Tests were conducted to highlight the effects of donor cell treatment (bovine G0- and non-G0 phase somatic cells refrigerated for 1–5 or 12–14 days at 4°C), fusion solution (with or without cytochalasin B) and age of the rabbit (young, 3–5-months old; or multiparous, 10–12 months old) as oocyte donor on the fusion and mortality rates of bovine–rabbit interspecies reconstructed eggs. Results showed that there were no significant differences (P>0.05) in fusion and mortality rates of eggs reconstructed with G0 cells or non-G0 cells as nuclear donor somatic cells. The same was also true when nuclear donor somatic cells (G0 cells or non-G0 cells) were treated at low temperature (4°C) for 1–5 or 12–14 days. The fusion rate of reconstructed eggs did not change significantly (P>0.05) when adding cytochalasin B into the fusion solution, but the mortality rate increased significantly (P<0.05). No significant effect (P>0.05) was found in fusion rate of the eggs reconstructed with young rabbits' and multiparous rabbits' oocytes as recipient, but the mortality rate decreased significantly (P<0.01) for young rabbit oocytes compared with multiparous rabbit ones.

Inter-simple sequence repeat (ISSR) analysis was carried out in Thatcher, 20 near-isogenic lines (NILs) containing respectively different genes conferring resistance against wheat leaf rust (Puccinia recondite f.sp. tritici), three materials carrying Lr37 and three materials without Lr37. All of the 100 ISSR primers showed clear amplification products. Two of them amplified the polymorphic DNA bands in the NILs, Thatcher and Lr37/6*Thatcher. The polymorphic bands were named UBC812-1200 and UBC848-700, respectively. The three materials with and without Lr37 were detected in tests using the two primers UBC812 and UBC848. Results also showed that only band UBC812-1200 was amplified in all resistant and absent in all susceptible materials. This suggests that UBC812-1200 marker is linked to the resistance gene Lr37. The genetic linkage of the polymorphic marker with Lr37 was tested using a segregating F2 population (128 plants) derived from a cross between the leaf rust-resistant Lr37/6*Thatcher and the susceptible cultivar Thatcher. The ISSR marker UBC812-1200 showed co-segregation to the Lr37 resistance gene. It could be used in molecular marker-assisted selection in a wheat breeding programme for leaf rust resistance.

Wheatgrass, Thinopyrum Löve (Elytrigia Nevski), is one of the most important alien resources for wheat improvement. To determine the transferability of wheat microsatellite primers to Thinopyrum spp., 40 wheat microsatellite primer pairs were selected to perform PCR analysis on 17 accessions of Thinopyrum spp. and two common wheat (Triticum aestivum) cultivars. Among the 40 primer pairs used, 25 pairs could produce PCR products on all or most of the alien species. Moreover, all of these 25 primer pairs, except Xgwm325, showed polymorphism in the Thinopyrum accessions. In total, 108 alleles were detected by the 25 primer pairs. The number of alleles per primer pair varied from 1 to 8, with an average of 4.3 alleles per primer. Although primer Xgwm325 did not detect polymorphic alleles in Thinopyrum spp., it could be used as a specific SSR marker for them. It amplified four specific DNA fragments in Thinopyrum spp. of length approximately 1400, 440, 120 and 100 bp. To verify the validity of these molecular markers, a further PCR programme with primer Xgwm325 was conducted on a full set of seven wheat–Thinopyrum elongatum disomic addition lines and 15 disomic substitution lines. This showed that only the 100-bp fragment appeared in all materials possessing the alien chromosome, indicating that it was a molecular marker for the entire genome of Th. elongatum, although primer Xgwm325 was specific for chromosome 6D in wheat.

