Factors influencing reliable shoot regeneration from leaf explants of rapeseed (Brassica napus L.) were examined. Addition of AgNO3 to callus induction medium was significantly effective for shoot regeneration in all three genotypes initially tested. When 48 genotypes subsequently were surveyed, a large variation of shoot regenerability was observed, ranging from 100 to 0% in frequency of bud formation and from 7.5 to 0 in the number of buds per explant. A significant correlation (r=0.84) was observed between the frequency of bud formation and the number of buds per explant. The shoot regenerability from leaf explants was not related to that from cotyledonary explants (r=0.28). Histological observations showed that an organized structure developed from calluses produced at vascular bundle tissues after 7 days of culture on callus induction medium, and they developed shoot apical meristems one week after transfer onto shoot induction medium. Regenerated plantlets were obtained 2 months after the initiation of culture and they normally flowered and set seeds. No alterations of morphology or DNA contents were observed in regenerated plants and their S1 progenies.

Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype

Inheritance of microspore embryogenic ability in oilseed rape (Brassicanapus L.) and Chinese cabbage (Brassica campestris L. ssp. pekinensis) was examined by 4 × 4 diallel crosses using cultivars showing a different response. In both species, embryo yields of most F1 hybrids were similar to, or over, the high responsive parent and some F1s showed intermediate embryo yields between their parents. Diallel analysis showed that both additive and dominant effects were significant at the 1% level for the genetic control of microspore embryogenic ability in both species. Dominant genes had positive effects on microspore embryogenesis. In oilseed rape, the additive effects were important, while in Chinese cabbage the dominant effects were largely contributed. The broad- and narrow-sense heritabilities were 0.972 and 0.811 in oilseed rape, and 0.959 and 0.659 in Chinese cabbage, respectively. From the results of the segregation of embryo yields in the F2 population of ’Lisandra’×’Kamikita’, it is considered that the microspore embryogenic ability is controlled by two loci with additive effects in oilseed rape.

Inheritance of microspore embryogenic ability in Brassica crops

Doubled haploid (DH) genotypes from a genetic mapping population of Brassica oleracea were screened for ease of transformation. Candidate genotypes were selected based on prior knowledge of three phenotypic markers: susceptibility to Agrobacterium tumefaciens, shoot regeneration potential and mode of shoot regeneration. Mode of regeneration was found to be the most significant of the three factors. Transgenic plants were successfully obtained from genotypes that regenerated multiple shoots via a distinct swelling or callus phase. The absence of tissue culture blackening (associated with genotypes that formed callus) was found to be critical for transformation success. Transgenic shoots were obtained from genotypes that regenerated via an indirect callus mode, even when susceptibility to Agrobacterium was low. The most efficient genotype (DH AG1012) produced transgenic shoots at an average rate of 15% (percentage of inoculated explants giving rise to transgenic plants). The speed and efficiency of regeneration enabled the isolation of transgenic shoots 5–6 weeks after inoculation with A. tumefaciens. This line was also self-compatible, enabling the production of seed without the need for hand-pollination. A genetically uniform DH genotype, with an associated genetic map, make DH AG1012 highly desirable as a potential model B. oleracea genotype for studying gene function. The possibility of applying the same phenotypic tissue culture markers to other Brassica species is discussed.

The use of phenotypic markers to identify Brassica oleracea genotypes for routine high-throughput Agrobacterium-mediated transformation.

Diallel analysis was used to investigate the genetic control of in vitro shoot regeneration in Brassica oleracea. Twelve doubled haploid (DH) lines, selected to include a range of genotypes with differing shoot regeneration potentials, were crossed reciprocally to produce 132 F1 and 12 selfed, DH families. Cotyledonary petioles from 4-day-old seedlings, from all families, were excised and maintained on MS medium supplemented with 2 mg/l BAP. Explants were scored after 44 days for both the presence or absence of shoots and the number of regenerating shoots per explant. Diallel analysis showed both shoot regeneration and the production of multiple shoots to be controlled by additive and dominant gene effects, with additive effects being more important. Additive gene effects accounted for 71% and 77% of the genetic variation observed within the diallel for shoot regeneration and multiple shoot regeneration, respectively. By investigating the shoot regeneration potential of subsequent backcross and F2 populations, the ability to introduce and increase shoot regeneration potential into otherwise recalcitrant lines was demonstrated.

Genetic analysis of in vitro shoot regeneration from cotyledonary petioles of Brassica oleracea.

A microcomputer program DIALL for diallel analysis of quantitative characters.

Genetic analysis of shoot regeneration from cotyledonary explants of Brassica napus was carried out by 7×7 diallel crosses using cultivars showing a different ability for regeneration. Both additive and dominant effects were significant, with the additive effect being more important than the dominant one. Dominant genes had a positive effect on shoot regeneration. Non-allelic interaction and average maternal effects were not detected, while specific the maternal one was significant. In the 5×5 sub-diallel table, the maternal effect became nonsignificant. The mean degree of dominance was 0.759. Broad- and narrow-sense heritabilities were 0.973 and 0.819, respectively, indicating that shoot regenera- tion ability can be easily transferred into economically important cultivars showing a low or an unresponsive ability.

Genetic analysis of shoot regeneration from cotyledonary explants in Brassica napus.

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Genetic analysis of shoot regeneration from cotyledonary explants in Brassica napus.