly upregulated at 20 DAA while those related to carotenoid biosynthesis were highly expressed in the last period of fruit maturation (40-60 DAA). Our RNA-Seq data was validated by examining the expression of nine genes involved in carotenoid biosynthesis by qRT-PCR. Conclusions: In general, more profound changes in the chili fruit transcriptome were observed in the intervals between 10 to 20 and 40 to 60 DAA. The last interval, between 40 to 60 DAA, included 49% of all significant changes detected, and was characterized predominantly by a global decrease in gene expression. This period signals the end of maturation and the beginning of senescence of chili pepper fruit. The transcriptome at 60 DAA was the most specialized and least diverse of the four states sampled.

Biologia Plantarum, 2005

The activities of sucrose phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (N... more The activities of sucrose phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (NI) and soluble acid invertase (SAI) were measured in callus cultures of four Mexican sugarcane cultivars (Saccharum spp.) with a different capacity to accumulate sucrose in stem parenchyma cells. The results indicated that sucrose accumulation in callus was positively correlated to the activity of SPS and SUSY and negatively to the activity of SAI and NI while SPS explained most of the variation found for sucrose accumulation and NI least.

Acta Physiologiae Plantarum, 2002

The activities of sucrose-phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (N... more The activities of sucrose-phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (NI) and soluble acid invertase (SAI) regulates sucrose activity in sugarcane were studied. Micropropagated sugarcane plants were obtained from callus cultures of four Mexican commercially available sugarcane varieties characterized by differences in sugar production, and activities of SPS, SUSY, NI, SAI and concentrations of sucrose were monitored in the sugarcane stem. The resuits indicated that sucrose accumulation was positively and significantly related to an increase in activity of SPS and SUSY and negatively to a reduction in activity of SAI and NI (P<0.05). SPS explained most of the variations found for sucrose accumulation and least for NI. The relationship between activity of SPS, SUSY, NI and SAI in sugarcane stem was similar in each variety. Su L.Y., Dela Cruz A., Moore P.H., Maretzki A. 1992. The relationship of glyphosate treatment to sugar metabolism in sugarcane: new physiological insights. J. Plant Physiol., 140: 168-173. van Handel E. 1968. Direct microdetermination of sucrose. Ann. Biochem., 22: 280-283. Veith R., Komor E. 1993. Regulation of growth, sucrose storage and ion content in sugarcane cells, measured with suspension cells in continuous culture growth under nitrogen, phosphorus or carbon limitation. J. Plant Physiol., 142: 414-424. Wendler R., Veith R., Dancer J., Stitt M., Komor E. 1990. Sucrose storage in cell suspension cultures of Saccharum sp (sugarcane) is regulated by a cycle of synthesis and degradation. Planta, 183:31-39. Zhu Y.J., Komor E., Moore P.H. 1997. Sucrose accumulation in the sugarcane stem is regulated by the difference between the activities of soluble acid invertase and sucrose phosphate synthase. Plant Physiol., I 15:609-616.

Planta, 2008

Capsaicinoids are responsible for the pungent taste of chili pepper fruits of Capsicum species. C... more Capsaicinoids are responsible for the pungent taste of chili pepper fruits of Capsicum species. Capsaicinoids are biosynthesized through both the phenylpropanoid and the branched-fatty acids pathways.

Plant Cell Reports, 1992

A protocol for the Agrobacterium-mediated transformation of tomatillo was developed. Up to 40 tra... more A protocol for the Agrobacterium-mediated transformation of tomatillo was developed. Up to 40 transgenic plants could be obtained in experiments using 60 cotyledon explants. The transformed nature of the regenerated plants was confirmed by NPT II and Southern blot hybridization analysis. Using the b-glucuronidase system the tissue specific and developmental patterns of expression of the Cauliflower Mosaic Virus 35S promoter were determined in transgenic tomatillo plants. It was found that this promoter is developmentally regulated during fruit and seed fornmtion.

Food Science and Technology, 2005

Scientia Horticulturae, 2001

The average number and average fresh weight of Spathiphyllum floribundum cv. Petite axillary shoo... more The average number and average fresh weight of Spathiphyllum floribundum cv. Petite axillary shoots grown on MS medium with three concentrations of BA (1, 2 and 5mgl− 1) in the absence of auxin or in combination with 0.05 mgl− 1 NAA, 0.5 and 1.0 mgl− 1 IAA or ...

