Improvements of doubled haploid production protocol for white cabbage (Brassica oleracea var. capitata L.)

Abstract

Protocols leading to the development of doubled haploid (DH) lines by microspore culture are widely used in white cabbage (Brassica oleracea var. capitata L.), but efficiency varies according to the cultivar and induction procedure. Forty different genotypes consisting of F1 cultivars and their crosses with responsive doubled haploid lines were tested to evaluate the androgenic response. In total, 20,032 embryos were produced. On average, the haploid induction response of F1 cultivars was 7.0 embryos/Petri dish, but the average of these hybrids crossed to responsive DH lines was 26.6 embryos/Petri dish. In seven reciprocal crosses, a difference was observed in just one, meaning that the maternal effect probably has a minor influence on haploid embryogenesis in cabbage. Addition of 0.02% activated charcoal (AC) to the induction media increased embryo formation in several low-responsive genotypes, but its effect on embryo formation of high-responsive genotypes was predominantly negative, although larger embryos were formed on media containing AC than without AC. Further development into plantlets was tested by two procedures. Formed embryos were either transferred directly to regeneration medium or treated with abscisic acid and desiccated for 4 weeks. Regrowth and further development reached on average 15.5 and 57.6%, for the first and second procedures, respectively. Plantlets developed by direct transfer often exhibited abnormal development or hyperhydricity, unlike the desiccated embryos. Spontaneous diploidisation of embryos reached 42.5% in total and was not affected by AC added to the induction media.

Proposal of a New Hybrid Breeding Method Based on Genotyping, Inter-Pollination, Phenotyping and Paternity Testing of Selected Elite F1 Hybrids

“…Cabbage cultivars and DH were obtained from our cabbage hybrid breeding program, the list of genotypes included is given in Supplementary Tables S1 , S2 , S3 and S4 . In 2016, doubled haploid lines were induced from highly heterozygous donor plants ( Supplementary Tables S1 and S2 ) by microspore culture, as described earlier (Rudolf-Pilih et al, 2018). Genetically diverse DH cabbage seedlings were cultivated on a sterilised clay substrate (Klasmann-Deilmann) fertilised with Osmocote exact (15-9-12+2MgO+TE) in a greenhouse and exposed to low temperatures (0–5°C) for three months.…”