The conditions for protoplast isolation and regeneration from Trichoderma viride were studied. Protoplasts were optimally isolated when mycelia of T. viride that had been cultured for 24 h were digested with 4 mg/ml Glucanex in phosphate buffer (pH 6.98) for 4 h at 30°C, resulting in a protoplast yield of 4.7×107 cfu/mg. The maximum regeneration ratio (14.5%) was obtained in mycelia culture medium containing 0.3 mol/l KCl and 0.3 mol/l inositol. In addition, a plasmid pCSSNCC1 carrying a hygromycin resistance gene and an elicitor-producing gene was transformed into T. viride protoplasts, with an efficiency of 1–2 transformants/μg DNA. The hygromycin-resistant transformants were determined by PCR and the elicitor protein was detected by ELISA. The results indicate that the elicitor protein was expressed stably in T. viride.

Genetic diversity and relationships among 21 accessions of Secale L., including three species and 10 subspecies, were evaluated using RAMP markers. Forty-one out of 80 (50.5%) RAMP primers, which produced clear and polymorphic bands, were selected for PCR amplification of genomic DNA. A total of 446 bands were amplified from the 41 primers, and 428 of these bands (about 96%) were polymorphic. Three to 19 polymorphic bands could be amplified from each primer, with an average of 10.4 bands. The RAMP-based genetic similarity (GS) values among the 21 Secale accessions ranged from 0.266 to 0.658, with a mean of 0.449. A high level of genetic variation was found between or within the wild populations and the cultivars. Based on the GS matrix, a dendrogram was constructed using the unweighted pair group method with arithmetic average (UPGMA). All 21 accessions could be distinguished by RAMP markers. Clustering results showed that the genetic diversity of Secale based on RAMP markers was correlated with geographical distribution. Six rye cultivars, originating from Poland, Portugal, Mexico, Hungary, Armenia and Ukraine, were clustered into one group. The six countries are all located in the transitional region of broad-leaf forests between maritime and continental temperate zones, with narrow latitude span. In comparison, the other five cultivars from countries scattered over a region with large latitude span were distributed within different groups or subgroups. Genetic relationships based on RAMP markers had great deviation from the original taxonomy. Some subspecies of the same species were distributed within different groups, while some accessions of different species were closely clustered into one subgroup. These results suggest that RAMP markers could be an effective technique for detecting genetic diversity among Secale and give some useful information about its phylogenic relationships.

Cili (Rosa roxburghii Tratt), characterized by containing the highest vitamin C content among fruits and showing attractive senescence-retarding and cancer-preventing effects, has gained widespread interest. RAPD markers were applied to identify the seven genotypes of cili and to evaluate the genetic relationships within cili, as well as among its relatives. Sixteen arbitrary primers screened from 154 were adopted to analyse polymorphism in RAPD profiles for the 15 samples. A total of 137 RAPD bands ranging in size from 480 bp to 3.3 kb were obtained, among which 95 were polymorphic, covering 69.3% of the total bands obtained; and an average of 8.6 bands/primer was scored. The genotypes of cili and seedless cili could be identified efficiently by 14 genotype-specific bands, which were obtained from the polymorphic primers OPB-11, OPAF-16 and OPW-02. Additionally, using the unweighted pair group method with arithmetic mean, a dendrogram showing genetic relationships among the 15 samples was constructed based on cluster analysis of genetic distance. The possible origin of seedless cili and multiple-corolla cili is also discussed.

The plant expression vector pCAMBIA1300-AtNHX1-als was constructed by inserting the herbicide resistance gene als of Arabidopsis thaliana into the plasmid pCAMBIA1300-AtNHX1, which contains the AtNHX1 gene encoding the Na+/H+ antiport from the vacuolar membrane of A. thaliana. Transgenic tobacco plants were obtained via Agrobacterium-mediated transformation. PCR and Southern blot assay indicated that genes als and AtNHX1 had been integrated into the genome of the transgenic plants. The herbicide resistance and salt tolerance of transgenic plants increased by about 1000-fold and by 1.5% NaCl concentration, respectively, compared with controls. Herbicide resistance of the T1 progeny was evaluated by spraying transgenic plants with different concentrations of Luhuanglong at the four-leaf stage. Controls gradually died under 100 mg/l Luhuanglong whereas 73% of the T1 plants still survived at 500 mg/l Luhuanglong. Thus the plant expression vector pCAMBIA1300-AtNHX1-als could be used to improve the herbicide resistance and salt tolerance of crops.

