M. Landgren

Gene transfer via somatic hybridization in plants

Abstract Successful plant protoplast culture and fusion techniques have now been developed for many crop species. This has stimulated hopes that the transfer of desirable traits between sexually incompatible species will prove to be a practical approach for the genetic improvement of crops.

Fertility and chromosome stability in Brassica napus resynthesised by protoplast fusion

SummaryFertile somatic hybrids between Brassica campestris and B. oleracea have been produced by protoplast fusion. Fusion products were identified by their intermediate protoplast morphology. Heterokaryons were isolated either with micropipettes using a micromanipulator or by flow sorting. About 2% of the obtained calli differentiated to shoots. Of the shoots obtained from manually selected heterokaryons, 100% were true hybrids as confirmed by isozyme analysis while 87% of the flow sorted ones showed a hybrid pattern. Ploidy level of the hybrid plants was determined by chromosome counting and relative DNA-content analysis. The sum of the chromosome number (38) from the two fusion partners were found in 30% of the hybrids; 9% had fewer and 61% had more chromosomes. Pollen viability and seed set varied with ploidy level. Compared to natural B. napus, a pollen viability of 52%–93% and a fertility of 1%–40% was found for the somatic hybrids with normal chromosome number. Restriction enzyme analysis of chloroplast-DNA showed that either B. campestris or B. oleracea chloroplasts were present in the somatic hybrid plants. Of 11 hybrid plants 5 had the campestris and 6 had the oleracea type (1∶1 ratio).

Brassica napus lines with rearranged Arabidopsis mitochondria display CMS and a range of developmental aberrations

Abstract. Numerous Brassica napus (+) Arabidopsis thaliana somatic hybrids were screened for male sterility and aberrant flower phenotypes. Nine hybrids were selected and backcrossed recurrently to B. napus. The resulting lines displayed stable maternal inheritance of flower phenotypes. Nuclear and organellar genomes were characterized molecularly using RFLP analysis. No DNA from A. thaliana was found in the nuclear genome after six back-crosses, whilst the mitochondrial genomes contained rearranged DNA from both A. thaliana and B. napus. Each line tested had a unique RFLP pattern of the mitochondrial DNA (mtDNA) that remained unchanged between the BC3 and BC6 generation. The plastid genomes consisted of B. napus DNA. Five lines of the BC5 generation were subjected to more comprehensive investigations of growth, morphology and fertility. On the basis of these investigations, the five CMS lines could be assigned to two groups, one represented by three lines displaying reduced vegetative development, complete male sterility, and homeotic conversions of stamens into feminized structures. The second group, represented by the other two lines, were not completely male-sterile but still displayed severely affected flower morphologies. These two lines did not display any reduction in vegetative development. For both groups only stamens and petals suffered from the morphological and functional aberrations, while the sepals and pistils displayed normal morphology. All plants were fully female-fertile. Different rearrangements of the mitochondrial genome disturbed nuclear-mitochondrial interactions and led to various types of aberrant growth and flower development. The existence of numerous CMS lines with different mitochondrial patterns involving a species with a sequenced genome offers new opportunities to investigate the genetic regulation of CMS and its associated developmental perturbations.

ALLOPLASMIC MALE-STERILE BRASSICA LINES CONTAINING B. TOURNEFORTII MITOCHONDRIA EXPRESS AN ORF 3' OF THE ATP6 GENE AND A 32 KDA PROTEIN

Analyses of mitochondrial transcription and in organello translation were performed with the Brassica tournefortii cytoplasm. This cytoplasm causes alloplasmic male sterility when combined with the nuclear genomes of B. napus and B. juncea. Mitochondrial RNA and protein banding patterns were compared between the fertile wild species B. tournefortii, an alloplasmic male-sterile B. juncea line, an alloplasmic male-sterile B. napus line and an alloplasmic B. napus line with restored fertility. The analyses were carried out to identify differences in gene expression and to investigate whether alterations in gene expression accompanied male sterility. A difference in transcription patterns between the fertile B. tournefortii and the alloplasmic lines was found for the atp6 gene. The atp6 region was investigated further, since a similar alteration in atp6 transcription has been observed in two other Brassica cytoplasms which are associated with cytoplasmic male sterility (CMS). The additional longer atp6 transcript detected in the alloplasmic lines in the present study was found to contain an open reading frame (ORF) located downstream of the atp6 gene. DNA sequencing revealed that the ORF, orf263, could encode a protein with a predicted molecular weight of about 29 kDa. In organello analysis detected two proteins, of 29 and 32 kDa respectively, which were found only in the alloplasmic lines. Furthermore, the 32 kDa protein accompanied male sterility since it was absent in alloplasmic plants restored to fertility. The protein analysis might indicate that orf263 is translated and causes CMS.

