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.

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.

[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.

[RAPD analysis for the genetic diversity of Brassica rapa in Tibet].

Tibet, a most beautiful place, locating in southwestern China. She has been called as the third pole of the earth. Unique geological history, complex land surface and climatic zones, various soil types, all different wild vegetations etc., all of these make Tibet a very typical area of vertical agricultural ecosystem. The ecosystem in Tibet may be the most complex in the world, which varies from place to place. Genetic differentiation of 107 accessions of Brassica rapa from Tibet plateau was studied by DNA PAPD analysis using 2210 bp random primers, the genetical distribution in 107 accession of Brassica rapa from Tibet plateau was found. The results are as follows: (1) Total 236 bands were produced from 107 Tibet oilseed accession of B. rapa germaplasm resource in Tibet, of 210 bands amplified from B. rapa germaplasm resource showed polymorphism, with the ratio 88.98%. The result showed that oilseed accession of B. rapa in Tibet has richer genetic diversity; (2) Dendrogram constructed from DNA RAPDs showed that 107 accessions of B. rapa from Tibet plateau were divided into 11 cluster by calculating genetic distance, the cluster analysis showed that the genetic variation among oilseed accessions of B. rapa was closely related with their eco-geographic distribution; extensive variation existed among the accessions from Tibet Province. Based on the analysis of unique geological history, complex land surface and climatic zones, various soil types, complex growing environments, long agricultural history, different cropping systems, and natural and artificial selection as well as plant geography, plant evolution theory, it concludes that Tibet is one of the oil seed gene centers in the word.