‘FT’, A Spontaneous Doubled Haploid Recipient of Transient Transformation in Chinese Cabbage

Chinese cabbage is relatively recalcitrant to genetic transformation. Microspore is an ideal haploid single cell recipient for genetic m anipulation, whereby homozygous Doubled Haploid (DH) transformants can be obtained to accelerate the breeding process. In this study, Microspore Derived Embryo (MDE) productive material DH ‘FT’ is screened out from 10 candidates; microspore culture parameters were optimized: bud size ranged from 2.4mm to 2.7mm and microspore density 20000~40000/mL shows highest MDE yield. Antibiotic (chemical) sensitivity assay indicates that 100mg/L Carbenicillin or 300mg/L Timentin can be applied to inhibit the overgrowth of Agrobacterium after transformation; microspores are sensitive to cellulase and lysozyme treatments when liquid cultured, kanamycin slight below 5mg/L can be used as selection marker. ‘FT’ microspore and MDE were successfully transfected by Agrobacterium. Transient expression of embryogenic transcription factor LEC1-GFP in nucleus, rather than cytoplasm, was clearly detected by confocal microscopy. ‘FT’ microspores, 14/28d MDEs can be used as transient transfection recipients in Chinese cabbage. This study provides an efficient and spontaneous doubled haploid recipient system in Brassica rapa.