Research Progress of Heat-tolerance of Brassica campestris ssp. Chinensis

Brassica campestris ssp. chinensis, also known as non-heading Chinese cabbage, is an important vegetable widely distributed in southern China. High temperature is the most common adversity factor in vegetable production, because Brassica campestris ssp. chinensis is a thermophilic vegetable, which can＇t well grow at high temperature. In summer and autumn, high temperature stress would prevent Brassica campestris ssp. chinensis from growing and result in poor quality of its seedlings, seriously influencing yield and quality of the vegetable in later period. In this paper, present situation and latest advances in heat resistance research of Brassica campestris ssp. chinensis in recent years were reviewed at home and abroad. The research tendency in Bassica campestris ssp. chinensis was also described, providing reference for breeding of heat-resistant Brassica campestris ssp. chinensis.

Study on Agrobacterium tumefaciens-mediated Transformation of Brassica campestris L. with Fusion Gene Ycoil-bFGF

[ Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Methed] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF was introduced into the rapeseed （ Brassica campestris L. ） by Agrobacterium tumefaciens-mediated transformation; meanwhile regeneration conditions of rapeseed were also optimized, and the regenerated resistant plantlets were detected by PCR and Southern blot. [ Result] This fusion gene had been integrated into rapeseed genome successfully, and the optimized conditions of transformation and regeneration were as follows： explants pre-culture for 2 d, co-culture for 3 d, bacteria solution OD600 for 0.3 and infection time for 5 min. [ Conclusion] The results laid a solid foundation for extraction, isolation and purification of protein in transgenic plant seeds.

Genetic Inheritance and Molecular Marker of Clubroot Resistance Genes in Brassica campestris ssp. chinensis

Objective] This study was conducted to investigate the genetic inheritance of clubroot resistance in Chinese non-heading cabbage （Brassica campestris ssp. chinensis）. [Method] The clubroot resistance gene was introduced from a Brassica campestris ssp. pekinensis cultivar to non-heading Chinese cabbage, and the inheri-tance and molecular markers of clubroot resistance gene in parental lines, F1, F2 and BC1 of non-heading Chinese cabbage were studied through pathogen inoculation at seedling stage and ISSR-PCR. [Result] Clubroot resistance in non-heading Chi-nese cabbage was control ed by a single dominant gene. ISSR molecular markers with Bulk segregant analysis （BSA） found that primer-873 was linked to resistance gene, named CR-873, and the genetic distance between the marker and the resis-tance gene was 9.72 cM. [Conclusion] The results provide references for the molecular marker assisted breeding of non-heading Chinese cabbage.

Effect of Combined Use of Boron and Manganese Fertilizers on the Nutritional Quality and Physiological Indices of Brassica campestris L.ssp.chinensis L.var.utilis

[Objective] The purpose of this study is to determine the effects of com-bined use of boron and manganese fertilizers on the nutritional quality and physio-logical indices of Brassica campestris. [Method] In the nutrient solutions for growing B. campestris by hydroponics, boric acid and manganese sulfate were added at 0.5, 2.5, and 7.5 mg/L respectively. Another treatment without boron and manganese was prepared as the control. Quality and physiological indices of B. campestris in the 10 treatments were measured. [Result] Boron and manganese shared obvious in-teraction in improving the quality and physiological indices of B. campestris. To cul-tivate B. campestris with high quality and strong resistance, the optimum concentra-tions of boron and manganese in the nutrient solution should be 2.5 mg/L boric acid and 2.5-7.5 mg/L manganese sulfate. [Conclusion] The findings wil provide refer-ence for studying effects of trace elements on nutrient composition of vegetables.

