Fang XiaoPing

Transgene directionally integrated into C-genome of Brassica napus

Integration of a transgene into a C-genome chromosome plays an important role in reducing ecological risk of transgenic Brassica napus. To obtain C-genome transgenic B. napus, herbicide-resistant bar gene was firstly transferred into B. oleracea var. alboglabra mediated by Agrobacterium tumefaciens strain LBA4404. Then using the transgenic B. oleracea as paternal plants and 8 non-transgenic varieties of B. rapa as maternal plants, C-genome transgenic B. napus with bar gene was artificially resynthesized by means of ovary culture and chromosome doubling. Among 67 lines of the resynthesized B. napus, 31 were positive, and 36 were negative according to PCR test for bar gene. At least 2 plants from each line were kept for PPT spray confirmation. The result was in consistence with the PCR test. Genomic Southern blotting of three randomly chosen lines also showed that bar gene had been integrated into the genome of resynthesized B. napus lines.

Resynthesis of Brassica napus with Brassica oleracea or Brassica rapa Cytoplasm

Nuclear genome is the primary genetic material,which plays a key role in the individual development and the system evolution of plant.Although relatively independent genetic system in cytoplasm is existed,the characteristics of plant is more often determined by the nuclear genes and sometimes affected by plasmagenes.To compare genetic effect of different plasmagenes of B.napus and study interactions between nuclear gene and cytogene,we artificially synthesized B.napus with B.oleracea or B.rapa cytoplasm by means of hybrid embryo and ovary culture of reciprocal crosses between 12 cultivated varieties of B.rapa and one cultivated variety of B.oleracea var.alboglabra.The average frequencies of hybrid plantlets obtained were 2.32% from ovary culture of B.rapa × B.oleracea var.alboglabra and 1.16% from embryo culture of B.oleracea var.alboglabra × B.rapa,respectively.The induction rate of hybrid plantlets differed greatly between different crosses,but the trends of induction easily or hardly were the same between reciprocal crosses with the same parents.The haploidic hybrid seedlings were pre-cultured in MS medium with 0.01% colchicine for 10 days which was the best way for chromosome doubling.The percentage of hybrid plantlets with doubled chromosomes was 59.32%.Agronomic traits of synthesized B.napus were similar to those of cultivated B.napus and mediated between parents in morphology,but more similar to maternal plants.The average pollen fertility for the most synthesized lines ranged from 40.53% to 88.95%.

Analysis of F1 hybrid and BC1 monosomic alien addition line plants from Brassica oleracea × Sinapis alba by GISH

Sterile and semi-fertile F1 plants were obtained by intergeneric sexual hybridization between paternal Brassica oleracea var. alboglabra (genome CC, 2n=18) and maternal Sinapis alba (genome SS, 2n=24), BC1 plants were obtained by backcrossing between paternal B. oleracea and maternal semi-fertile F1 plants. Genomic in situ hybridization (GISH) combined with dual-colour fluorescence in situ hybridization (dcFISH) showed that sterile F1 plants contained 21 chromosomes consisting of one B. oleracea chromosome set and one S. alba chromosome set, belonging to expected hybrids, and semi-fertile F1 plants contained 30 chromosomes consisting of two B. oleracea chromosome sets and one S. alba chromosome set. It is obvious that the semi-fertile F1 plants belong to unexpected hybrids. 1–3 trivalents were detected at meiotic metaphase I of semi-fertile F1 pollen mother cells (PMCs). Different separation ratios of S chromosomes were detected at anaphase I. A monosomic alien addition line (MAAL) was identified by GISH-dcFISH from BC1 plants; it contained 19 chromosomes consisting of 18 C chromosomes and 1 S chromosome. At meiotic metaphase I, 9 divalents from B. oleracea and one univalent from S. alba could be detected. Sometimes, one putative C-S trivalent could also be detected. The achievement of B. oleracea-S. alba monosomic alien addition lines lays a foundation for gene introgression, location and cloning.