Somatic hybrids with substitution type genomic configuration TCBB for the transfer of nuclear and organelle genes from Brassica tournefortii TT to allotetraploid oilseed crop B. carinata BBCC

Oilseed crop Brassica carinata BBCC is a natural allotetraploid of diploid species B. nigra BB and B. oleracea CC. To transfer the nuclear and organelle genes in a concerted manner from an alien species, B. tournefortii TT, to B. carinata, we produced somatic hybrids with genomic configuration TCBB using B. nigra and B. oleracea stocks that carried selectable marker genes. B. tournefortii TT was sexually crossed with hygromycin-resistant B. oleracea CC. Protoplasts isolated from shoot cultures of hygromycin-resistant F₁ hybrids of B. tournefortiixB. oleracea TC were fused with protoplasts of kanamycin-resistant B. nigra BB. In two different fusion experiments 80 colonies were obtained through selection on media containing both hygromycin and kanamycin. Of these, 39 colonies regenerated into plants. Analysis of 15 regenerants by random amplified polymorphic DNA (RAPD) markers showed the presence of all three genomes, thereby confirming these to be true hybrids. Restriction fragment length polymorphism (RFLP) analysis of organelle genomes with heterologous chloroplast (cp)and mitochondrial (mt) DNA probes showed that the chloroplast genome was inherited from either of the two parents while mitochondrial genomes predominantly showed novel configurations due to either rearrangements or intergenomic recombinations. We anticipate that the TCBB genomic configuration will provide a more conducive situation for recombination between the T and C genomes during meiosis than the TTCCBB or TCCBB type configurations that are usually produced for alien gene transfer. The agronomic aim of producing TCBB hybrids is to transfer mitochondrial genes conferring cytoplasmic male sterility and nuclear genes for fertility restoration from B. tournefortii to B. carinata.     

Genetic analysis of morphological variation in Brassica oleracea using molecular markers

A cross between the open-pollinated Brassica oleracea cabbage cultivar Wisconsin Golden Acre and the hybrid broccoli cultivar Packman was used with molecular markers to investigate the genetic control of morphological variation. Twenty-two traits derived from leaf, stem, and flowering measurements were analyzed in 90 F₂ individuals that were also classified for genotype by restriction fragment length polymorphism (RFLP) markers. Seventy-two RFLP loci, which covered the mapped genome at an average of 10 map-unit intervals on all nine linkage groups, were tested individually for associations to phenotypic measurements by single factor ANOVA, and markers with significant associations (P<0.05) were used to develop multilocus models. These data were utilized to describe the location, parental contribution of alleles, magnitude of effect, and the gene action of trait loci. Single marker loci that were significantly associated (P<0.05) with trait measurements accounted for 6.7–42.7% of the phenotypic variation. Multilocus models described as much as 60.1% of the phenotypic variation for a given trait. In some cases, different related traits had common marker-locus associations with similar gene action and genotypic class ranking. The numbers, action, and linkages, of genes controlling traits estimated with marker loci in this population corresponded to estimates based on classical genetic methods from other studies using similar, or similarly-wide, crosses. There was no evidence that genome duplication accounted for a significant portion of multiple genes controlling trait loci over the entire genome, but possible duplications of trait loci were identified for two regions with linked, duplicated marker loci.     

Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes

Genetic similarity among 45 Brassica Oleracea genotypes was compared using two molecular markers, random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLPs). The genotypes included 37 broccolis (var. italica), five cauliflowers (var. botrytis) and three cabbages (var. capitata) which represented a wide range of commercially-available germplasm, and included open-pollinated cultivars, commercial hybrids, and inbred parents of hybrid cultivars. Fifty-six polymorphic RFLP bands and 181 polymorphic RAPD bands were generated using 15 random cDNA probes and 62 10-mer primers, respectively. The objectives were to compare RFLP and RAPD markers with regard to their (1) sampling variance, (2) rank correlations of genetic distance among sub-samples, and (3) inheritance. A bootstrap procedure was used to generate 200 random samples of size n (n=2,3,5,... 55) independently from the RAPD and RFLP data sets. The coefficient of variance (CV) was estimated for each sample. Pooled regressions of the coefficient of variance on bootstrap sample size indicated that the rate of decrease in CV with increasing sample size was the same for RFLPs and RAPDs. The rank correlation between the Nei-Li genetic similarity values for all pairs of genotypes (990) based on RFLP and RAPD data was 0.745. Differences were observed between the RFLP and RAPD dendrograms of the 45 genotypes. Overlap in the distributions of rank correlations between independent sub-samples from the RAPD data set, compared to correlations between RFLP and RAPD sub-samples, suggest that observed differences in estimation of genetic similarity between RAPDs and RFLPs is largely due to sampling error rather than due to DNA-based differences in how RAPDs and RFLPs reveal polymorphisms. A crossing algorithm was used to generate hypothetical banding patterns of hybrids based on the genotypes of the parents. The results of this study indicate that RAPDs provide a level of resolution equivalent to RFLPs for detemination of the genetic relationships among genotypes.     