The expression patterns of Cry1Ac protein and the resistance to striped stem borer Chilo suppressalis (Walker) of cry1Ac/CpTI transgenic rice (Oryza sativa L.) were investigated by ELISA and laboratory bioassay. ELISA results showed that Cry1Ac protein could be detected in all organs during the development process of cry1Ac/CpTI transgenic rice. The content of Cry1Ac protein varied markedly in different tissues at different stages: it was higher in the earlier stage than in the later stage. The highest level of Cry1Ac protein in terminal leaves of the main stem was found at the jointing stage. Thereafter, it decreased at the booting stage, and a gradual reduction to the lowest level was recorded from the flowering to the filling stage, before a slight increase was shown at the maturing stage. In addition, higher concentrations of this protein were detected in the main stem at the seedling, jointing and flowering stages than at other stages. Relatively lower levels of Cry1Ac protein were found in seeds. The results from laboratory bioassay showed high resistance of terminal leaves of the main stem in all developmental stages to C. suppressalis larvae. Corrected mortality values of C. suppressalis at 4 days after feeding on the detached leaves of cry1Ac/CpTI transgenic rice reached 90% before the filling stage and decreased to nearly 60% after the filling stage. Interestingly, results of whole plant bioassay coincided with those obtained in the detached leaf bioassay. The tendency of larval morality in the detached leaf bioassay and whole plant bioassay was consistent with that of Cry1Ac protein content in the different growing periods and organs.

The influence of calcium and ABA on ethylene biosynthesis was investigated at different stages and patterns of tomato (Lycopersicon esculentum cv. Lichun) fruit to analyse the relationship between calcium and ABA in relation to the ethylene biosynthesis system. The ethylene production of discs excised from mature green tomato fruits increased rapidly with 100 μmol/l ABA or 100 mmol/l calcium treatment. The increase with the two treatments combined was more than that after application of a single chemical, which suggests that these two chemicals could play synergistic roles in the ethylene synthesis of mature green tomato fruits. It was also found that application of the calcium chelator EGTA inhibited ethylene synthesis, and the production of ethylene after application of EGTA plus ABA was higher than that after treatment with EGTA alone. However, the effect changed when immature fruits were treated with these chemicals. ABA inhibited ethylene synthesis obviously and calcium still promoted it. When we treated immature fruit discs with both calcium and ABA simultaneously, ethylene production was more than that of ABA and less than that of calcium, which indicates that ABA and calcium play agonistic roles in ethylene biosynthesis of the immature tomato fruit discs. Furthermore, ethylene release from transgenic antisense ACS immature tomato fruit discs with application of both calcium and ABA was inhibited markedly, but was stimulated when the mature fruits were treated with ABA and calcium. All of these data suggest that there are different relationships between calcium and ABA in the ethylene synthesis system in different patterns and stages of tomato fruit ripening.

Tubulin from fresh pig brain was extracted through two cycles of polymerization and depolymerization, purified by chromatography on an ion-exchange column and labelled with 5(6)-carboxytetramethylrhodamine succinimidyl ester. We prepared 10.8 mg/ml rhodamine-labelled tubulin probe (dye/protein=0.6) and then microinjected it into living guard cells in leaves of Vicia faba L. After 5–10 min, a network of microtubules (MTs) could clearly be observed in the guard cells under a confocal laser scanning microscope. In opening guard cells, the cortical MTs radiated from the ventral wall to the dorsal wall and in closed guard cells the radial MTs were completely depolymerized into random and diffuse fragments. This shows that pig brain tubulin can easily be incorporated into MTs in guard cells, and that the dynamic behaviour of MTs can be examined directly in vivo during stomatal movement.

A rice cDNA library was screened using OSK3 protein kinase as bait in a yeast two-hybrid system. The gene encoding oxalate oxidase was one of the positive clones interacting with OSK3 protein kinase. The interactions were verified by detecting expression of the reporter gene lacZ. The results suggest that oxalate oxidase is a downstream element in the disease resistance signal cascade mediated by OSK3 protein kinase in rice.

Three Porcine reproductive and respiratory syndrome virus (PRRSV) isolates (HB-1(sh)/2002, HB-2(sh)/2002 and JX-1/2002) were obtained from pig farms in Hebei and Jiangxi provinces, China. The complete ORF5 gene of the isolates was amplified using RT-PCR and sequenced. It was shown that ORF5 genes of all isolates encoded 200 amino acids. Comparing ORF5 genes of the three isolates and published sequences for five other PRRSV isolates in China, variation analysis showed that all of the isolates were of the American genotype, with 88.2–99.0% amino acid identity. ORF5 genes among BJ-4, S1 and J1 had higher similarity, sharing 98–99% identity of the deduced amino acids. HB-1(sh)/2002, HB-2(sh)/2002 and JX-1/2002 and CH-1a presented 92–96% identity among their ORF5 genes. Phylogenetic analysis revealed that these isolates could be divided into two subgroups based on the genetic distance of their ORF5 gene: the first subgroup comprised BJ-4, S1 and J1 and was closer to VR2332 and vaccine strains; the second included HB-1(sh)/2002, HB-2(sh)/2002, JX-1/2002 and CH-1a.

