Ana M. Vázquez

Somatic embryogenesis and plant regeneration from leaf tissues of rye (Secale cereale L.)

Abstract Leaf explants of rye ( Secale cereale L.), obtained from 3–4-week-old plants growing in aseptic conditions were cultured on the Murashige and Skoog medium (MS) with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). Embryogenic calluses from which plantlets could be obtained were formed in most of the cases.

Somatic embryogenesis from immature inflorescences of rye.

Abstract Immature inflorescences of four cultivars of rye were cultured on Murashige and Skoog medium (MS) with different concentration of 2,4-dichlorophenoxyacetic acid (2,4-D). Embryogenic calluses were obtained in most cases. However the frequency varied in regard to inflorescence length, 2,4-D concentration and donor cultivar. Evidence is provided that the genotypic effect on the in vitro response of an allogamous cultivar in rye is due to two factors: the number of competent plants and the response level of each individual.

Influence of time on the genetic heterogeneity of Spanish porcine reproductive and respiratory syndrome virus isolates.

The aim of the present study was to establish the degree of diversity of porcine reproductive and respiratory virus (PRRSV) isolates that circulate in the same geographical area in different years. Nucleotide sequences of open reading frame (ORF) 5 were determined for 28 Spanish field PRRSV isolates from different years and three European-type modified live virus vaccines. Sequences were aligned using Clustal W software and a phylogenetic tree constructed using the neighbour joining method. The results of pairwise homology comparisons of nucleotide and deduced amino acid sequences of these PRRSV isolates indicate a tendency for heterogeneity to increase with time. The study of the phylogenetic tree revealed that Spanish PRRSV isolates constitute two well-defined clades and a group of unrelated sequences. The observed heterogeneity does not appear to be due to temporal evolution exclusively. Early and recent isolates group themselves into different clusters independently of the time of isolation, indicating the co-circulation of different variants and the maintenance of variants of the original isolates in the field.

Insight into somaclonal variation

ABSTRACT Somaclonal variation (S.V.) refers to mutational events occurring in tissue culture, although some permanent methylation processes should possibly also be included under this name. In this review, the possible causes as well as the mechanisms implicated in the induction of mutation in cultured cells are discussed. The needs for an easy assay to assess S.V. is pointed out.

Somatic embryogenesis, plant regeneration and somaclonal variation in barley

In vitro culture of immature embryo and young leaf tissues was carried out with five cultivars of barley, Hordeum vulgare. Two cultivars (Albacete and Porthos) responded poorly from both types of explants, while the three others (Dissa, Golden Promise and Ingrid) produced a high frequency of embryogenic callus from these explants (25–60%). For ‘Dissa’ and ‘Ingrid’, young leaf explants were slightly better than immature embryo explants for embryogenic callus induction, while immature embryo cultures of ‘Golden Promise’ responded better than young leaf explants. Thus, there appears to be a significant genotype × explant interaction in the initiation of embryogenic callus in barley.Some phenotypic variants were detected among the regenerated plants of ‘Golden Promise’ and ‘Ingrid’, most originating by epigenetic changes. Only in one case was the variant phenotype heritable, probably due to a mutation in the chloroplast DNA. Mitotic alteractions were not detected. Consequently, somaclonal variation did not appear to be a very frequent event in plants regenerated from 1- to 6- month-old cultures of barley.

RYS1, a foldback transposon, is activated by tissue culture and shows preferential insertion points into the rye genome

The study of two variable amplicons of rye indicates that RYS1, a mobile element, is activated during tissue culture. We propose that RYS1 could be a foldback (FB) transposon. The FB transposons have been rarely reported in plants; RYS1 is the first described in rye and also the first active plant FB transposon reported. Preferential integration points in the rye genome exist, because the new insertions seem to be located, in all studied cases, in the same genome positions. We assume that RYS1 became active in rye very recently, as different plants from in vivo-growing cultivars showed that these elements were present or absent in the same genomic position in which the in vitro-activated element was found. This high rate of modification in these particular loci, both in the in vivo and in vitro populations, could indicate that probably the mechanisms promoting genetic variability in nature are the same that induce variation in vitro, and the modifications induced by somaclonal variation could be already present in vivo populations

Shoot regeneration in four Begonia genotypes

In vitro regeneration of four Begonia genotypes, B. semperflorens, B. rex, B.×elatior, and hybrid of Begonia with unknown parents ‘Tiger’ was carried out starting from leaf and petiole segments as explants. Five Murashige and Skoogs derived media were tested, three of them supplemented with α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), and the other two with NAA and kinetin (KIN) in different concentrations. Shoot regeneration was preferentially induced on the BA containing media, quantitative differences being observed among explants and genotypes.

Genetic and epigenetic relationship in rye, Secale cereale L., somaclonal variation within somatic embryo-derived plants

In vitro regenerated plants of rye, Secale cereale L., Ailés and Merced cultivars, were studied to verify if genetic and/or epigenetic changes were promoted by in vitro conditions. Inter-simple sequence repeat (ISSR) fingerprints on HpaII/MspI-digested and uncut DNA were generated. DNA digested with methylation-sensitive isoschizomers revealed epigenetic modifications, while modification of ISSR patterns obtained with undigested DNA indicated genetic changes. With this technique, it was possible to study both genetic and/or epigenetic changes within the same DNA sequences. The frequency of plants with at least one variation was high: 73% and 30% of rye plants showed at least one genetic change, and 50% and 73% carried at least one methylation change, in the Ailés and Merced cultivars, respectively. Further analyses revealed that a considerable number of variable markers showed both types of modifications, indicative of both genetic and epigenetic changes. Moreover, genetic variation was related to the presence of the CCGG target in the analyzed bands. These results indicate the possible existence of a common mechanism connecting both types of variation.

Somatic embryogenesis in polyembryonic Secale cereale L.

SummaryThe progeny of polyembryonic Secale cereale L., was used to study the in vitro response of the immature embryos. The formation of embryogenic calli was very high, and this response and its distribution was statistically different to that shown by the normal regenerated plants and the original population. This behaviour seems to be related to a genetic condition which favours the presence of supernumerary embryos, in vivo as well as in vitro.

A comparative study of somatic embryogenesis in Secale vavilovii

SummaryThe ability of immature embryos, inflorescences and leaves of Secale vavilovii to form embryogenic callus was tested on Murashige and Skoog (1962) medium supplemented with different concentrations of 2,4-D. All cultured immature embryos formed calluses. The highest percentage of embryogenic callus production was from 1–2 mm embryos. Young leaves also formed calluses, mainly from the 10–15 mm basal segment, the percentages of embryogenic calluses being higher when cultures were maintained in darkness. Embryogenic calluses were obtained also from all the cultured immature inflorescences, in the three cases, rooted green plants were obtained and grown in soil. Comparison of the responses of the three explants used indicates that immature inflorescence is the most useful explant for obtaining regenerated plants in Secale vavilovii.