Role of nitrification inhibitor DMPP(3,4-dimethylpyrazole phosphate) in NO^-_3-N accumulation in greengrocery( Brassica campestris L. ssp. chinensis ) and vegetable soil

The influence of nitrification inhibitor(NI) 3,4 dimethylpyrazole phosphate(DMPP) on nitrate accumulation in greengrocery( Brassica campestris L. ssp. chinensis ) and vegetable soil at surface layer were investigated in field experiments in 2002 and 2003 Results showed that NI DMPP took no significant effect on yields of edible parts of greengrocery, but it could significantly decrease NO - 3 N concentration in greengrocery and in vegetable soil at surface layer. In addition, NI DMPP could reduce the NO - 3 N concentration during the prophase stage of storage.

Analysis on Cloning and Expression of WRKY Gene Segment in Brassica campestris L.

[Objective]The aim was to explore the relative expression trends of WRKY transcription factor gene under ABA treatment in Brassica campestris.[Method]Actin gene and WRKY gene were cloned by using the homology cloning method.The sequences of nucleic acid and amino acid were analyzed using BLAST and DNAMAN software.Relative expression trends of WRKY gene were detected by applying real-time relative quantification PCR（RT-qPCR）under ABA（100 μmol/L）treatment.[Result]A 680 bp WRKY gene segment and a 933 bp β-actin gene segment were acquired in Brassica campestris.The result of RT-qPCR analysis revealed that BcWRKY expression could be induced by ABA and that the relative expression of WRKY gene reached the peak at 1 h with ABA treatment.[Conclusion]Actin and WRKY gene in B.campestris were cloned successfully,which was proved to play an important role in ABA signal pathway.

Effect of Nitrogen and Sulfur Supply on Glucosinolates in Brassica campestris ssp.chinensis

Glucosinolates （GSs） are a group of plant secondary metabolites containing abundant nitrogen （N） and sulfur （S） mainly in Brassica and have the beneficial effects on human health including anti-carcinogenic, cholesterol-reducing and other pharmacological effects. The objective of this study was to investigate the effect of different concentrations of N （5, 10, and 20 mmol L-a, denoted by N5, N10 and N20） and S （0,5, 1, and 2 mmol L^-1, denoted by S0.5, S1 and S2） on the yield and GSs in pakchoi （Brassica campestris L. ssp. chinensis var. communis） in hydroponics. Results showed that N10 and N20 significantly enhanced the yield compared with N5, however, N20 had a negative effect relative to N10. Only with N10 and N20 low S supply （S0.5） reduced the yield. The concentrations of aliphatic GSs, aromatic GS and total GSs were enhanced by N5 and indolyl GSs were enhanced by N20. S2 enhanced the concentration of individual GS and total GSs. The concentrations of indolyl GSs were maximized in N20S2 treatment, whereas the highest concentrations of aliphatic GSs, aromatic GS and total GSs were found in N5S2 treatment. Effects of N and S on aliphatic GSs were higher than on indolyl GSs. The results suggest that the accumulation of aliphatic GSs and aromatic GS could be enhanced by low N and high S and restricted by high N while that of indolyl GSs could be enhanced by high N and high S.

A Genetic Linkage Map of Brassica campestris L.ssp. pekinensis (syn. B. rapa L. ssp. pekinensis)

A molecular genetic map of Chinese cabbage was constructed with a 102 recombinant inbred (RI) population from a cross of two cultivated Chinese cabbage lines 177 and 276, using AFLP and RAPD markers. 352 markers including 265 AFLP markers and 87 RAPD markers were integrated into 17 linkage groups. It covered a total of 2 665. 7 cM with an average interval of 7. 6 cM. AFLP marker is efficient for map construction while it easily forms clusters to cause big gaps in map. A total of 13.92 % abnormal segregation markers distributed in the map. The molecular genetic map is fundamental for gene localization, comparative genomics, and QTL mapping of important agronomic traits.

