Jiashu Cao

The polygalacturonase gene BcMF2 from Brassica campestris is associated with intine development

Brassica campestris Male Fertility 2 (BcMF2) is a putative polygalacturonase (PG) gene previously isolated from the flower bud of Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis). This gene was found to be expressed specifically in tapetum and pollen after the tetrad stage of anther development. Antisense RNA technology was used to study the function of BcMF2 in Chinese cabbage. Scanning and transmission electron microscopy revealed that there were deformities in the transgenic mature pollen grains such as abnormal location of germinal furrows. In addition, the homogeneous pectic exintine layer facing the exterior seemed to be overdeveloped and predominantly occupied the intine, thus reversing the normal proportional distribution of the internal endintine layer and the external exintine layer. Since it is a continuation of the intine layer, the pollen tube wall could not grow normally. This resulted in the formation of a balloon-like swelling structure in the pollen tube tip in nearly 80% of the transgenic pollen grains. Premature degradation of tapetum was also found in these transgenic plants, which displayed decreased expression of the BcMF2 gene. BcMF2 might therefore encode a new PG with an important role in pollen wall development, possibly via regulation of pectins dynamic metabolism.

Pollen wall development: the associated enzymes and metabolic pathways

Pollen grains are surrounded by a sculpted wall, which protects male gametophytes from various environmental stresses and microbial attacks, and also facilitates pollination. Pollen wall development requires lipid and polysaccharide metabolism, and some key genes and proteins that participate in these processes have recently been identified. Here, we summarise the genes and describe their functions during pollen wall development via several metabolic pathways. A working model involving substances and catalytic enzyme reactions that occur during pollen development is also presented. This model provides information on the complete process of pollen wall development with respect to metabolic pathways.

Functional analysis of a novel male fertility CYP86MF gene in Chinese cabbage (Brassica campestris L. ssp. chinensis makino)

In our earlier work, a cytochrome P450 CYP86MF gene was isolated from floral bud of Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa L.) by mRNA differential display PCR (DD-PCR) and rapid amplification of cDNA ends (RACE). To unravel the biological function of CYP86MF gene, the antisense fragment from the CYP86MF gene was transferred into Chinese cabbage pak-choi (B. campestris ssp. chinensis var. communis Tsen et Lee). Out of 22 plants transformed with the antisense gene constructed from the CYP86MF, 20 reached to flowering stage. Morphological investigations showed that the transgenic plants developed the normal floral organ. However, they remained self-infertile, even when artificial self-pollination was performed in the bud stage. Pollen germination test indicated that the pollen from the transgenic line TB-2 could not germinate normally. Further physiological, biochemical and cytological analyses showed that only significant difference was detectable in contents of the endogenous hormones, and a layer of unknown material adhered to the surface of microspore. The present studies thus provided valuable clues for understanding the biological function of the CYP86C subfamily genes. Furthermore, our studies also demonstrate a novel method for obtaining artificial male sterility line of Chinese cabbage.

Transcriptional differences between the male-sterile mutant bcms and wild-type Brassica campestris ssp. chinensis reveal genes related to pollen development

A novel male-sterile mutant which lacks mature pollen, Brassisa campestris male sterile (bcms), was identified in Brassica campestris L. ssp. chinensis Makino (syn. B. rapa ssp. chinensis). Genetic analysis revealed that bcms was controlled by a single recessive mutation locus. Genome-wide transcriptional profiling was performed on the flower buds of both the bcms mutant and the wild-type from which it originated, and profiling analysis indicated that there were numerous changes in gene expression attributable to the gene mutation. This mutation resulted in down-regulation of a variety of genes and up-regulated expression of a few other genes. A total of 51 transcript-derived fragments (TDFs) were isolated: 32 specifically and 12 predominantly accumulated in wild-type flower buds, and two specifically and five predominantly accumulated in bcms flower buds. Sequence analysis showed that some of these TDFs share significant similarities with genes involved in different aspects of cellular development, such as signal transduction, cell wall biosynthesis and regulation. Most other TDFs showed no or very poor sequence similarities to entries in any database and might represent new candidate proteins involved in pollen development. Furthermore, spatial and temporal expression pattern analysis of 20 genes derived from cDNA-amplified fragment length polymorphism in different tissues of both the bcms and wild-type plants revealed their complex and dynamic expression patterns. The bcms mutant and the genes isolated in this paper provide excellent material for future studies on the molecular mechanism of male sterility.