Analysis of chloroplast and mitochondrial segregation in three different combinations of somatic hybrids produced within Brassicaceae

SummaryMitochondrial and chloroplast DNA were characterized in three different combinations of somatic hybrids produced between different species within Brassicaceae. The fusions were made between B. campestris and B. oleracea, B. napus and B. nigra and between B. napus and Eruca sativa. The combinations represent interspecific hybridizations, but the phylogenetic distance between the species used in each instance is different. Whereas the B. campestris (+) B. oleracea and the B. napus (+)B. nigra hybrids are both examples of intrageneric hybrids, B. campestris is more closely related to B. oleracea than B. napus is to B. nigra. The fusion of B. napus and E. sativa represents an intergeneric hybridization. Since hybrids were produced with reproducible and uniform fusion and culture methods, a comparison of chloroplast and mitochondrial segregation and mitochondrial DNA (mt-DNA) rearrangements could be made between the combinations. The segregation of both chloroplasts and mitochondria was biased in the B. napus (+)B. nigra and the B. napus (+)E. sativa combination. The nonrandom segregation of chloroplasts and mitochondria could be due to the different ploidy levels of the fusion partners and/or reflect differences in organelle replication rate. Furthermore, segregation of mitochondria was correlated to the differences in phylogenetic distance between the species used in the fusions. However, mitochondrial segregation, in contrast to chloroplast segregation, could in all combinations also have been affected by the cell type used as protoplast source in the fusions. All different chloroplast types could be established within each combination. Hybrids containing chloroplast from one parent together with mitochondria from the other parent were found in two of the combinations, although the majority of the hybrids had mt-DNA that was altered compared to the parental species. The rearranged mt-DNA found in most hybrids was an effect of the heteroplasmic state following protoplast fusion rather than of the tissue culture methods, since no mt-DNA rearrangements were found in B. napus plants regenerated from protoplast culture. The mtDNA restriction patterns of the hybrids with rearranged mt-DNA indicated that specific regions of the mt-DNA were involved in the rearrangements following protoplast fusion.

Intertribal somatic hybrids between Brassica napus and Thlaspi perfoliatum with high content of the T. perfoliatum-specific nervonic acid

Protoplast fusions were performed between hypocotyl protoplasts of Brassica napus and mesophyll protoplasts of Thlaspi perfoliatum. The two species are members of the Lepidieae and Brassiceae tribes, respectively, in the family of Brassicaceae. Seeds of T. perfoliatum are rich in the fatty acid C24∶1 (nervonic acid), an oil valuable for technical purposes. In the search for renewable oils to replace the mineral oils, plant breeders have been trying to develop oil crops with a high content of long-chain fatty acids. After fusion of B. napus protoplasts with non-irradiated as well as irradiated protoplasts of T. perfoliatum selection was carried out by flow cytometry and cell sorting. Of the shoots regenerated from different calli 27 were verified as hybrids or partial hybrids using the isoenzyme phosphoglucose isomerase (PGI) as a marker. Another 6 plants were identified as partial hybrids using a T. perfoliatum-specific repetitive DNA sequence. Slot blot experiments were performed to estimate the copy number of the repetitive DNA sequence in the parental species and in the hybrids. In T. perfoliatum there were approximately 105 copies per haploid genome, and the range in the hybrids was 1–37% of the value in T. perfoliatum. When the nuclear DNA content of the regenerated shoots was analysed we found partial as well as symmetric hybrids. Even though the rooting and establishment of hybrid shoots in the greenhouse were difficult, resulting in the death of many plants, 19 plants were cultured to full maturity. Seeds obtained from 15 plants were analysed to determine whether they contained nervonic acid, and 5 of the hybrids were found to contain significantly greater amounts of nervonic acid than B. napus.

Fertile somatic hybrids between Brassica napus and Arabidopsis thaliana

Abstract Fertile somatic hybrids between Brassica napus and Arabidopsis thaliana were produced by protoplast fusion and enriched by flow sorting. Isoenzyme analysis and hybridization of repetitive DNA sequences from the two parental species to total DNA from the hybrids were used to verify hybrid characteristics of the nuclear genomes. Of 29 shoots, each from a different callus, 25 had a hybrid pattern for at least one of the markers investigated. Analysis of chromosome number revealed that 11 out of 18 hybrids had the sum of the two parental chromosome numbers. Organelle analysis of 18 hybrids showed that ten hybrids had mitochondrial (mt) restriction patterns identical to those of B. napus, while eight displayed a rearranged mtDNA restriction fragment pattern. B. napus chloroplasts (cp) were favoured in the hybrids, and out of 21 analysed hybrids only one plant had A. thaliana cp. Most of the hybrids had intermediated morphological features compared with those of the parental species and 14 out of the 19 tested hybrids were female fertile and set seed after backcrossing to B. napus. Of the female-fertile hybrids, two were also self-fertile.

