Chloroplast transformation of rapeseed (Brassica napus) by particle bombardment of cotyledons.

A protocol for chloroplast transformation of an elite rapeseed cultivar (Brassica napus L.) was developed based on optimized conditions for callus induction and regeneration from cotyledonary tissues. Comparison of six different media with three elite cultivars showed that B5 medium plus 3 mg/l AgNO(3) supplemented with 0.6 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-furfurylaminopurine was optimal for callus formation and maintenance without differentiation, while the medium suitable for regeneration was B5 medium supplemented with 1 mg/l 6-benzylaminopurine, 1 mg/l 6-furfurylaminopurine and 0.5 mg/l alpha-naphthaleneacetic acid. A rapeseed-specific chloroplast transformation vector was constructed with the trnI and trnA sequences amplified from the rapeseed chloroplast genome using two primers designed according to Arabidopsis homologs. The aadA gene was used as a selection marker regulated by the ribosome-binding site from the bacteriophage T7 gene 10L, the tobacco 16S rRNA promoter and the psbA terminator. After bombardment, cotyledonary segments were cultured for callus formation on media containing 10 mg/l spectinomycin and regeneration was carried out on medium with 20 mg/l spectinomycin. Heteroplasmic plastid transformants were isolated. An overall efficiency for the chloroplast transformation was one transplastomic plant per four bombarded plates. Southern blot analyses demonstrated proper integration of the target sequence into the rapeseed chloroplast genome via homologous recombination. The expression of the aadA gene was confirmed by Northern blot analysis. Analysis of T1 transplastomic plants revealed that the transgenes integrated into the chloroplast were inheritable with a ratio of about 8%. These results suggest that rapeseed may be a suitable crop for chloroplast transformation with cotyledons as explants under appropriate conditions.

Cloning and characterization of microRNAs from Brassica napus.

A library containing approximately 40,000 small RNA sequences was constructed for Brassica napus. Analysis of 3025 sequences obtained from this library resulted in the identification of 11 conserved miRNA families, which were validated by secondary structure prediction using surrounding sequences in the Brassica genome. Two 21 nt small RNA sequences reside within the arm of a pre-miRNA like stem-loop structure, making them likely candidates for novel non-conserved miRNAs in B. napus. Most of the conserved miRNAs were expressed at similar levels in a F1 hybrid B. napus line and its four double haploid progeny that showed marked variations in phenotypes, but many were differentially expressed between B. napus and Arabidopsis. The miR169 family was expressed at high levels in young leaves and stems, but was undetectable in roots and mature leaves, suggesting that miR169 expression is developmentally regulated in B. napus.

Molecular validation of multiple allele inheritance for dominant genic male sterility gene in Brassica napus L.

Dominant genic male sterility (DGMS) has been playing an increasingly important role, not only as a tool for assisting in recurrent selection but also as an alternative approach for efficient production of hybrids. Previous studies indicate that fertility restoration of DGMS is the action of another unlinked dominant gene. Recently, through classical genetic analysis with various test populations we have verified that in a DGMS line 609AB the trait is inherited in a multiple allelic pattern. In this study, we applied molecular marker technology to provide further validation of the results. Eight amplified fragment length polymorphism (AFLP) markers tightly linked to the male sterility allele (Ms) were identified in a BC1 population from a cross between 609A (a sterile plant in 609AB) and a temporary maintainer GS2467 as recurrent parent. Four out of the eight markers reproduced the same polymorphism in a larger BC(1) population generated with microspore-derived doubled haploid (DH) parents (S148 and S467). The two nearest AFLP markers SA12MG14 and P05MG15, flanking the Ms locus at respective distances of 0.3 centiMorgan (cM) and 1.6 cM, were converted into sequence characterized amplified region (SCAR) markers designated SC6 and SC9. Based on the sequence difference of the marker P05MG15 between S148 and a DH restorer line S103, we further developed a SCAR marker SC9f that is specific to the restorer allele (Mf). The map distance between SC9f and Mf was consistent with that between SC9 and Ms allele. Therefore, successful conversion of the marker tightly linked to Ms into a marker tightly linked to Mf suggested that the restoration for DGMS in 609AB is controlled by an allele at the Ms locus or a tightly linked gene (regarded as an allele in practical application). The Ms and Mf-specific markers developed here will facilitate the breeding for new elite homozygous sterile lines and allow further research on map-based cloning of the Ms gene.