Genetic linkage map of Brassica campestris L.using AFLP and RAPD markers

A genetic linkage map comprised of 131 loci was constructed with an F 2 population derived from an inter subspecific cross between Brassica campestris L. ssp. chinensis cv. 'aijiaohuang' and ssp. rapifera cv. 'qisihai'. The genetic map included 93 RAPD loci, 36 AFLP loci and 2 morphological loci organized into 10 main linkage groups (LGs) and 2 small groups, covering 1810.9cM with average distance between adjacent markers being approximately 13.8cM. The map is suitable for identification of molecular markers linked to important agronomic traits, QTL analysis, and even for marker assisted selection in breeding programs of Chinese cabbage and turnip.

Differential Expression Analysis of Genie Male Sterility A/B Lines in Chinese Cabbage-Pak-Choi (Brassica campestris ssp. chinensis Makino)

To determine differential expression of genie male sterility A/B lines in Chinese cabbage-pak-choi (Brassica campestris ssp. chinensis Makino var. communis Tsen et Lee), we used the RNA fingerprinting technique, cDNA-AFLP analysis, in different developmental stages and different tissues. While no obvious differential expressions were observed in rosette leaves, florescence leaves, and scapes, some differential expressions were found in alabstrums of A/B lines and among leaves, scapes and alabstrums. We analyzed the al-abstrums collected in different developmental stages with 10 primer combinations. We got a unique band between middle size alabstrums and large alabstrums in B line in one of the ten pair primers, and in another one pair, one band reflecting a higher gene-expression level in A line than that in B line was obtained. No unique bands were found with the other primer combinations. The bands reflecting different gene-expression level were confirmed by Northern hybridization. The results indicated that cDNA-AFLP was a suitable tool for studying differential expression of genie male sterility in plants. SDS-polyacrylamide gel electrophoresis patterns of soluble proteins further verified the difference in A/B lines.

Nitrogen Dioxide-Induced Responses in Brassica campestris Seedlings: The Role of Hydrogen Peroxide in the Modulation of Antioxidative Level and Induced Resistance

This article investigates the responses of Brassica campestris seedlings to an acute level of nitrogen dioxide （NO2） exposure in a plant growth chamber, and examines whether pretreating plants with hydrogen peroxide （H2O2） will alleviate NO2-caused injury. Twenty-eight-day-old B. campestris plants sprayed with 10 mmol L^-1 H2O2 aqueous solution （corresponding to approximate 1.0 mg H2O2 per single plant） were exposed to different concentrations of NO2 （0.25, 0.5, 1.0, and 2.0 μL L^-1, respectively） for 24 h under controlled environment. To measure the plant biomass, the plants were fumigated with the same NO2 concentrations as mentioned above for 7 h per day （8.00-15.00） for 7 days. As a control, charcoal filtered air alone was applied. Data were collected on plant biomass, total chlorophyll, photosynthetic rate, stomatal conductance, nitrate and nitrate reductase （NR）, antioxidative enzymes, ascorbate （ASA）, and malondialdehyde （MDA）, immediately after exposure. The results showed that exposure to a moderate dose of NO2 （e.g., 0.25 μL L^-1） had a favorable effect on plants, and the dry weight of the above-ground part increased, whereas the exposure to high NO2 concentrations （e.g., 0.5 μL L^-1 or higher） caused a reduction in the plant biomass and the total chlorophyll, when compared with the control. In addition, at 0.5 μL L^-1 or higher NO2 concentrations, prominent increases in the MDA level and superoxide dismutase （SOD） and NR activities were observed. Exposure to 1 μL L^-1 and higher NO2 resulted in necroses appearing on older leaves, and an increase in catalase （CAT） activity, decrease in ASA content, increased accumulation of NO3^-, and reduction in photosynthesis, when compared with the controls. No changes were detected in stomatal conductance under NO2 fumigation. The pretreatment with 10 mmol L^-1 H2O2 alleviated significantly NO2- caused biomass decrease and photosynthetic inhibition when compared with H2O2-untreated plants. Under NO2 fumigation, further induction in SOD and CAT activities occurred in H2O2 treated plants when compared with H2O2- untreated plants. The effect of NO2 on the ASA and MDA contents was also absent in H2O2-treated plants. However, the H2O2 treatment did not alter the nitrate content and NR activity in plants under NO2 fumigation. The H2O2 treatment caused a lower rate of stomatal conductance. Taken together, these data suggest that fumigation with an acute level of NO2 causes oxidative damage to B. campestris seedlings. The H2O2 pretreatment markedly protects plants against NO2 stress and this may be associated with inducible antioxidative level. NO2 fumigation contributes, at least in part, to the enhanced levels of nitrate in B. campestris leaves.