The levels of drought tolerance and nucleotide polymorphism at the CBF4 locus were examined in a world-wide sample of 17 core accessions of Arabidopsis thaliana. The results showed that different accessions exhibited considerable differences in adaptation to drought stress. Compared with Columbia accession, the frequency of nucleotide polymorphism at the CBF4 locus of 25av, 203av and 244av accessions, including single nucleotide polymorphism (SNP) and insertion/deletion (Indel), was high, on average 1 SNP per 35.8 bp and 1 Indel per 143 bp. No significance in all regions of Tajima's D test indicated that the neutral mutation hypothesis could explain the nucleotide polymorphism in this CBF4 gene region. The higher polymorphism was the result of purification selection. Nucleotide polymorphism in the non-coding region was three times higher than in the coding region. This might indicate a recent relaxation of selection pressures on the non-coding region of CBF4 gene. In the coding region of CBF4, SNP frequency was 1 SNP per 96.4 bp and one non-synonymous mutation was detected from 25av, 203av and 244av accessions: the amino acid variation gly↔val at position 205, caused by the nucleotide variation G↔T at position 1034 (corresponding to the nucleotide at position 19 696 of GenBank accession no. AB015478 as 1). Furthermore, four differential SNPs were discovered in haplotype 6 constituted by 203av, one of them located in the 3′ non-coding region (A↔C at position 1106) and the others in the 5′ non-coding region (A↔G, A↔C and G↔A at positions 27, 129 and 171, respectively). The drought tolerance assay indicated that accession 203av was the best at tolerating water deficiency. We propose that haplotype 6 is consistent with its drought tolerance.

Embryonic germ (EG) cells are pluripotent cells derived from the primordial germ cells of gonads, gonadal ridges and mesenteries, and analogies of foetuses, with the ability to undergo both self-renewal and multiple differentiation. These cells can differentiate into derivatives of all three embryonic germ layers when transferred to an in vitro environment and have the ability to form any fully differentiated cell of the body. The present paper investigates some factors influencing the efficiency of isolation and culture of human EG cells, such as foetus age, culture serum, added cytokines and feeder cells. The results demonstrate that foetuses of 7–12 weeks are optimal for in vitro culture of human EG cells. The basic medium consisted of DMEM, 1×non-essential amino acids, 2 mM l-glutamine and 1 mM sodium pyruvate. Supplementation with 15% foetal bovine serum, 4 ng/ml human recombinant leukaemia inhibitory factor, 4 ng/ml basic fibroblast growth factor and 20 ng/ml stem cell factor clearly improved the efficiency of isolation and culture of human EG cells. Murine embryonic fibroblasts were better feeder cells than human embryonic fibroblasts, bovine embryonic fibroblasts or STO cell line.

The effects of some factors on the isolation of protoplasts from Penicillium digitatum were studied, including the appropriate material (young mycelia and generating spores), the concentrations of enzyme and osmotic pressure stabilizers, reaction time and reaction temperature. Results demonstrated that germinating spores were an ideal material resource for the isolation of P. digitatum protoplasts. Highest yield and quality of P. digitatum protoplasts were obtained by shaking germinating spores suspended in a solution of 10 mg/ml Lywallzyme™ dissolved in 0.7 M NaCl as osmotic pressure stabilizer at 80 rev/min and 30°C for 3.0–3.5 h. The regeneration rate of the isolated protoplasts was as high as 24.9% on double-layer Czapek medium containing 0.7 M NaCl. Additionally, observation of the protoplast release pattern showed that the protoplasts of P. digitatum were released primarily from the hyphal apex and occasionally from the subapical or original sites of germinating tubes. The protoplasts of P. digitatum were regenerated in a direct manner of either yeast-like cell development or mycelium formation.