Claudete Aparecida Mangolin

Isozyme patterns in callus cultures and in plants regenerated from calli ofCereus peruvianus (Cactaceae)

Electrophoretic patterns for isocitrate dehydrogenase (IDH; EC 1.1.1.42), acid phosphatase (ACP; EC 3.1.3.2), peroxidase (PER; EC 1.11.1.7), and esterase (EST; EC 3.1.1.1) isozymes were determined inCereus peruvianus tissues and used as markers of genetic uniformity of calli and of the plants regenerated from callus cultures. One IDH, six ACP, six PER, and six EST isozymes were induced in cultured callus tissues in medium containing three 2,4-dichlorophenoxyacetic acid and kinetin combinations. Four ACP, two PER, and three EST isozymes were still present in all regenerated plantsin vitro and therefore can be used as markers of theC. peruvianus plants regenerated from callus tissues. The differential patterns of ACP and IDH isozymes and the similar zymograms for PER and EST isozymes presented by callus tissues were used in a comparison of callus tissues cultured for 2 years. The comparative analysis of zymograms within each enzyme system indicated a mean heterogeneity coefficient of 0.33 forC. peruvianus calli cultured for 2 years. Because of the isozyme variations, which developed in culture medium and were transferred to the regenerated plants, the IDH, ACP, PER, and EST enzyme systems can be considered to be good markers for investigating possible genetic variations in plant populations ofC. peruvianus obtainedin vitro from callus culture.

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes in tissues and callus cultures ofCereus peruvianus (cactaceae)

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes were investigated in seeds and in seedlings and calli cultures ofC. peruvianus to determine if the changes in MDH isozyme banding patterns could be used as biochemical markers to identify the origin of regenerated plants from callus tissues. Four cytoplasmic MDH isozymes (sMDH), five mitochondrial MDH isozymes (mMDH), and one glyoxysomal MDH isozyme (gMDH) were detected and showed tissue- and stage-specific expression. A relationship of mMDH and gMDH isozyme patterns with callus tissues subcultured in three hormonal combinations and with the plants regenerated from these callus tissues was demonstrated. Furthermore, temperature and mechanical stress were found to be closely related to mMDH-1 activity in callus culture. Therefore, the different patterns of MDH isozymes in the various tissues ofC. peruvianus can be used as biochemical markers for the study of gene expression during development and as powerful tools in monitoring studies on callus cultures.

Isozyme Variability in Plants Regenerated from Calli of Cereus peruvianus (Cactaceae)

Morphological and isozyme variation was observed among plants regenerated from callus cultures of Cereus peruvianus. Different morphological types of shoots (68%) were observed in 4-year-old regenerated plants, while no distinct morphological variants were observed in plants grown from germinated seeds. Isozyme patterns of 633 plants regenerated from calli and of 261 plants grown from germinated seeds showed no variation in isocitrate dehydrogenase isozyme, and the differential sorbitol dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, acid phosphatase, and peroxidase isozyme patterns observed in regenerated plants were attributed to nonallelic variation. Allelic variation was detected at three isoesterase loci. The proportion of polymorphic loci for both populations was 13.6% and the deviation from Hardy–Weinberg equilibrium for the Est-1 and Est-7 loci observed in somaclones was attributed to the manner in which the regenerant population was established. The high values for genetic identity among regenerant and seed-grown plant populations are in accordance with the low levels of interpopulation genetic divergence. In somaclones of C. peruvianus, morphological divergence was achieved within a short time but was not associated with any isozyme changes and also was not accompanied by biochemical genetic divergence.

In vitro propagation ofCereus peruvianus mill. (cactaceae)

SummaryCereus peruvianus seedlings were used as a source of stem explants to determine the effective conditions for inducing and maintaining callus tissues in a state of rapid growth, as well as to obtain plants regenerated from callus cultures. Factorial combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin in MS medium were tested, and we concluded that the 18.1µM 2,4-D and 18.6 or 27.9µM kinetin combinations were suitable for callus induction. The cactus shoots were produced from the friable callus; root elongation occurred within 2 wk in medium without 2,4-D and with 18.6µM kinetin. This method can be used to rapidly produce manyC. peruvianus plants.Cereus peruvianus seedlings were used as a source of stem explants to determine the effective conditions for inducing and maintaining callus tissues in a state of rapid growth, as well as to obtain plants regenerated from callus cultures. Factorial combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin in MS medium were tested, and we concluded that the 18.1µM 2,4-D and 18.6 or 27.9µM kinetin combinations were suitable for callus induction. The cactus shoots were produced from the friable callus; root elongation occurred within 2 wk in medium without 2,4-D and with 18.6µM kinetin. This method can be used to rapidly produce manyC. peruvianus plants.

Genetic diversity of breeding popcorn lines determined by SSR markers

P1-3 and P8-1 was lowest, while P3-3 and P8-2 were genetically more similar. The cophenetic correlation showed that the Unweighted Pair-Group Method Using Arithmetic Averages (UPGMA) was reliable to discriminate the genotypes in five groups. The clusters were consistent with the estimates of genetic identity. There was a moderate coincidence degree between the groups and genealogy of lines. Higher levels of heterozygosity are expected from crosses between the group containing lines P3-3 and P7-3 with that of P1-3 and P7-4. Crosses between lines P1-3 and P8-1 are also promising.