This paper has been retracted by Dai Mei-Xue

Regeneration of sweet potato (Ipomoea batatas cv. Lizixiang) transgenic plants with oryzacystatin-I (OCI) gene was achieved using an Agrobacterium tumefaciens-mediated method. A. tumefaciens strain LBA4404, harbouring a binary vector pBinh with neomycin phosphotransferase II and OCI genes, was used. After 3 days of subculture, Lizixiang embryogenic suspension cultures were co-cultivated with LBA4404 (OD600nm=0.5) for 4 days. Next, the infected suspension cultures were first cultured for 5 days in MS medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 300 mg/l carbencillin (Carb) but without kanamycin (Kan) and then transferred to MS medium supplemented with 2 mg/l 2,4-D, 50 mg/l Kan and 300 mg/l Carb for the selection culture. Four weeks after selection, 200 Kan-resistant cell aggregates (∼1 mm in size) from the embryogenic suspension cultures were transferred to MS solid medium supplemented with 2 mg/l 2,4-D, 50 mg/l Kan and 300 mg/l Carb, and eight embryogenic calluses were obtained. After transferring to MS medium supplemented with 1 mg/l ABA, 50 mg/l Kan and 100 mg/l Carb, these embryogenic calluses formed 13 plantlets via somatic embryogenesis. PCR and PCR–Southern blot analysis indicated that seven of the 13 plantlets were transgenic.

The indica rice Xieqingzao eB1 (XQZ eB1), a mutant with an elongated uppermost internode, was produced by mutagenesis from elite maintainer line Xieqingzao B (XQZ B) using γ-radiation. Compared with the wild-type XQZ B, plant height and the length of the rice panicle neck, second internode and third internode from the panicle were elongated significantly in XQZ eB1. Genetic analysis indicated that the trait for uppermost internode elongation of XQZ eB1 is controlled by a single recessive gene eui1, which is allelic to that of IR50eui (derived from 76:4512). Further studies were carried out using the bulk segregated analysis approach. An F3 population, derived from the cross XQZ eB1×Aijiaonante, was used to map the mutant gene eui1. Two SSR makers, RM164 and AC9, were identified to link to eui1, their genetic distances to eui1 being 18.4 and 7.9 cM, respectively. The eui1 gene was also mapped to the long arm of chromosome 5 in this research.

Differences in immunostimulation activity of CpG oligodeoxynucleotides (ODNs) between pigs and mice were studied. In accordance with current research data, three kinds of CpG ODN – 2006, D19 and 1826, which were identified as optimally active in humans, pigs and mice, respectively – and control ODN D48, a non CpG-containing ODN, were selected and synthesized. Results indicated that CpG ODN D19 strongly stimulated the proliferation of porcine peripheral blood mononuclear cells (PBMCs), and the amount of γ-interferon in the supernatant of activated cells increased, exceeding that of all other ODNs tested, while mouse splenocytes did not respond. CpG ODN 1826 strongly stimulated the proliferation of mouse splenocytes, but porcine PBMCs remained unresponsive. CpG ODN 2006 effectively stimulated proliferation of both porcine PBMCs and mouse splenocytes. Therefore some specific CpG ODNs, D19 and 1826, selectively stimulated animal species, while CpG ODN 2006 possessed a common stimulation effect. In addition, CpG ODN D19 predominantly induced the response of type 1 T-helper cells in vivo.

The expression pattern of genes encoding putative transcription factors in ethylene signal transduction was investigated, after treatment of tomato (Lycopersicon esculentum cv. UC-82) seedlings with solutions of differing NaCl concentration for 12, 24 and 48 h. The role of ethylene signalling in tomato seedlings adapting to salt stress is discussed. Le-EIL1 gene expression in tomato seedlings was promoted after 12 h of treatment under different NaCl concentrations. The amount of Le-EIL2 transcript increased after 12 and 24 h in 0.08 mol/l NaCl, and the same effect was observed for Le-EIL3 gene expression in tomato seedlings grown in 0.04 and 0.08 mol/l NaCl. After 12 h, Le-ERF1 gene expression was significantly enhanced by 0.04 and 0.08 mol/l NaCl treatments. Le-ERF2 gene transcriptional expression increased with the application of 0.04, 0.08 and 0.16 mol/l NaCl to seedlings. However, Pti4 gene expression did not change remarkably. These results suggest that different temporal gene expression patterns exist in Le-EIL and Le-ERF family members after NaCl stress induction.

