A modified protocol for the comet assay allowing the processing of multiple samples.

In the present study, we developed a modified protocol for the basic comet assay that increased efficiency without sacrificing assay reliability. A spreader was used to spread agarose-embedded cells on a slide, making the manipulation and processing of multiple samples easier. Using this technique, we are able to rapidly prepare five or more comet assay samples on one slide. To demonstrate the effect of the protocol modifications on assay reliability, we present an example of how the comet assay was used in our laboratory to analyze the effect of melatonin (N-acetyl-5-methoxitryptamine; MEL) on the DNA repair ability of Gentiana macrophylla Pall. protoplasts after irradiation with different doses of ultraviolet-B radiation. A slight, but statistically significant (P<0.01), dose-related protective effect of MEL was observed in our experiments. The first use of the comet assay was to confirm the antioxidant and DNA repair functions of MEL in plants. The modified protocol is cost-effective and provides substantial advantages over the conventional comet assay.

Effect of CO2 laser radiation on physiological tolerance of wheat seedlings exposed to chilling stress.

To determine the effect of CO(2) laser pretreatment of wheat seeds on the physiological tolerance of seedlings to chilling stress, wheat seeds were exposed to CO(2) laser radiation for 300 s. After being cultivated for 48 h at 25 degrees C, the wheat seedlings were subjected to chilling stress for 24 h. Selected physiological and biochemical parameters were measured in 6-day-old seedlings. We observed that chilling stress enhanced the concentrations of malondialdehyde and oxidized glutathione while decreasing the activities of nitric oxide synthase, catalase, peroxidase, superoxide dismutase and the concentrations of nitric oxide and glutathione in the wheat leaves compared with controls. When the chilling stress was preceded by CO(2) laser irradiation, the concentrations of malondialdehyde and oxidized glutathione were decreased while the activities of nitric oxide synthase, catalase, peroxidase, superoxide dismutase and the concentrations of nitric oxide and glutathione increased. Furthermore, chilling stress decreased the biomass, biophoton intensity and GHS/GSSG ratios of seedlings while these parameters increased when the seedlings were treated with CO(2) laser irradiation prior to the chilling stress. The results suggest that a suitable dose of CO(2) laser stimulation can enhance the physiological tolerance of wheat seedlings to chilling stress.

Weak microwave can alleviate water deficit induced by osmotic stress in wheat seedlings.

The aim of the investigation is to determine the effect of microwave pretreatment of wheat seeds on the resistance of seedlings to osmotic stress. Changes in biophysical, physiological and biochemical characters were measured. The results showed: (1) The magnetic field intensity and seeds temperature increased progressively with microwave pretreatments of 5, 10, 15, 20 s and 25 s compared with controls. Although each microwave pretreatment resulted in an increase in alpha-amylase activity and photon emission intensity, the increase of alpha-amylase activity and photon emission intensity was maximal at a microwave pretreatment of 10 s. (2) Osmotic stress induced by PEG treatment enhanced the concentration of malondialdehyde, while decreasing the activities of nitricoxide synthase, catalase, peroxidase, superoxide dismutase and the concentration of nitric oxide, ascorbic acid, glutathione in the seedlings compared with controls. However, compared to osmotic stress alone, in the seedlings treated with microwave irradiation plus osmotic stress the concentration of malondialdehyde decreased, while the activities of nitricoxide synthase, catalase, peroxidase, superoxide dismutase and the concentration of nitric oxide, ascorbic acid and glutathione increased. These results suggest that a suitable dose of microwave radiation can enhance the capability to eliminate free radicals induced by osmotic stress in wheat seedlings resulting in an increase in resistance to osmotic stress.

[Transformation of Astragalus melilotoides Pall with AtNHX1 gene and the expression of salinity tolerance of transformants].