Genetic analysis of oil content in Brassica napus L. using mixed model of major gene and polygene.

The joint segregation analysis of a mixed genetic model of major gene plus poly-gene was conducted to study the inheritance of oil content in Brassica napus L.. Five populations, i.e the populations of 2 parents (P1 and P2), F1, F2 and F2:3 (derived from F2) family, from each of the two crosses (1141B x Ken C-1, 32B x Ken C1-2) were investigated.The frequency distributions of oil content in F2 and F2:3 family populations show characteristics of a mixed normal distribution, which indicated that the inheritance of oil content followed a major gene plus poly-gene model. Twenty-one genetic models were established, which could be classified into five types: one and two major genes, polygenes, one and two major genes plus polygenes. The most suitable genetic model could be selected using Akaike's Information Criterion and the fitness of the selected one could be examined by a set of tests. Results show that genetic model D-2 is the most fitting genetic model for the trait. In other words, oil content in oilseed rape is controlled by one additive major gene plus additive and dominance polygenes. For cross 1 (1141B x Ken C1-1) the heritabilities of major gene and poly-genes in F2 are 68.21% and 27.17%, respectively, and in F2:3 are 81.70% and 16.80%, respectively. The additive effect of major gene is -1.74, which indicates that the locus of the allele in parent 1141B may decrease the oil content, but that in parent Ken C1-1 may increase it. The additive and dominance effects of the polygenes are 1.20 and -1.93, respectively. For cross 2 (32B x Ken C1-2) the heritabilities of major gene and polygenes in F2 are 66.20% and 28.10%, respectively, and in F2:3 were 81.00% and 14.90%, respectively. The additive effect of major gene was -3.74, which also indicates that the locus of the allele in parent 32B may decrease the oil content, but that in parent Ken C1-2 may increase it. The additive and dominance effects are -1.99 and 0.93, respectively. The heritability of the major gene in F2:3 is higher than that in F2 in both crosses, so it would be more efficent to conduct selection in F2:3 families for high oil content in breeding.

Identification of the linkage relationship between the flower colour and the content of erucic acid in the resynthesized Brassica napus L.

Brassica napus variety Quantum (yellow flower and low erucic content) as the female parent was crossed with a resynthesized Brassica napus line No. 2127-17 (white flower and high erucic content). The segregation ratios of the flower colour and the erucic acid content were analyzed in the F1, BC1, F2, and DH (doubled haploid) populations. The results indicated that the white flower was dominant over yellow and the erucic acid content was additively inherited. Both traits fit with a monogenic inheritance model, respectively. There were strong evidences to support the linkage relationship between the flower colour and the erucic acid content in the C-genome of B. napus with a recombination frequency of 5.8% in the DH population. The BSA (bulked segregant analysis) strategy was employed to identify random amplified polymorphic DNA (RAPD) marker linked to the genes for the flower colour and erucic acid content in the DH population. Of 685 arbitrary 10-base pair (bp) primers, one primer S92 generated a RAPD marker S92(-1400) that was tightly linked to the genes for the yellow flower and the low erucic acid content in the C-genome of B. napus L. The genetic distance of the genes for yellow flower and the low erucic acid content was 2.2 cM and 5.4 cM from the marker S92(-1400), respectively.

[Hypoploid groups and their cytogenetic analysis in the progenies between Brassica napus and Orychophragmus violaceus].