Effects of Different Simple Cultivation Facilities on Yield and Quality of Over-summer Brassica campestris L. in South China

The effects of different simple cultivation facilities on yield and quality of Brassica campestris L. were compared to explore the optimal facility type for pro- duction of B. campestris, thereby providing a technical reference for efficient cultiva- tion of B. campestris during hot season. A total of four treatments were designed, open field cultivation （CK）, ordinary anti-insect net shed cultivation, ordinary plastic film greenhouse cultivation and Japanese translucent anti-insect net shed cultivation. The results showed that compared with those in open field cultivation, the plant height, leaf area, yield, Vc content, soluble sugar content and soluble protein con- tent of B. campestris in Japanese translucent anti-insect net shed cultivation were increased by 22%, 63%, 49%, 14%, 13% and 10%, respectively, and Japanese translucent anti-insect net shed was the optimal facility type for cultivation of B. campestris in hot season.

Effect of the Antisense BcMF12 Driven by the BcA9 Promoter on Gene Silencing in Brassica campestris L.ssp.chinensis

The study analyzed the silencing of BcMF12 gene regulated by BcA9 promoter in the transgenic pakchoi and confirmed the effect of antisense BcMF12 gene on the pollen development. A conserved BcMF12 gene fragment was amplified from the cDNA of flower buds in pakchoi （Brassica campestris L. ssp. chinensis, syn. B. rapa L. ssp. chinensis） and was fused to the anther specific BcA9 promoter. The plant antisense expression vector was constructed and then introduced into pakchoi via Agrobacterium-mediated transformation. The transgenic plants were screened by antibiotics and molecular analysis. PCR and Southern blot revealed that the antisense BcMF12-GUS fusion gene regulated by BcA9 promoter was integrated into transgenic plants. Northern blot suggested that the expression of BcMF12 gene was down-regulated significantly. The pollen germination rate of transgenic plants with antisense BcMF12 gene decreased as compared with that of the control plants. The expression of the gene BcMF12 related to the pollen development was inhibited by the antisense BcMF12 driven by BcA9 promoter, which consequently affected the pollen development in pakchoi.

Interspecific Hybridization of Brassica campestris × B.oleracea Through Ovary Culture

Using three varieties of Brassica campestris, Hauarad (708), Maoshan-3 (714) and Youbai (715),as the maternal plants and one variety of Brassica oleracea Jingfeng-1 (6012) as paternalplants, crosses were made to produce interspecific hybrids through ovary culture techniques.The ovaries from the cross between B. campestrisB.oleracea (7086012 and 7146012) werecultured and ovary culture was more effective in terms of obtained seeds when ovaries werecultured in vitro at 9 d after pollination (DAP). While for the cross of 7156012, it wasbetter when ovaries in vitro cultured at 12 DAP. Among three cross combinations, the cross of7146012 showed the best response and 43 seeds per ovary were obtained. Among the mediastudied, the ovaries from the cross of 7086012 cultured on MS media supplemented with 3.0 mgL-1 BA0.1 mg L-1 NAA showed better response, and its rate of seeds per ovary reached 44.0%.While the ovaries from the other two crosses (7146012 and 7156012) showed the best responsewhen cultured on B5 media supplemented with 3.0 mg L-1 BA + 0.2 mg L-1 NAA, and the rates of seedsper ovary reached 72.0 and 60.0%, respectively. All seeds obtained from the three crosscombinations were cultured on the MS media supplemented with 1.0 mg L-1 BA + 0.05 mg L-1 NAA,and the seeds from the cross of 7156012 showed the best germination response and thepercentage of germinations reached 66.7%. The regenerated plantlets were obtained from theseseedlings after cultured on the MS media supplemented with 0.05 mg L-1 NAA. Cytological studyshowed that these regenerated plants were all true hybrids of B.campestrisB.oleracea.

