M. Candela

Elicitation of peroxidase activity and lignin biosynthesis in pepper suspension cells by Phytophthora capsici

Summary Cell suspension cultures of three varieties of Capsicum annuum L., each with a different degree of sensitivity to the fungus Phytophthora capsici, responded to elicitation by both lyophilized mycelium and fungus filtrate with a hypersensitive reaction. They showed the synthesis or accumulation of PR-proteins with peroxidase (EC 1.11.1.7) activity and the accumulation of lignin-like polymer (as measured by derivatization with thioglycolic acid). The cultivation medium was optimised for both plant and fungus growth in order to avoid any stress during their combination. The resistant pepper variety, Smith-5, showed a more intense response to the elicitor preparations than the sensitive varieties, Americano and Yolo Wonder. This was particularly evident when the cell suspensions were elicited with the filtrate. After elicitation, the cell walls thickened through the accumulation of lignin, as can be observed by staining microscope preparations with methylene blue. Elicitation also reduced the level of total peroxidase activity in the susceptible varieties, while such activity increased in resistant varieties, and was accompanied by de novo expression of acidic peroxidase isoenzymes in the extracellular and cell wall fractions. Of note was the PR protein of pI 5.7 showing peroxidase activity, which was induced by both elicitor types in the elicited cell suspensions of the resistant variety alone, making it a marker of resistance. The increases in the activity of these peroxidases in the resistant variety are in concordance with the accumulation of lignin observed 24 h after inoculation by both elicitors from the fungus. The possible role of these isoenzymes in lignin biosynthesis, used to reinforce the cell walls against fungal penetration of the cells, is discussed. These results are in accordance with those previously observed in plant stem sections.

Use of recombinant inbred lines (RILs) to identify, locate and map major genes and quantitative trait loci involved with in vitro regeneration ability in Arabidopsis thaliana

Abstract The Landsberg erecta× Columbia recombinant inbred lines (RILs) of Arabidopsis have been used in order to identify and localize chromosome regions involved in the genetic control of the in vitro regeneration ability. Callus morphology (CM) and shoot regeneration (SR) traits have been considered for both leaf and root explants. The MAPMAKER analysis of leaf culture data has revealed at least one chromosome region involved with CM and several with SR, the 29–30 region of chromosome 1 being common for the two traits. Root explants did not segregate for CM but several QTLs have been detected for SR. The chromosome regions involved with leaf culture regeneration seem to be different from those of root cultures, although the regeneration of abnormal shoots in leaf explants share two chromosome regions with the regeneration of normal shoots in root cultures.

Maintenance of interchange heterozygosity in cultivated rye, Secale cereale L.

SummaryThe maintenance for several generations of interchange heterozygosity in both experimental and cultivated fields of rye is analysed. An experimental analysis of interchange heterozygotes has been carried out to ascertain whether only one or several translocations exist in the population. Interchange heterozygosity is discussed on the basis of the karyotypic orthoselection model. The maintenance of a constant frequency (20 per cent) of structural heterozygotes for several generations suggests the existence of an equilibrium. The possible mechanisms involved in such an equilibrium are discussed.

Differences in in vitro plant regeneration ability among four Arabidopsis thaliana ecotypes

SummaryThe Arabidopsis ecotypes Columbia (Col), Landsberg erecta (Ler), Cape Verde Island (Cvi) and Wassilewskija (WS) have been tested for their regeneration response in vitro. A characteristic morphology of leaf-derived calluses has been found for each ecotype. Differences in regeneration ability have been detected depending on the plant strain. the explant source and on the culture medium composition. In CIR/SIR media, which contain 0.5 mg l−1 (2.26 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D) and glucose, root explants from the four ecotypes are able to reach a considerable regeneration level, while leaf explants do not regenerate beyond a basal level (5% approximately). In CIH/SIH media, which contain 2.2 mg l−1 (9.95 μM) of 2,4-D and suerose, leaf explants from all the ecotypes, with the exception of Col, are able to regenerate, but they do it at variable levels (Ler 5.75%, WS 75.09%, and Cvi 27.53% as regeneration rates). With these media all root explants are able to regenerate, but again the four ecotypes show different rates (Col 27.7%, Ler 57.25%, WS 98.54%, and Cvi 42.25%). The variation of the different medium components affects differentially the regeneration ability of the four ecotypes depending also on the kind of explant. Thus, when the 2,4-D concentration is raised WS duplicates its regeneration rate in both leaf and root explants. Changing glucose for sucrose in CIR/SIR media diminishes to the basal level the regeneration of Cvi root explants, while the CIH/SIH salts and vitamin concentration permit the regeneration of leaf explants from all the ecotypes except Col. The genes responsible for those observed differences in regeneration ability could be identified and mapped by analyzing the in vitro regeneration behavior of the recombinant inbred lines (RILs) obtained by crossing these ecotypes.

Reciprocal translocations in Spanish and Potuguese natural populations of cultivated rye, Secale cereale L.

