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Genetic structure of an endangered plant, Antirrhinum microphyllum (Scrophulariaceae): allozyme and RAPD analysis.

Thirteen allozyme loci and 68 random amplified polymorphic DNA (RAPD) markers were analyzed to assess the genetic diversity and population structure of threatened Antirrhinum microphyllum (Scrophulariaceae), a narrow endemic of central Spain known from only four populations. According to allozyme data, species genetic diversity (p = 46.15%, A = 2.61, and H(e) = 0.218), as well as within-population genetic diversity (p = 44.23%, A = 2.10, and H(e) = 0.204), were high when compared to average estimates for other narrowly distributed plant species. Ninety-four percent of species genetic diversity corresponded to within-population genetic diversity. Nevertheless, significant differences were found among populations in allele frequencies of four of the six polymorphic loci, and three private alleles were detected. Inbreeding coefficients (F(IS)) suggest that populations are structured in genetic neighborhoods. The RAPDs also showed high levels of genetic diversity (p = 89.71% and H(e) = 0.188 at the species level, and p = 67.65% and H(e) = 0.171 at the population level). Neis genetic distances estimated both from allozymes and RAPDs indicated low differentiation among populations. In spite of this, the low frequencies of certain alleles and the presence of private alleles indicate that efforts should be made to conserve all four remaining populations.

Application of RAPD markers in the characterisation of Chrysanthemum varieties and the assessment of somaclonal variation

Characterisation of fifteen commercial varieties of Chrysanthemum was carried out through RAPD analysis. Varieties could be distinguished from each other and the level of similarity between varieties seemed to be not very high. In vitro cultures were establish from four varieties and were subjected to different proliferation conditions. Five individuals from each variety and treatment were analysed using RAPD at the beginning of the treatment and after a month of culture. Variation was detected at both stages of the culture period. The rate of variation found showed differences between varieties, but no significant difference was found between culture conditions.

GERMINATION STUDIES IN ENDEMIC PLANT SPECIES OF THE IBERIAN PENINSULA

The germinative behavior of nine plant species of the Iberian Peninsula, four of them catalogued as vulnerable or endangered by the International Union for the Conservation of Nature, was studied under controlled laboratory conditions. Most of the species studied germinated better at relatively low temperatures (15 °C and 20 °C) and, in general, high germination rates were also obtained at alternating temperatures (25/15 °C). Coronopus navasii germinated over 60% at 25 °C, the highest temperature used in this study. Lavatera oblongifolia did not surpass 20% germination under any of the conditions assayed. Seed coat scarification increased germination in Helianthemum squamatum, while seed pretreatments with hot water or sulfuric acid were effective in H. polygonoides. Less favorable results were obtained in Lavatera oblongifolia, whereas scarification was completely ineffective in Onobrychis peduncularis ssp. matritensis.

Vulnerability and determinants of reproductive success in the narrow endemic Antirrhinum microphyllum (Scrophulariaceae)

The breeding system and flowering phenology of the narrow endemic Antirrhinum microphyllum (Scrophulariaceae) were studied in order to assess the main factors affecting female reproductive success and to identify existing or potential threats to the viability of its populations. Hand-pollination experiments showed that A. microphyllum is an allogamous self-incompatible species. In both populations studied, the flowering season was 4 mo long and mean flowering duration per plant was about 1 mo. Peak flower production took place between mid-April and mid-May and overlapped with the period of activity of the main pollinator, Rhodanthidium sticticum (Megachilidae). Estimated mean number of seeds produced per plant was 9391, showing that population viability is not presently limited by seed output. The study of the direct and indirect effects of plant size, phenological traits (first flowering date, flowering duration, and flowering synchrony), and distance to neighbor plants on reproductive success was performed using structural equation modeling (SEM). In both populations, number of flowers and plant size were the main factors that determined the total number of fruits produced by a plant. First flowering date and flowering synchrony also affected fruit production. Multisample comparison of path coefficients for the two populations rejected the possibility that reproductive patterns could be described by one single model.

ALTERNATIVE STATISTICAL ANALYSES FOR MICROPROPAGATION: A PRACTICAL CASE OF PROLIFERATION AND ROOTING PHASES IN VIBURNUM OPULUS

SummaryThe kinds of data obtained in micropropagation studies are very often problematic, since they do not follow continous distribution and observations through culture vessels complicate measurement. Accordingly, standard analyses are often used, leading to misinterpretation of results. In this paper, we present a study of Viburnum opulus micropropagation using planned contrasts and fitting regression models in generalized linear models as an alternative statistical analysis of micropropagation results, and compare the results with that of traditional ANOVA. The advantages and possibilities of the alternative data analyses in plant tissue culture are discussed.

MICROPROPAGATION AND IN VITRO STORAGE OF CENTAURIUM RIGUALII ESTEVE (GENTIANACEAE)

Centaurium rigualii Esteve, an endangered plant species from southeastern Spain with potentially useful secondary products, was successfully micropropagated from shoot tips using MS supplemented with 0.44 μM 6- benzylaminopurine (BA) and 0.05 μM 1-naphthaleneacetic acid (NAA) at the proliferation step, and MS medium alone or with varying concentrations of indole-3-butyric acid (IBA) at the rooting step. No incidence of somaclonal variation was detected among randomly chosen shoots from ten subclonal lines in eight isozyme systems studied and in chromosome counts. However, this does not preclude the existence of somaclonal variation at other levels of the genome. A 90% survival rate was obtained when shoots were stored for three years in MS medium supplemented with 4.92 μM IBA at 5 °C and with a 16-h photoperiod.

Evaluation of genetic variability in Iberian Colutea spp. using isozyme electrophoresis

Twenty-three populations from Colutea arborescens ssp. gallica and C. atlantica, were studied by polyacrylamide gel electrophoresis to estimate variation at ten enzyme systems. Seed isozyme data were analysed by multivariate techniques including principal coordinates and cluster analyses based on Jaccards similarity coefficient. The results indicated the existence of two major gene pools that did not coincide with the current taxonomic delimitation of Colutea species in the Iberian peninsula. If the regional origins were taken into account, a prevailing geographic pattern was detected. The eastern area of Colutea distribution is discussed as an origin of variability of this genus in the Iberian peninsula.

Effects of accelerated ageing and cryopreservation on seeds and embryonic axes of Phaseolus vulgaris L and Arachis hypogaea L. Germination and seedlings vigor

The effects of artificial aging and cryopreservation methods on the germination of seeds and embryonic axes of Phaseolus vulgaris and Arachis hypogaea , were studied. Aging and cryopreservation treatments in bean seeds not affect the germination percentage, reaching values not significantly different from control. Germination percentage was higher when isolated embryonic axes were studied. Aging treatments gave rise to larger bean seedlings than the controls and cryopreservation treatments affected the size depending on the explant used. In groundnut seeds, aging treatments and cryopreservation positively affected germination and vigor of seedlings, but embryonic axes from not rehydrated aging seeds not germinate. Undried groundnut seeds not survive cryopreservation, while the dried ones showed a germination behavior superior to the control. Embryonic axes tolerate immersion in liquid nitrogen but when droplet-vitrification was used, formed callus instead of seedlings. Electrolyte leakage was always higher in embryonic axes than in seeds, both in beans and groundnut, and not to have a significant effect, under the conditions studied, on germination and vigor of seedlings. The present study evidences the possibility of cryopreserving, by simple procedures, germplasm of beans and groundnut, and to verify the aging tolerance of the seeds and embryonic axes of these important species.