Y. P. Wang

Behaviour of Sinapis alba chromosomes in a Brassica napus background revealed by genomic in-situ hybridization

Genomic in-situ hybridization (GISH) was applied to study the behaviour of addition chromosomes in first and second backcross (BC) progenies of hybrids between Brassica napus ssp. napus L. (AACC, 2n = 38) and Sinapis alba L. (SS, 2n = 24) produced by electrofusion. With GISH using genomic DNA of S. alba was used as probe it was possible to clearly distinguish both of the parental genomes and effectively monitor the fate of S. alba chromosomes in the BC1 and BC2 progenies. GISH analysis confirmed the sesquidiploid genome composition (AACCS) of the BC1 progenies, which contained 38 chromosomes from B. napus and 12 chromosomes from S. alba. Genome painting in the pollen mother cells (PMCs) of the BC1 plants revealed intergenomic association between B. napus and S. alba chromosomes, whereby a maximum of 4 trivalents between AC and S chromosomes were identified at metaphase I. In the BC2 progenies, aneuploids with different numbers of additional chromosomes from S. alba, ranging from 1 to 7, were confirmed. Three putative monosomic alien addition lines were characterized, and the results are discussed with respect to the potential for intergenomic chromosome recombination.

Intertribal somatic hybrids between Brassica napus and Camelina sativa with high linolenic acid content

Intertribal somatic hybrids of Brassica napus and Camelina sativa were developed by protoplast electrofusion. Hybrid identity of the regenerants was determined using flow cytometric analysis of nuclear DNA content and simple sequence repeat (SSR) marker analysis. Three hybrids exhibited specific bands for B. napus and C. sativa. These hybrids showed intermediate leaf, flower and seed morphology compared with the two parental species. The seeds of these three hybrids had a modified fatty acid profile, indicating higher level of linolenic and eicosanoic acids than those of B. napus. Our results suggest that somatic hybridization offers opportunities for transferring entire genomes between B. napus and C. sativa in improving rapeseed breeding.

Phenotypic variation in progenies from somatic hybrids between Brassica napus and Sinapis alba

A number of variant phenotypes, such as long siliques, high density of pods, increased seed number per pod and yellow seed color resembling Sinapis alba, were selected from intergeneric somatic hybrids between Brassica napus and Sinapis alba through successive backcrosses. Resistance to Sclerotinia sclerotiorum among 24 BCF4 lines was also tested by inoculation with mycelia, and four lines showed stronger resistance than the resistant rapeseed variety ‘Zhongshuang 9’. Comparative anatomy studies on mature seed revealed that the seed coat pigments were mainly distributed in the palisade layer, which is considerably thinner in S. alba compared to B. napus. The area index of protein bodies in cotyledon cells was highest in S. alba, lowest in B. napus and intermediate in the intergeneric progenies. This study demonstrates that wide hybridization can enable exploitation of valuable trait diversity from S. alba for broadening the genetic basis for rapeseed breeding.

GISH analysis of disomic Brassica napus-Crambe abyssinica chromosome addition lines produced by microspore culture from monosomic addition lines

Two Brassica napus--Crambe abyssinica monosomic addition lines (2n=39, AACC plus a single chromosome from C. abyssinca) were obtained from the F2 progeny of the asymmetric somatic hybrid. The alien chromosome from C. abyssinca in the addition line was clearly distinguished by genomic in situ hybridization (GISH). Twenty-seven microspore-derived plants from the addition lines were obtained. Fourteen seedlings were determined to be diploid plants (2n=38) arising from spontaneous chromosome doubling, while 13 seedlings were confirmed as haploid plants. Doubled haploid plants produced after treatment with colchicine and two disomic chromosome addition lines (2n=40, AACC plus a single pair of homologous chromosomes from C. abyssinca) could again be identified by GISH analysis. The lines are potentially useful for molecular genetic analysis of novel C. abyssinica genes or alleles contributing to traits relevant for oilseed rape (B. napus) breeding.