Plant Molecular Biology Reporter, 2002

High-quality RNA is important in studying gene expression. This report describes an improved meth... more High-quality RNA is important in studying gene expression. This report describes an improved method for isolating intact purified RNA from dehydrated organs of chili pepper plants. Common RNA extraction protocols have produced poor yields because dehydrated leaves accumulate polysaccharides and RNases. Our protocol is based on a guanidine thiocyanate extraction combined with additional purification steps using butanol and the ionic detergent CTAB (cetyltrimethylammonium bromide). Using this protocol, RNA yields ranged from 40-70 ktg of total RNA per 200 mg of fresh tissue. This method can be adapted to large-scale isolations, allowing the recovery of larger amounts of intact RNA (up to 250 ktg per gram of fresh tissue).

Plant Cell, Tissue and Organ Culture, 2008

Page 1. ORIGINAL PAPER In vitro propagation of three Agave species used for liquor distillation a... more Page 1. ORIGINAL PAPER In vitro propagation of three Agave species used for liquor distillation and three for landscape Rafael Ramırez-Malagón Æ Anatoli Borodanenko Æ Luis Pérez-Moreno Æ Manuel Darıo Salas-Araiza ...

Plant Cell, Tissue and Organ Culture, 2006

Presence of potyvirus in single garlic (Allium sativum L.) cloves from the same bulb, and in five... more Presence of potyvirus in single garlic (Allium sativum L.) cloves from the same bulb, and in five single leaves excised from commercial field-grown individual plants was studied using ELISA. It was found that the viruses were not present in all organs of the same plant, since some cloves of the same bulb were infected with potyvirus but some others were

Plant Cell, Tissue and Organ Culture, 1994

Cell cultures of chili pepper (Capsicum annuum L.) were established from callus tissue inoculated... more Cell cultures of chili pepper (Capsicum annuum L.) were established from callus tissue inoculated in MS liquid medium supplemented with 6.25 laM 2,4-D and 0.44 ktM BA. Cell clones were isolated by plating the cell suspension on filter paper discs supported by polyurethane foam that were bathed with culture medium containing 15% PEG. The cell clones T6 and T7 were chosen based on their characteristics of growth and friability. These cell clones were established as cell suspensions in the presence of 15% PEG and subsequently subcultured in increasing concentrations of osmoticum. By this approach the cell clones T7 and T6 were capable of growing in the presence of 20 and 25% PEG, respectively. The cell clone T7 was found to grow better in the presence of 5-10 % PEG after a period of subculturing in the absence of osmoticum indicating that the tolerance trait was stable. The tolerant cell clones exhibited a 3 to 3.5-fold decrease in the osmotic potentials in comparison with the nonselected cells suggesting that osmotic adjustment occurred. K + was the major contributing solute to the osmotic potential in all the cell cultures among those tested and was found to be higher in concentration in the PEG-tolerant clones (1.3-3 times higher than nonselected cells). Proline and glycine betaine levels showed a positive correlation with the degree of tolerance to water deficit in the PEG-tolerant cell clones. The levels of proline in the cell clone T7 subcultured in the absence of PEG in the culture medium decreased to values similar to those of nonselected cells, whereas the contents of glycine betaine in the same conditions were maintained at high levels.

Plant Cell, Tissue and Organ Culture (PCTOC), 2009

Eight cultivars and two accessions of Physalis ixocarpa Brot. were tested for their capacity to r... more Eight cultivars and two accessions of Physalis ixocarpa Brot. were tested for their capacity to regenerate embryos and plants from anther cultures. Anthers were pretreated at 4°C for 2 days and then at 35°C for 8 days in the dark while cultured on MS medium supplemented with 0.045 lM 2,4-D ? 0.03 mg l -1 vitamin B 12 (MS1) or with 2.26 lM 2,4-D ? 0.1 mg l -1 vitamin B 12 (MS3). Anther incubation proceeded under a 16 h photoperiod at 25 ± 2°C. Embryo formation occurred after 6 weeks of incubation in these conditions. Androgenetic responses were cultivar-and culture medium-dependent, with the greatest embryo yields recorded for cv. Chapingo (36.3%) on MS1 medium, and with wild-type 2 (21.8%) on MS3. Further development of regenerated embryos was promoted on MS medium supplemented with 0.54 lM NAA, 8.88 lM BA and 50 mg l -1 casein hydrolysate. The regenerated plants were cultured on half-strength mineral salts MS medium with 2.85 lM IAA to enhance root formation. Rooted plantlets were transferred to pots and acclimatized to the greenhouse. Ploidy analysis of regenerated plants using flow cytometry revealed 72% diploids, 15% haploids and 7% triploids. AFLP analysis of regenerated plants from anthers of a single parental plant showed different polymorphic patterns indicating their gametophytic origin.