RAPD Markers to Evaluate Callus Tissue of Cereus peruvianus Mill. (Cactaceae) Maintained in Different Growth Regulator Combinations

RAPD markers were used to detect DNA polymorphisms in callus tissues maintained at different auxin and cytokinin combinations. There is a higher level of genetic variablity in callus tissue maintained with the highest kinetin versus2, 4-D concentration. Callus tissues subcultured in a 4.0 mg/L 2,4-D and 4.0 mg/L kinetin combination showed high similarity and can be recommended as more suitable sources for industrial procedures of extraction of natural products such as secondary metabolites since extraction protocols can be easily standardized using genetically uniform materials. The higher genetic diversity in callus tissues of C. peruvianus cultured at 4.0 mg/L 2,4-D and 8.0 mg/L kinetin indicates this tissue as a matrix for in vitro selection of cell lines for higher natural products production. RAPD markers are, therefore, effective tools useful for detecting DNA polymorphism in callus tissue as well as in the DNA identification of callus tissues maintained in different auxin and cytokinin combinations.

Esterase Polymorphism for Analysis of Genetic Diversity and Structure of Wild Poinsettia (Euphorbia heterophylla) Populations

Abstract Native polyacrylamide gel electrophoresis was used in the current study to identify polymorphism in α- and β-esterase loci in leaf tissues of wild poinsettia plants for the analysis of genetic diversity and structure of populations. Seeds were collected from different plants in 12 different populations. Two to three allelic variants were at Est-1, Est-2, Est-3, Est-4, Est-5, Est-6, and Est-7 loci. The estimated proportion of polymorphic loci in populations is 87.5%. High and low values of observed and expected proportion of heterozygous loci in 12 populations confirm our suspicion that the populations are genetically structured (FST = 0.1663). The heterozygous deficiencies are evidenced by the positive value of FIS (0.1248). The positive FIS value indicates a deficit of heterozygous (12.48%) or an excess of homozygous plants, which could be the result of frequent herbicide application in areas where seeds were collected and/or the result of self-pollination. Overall inbreeding or nonrandom breeding, according to the significant FIT value (0.2703), did play a major role in shaping the genetic structure of these populations. Identity values represented in the dendrogram should play a more central role in developing policies to manage and control this species. Nomenclature: Wild poinsettia, Euphorbia heterophylla L. EPHHL

Two‐dimensional electrophoresis of Cereus peruvianus (Cactaceae) callus tissue proteins

Two‐dimensional electrophoresis of Cereus peruvianus callus tissues grown in culture media containing two different 2,4‐dichlorophenoxyacetic acid (2,4‐D) and kinetin combinations was used to identify minor differences in polypeptide composition of these cell clones. Altered expression during growth in the two 2,4‐D and kinetin combinations was apparent for 13 polypeptides when calluses in the two media were compared. The number of proteins with differential expression (presence or absence of specific spots) was higher in callus tissues cultured in the 4.0 mg/L 2,4‐D and 8.0 mg/L kinetin combination than in callus tissues cultured in the 4.0 mg/L 2,4‐D and 4.0 mg/L kinetin combination. The present results show that the callus tissues maintained at 4.0 mg/L 2,4‐D and 8.0 mg/L kinetin can be used as a matrix for in vitro selection programs.

Microsatellite analysis of the parental contribution of Piaractus mesopotamicus to the production of offspring in the semi-natural system of reproduction

The objective of this study was to evaluate the genetic diversity and the parental contribution of Piaractus mesopotamicus in the production of offspring in the semi-natural system of reproduction. Twenty parental fishes (eleven males and nine females) and the total of 100 larvae were evaluated by microsatellite marker. The parents and offspring had thirty-one alleles and heterozygosity of 0.550 and 0.563, respectively. The females were fertilised by two up to six males while the males fertilised three up to five females. The contribution of the females and males to the offspring were 66.6 and 58%, respectively. Such results indicated no loss in the genetic variability in the offspring, and the parents had multiple paternity and reasonable contribution to the offspring production.

Heterozygosity following half-sib recurrent selection in popcorn using isoenzyme markers

Isozyme biochemical marker may be useful tool for genomic analysis of maize populations undergoing recurrent selection. Thus, isozymes markers was utilized for assess the changes in the genetic variability and distance in a Brazilian composite population of popcorn following four cycles of recurrent selection for yield. One hundred and ninety-six half-sib families were evaluated from each cycle and the ten highest-yielding families (5.2%) were recombined to produce the next cycle. Isozyme analysis considered 80 seedlings per cycle. Simple linear regression equations were estimated among the allele frequencies in each locus in function of the selection cycles, the genetic distances among the cycles and the average heterozygosity per locus for each cycle. Regression analysis did not reveal any common trend for changes in allele frequencies presumably due to selection. The estimates of the number of polymorphic locus, of the mean of allele per locus and the mean heterozygosity did not reveal any reduction in variability. It was concluded that four selection cycles did not cause relevant changes in the variability or genetic distance among the selection cycles of CMS-43 popcorn population. Isozymes markers analysis showed that the number of recombined half-sib families in recurrent selection was suitable.