A high frequency of intergenomic mitochondrial recombination and an overall biased segregation of B. campestris or recombined B. campestris mitochondria were found in somatic hybrids made within Brassicaceae

Mitochondrial segregation and rearrangements were studied in regenerated somatic hybrids from seven different species combinations produced using reproducible and uniform methods. The interspecific hybridizations were made between closely or more distantly related species within the Brassicaceae and were exemplified by three intrageneric, two intergeneric and two intertribal species combinations. The intrageneric combinations were represented by Brassica campestris (+) B. oleracea, B. napus (+) B. nigra and B. napus (+) B. juncea (tournefortii) hybrids, the intergeneric combinations by B. napus (+) Raphanus sativus and B. napus (+) Eruca sativa hybrids, and the intertribal combinations by B. napus (+) Thlaspi perfoliatum and B. napus (+) Arabidopsis thaliana hybrids. In each species combination, one of the two mitochondrial genotypes was B. campestris since the B. napus cultivar used in the fusions contained this cytoplasm. Mitochondrial DNA (mtDNA) analyses were performed using DNA hybridization with nine different mitochondrial genes as probes. Among the various species combinations, 43–95% of the hybrids demonstrated mtDNA rearrangements. All examined B. campestris mtDNA regions could undergo intergenomic recombination since hybrid-specific fragments were found for all of the mtDNA probes analysed. Furthermore, hybrids with identical hybrid-specific fragments were found for all probes except cox II and rrn18/rrn5, supporting the suggestion that intergenomic recombination can involve specific sequences. A strong bias of hybrids having new atp A-or atp9-associated fragments observed in the intra- and intergeneric combinations could imply that these regions contain sequences that have a high reiteration number, which gives them a higher probability of recombining. A biased segregation of B. campestris-or B. campestris-like mitochondria was found in all combinations. A different degree of phylogenetic relatedness between the fusion partners did not have a significant influence on mitochondrial segregation in the hybrids in this study.

Biased mitochondrial segregation, independent of cell type used for fusion and of hybrid nuclear DNA content, was found in Brassica napus (+) B. oleracea somatic hybrids

Abstract To determine whether the cell type used for protoplast fusion influenced the mitochondrial segregation in somatic hybrids, mtDNA analysis was performed in Brassica napus (+) B. oleracea hybrid plants obtained from four different fusion combinations of hypocotyl and mesophyll protoplasts. In these fusions B. oleracea mitochondria were combined with B. campestris mitochondria since the B. napus cultivar had a cytoplasm derived from B campestris . Two mitochondrial gene probes, atp9 and atpA , were used to analyse mtDNA in the regenerated hybrids. In total, 96 plants were analysed. In all fusion categories, an overall biased segregation of mtDNA demonstrating a B. campestris specific pattern was found. No significant differences in segregation pattern of atp9 or atpA were found between the fusion categories. Thus, no major influence of the cell type used for protoplast fusion on mitochondrial segregation in the hybrids was found. Furthermore, nuclear DNA content analyses were available for 74 hybrids and we investigated whether the mitochondrial segregation patterns were different in hybrids exhibiting different ploidy levels. No significant difference in mitochondrial segregation was found between symmetric hybrids and hybrids with higher or lower ploidy levels. The overall strong bias of hybrids exhibiting a B. campestris RFLP pattern indicates that inherent differences of mitochondrial genomes from the different species exist, which affect the segregation in biparental cytoplasms.

Characterization of sexual progenies of male-sterile somatic cybrids between Brassica napus and Brassica tournefortii.

Abstract Cytogenetic studies were performed on four male-sterile progenies derived from four different cybrids produced between Brassica napus and B. tournefortii using the donor-recipient protoplast fusion method. The objective of these studies was to characterize the nuclear constitution of the plants. Mitotic investigation revealed that three of the four male-sterile lines had 38 chromosomes, which is equal to that of B. napus. The fourth line, C6, had variable chromosome numbers, ranging from 39 to 42 in different plants. The meiotic behavior in each progeny varied distinctly. Of the plants having 38 chromosomes, fairly high chromosome pairing, on average 18.08 bivalents per cell, was detected at metaphase-I. However, univalents with an average of 1.39 per cell, and very low frequencies of trivalents and/or tetravalents, were also observed in the lines. These results revealed that male-sterile cybrid lines were obtained with 38 chromosomes and a relatively high level of chromosome-pairing ability, indicating their potential for establishing a stable male-sterile rapeseed line.