Shoot regeneration and the relationship between organogenic capacity and endogenous hormonal contents in pumpkin

To induce multiple shoots from pumpkin (Cucurbita moschata Duch.), cotyledon explants excised from various ages of seedlings after inxa0vitro germination were cultured on MS augmented with different concentrations of BA (0, 0.5, 1.0 or 2.0xa0mgxa0l−1). The highest frequency of shoot regeneration (63.7%) was observed from seven-day-old cotyledon explants cultured on MS containing 0.5xa0mgxa0l−1 BA. The frequency and duration of shoot formation showed close correlation with the donor seedling age. By contrast, BA supply was necessary to promote shoot formation but no differences were observed in relation to different concentrations. Multiple shoots elongated on MS supplemented with 0.1xa0mgxa0l−1 BA and 5–7 shoots per regenerated explant were recovered. Elongated shoots were rooted on MS, which was easier than that on 2/3MS, 1/2MS, or MS supplemented with 0.1xa0mgxa0l−1 NAA. The rooted shoots were then transferred to greenhouse where they grew and flowered normally. Quantitative analysis of endogenous auxin (IAA) and cytokinins (iPA and ZR) in initial cotyledon explants of different aged seedlings showed that the regeneration ability of cotyledon explants varied dependently on their endogenous iPA contents. This study therefore deduces that the various organogenic capabilities of cotyledon explants from pumpkin are the result of their endogenous hormonal contents.

Functional analysis of a pollen-expressed polygalacturonase gene BcMF6 in Chinese cabbage (Brassica campestris L. ssp. chinensis Makino)

In our earlier work, a pollen-expressed polygalacturonase gene BcMF6 was isolated from floral bud of Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) by cDNA-amplified fragment length polymorphism (cDNA-AFLP) transcript profiling and rapid amplification of cDNA ends (RACE). To unravel the biological function of BcMF6 gene, the antisense fragment from the BcMF6 gene with A9 promoter and CaMV35S promoter was transferred into flowering Chinese cabbage (B. campestris ssp. chinensis var. parachinensis). Out of transgenic plants transformed with the antisense gene constructed from the BcMF6, transgenic line with A9 promoter have a similar appearance to that with CaMV35S promoter. Morphological investigations showed that the transgenic plants developed the smaller floral organ with thin anther and less pollen. Pollen germination test indicated that only near 50% the pollen from the transgenic line could normally germinate. Further scanning electron microspore analysis of transgenic plants confirmed that half of pollen was abnormal. Cytological comparisons of microspore development also demonstrated that process of microsporogenesis was held up, microspores maturation was disrupted and pollen grain fail to separate, finally. In a word, the present study revealed that BcMF6, as a polygalacturonase gene, has a role in pollen maturation and pollen tube growth.

Construction of an antisense CYP86MF gene plasmid vector and production of a male-sterile Chinese cabbage transformant by the pollen-tube method

Summary A plant expression vector pBIA9-AMF containing an antisense fragment of the CYP86MF gene and the tapetum-specific A9 promoter was constructed. Plasmid vectors were introduced by floral-dipping and pollen-tube transformation methods to Chinese cabbage pak-choi (Brassica campestris ssp. chinensis (L.) Makino var. communis Tsen et Lee, syn. B. rapa ssp. chinensis (L.) Makino var. communis Tsen et Lee) and flowering Chinese cabbage (B. campestris ssp. chinensis (L.) Makino var. parachinensis (Bailey) Tsen et Lee). Results showed that KanR seedlings could be obtained by the pollen-tube method through germination tests of T1 progeny seeds, but not by the floral-dipping method. One of the two KanR seedlings proved that the antisense fragment of the CYP86MF gene was integrated into the Chinese cabbage genome by PCR amplification and Southern blotting. Northern hybridization indicated that the CYP86MF gene, under the A9 promoter, was inhibited in the transformant, and self-infertility was found in the transgenic plantlets after self-pollination. Comparison of floral organs and the morphology of microspores by scanning electron microscopy of transformant and wild-type plantlets, showed no significant differences except that microspores from the transgenic plant were covered in a layer of an unknown substance. We suggest this layer might be the cause of the male-sterile phenotype induced by the antisense fragment of the CYP86MF gene.