Preliminary Study on Tissue Culture Technique of Brassica campestris L. ssp. chinensis (L.) Makino var. utilis

In order to establish a rapid propagation system for Brassica campestris L. ssp. chinensis var. utilis, the anther and cotyledon-cotyledonary petioles were used as explants to conduct tissue culture research. The results showed that not bloomed flower buds with higher styles than petals were appropriate for anther cul- ture. Moreover, most microspores were at the mid-late uninucleate stage at that time. The pollen germination rates of Brassica campestris L. ssp. chinensis var. u- tills were low and the germination rates in autumn and winter were higher than that in summer. The induction medium of anther callus of Brassica campestris L. ssp. chinensis var. utilis was MS＋1.0 mg/L KT＋1.0 mg/L 2, 4-D＋3% sugar＋6 g/L agar＋ 8% coconut milk and the induction media of adventitious bud were MS＋2.0 mg/L 6-BA＋ 0.5 mg/L NAA＋I.0 g/L activated carbon＋2% sugar＋6 g/L agar or MS＋2.0 mg/L ZT＋ 0.5 mg/L IAA＋0.5 g/L AgNO3＋1.0 g/L activated carbon＋2% sugar＋6 g/L agar. The induction percentage of adventitious bud by anther culture was 36.7%. Browning phenomenon appeared during the culture of adventitious bud and regeneration plant could not be formed. The plant regeneration rate reached 80% when cotyledon- cotyledonary petioles were used as explant.

Effects of Different Cadmium Levels on Active Oxygen Metabolism and H_2O_2-Scavenging System in Brassica campestris L.ssp.chinensis

The effects of different Cd (Cadmium) levels on generation of active oxygen speceies(AOS) and H2O2-scavenging system in the leaves of Brassica campestris L. ssp. chinensiswere studied. The results showed that generation rate, and H2O2 content were enhancedand malondialdehyde (MDA) content increased with the increase of Cd concentrations inthe growth medium. The activities of ascorbate peroxidase (APX), dehydroascorbatereductase (DR) and glutathione reductase (GR) were promoted by the addition of Cd.Exposed to Cd also increased the contents of ascorbate (AsA) and glutathione (GSH) in theleaves.

Identifying Cytoplasmic Male Sterile Types of Chinese Cabbage(Brassica campestris L.)by Molecular Markers

Different sterile cytoplasm types of nine cabbage cytoplasmic male sterile materials were identified by molecular marker in the study, in order to better use molecular marker to conduct the assisted breeding in the future. Genomic DNA was isolated from Chinese cabbage by CTAB method. The design of two pairs of specific primers was performed on conserved flanking region of orf138 gene in the GenBank. PCR was performed with genomic DNA of the nine Chinese cabbage materials. The bands were sequenced. The homologous comparison was conducted in NCBI, and finally, the type of sterile cytoplasm was determined. The results showed that the bands were amplified only in four Chinese cabbage male sterile materials with two pairs of specific primers PUPIl and PIII/PIV, while the other five materials did not obtain the relative bands. The result was consistent with the field sterility identification. And then four molecular markers of Chinese cabbage Ogura cytoplasmic male sterility （CMS） were obtained. After conducting a homologous comparative analysis with BLAST in GenBank, it was found that the homologous degree was 100% in specific segments of tbe tbree sterility materials （L1-CI, L3-CI and L3- F1 ） and Ogu orf138 gene （GenBank accession No. ： HQ149728） of the reported broccoli Ogu CMS. The homologous degree of L1-F1 was 99% with a variation point. The type of cytoplasmic male sterility of the other five materials needed further research. Four materials of the nine were identified as the radish cytoplasmic male sterility materials and four molecular markers were obtained.