SummaryTwenty populations (cultivars) of rye Secale cereale L., cultivated in Spain and Portugal have been analyzed to ascertain if they are polymorphic for the chromosome structure. In 9 Spanish cultivars interchange heterozygous individuals have been found with frequencies ranging from about 1 to 10 per cent. All the heterozygotes analyzed showed similar cytogenetic characteristics. The evolutionary significance of the intraspecific chromosomal polymorphism found in Secale cereale populations is discussed from the karyotypic orthoselection point of view. Likewise, the necessity of a reappraisal of the role of reciprocal translocations in cytotaxonomic studies of Gramineae is suggested.

Chromosomal identification and meiotic behavior of reciprocal translocations in a rye polymorphic population. Evolutionary implications.

SummaryThe spontaneous interchange polymorphism of rye cultivar “Ailés” is composed, as can be deduced from the chromosomal identification of the interchanges analyzed, of several different reciprocal translocations in which the chromosomes of its haploid complement are involved with a similar frequency, except for chromosomes 4R and 6R. Several features of chromosome behavior at metaphase I, such as configuration and orientation of quadrivalents and frequency of chiasmata, were analyzed in structural heterozygotes for different interchanges. The two main factors affecting the orientation of quadrivalents at metaphase I proved to be the morphology of these chromosome associations at metaphase I and, in particular, the frequency of bound chromosome arms that they showed. A genotypic control of alternate orientation of quadrivalents independent of chiasmata frequency was not detected. In addition, the frequency of alternate orientation shows no relation to the fitness. Possible evolutionary implications of the results obtained are discussed.

Genic heterozygosity maintained by chromosomal interchanges in rye

Six different rye cultivars were electrophoretically analysed. Three of them showed chromosome polymorphism for reciprocal translocations at various frequencies, while the other three were chromosomally homogeneous. The cultivars were shown to be polymorphic for isozymes at the loci Got-3, Pgm-1, Gpi-1, Acph, Mdh-1 and Mdh-2b. The results obtained seem to indicate that the frequency of polymorphic loci, heterozygosity per locus and mean heterozygosity per cultivar are higher in those cultivars having chromosomal interchanges. In the “Ailés” cultivar, plants homozygous and heterozygous for interchanges were analysed, both in open- and self-pollination. The structural heterozygotes showed a higher genic heterozygosity mainly for the Pgm-1 and Mdh-2b loci. Possible explanations of this phenomenon are discussed.

Analysis of natural allelic variation in in vitro organogenesis of Arabidopsis thaliana

We have evaluated the regeneration ability of leaf and root explants in 26 natural populations with the aim of analyzing the naturally occurring variation of Arabidopsis in vitro shoot and root organogenesis. All the accessions have been used as parental lines of available or under construction recombinant inbred lines (RILs) under construction. Phenotypic differences found between Ler and Cvi ecotypes were mapped in a population of 162 RILs. Several involved quantitative trait loci (QTL) have been identified. The analysis of near isogenic lines (NILs) would allow us to assign the effect of the QTLs identified and also possible candidate genes will be tested among those already described in the literature mapping into those regions.

Differential effects of gamma irradiation on rye cultivars with or without spontaneous translocation polymorphism

Abstract The effects of gamma-rays on dry seeds of two rye cultivars, a polymorphic cultivar for reciprocal translocations (cv. “Ailes”) and a non-polymorphic one (cv. “Elbon”), were analysed. Reduction in germination and survival was greater in “Ailes” than in “Elbon”. Reciprocal translocation frequencies observed in mitotic and miotic analyses were also higher in “Ailes” than in “Elbon”. Cytomorphological features and meiotic behavior of gamma-ray-induced structural heterozygotes were similar in both cultivars. The results agree with those observed for spontaneous heterozygotes. The higher radiosensitivity of “Ailes” relative to that of “Elbon” might be attributed to a chromosomal mutation which resulted in a stable chromosomal polymorphism for reciprocal translocations with a structural heterozygote frequency of 15.3%.

Genic heterozygosity, chromosomal interchanges and fitness in rye: any relationship?

Relationship between heterozygosity at allozyme loci, chromosomal interchanges and fitness was analyzed in a rye cultivar showing a polymorphism for such rearrangements. Nine allozyme systems (ACO, ACPH, GOT, GPI, LAP, MDH, PER, PGD and PGM) and five components of fitness (number of fertile tillers, total offspring, egg cell fertility, flowers/ear and seeds/ear) were studied. The estimated selection coefficients against interchange heterozygotes ranged from s = 0.12 to s = 0.34. A significant effect of the genic heterozygosity on some fitness components was observed in interchange heterozygotes (tillering and total offspring), in their standard homozygous sibs (flowers/ear and seeds/ear) and in the descendants of the crosses between standard karyotypes (flowers/ear, seeds/ear and egg cell fertility). However, the main effect was linked to genetic background associated to different crosses. Significant differences for Acph-1, Gpi-1, Lap-1, Mdh-1, Mdh-4, Pgd-2 and Pgm-1 loci were also found in some of these crosses although these differences were inconsistent. This suggests that probably the allozyme loci analyzed were not directly contributing to the fitness and that they are linked, in some cases, to different deleterious alleles depending on both cross and locus. This fact could support the local effect hypothesis as explanation although we do not discard the existence of some inbreeding level (general effect hypothesis) since all crosses and loci studied show a overall consistent trend of increased fitness with increased heterozygosity.