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter--glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.     

Effects of an antisense napin gene on seed storage compounds in transgenic Brassica napus seeds

To manipulate the quantity and quality of storage components in Brassica napus seeds, we have constructed an antisense gene for the storage protein napin. The antisense gene was driven by the 5-flanking region of the B. napus napin gene to express antisense RNA in a seed-specific manner. Seeds of transgenic plants with antisense genes often contained reduced amounts of napin. In some transgenic plants, no accumulation of napin was observed. However, the total protein content of transgenic and wild-type seeds did not differ significantly. Seeds lacking napin accumulated 1.4 to 1.5 times more cruciferin than untransformed seeds, although the oleosin content was not affected. Fatty acid content and composition in the seeds of transgenic plants were also analyzed by gas chromatography. Though the total fatty acid content of the transformants was the same as that of non-transformants, there was a reduction in 18:1 contents and a concomitant increase of 18:2 in seeds with reduced napin levels. This observed change in fatty acid composition was inherited in the next generation.     

Phytohormonal changes in intact shoots of wheat and oilseed rape treated with the acylcyclohexanedione growth retardant prohexadione calcium

The effects of the acylcyclohexanedione-type growth retardant prohexadione calcium on seedling growth and endogenous levels of immunoreactive phytohormone-like substances in shoots of wheat ( Triticum aestivum L. cv. Kanzler) and oilseed rape ( Brassica napus L. ssp. napus cv. Lirajet) were studied. After treatment of seedlings with increasing retardant concentrations in hydroponics, plant height and fresh weight of shoots were reduced by up to 40%. Concomitantly, the amount of immunoreactive gibberellins decreased, on a fresh weight basis, when compared with levels in the shoots of control plants. In contrast, the levels of abscisic acid and dihydrozeatin riboside and isopentenyladenosine-type cytokinins were considerably elevated by the growth retardant. The content of 3-indoleacetic acid decreased slightly. These results suggest that, in addition to its effect on gibberellin content, prohexadione calcium also influences the levels of endogenous abscisic acid and cytokinins.     

Opportunities for gene transfer from transgenic oilseed rape (Brassica napus) to related species

Before novel transgenic plant genotypes are grown outside containment facilities and evaluated under field conditions, it is necessary to complete a risk assessment to consider the possible consequences of that release. An important aspect of risk assessment is to consider the likelihood and consequences of the transgene being transferred by cross-pollination to related species, including other crops, weeds and ruderal populations. The purpose of this report is to review the literature to assess the ease with whichBrassica napus can hybridize with related species. The evidence for hybridization is considered at three levels: a) by open pollination, b) by hand pollination and c) by the use ofin vitro ovule and embryo rescue techniques; and also examines the fertility and vigour of the F₁, F₂ and backcross generations. Four species are reported to hybridize withB. napus by open pollination:B. rapa andB. juncea using fully fertile parents; andB. adpressa andR. raphanistrum using a male-sterileB. napus parent. Seventeen species are reported to form hybrids (including the four species above) withB. napus when pollination is carried out manually. At least 12 of these species were unable to form F₂ progeny, and eight were unable to produce progeny when the F₁ was backcrossed to one of the parental species. Many factors will influence the success of hybridization under field conditions, including: distance between the parents, synchrony of flowering, method of pollen spread, specific parental genotypes used, direction of the cross and the environmental conditions. Even where there is a possibility of hybridization betweenB. napus and a related species growing in the vicinity of a release, poor vigour and high sterility in the hybrids will generally mean that hybrids and their progeny will not survive in either an agricultural or natural habitat.     

甘蓝型油菜下胚轴原生质体培养的研究

从甘蓝型油菜下胚轴分离纯化原生质体。种子萌发时进行光照处理对原生质体产率影响不大,但可提高其活力。对液体浅层、固体平板和“琼脂岛”3种培养方法进行比较,结果“琼脂岛”法效果最好,不但细胞分裂速度快,而且克隆形成频率高。再生的愈伤组织转到分化培养基上后迅速分化出芽。诱导生根后进行移栽,生长状况良好。