A cDNA library was constructed from the heading leaf in the early phase of the heading stage of Chinese cabbage (Brassica rapa L. ssp. pekinensis). By sequencing the randomly selected clones, 1363 sequences longer than 200 bp were found, with better trace data. After removing the poly(A) and contamination sequences, 1162 ESTs longer than 150 bp were obtained, of which 1102 shared significant similarity with known sequences in protein and nucleotide databases of the National Center for Biotechnology Information (NCBI) as revealed by searches using the BLASTX and BLASTN engines. Functional assignment of the ESTs was based on the method used in the Arabidopsis thaliana genome-sequencing project. About 77% of the putative protein sequences with known biological functions best matched with those of A. thaliana deposited in the non-redundant database of NCBI. These data suggest that Chinese cabbage is closely related to A. thaliana. This result is different from that reported in other Brassica species. At nucleotide level, however, 51% of the ESTs were homologous to those deposited for A. thaliana when all ESTs were searched against the est-others database. In addition, 60 ESTs had no homology with any of the plant gene sequences deposited in GenBank. These ESTs are very important for understanding the unique developmental process of Chinesecabbage and elaborating its genetic mapping. Among the genes with assigned functions, the most abundant representatives were those involved in protein synthesis and energy metabolism. With the 1162 ESTs, 895 non-redundant contigs were generated after being aligned using the Seqman II module of DNAStar software at the threshold of more than 80% homology over a minimum of 40 base pairs. Of these, 723 were singletons containing only one EST sequence, indicating that many kinds of such genes are expressed in the heading leaf of Chinese cabbage. An expression profile of Chinese cabbage heading leaf with the 1162 ESTs was therefore acquired in this work. This could be very useful for uncovering the mechanism of the heading process, which is the most obvious characteristic of Chinese cabbage and perhaps other related species, such as Brassica oleracea. This work could accelerate the finding and characterization of genes specifically expressed in the heading stage of Chinese cabbage.

The cryptogein (Crypt) gene was obtained by PCR amplification of genomic DNA from Phytophthora cryptogea and confirmed by DNA sequencing. A promoter of the rice phenylalanine ammonia-lyase (PAL) gene was used to regulate the expression of Crypt, because this promoter has a low level of constitutive expression, is strongly induced by pathogen infection and is expressed in epidermal tissues. These promoter characteristics may be suitable for potential inhibition of pathogen attack on epidermal tissues. For functional interaction of Crypt with its outer plasma membrane binding sites, Crypt was led by a signal sequence of the extracellular pathogenesis-related protein (PR1b) of tobacco (Nicotiana tabacum). The fused gene was constructed into a binary vector and the final plasmid (CHF-PAL::Crypt) was transformed into tobacco using the Agrobacterium-mediated transformation method. Twenty-two lines of transformants were obtained from selection medium containing 100 mg/l of kanamycin. Results from PCR amplification and Southern blot analysis demonstrated that Crypt was integrated into the tobacco genome. In the assay of pathogen challenge, nearly two-thirds (68.2%) of the transgenic plants showed significantly enhanced resistance against black shank fungal (Phytophthora parasitica var. nicotianae), brown spot fungal (Alternaria alternata) and wild fire bacterial (Pseudomonas syringae pv. tabaci) diseases. This observation indicates that low-level constitutive expression of Crypt gene in tobacco could have potential use in generating broad-spectrum disease-resistant plants.

Membranous cDNA microarrays containing 2200 unique rice transcripts were designed for screening the characteristics of spatially expressed genes in post-germination rice seedlings. By comparing the profiles obtained, 31 genes were identified as expressed specifically in the plumule, 36 in the mesocotyl and 73 in the radicle. Several genes, such as polyubiquitin, UDP-glucose pyrophosphorylase, sucrose synthase and phosphoglycerate kinase, which encode components of the carbohydrate or protein metabolic reaction cascades, were expressed specifically in the mesocotyl, indicating that degradation reactions of the endospermous reserve starch and proteins occur mainly in the mesocotyl during the post-germination stage. A number of genes involved in defence mechanisms or in the processes of replication, transcription and translation were identified as expressed specifically in the plumule or radicle. Among plumule specifically expressed genes, translation initiation factor 5a, 40s ribosomal protein s28 and ribosomal protein 136 are considered to have a critical role in protein biosynthesis; while allergenic protein, β-D-glucan exohydrolase and actin 11 are genes with defending functions. Among the catalogue of radicle specifically expressed genes, EF-1a, Tat binding protein, replication protein A2, histone h3.2, ribosomal protein s29a and 40s ribosomal protein s19 are genes that function in the process of replication, transcription or translation; whereas glycine-rich protein, wound-induced basic protein, Bowman-Birk proteinase inhibitor and lipid transfer protein-2 are genes involved in defence responses. Results of this experiment have provided insight into post-germination molecular physiology at the genomic level of gene expression.