DNA methylation analysis during the optimization of Agrobacterium-mediated transformation of soybean

Soybean is recognized as one of the plants which are very difficult to be transformed. Considering the low transformation efficiency of soybean, we aimed to determine the effect of 6-benzylaminopurine (6-BA), shoot induction time, and infection time of Agrobacterium on the clonal propagation of Glycine max. Results showed that 1.6 mg/L 6-BA could be optimal to promote the induction of adventitious shoots. An induction time of 15 d was considered optimal for the actual experiment involving soybean shoot induction. Agrobacterium was cultured until an OD600 = 0.8 was reached for an infection time of 30 min; this infection time may be optimal to promote soybean transformation. Whole genome DNA methylation was analyzed by high-performance liquid chromatography (HPLC)-assisted quantification, and DNA methylation result is consistent with the phenotypic data of shoot development. In addition, two methylation-related genes (decrease in DNA methylation 1 and DNA methyl transferases chromomethylase 2) were analyzed to determine expression differences by qRT-PCR in the shoots that were developed under different experimental conditions. In general, the expression values of these genes were normally downregulated under the recommended experimental conditions of soybean regeneration. This study showed the overall methylation changes in the in vitro culture of soybean, as affected by several variable parameters, which is useful to promote the transformation efficiency of soybean.

Chromosomal distribution of 18S-25S rDNA in four Lupinus species visualized by fluorescence in situ hybridization

The number and position of 18S–25S rDNA sites in 4 selected Lupinus species are reported for the first time. L. atlanticus, L. subcarnosus and L. paniculatus had two rDNA loci, while L. albus exhibited only one loci. Among these 4 species, all of them exhibited one large pair of strong signals that extends from the short arm to a NOR on a chromosome satellite. L. atlanticus, L. subcarnosus, L. paniculatus had one more locus of 18–25S rDNA, but a pair of weak hybridization signals were observed in L. paniculatus when 18S–25S rDNA was used as probe. The results are discussed in terms of the evolutionary relationships among these species.

Epigenetic variation in the callus of Brassica napus under different inducement conditions

Tissue culture, a traditional technique broadly used for the genetic transformation and functional verification of target genes, induces epigenetic variations in transgenic acceptors of plants. This study compared the DNA methylation patterns during the callus formation of Brassica napus induced by different concentrations of 6-BA and 2,4-D through methylation-sensitive amplification polymorphism. The highest induction rate (85%) was observed in the hypocotyls cultured with 0.1 mg/L 2,4-D and the lowest methylation rate (25.09%) was detected in the hypocotyls cultured with 1.0 mg/L 6-BA. The methylation rates of the callus cultured with 0.2 and 0.05 mg/L 2,4-D were 29.99 and 28.31%, respectively. The callus induction rates were reduced to 79 and 80%. The methylation rates of the callus induced by 2.0 and 0.5 mg/L 6-BA were 28.17 and 33.98%, respectively. The callus induction rates were reduced to 76 and 74%. The expression analysis of methyltransferase under different induction conditions agreed with methylation modifications; therefore, the effects of hormones on callus induction may be partially indicated by methylation changes in B. napus genome.

Transformation of LRP gene into Brassica napus mediated by Agrobacterium tumefaciens to enhance lysine content in seeds.

Lysine rich protein (LRP) gene derived from the seed of Psophocarpus tetragonolobus was transformed into Brassica napus, employing cotyledon petiole as explants and by using the Agrobacterium tumefaciens strain LBA4404. Transformation efficiency was found to be closely related with phytohormone concentration, infection incubation, and co-cultured time. A medium containing 4 mg/l 6-benzyladenine (6-BA) and 0.3 mg/1 naphthalene acetic acid (NAA) was used for plant regeneration. With infection incubation of A. tumefaciens (OD600 = 0.4) for 20 min and co-culture of infected cotyledon petiole for 3 days, the highest transformation efficiency of 8.5% was obtained. To confirm LRP gene expression, PCR and Southern blot analysis were performed on leaf-isolated DNA from regenerated plants resistant to kanamycin. All transgenic plants of the generation T0 formed fertile seeds, which were sowed for the inheritance study of generational T1 and amino acid analysis. It was found that the lysine content of seeds from T1 generation increased by 16.7% compared with non-transgenic lines.