A. Rode

Identification of new mitochondrial genome organizations in wheat plants regenerated from somatic tissue cultures

SummaryPlants have been regenerated from short-and long-term in vitro somatic tissue cultures made from immature embryos of the hexaploid wheat cultivar “Chinese Spring”. The mitochondrial genome organization of each regenerated plantlet was studied, after one selfing, by probing Sal I-restricted total DNA with cloned Sal I fragments of wheat mitochondrial DNA derived from a segment of the genome, which displays marked structural changes in response to in vitro culture. Short-term in vitro cultures give rise to regenerated plants whose mitochondrial genome organization is either close to that of the parental cultivar or to that of embryogenic callus cultures, except for a single plant which has an organization resembling that of short-term non-embryogenic cultures. In contrast, all but one of the plants regenerated from long-term cultures exhibited a mitochondrial genome organization similar to that of long-term nonembryogenic cultures. In addition, extra labelled bands were detected in some of the regenerated plants with two of the probes used. These results emphasize the importance of the duration of the in vitro step preceding the regeneration process: the longer it is, the higher the probability is of obtaining mitochondrial DNA variability in regenerated plants. Furthermore, since increasing the duration of the in vitro stetp results in the production of regenerated plants with a mitochondrial genome organization resembling that of non-embryogenic tissue cultures, the question is thus raised as to whether regeneration from long-term cultures is suitable for use in plant breeding.

Characterization of the expression of a wheat cystatin gene during caryopsis development

A cDNA coding for phytocystatin, a protease inhibitor, was isolated from wheat embryos by differential display RT-PCR and the corresponding full-length cDNA (named WC5 for wheat cystatin gene 5) subsequently obtained by RACE. The deduced primary sequence of the protein suggests the presence of a 28 amino acid N-terminal signal sequence and a 100 amino acid mature protein containing the three consensus motifs known to interact with the active site of cysteine peptidases. Northern and western analysis revealed a spatio-temporal pattern of the cystatin gene expression during caryopse development. In the embryo, WC5 was only expressed during early embryogenesis whereas, in seed covering layers, WC5 expression was restricted to the maturation stage of grain development. In addition, immunolocalization experiments showed that cystatin accumulated in the aleurone layer of the maturating seed and in the parenchymal tissues of the embryo scutellum. A recombinant form of the wheat cystatin was shown to be able to inhibit peptidase activities present in whole seed protein extracts. In addition, immunological techniques allowed us to identify two putative target peptidases. The possible roles of the cystatin protein are discussed in relation with tissular localization and putative peptidase targets during seed maturation.

Extensive mitochondrial DNA variation in somatic tissue cultures initiated from wheat immature embryos

SummaryWheat mitochondria) DNA has been isolated from callus cultures initiated from both immature embryos and the corresponding parental cultivar. A Sall restriction pattern study has shown that the organization of callus culture mitochondria) DNA underwent extensive change, characterized by either the disappearance or the decrease in the relative stoichiometry of several restriction bands. Hybridization of labelled mitochondrial fragments obtained from a recombinant cosmid library to Southern blots of callus and parental line restricted mitochondria) DNAs has shown that a fraction of the mitochondria) genome was lost in callus cultures. Data from a Sall + HindIII restriction map of a defined part of the wheat mitochondria) genome concerned with some of these variations strongly suggest that the observed variations correspond to the disappearance of at least one mitochondria) DNA subgenomic molecule in callus cultures.

Gametoclonal variation detected in the nuclear ribosomal DNA from doubled haploid lines of a spring wheat (Triticum aestivum L., cv. 'César').

SummaryThe organization of the nuclear ribosomal DNA from a parental line of wheat (Triticum aestivum L., cv. ‘César’) and its anther-derived first cycle and second cycle doubled haploid lines has been analyzed by DNA-DNA molecular hybridization. Restricted DNA has been probed by three subclones of wheat nuclear ribosomal DNA covering the entire repeat unit. No significant difference was detected in the extent of methylation of ribosomal DNA of the doubled haploid lines with respect to the parental line. On the other hand, a variation has been found in the organization of the nontranscribed spacer region of ribosomal DNA of the first cycle doubled haploid line. This variation remains stable after a second cycle of in vitro androgenesis. However, one out of five second cycle doubled haploid lines so far tested showed an additional hybridization band present in the parental line but lacking in the first cycle doubled haploid line.

Mitochondrial DNA variability detected in a single wheat regenerant involves a rare recombination event across a short repeat.

The mitochondrial genome of the selfed progeny of a plant regenerated from long-term somatic tissue culture displays specific structural rearrangements characterized by the appearance of novel restriction fragments. A mitochondrial DNA library was constructed from this selfed progeny in the SalI site of cosmid pHC79 and the novel fragments were subsequently studied. They were shown to arise from reciprocal recombination events involving DNA sequences present in the parental plant. The regions of recombination were sequenced and the nucleotide sequences were aligned with those of the presumptive parental fragments. We characterized an imperfect short repeated DNA sequence, 242 bp long, within which a 7-bb DNA repeat could act as a region of recombination. The use of PCR technology allowed us to show that these fragments were present in both parental plants and tissue cultures as low-abundance sequence arrangements.