Mapping QTLs for root morphological traits in Brassica rapa L. based on AFLP and RAPD markers

Root growth and thickening plays a key role in the final productivity and even the quality of storage roots in root crops. This study was conducted to identify and map quantitative trait loci (QTLs) affecting root morphological traits inBrassica rapa by using molecular markers. An F2 population was developed from a cross between Chinese cabbage (Brassica rapa ssp.chinensis) and turnip (B. rapa ssp.rapifera), which differed greatly in root characters. A genetic map covering 1837.1 cM, with 192 marker loci and 11 linkage groups, was constructed by using this F2 population. The F3 families derived from F2 plants were grown in the field and evaluated for taproot traits (thickness, length, and weight). QTL analysis via simple interval mapping detected 18 QTLs for the 3 root traits, including 7 QTLs for taproot thickness, 5 QTLs for taproot length, and 6 QTLs for taproot weight. Individually, the QTLs accounted for 8.4–27.4% of the phenotypic variation. The 2 major QTLs,qTRT4b for taproot thickness andqTRW4 for taproot weight, explained 27.4% and 24.8% of the total phenotypic variance, respectively. The QTLs for root traits, firstly detected inBrassica crops, may provide a basis for marker-assisted selection to improve productivity in root-crop breeding.

Effect of radiation on regeneration of Chinese narcissus and analysis of genetic variation with AFLP and RAPD markers

The aim of our study was to establish an efficient in vitro propagation protocol for Chinese narcissus (Narcissus tazetta var. chinensis) to obtain variants of this species using γ-radiation treatment and evaluate the effectiveness of this system for variant induction using amplification fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPD) analysis. Various doses (5–100xa0Gy) of gamma rays were applied to investigate the effect of radiation on adventitious bud formation from bulb-scales and the survival rate of plantlets. It was demonstrated that the regeneration of Chinese narcissus was very sensitive to gamma radiation even at low doses. The survival and multiplication rate significantly decreased with an increase of radiation dose. The optimal irradiation dose for survival and mutation induction was approximately 10xa0Gy. The genetic variations among the regenerants derived from irradiated explants were evaluated by DNA fingerprinting using RAPD and AFLP markers which detected a variation frequency of 8.33% and 15.48% respectively. The high frequency of mutants detected by molecular markers indicated that treatment of in vitro cultures with γ-rays may be an effective way to improve narcissus cultivars.

Characterization of a putative pollen-specific arabinogalactan protein gene, BcMF8, from Brassica campestris ssp. chinensis.

The BcMF8 (Brassica campestrismale fertility 8) gene, possessing the features of ‘classical’ arabinogalactan protein (AGP) was isolated from Brassica campestris L. ssp. chinensis, Makino syn. B.xa0rapa L. ssp. chinensis. This gene was highly abundant in the fertile flower buds but silenced in the sterile ones of genic male sterile A/B line (‘ZUBajh97-01A/B’) in B.xa0campestris. Expression patterns analysis suggested BcMF8 was a pollen-specific gene, whose transcript started to be expressed at the uninucleate stage and maintained throughout to the pollen at pollination stage. BcMF8 is highly homologous to the known pollen-specific AGP genes Sta 39-4 and Sta 39-3 from B.xa0napus. Isolation and multiple alignment of the homologs of BcMF8 gene in the family Cruciferae indicated that BcMF8 was highly conserved in this family, which reflect the conservation in biological function and importance of this putative AGP gene in plant development. Similarity analysis also demonstrated Sta 39-4 and Sta 39-3 may originate from different genomes.