Cloning and Characterization of the Microspore Development-Related Gene BcMF2 in Chinese Cabbage Pak-Choi （Brassica campestris L. ssp. chinensis Makino）

: For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages, as well as in rosette leaves, florescence leaves, and scapes was analyzed using cDNA amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile A and fertile B line of Chinese cabbage pak-choi. Following amplification of 125 pairs of primer combinations, 11 differential fragments were obtained, of which eight were from the B line and the other three were from the A line. Of 11 differential fragments, four were verified by Northern hybridization that were expressed preferentially in fertile flower buds. Results of GenBank BLAST showed that one fragment was with unknown function, whereas the other fragments have strong nucleotide sequence similarities with the polygalacturonase (PG) gene, the pectinesterase (PE) gene, and the polygalacturonase inhibitory protein (PGIP4) gene. Only full-length cDNA from the differential fragment BcMF-A18T16-1 was amplified by rapid amplification of cDNA ends (RACE) and Northern analysis showed that this fragment was expressed only in medium and large-sized flower buds of the B line. The full-length cDNA, designated as BcMF2 (Brassica campestris Male Fertile 2), was 1 485 bp long and was composed of a 1 263-bp open reading frame, which had 83% nucleotide similarity to a PG gene from Arabidopsis encoding polygalacturonase. Analysis of the basic structure of the protein revealed that it had one polygalacturonase active site (RVTCGPGHGLSVGS) at 256th site of amino acids and was classified as being a member of family 28 of the glycosyl hydrolases. The role of the BcMF2 gene on microspore development is discussed in the present paper.

PECTATE LYASE-LIKE10 is associated with pollen wall development in Brassica campestris

PECTATE LYASE‐LIKE10（PLL10） was previously identified as one of the differentially expressed genes both in microspores during the late pollen developmental stages and in pistils during the fertilization process in Chinese cabbage（Brassica campestris ssp. chinensis）. Here, antisense‐RNA was used to study the functions of BcPLL10 in Chinese cabbage. Abnormal pollen was identified in the transgenic lines（bcpll10‐4, ‐5, and ‐6）. In fertilization experiments, fewer seeds were harvested when the antisense‐RNA lines were used as pollen donor. In vivo and in vitro pollen germination assays less germinated pollen tubes were observed in bcpll10 lines. Scanning electron microscopy observation verified that the tryphine materials were over accumulated around the pollen surface and sticked them together in bcpll10.Moreover, transmission electron microscopy observation revealed that the internal endintine was overdeveloped and predominantly occupied the intine, and disturbed thenormal proportional distribution of the two layers in the non‐germinal furrow region; and no obvious demarcation existed between them in the germinal furrow region in the bcpll10 pollen. Collectively, this study presented a novel PLL gene that played an important role during the pollen wall development in B. campestris, which may also possess potential importance for male sterility usage in agriculture.

Differential expression of salt tolerance related genes in Brassica campestris L. ssp. chinensis (L.) Makino var. communis Tsen et Lee

We examined salt tolerance responsive genes in Pak-choi under salt stress and analyze their potential function. The LRNA differential display was used to screen the transcript derived fragments （TDFs） related to salinity tolerance in tolerant and Loderately tolerant Pak-choi germplasm. Seventy-eight primer combinations generated 101 differential eDNA fragments, which ere divided into 10 expression types. Seven cDNA sequences （GenBank accession Nos. DQ006915-DQ006921） obtained and ,~quenced were highly homologous to some known expression genes or the genes related to the signaling pathways in plants under ifferent abiotic stress.