Organelle genome stability in anther-derived doubled haploids of wheat (Triticum aestivum L., cv. ‘Moisson’)

SummaryChloroplast and mitochondrial compartments of a parental line of wheat (Triticum aestivum L., cv. ‘Moisson’) and its anther-derived doubled haploid lines have been analyzed and compared on the basis of their DNA restriction patterns. The results obtained show that no noticeable difference can be detected between doubled haploid lines and parental line at the level of ctDNA and mtDNA organization. It may be concluded that in vitro culture by itself does not systematically generate a cytoplasmic variation in germ cells.

Nuclear genes control changes in the organization of the mitochondrial genome in tissue cultures derived from immature embryos of wheat

SummaryAlthough the mitochondrial genomes of the Chinese Spring and Aquila varieties of wheat are normaly similar in organization, this is not so in tissue cultures initiated from their immature embryos where the mitochondrial genomes of both are rearranged and in different, characteristic, ways. However, the mitochondrial genomes of tissue cultures of reciprocal F1 crosses between these varieties were almost identical to one another, showing that nuclear genes control the rearrangement processes. These rearrangements are either due to the appearance of new structures or else result from changes in the relative amounts of subgenomic components. The severe reduction in the amount of certain molecular configurations in tissue cultures from reciprocal crosses is probably due to the presence of dominant information in the Aquila nuclear genome. Data obtained from tissue cultures initiated from F2 embryos of the cross Aquila x Chinese Spring suggest that at least two complementary genes are involved in this control. In contrast, the presence of new molecular arrangements appears to be under the control of a dominant allelic form of a Chinese Spring gene or genes. Thus, this study demonstrates that at least two sets of nuclear genes control the reorganization of the mitochondrial genome which occurs when tissue cultures are initiated from the immature embryos of wheat.

A mitochondrial molecular marker of resistance to Bayoud disease in date palm

Abstract We have previously shown that two circular plasmid-like DNAs (the S and the R plasmids) can be found in the mitochondria of date palm (Phoenix dactylifera L.), a dioecious monocotyledonous tree. The two plasmids differ essentially by the absence, in the R plasmid, of a 109-bp DNA segment. Using 36 date palm varieties and employing a PCR-based approach, we show that the simultaneous presence of the R plasmid and absence of the S plasmid can be considered as a reliable molecular marker of resistance to a vascular wilt (Bayoud disease) caused by the fungus Fusarium oxysporum f. sp. albedinis. Conversely, the simultaneous presence of the S plasmid and absence of the R plasmid is correlated to Bayoud disease susceptibility The availibility of this diagnostic tool for plasmid characterization should subsequently allow simple, rapid and efficient selection of Bayoud-resistant individuals from the large number of date palms obtained by natural crosses which display good date quality.

Evidence for a direct relationship between mitochondrial genome organization and regeneration ability in hexaploid wheat somatic tissue cultures

SummaryEmbryogenic and non-embryogenic long-term callus cultures of hexaploid wheat exhibit differences in the organization of their mitochondrial genome. Embryogenic and non-embryogenic fractions of callus cultures initiated from immature embryos of the wheat cultivar “Chinese Spring” have been isolated and subsequently subcultured. DNA-DNA hybridization experiments using labelled cloned wheat mitochondrial DNA fragments have shown that the mitochondrial DNA organization of embryogenic subcultures derived from embryogenic parts of “Chinese Spring” calli is closely related to that of the initial “Chinese Spring” calli, while non-embryogenic subcultures derived from non-embryogenic fragments of “Chinese Spring” calli exhibit a mitochondrial DNA organization similar to that found in non-embryogenic calli derived from cultivar “Aquila”. In addition, somatic tissue cultures initiated from three other non-embryogenic wheat cultivars (“Talent”, “Thésée” and “Capitole”) display mitochondrial DNA arrangements similar to those found in cultivar “Aquila”. These results strongly suggest that, in wheat callus cultures, a particular mitochondrial genome organization is correlated with the ability of cultured cells to regenerate whole plants.

Em gene expression during somatic embryogenesis in the monocot Triticum aestivum L.

We have used in vitro cultures initiated from immature zygotic embryos of the monocot Triticum aestivum, variety Chinese Spring, to investigate some features of the transcriptional regulation of the Em gene. A 210-bp cDNA fragment internal to the coding region of the Em gene was amplified by PCR, cloned and used as a probe in northern analyses. Em gene expression was found to be associated with cultures displaying an embryogenic potential and, more precisely, with the presence of somatic embryos. The time-course of Em gene expression was thereafter monitored, by PCR and northern analysis, from the initiation of cultures until the appearance of embryogenic structures and was shown to be temporally regulated during the somatic embryogenesis process. Finally, the Em protein was observed in both somatic embryos and embryogenic cultures whereas it was not detected in non-embryogenic cultures. Our results, obtained from a monocot, ask the question of whether Em gene expression could be used as a universal late marker of somatic embryogenesis.