J. De Buyser

Genetic analysis of in vitro plant tissue culture responses and regeneration capacities

SummaryThe in vitro development of a whole plant from a single cell (eg microspore or somatic cells) is a characteristic feature of plants. The amenability of a plant to in vitro culture is influenced by the genotype, which is thus of major importance in the plant tissue culture response. The differences observed between different cultivars during in vitro tissue culture with respect to embryogenesis and regeneration result from quantitative or qualitative genetic differences. We describe results obtained from quantitative genetic studies, from Mendelian genetic analysis and from gene mapping. It is less easy to study the influence of cytoplasmic genomes. Moreover, it is necessary to discriminate between maternal effects and cytoplasmic inheritance. A conclusion from this review is that the choice of parental strains for a breeding program should be realized on agronomic criteria rather than on compatibility with the tissue culture technique used. Fortunately, it is relatively easy to incorporate short-term tissue culture capacity into agronomically valuable genotypes. This is of major interest since tissue culture remains necessary for most aspects of crop plants biotechnology. Very little is known about the molecular events that trigger in vitro embryogenesis and regeneration. It is clear that genes involved in the tissue culture response are not specialised ‘tissue culture genes’.

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.

A comparison of male and female recombination frequency in wheat using RFLP maps of homoeologous group 6 and 7 chromosomes

A novel approach was used to compare male and female recombination rates in wheat. Doubled haploid lines were developed from an F1 using two distinct approaches: the anther-culture technique and the Hordeum bulbosum system, from which sets of lines were developed from “male” and “female” meioses, respectively. The genotype of the lines was established at RFLP and isozyme markers polymorphic on chromosomes of homoeologous groups 6 and 7, and “male” and “female” linkage maps were calculated using this information. The markers in one segment of chromosome 6B exhibited disturbed segregation frequencies in the anther-culture population. The “male” and “female” maps differed significantly in recombination frequency between some markers on two chromosomes, and these were consistent in direction within chromosomes and inconsistent in direction between chromosomes. In two of the four chromosomes studied the “male” map was much longer than the “female” map. These results suggest that significant differences may exist in male and female recombination frequencies in bread wheat which are specific to certain chromosomal segments but are inconsistent in direction between chromosomes. Other factors, such as environmental influences, may also be important in creating differences.

Wheat Anther Culture: Agronomic Performance of Doubled Haploid Lines and the Release of a New Variety “Florin”

The production of haploid plants from hybrids, followed by chromosome doubling, provides wheat breeders with a means of accelerating the process of true breeding line development. The interest of doubled haploid wheat plants has been understood since their discovery (Gaines and Aase 1926). Nevertheless, a useful technique for producing haploids started at the beginning of the 1970’s with anther culture (Chu et al. 1973; Ouyang et al. 1973; Picard and de Buyser 1973; Wang et al. 1973). Since that time, other techniques have been developed in order to produce haploid wheat plants: the bulbosum technique used for some particular genotypes (Barclay 1975), the Salmon method (Kobayashi and Tsunewaki 1978; Tsunewaki et al. 1984) and the wheat × maize crosses (Laurie and Bennet 1988). Limited numbers of haploids can also be produced using irradiated pollen (Natarajan and Swaminathan 1958; Snape et al. 1983) and unpollinated ovary culture (Zhu and Wu 1979; Yan et al. 1979).

Chromosomal location of genes controlling short-term and long-term somatic embryogenesis in wheat revealed by immature embryo culture of aneuploid lines.

The expression of essential genes during somatic embryogenesis can be analysed by inducing aneuploid cells to undergo embryogenesis during immature embryo culture and then determining whether defects occur. Triticum aestivum disomic and aneuploid stocks, including 36 ditelosomics and 7 nullitetrasomic ‘Chinese Spring’ wheats, were compared for their ability to undergo somatic embryogenesis after 2 months of in vitro immature embryo culture. Their regeneration capacity was observed after 4 and 14 months of in vitro culture to determine which chromosome arms influence the process. The large range of variation found among the tested aneuploids suggested that genetic control of the somatic tissue culture ability is polygenic. Our results indicate that genes affecting somatic embryo-genesis and regeneration are located in all of the homoeologous chromosome groups. The lack of chromosome arms 1AL (DT 1AS) and 3DL (DT 3DS) practically suppresses somatic embryogenesis, demonstrating that major genes on wheat chromosome arms 1AL and 3DL control regeneration capacity. Results suggest that plants were mainly produced from somatic embryo development. Although the control of somatic embryogenesis and regeneration is polygenic, the genes located on the long arms of homoeologous group 3 chromosomes have a major effect. We also have evidence of chromosome arms that determine the time required for regeneration.

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.

Genetic analysis of in vitro wheat somatic embryogenesis

SummaryA spring wheat genotype which produces somatic embryos in vitro, after short and long-term culture, was tested for its ability to sexually transmit this embryogenic trait. Reciprocal crosses were performed between a embryogenic line and a nonembryogenic variety.Immature embryos were cultured on Murashige and Skoog medium plus 2 mg/l 2,4-dichlorophenoxyacetic acid, gelled with 5.5 g/l agarose. Somatic embryogenesis was not expressed in the F1s. In contrast, from several hundred immature embryos of the F2 generation of one cross, 10.7% and 1.6% expressed somatic embryogenesis in short and long-term cultures respectively. These percentages of embryogenic: non-embryogenic fits a model of a few complementary genes. The embryogenic capacity of the F2 genotypes depends on the presence of recessive alleles at these gene loci. The long-term wheat somatic embryogenesis capacity requires a more complex mechanism than the short-term one.

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.

Multiple patterns of mtDNA reorganization in plants regenerated from different in vitro cultured explants of a single wheat variety

SummaryWe have previously shown that tissue cultures derived from various explants of the wheat variety Chinese Spring exhibit organ/tissue-specific changes in the organization of their mitochondrial genome. The aim of this work was to study the influence of passage out of in-vitro culture, and subsequent plant regeneration, on the in vitro “induced” reorganization of this genome. In all cases but one, subgenomic configurations present in both the donor parent and the tissue culture were evident, in corresponding regenerated plants. The presence, in regenerated plants, of subgenomic configurations found in tissue culture but undetectable in the donor parent appeared to be both timeand organ/tissue-dependent. Moreover, when present, these novel organizations were not systematically found in all regenerated plants. Finally, novel subgenomic configurations were specifically detected after passage out of in-vitro culture. As these results were obtained from a single plant variety, they clearly confirm the extreme plasticity of mitochondrial genome structure in response to in-vitro culture.

Changes in the molecular organization of the mitochondrial genome in albino tissue cultures derived from wheat pollen embryos and in plants regenerated from these cultures

Abstract Total DNA was isolated from albino short- and long-term tissue cultures prepared from pollen embryos of wheat cultivars Chinese Spring and Aquila respectively and from the corresponding albino plants regenerated from these cultures. The DNA was digested with Sall, fractionated and probed with cloned restriction fragments of wheat mitochondrial DNA. These probes hybridized to regions of the mitochondrial DNA known to vary in somatic tissue cultures initiated from immature embryos of Aquila and Chinese Spring and in green plants regenerated from Chinese Spring embryogenic tissue cultures. The hybridization patterns obtained showed that the variability detected in albino androgenic cultures was similar to that previously observed in a comparable somatic system. However, all the regenerated albino plantlets within each group had the same mitochondrial genome organization, contrary to plants regenerated from somatic tissue cultures.