Several groups of hypoploids were characterized in pentaploid (p) progenies of B. napus and O. violaceus. Cytological characterization of the P3 population revealed allosubstitution of one pair of B. napus chromosomes with O. violaceus chromosomes. This population had normal fertility and had three kinds of somatic cells with 36-38 chromosomes. Its somatic cells and pollen mother cells (PMC) mainly had 38 chromosomes. From a P4 population, three monosomic plants with 37 chromosomes were identified with differing morphology and fertility. One plant which grew vigorously was composed of various types of somatic cell and PMC, in which the cells with 37 chromosomes were observed mainly. The unpaired chromosome was small and with no negative effects on plant development. In the offsprings of the aneuploids (P3) with 41-44 chromosomes, there are four types of plants with 29-32 chromosomes. Except the plant with 29 chromosomes, others which appeared abnormal meiosis behavior and poor pollen fertility are mixoploid. The origin of these hypoploids and their prospects of application have been discussed.

[Linkage map construction and mapping of a dominant genic male sterility gene (Ms) in Brassica napus].

A backcross population derived from a cross between the dominant genic male sterile line Rs1046A and the double-low cultivar 'Samourai' was used for linkage map construction in Brassica napus. A linkage map with a total length of 2 646 cM was developed using 138 AFLP markers, 83 SSR markers and one morphology trait, which were distributed over 18 major linkage groups, two triplets and one linkage pair. 11.7% of the mapped markers distorted from the expected 1:1 ratio. The dominant genic male sterility gene (Ms) was mapped in LG10 and surrounded by five closely linked AFLP markers. Meanwhile,two evident marker segregation distortion regions were observed in both LG8 and LG16. The map constructed in the present study and the mapping of Ms gene are highly valuable in designing marker-assisted breeding program for the genic male sterile two-type line in Brassica napus. They are also important to map-based cloning of the Ms gene and to better understanding of the mechanism of genic male sterility at molecular level.

Transcriptional control of orf224/atp6 by the pol CMS restorer Rfp gene in Brassica napus L.

In order to investigate effects of fertility restorer gene Rfp on expression of orf224/atp6, Northern analyses of floral mitochondrial RNA (mtRNA) from polima (pol) CMS three lines were conducted using 10 probes of mitochondrial genes in this paper. Differences in transcriptional patterns were observed only with the probes atp6, orf224 and orf222. The atp6 probe detected a single, 1.1 kb transcript with strong abundance in the male fertile 1141B (nap cytoplasm). In the male sterile 1141A (pol cytoplasm) and fertile restorer line 1141R (pol cytoplasm), levels of this transcript appeared greatly reduced, and two longer transcripts of 2.2 and 1.9 kb were observed. Compared with 1141A, levels of the 2.2 and 1.9 kb transcripts appeared to be slightly reduced in the restorer line 1141R, and two additional transcripts of 1.3 and 1.4 kb were observed. The results suggested that the expression of orf224/atp6 gene region was associated with pol CMS and regulated by Rfp gene. In contrast to floral buds of the male sterile 1141A, the 1.4 kb transcript specific to fertility-restored plants was also observed in seedlings of 1141A. Possible mechanisms by which transcriptional alteration of orf224/atp6 between floral buds and seedlings of 1141A may be related to nuclear genotypes and relatively low temperature are discussed.

[Improvement of microspores culture techniques in Brassica napus.L].

The application of microspore culture technique was restricted because of its low frequency of embryogenesis and chromosome doubling. Two methods of enhancing the frequency of embryogenesis were employed in the study,namely,activated charcoal treatment in NLN-13 media and 6-BA treatment in NLN-16 media. The treatment with 0.05% activated charcoal produced 24 embryos per plate,which increased 1.7 embryos per plate, as compared with the treatment without activated charcoal. However,the analysis of T-test showed that it was not significant. After adding 0.1 mg/L 6-BA in NLN-16 media, the frequency of embryogeny was 38.3 embryos per plate,and it was 26 embryos more per plate than that of CK. Analysis of T-test is significant. This indicates that 6-BA promotes embryogeny in microspore culture. Adding 50 mg/L colchicines in NLN-16 media,the doubling frequency was 67.6%. The plantlets transplanted into field with two methods of light-covered net and plastic films were investigated. A survival rate of 87.6% was obtained using light-covered method whereas 57.7% survived using plastic film method.