Using RT-PCR method, the open reading frame (ORF) of AtNHX1-cDNA, encoding the vacuolar Na+/H+ antiportor, was cloned from Arabidopsis thaliana seedlings pretreated with 100 mmol/L NaCl for 24h. This ORF was inserted between CaMV35S promoter, a Omega fragment of TMV RNA 5'UTR and NOS polyA terminator in the T-DNA region of a binary expression vector pNT (Fig1). The recombinant plasmid, designated as pNT-AtNHX1, was then transformed into Agrobacterium tumefaciens LBA4404. Mediated by this engineering Agrobacterium, the AtNHX1 gene was transferred into T0 generation transgenic plant strains of A. melilotoides and 103 regenerated plants resistant to Kanamycin (Kan) were obtained. Some factors influencing the transformation efficiency, such as the concentration and infection duration of Agrobacterium, the concentration of Acetosyringone (AS), were optimized to establish a stable Agrobacterium mediated gene transformation protocol of A. melilotoides. PCR analysis, Southern blot and RT-PCR detection of some T0 transgenic plants showed that the AtNHX1 gene was evidently integrated into the genome of transgenic plants and couldl be transcripted properly. Under the same salt stress conditions, the detection of NaCl resistance revealed the difference between the wild-type calli and the transgenic calli that induced from the transgenic plants, i.e, the relative growth rates of the transgenic calli were remarkably higher than that of the wild-type calli. The K+ and Na+ contents and relative conductivity in the leaves of the transgenic plants and wild-type plants were estimated. It suggested that under the stress of different concentration of NaCl, K+/Na+ ratio in the transgenic plant cells were always higher than that in wild-type, however the situation of relative conductivity was on the opposite. From the facts above mentioned, the transformation of AtNHX1 gene not only enhanced the salt tolerance of transgenic A. melilotoides, but also reduced the cell membrane damage induced by salinity.

[Cold induced cDNA library construction of highland barley (Hordeum vulgare L. var. nudum Hk. f.) using suppression subtractive hybridization technology].

Cold-induced genes of highland barley (Hordeum vulgare L. var. nudum Hk. f.) were studied using suppression subtractive hybridization (SSH) technique. The cDNA from the materials treated with 4 degrees C was used as "tester", and that from the materials growing in green house (20+/-2 degrees C) as "driver". A subtractive library of highland barley including 640 cDNA clones was constructed in this study. Enzyme digestion of 32 clones chosen randomly from the library indicated that 87.5% of them contained inserts. The cDNA inserts of 16 clones were sequenced. Blast search analyses showed that these cDNAs were homologies to genes encoding the following proteins: metallothionein, protein kinase, ethylene signal transcription factor, bZIP transcription factor, zing finger transcription factor, ribulose-1,5-bisphosphate carboxylase, ribosomal protein, sodium: hydrogen antiporter, catalase, NADPH-cytochrome reductase, ascorbate peroxidase, DNA binding protein, and sugar transporter-like protein. These results indicated that the cDNA clones in the library were related to cold-induced genes, and suggested that the cold-tolerant mechanism of highland barley might be a complicated, interactive system involving multiple approaches and genes. Construction of subtractive cDNA library provided an advantage for further studies to isolate and clone cold-induced genes in highland barley.

[Hairy root induction and plant regeneration of Rehmannia glutinosa Libosch. f. hueichingensis (Chao et Schih) Hsiao transformed by Agrobacterium rhizogenes].

An efficient system of genetic transformation and plant regeneration was established in Rehmannia glutinosa Libosch. f. hueichingensis (Chao et Schih) Hsiao by infecting the segments of leaves, stems and petioles of young regenerated plantlets with Agrobacterium rhizogenes strain 15834. Hairy roots were produced directly from the wounded surface of the explants on hormone-free Murashige and Skoog (MS) medium after infection by A. rhizogenes. Transformed roots grew rapidly either on solid or on liquid 1/2 MS medium, and exhibited typical hairy root phenotypes. The highest transformation frequency of 46.7% was achieved by pre-treating the A. rhizogenes with 100 micromol/L acetosyringone at logarithmic phase (OD600 = 1.8). The calluses with 100% induction frequency were induced from hairy roots on 1/2 MS medium containing 0.2 mg/L KT and 3.0 mg/L 6-BA, from which the shoots with 51.49% differentiation frequency was produced. These shoots could take root at a percentage of 100% and develope into four transformed plantlets when transferred on 1/2 MS medium, which had differences in morphological characters such as dwarfing, shortened nodals and abundant literal branching roots, and which survived vigorously after transplantation. The content of catalpol in an transformed hairy root clone was 0.557 mg/g. FW by means of HPLC, 48.5% and 18% of that in fresh and dried Rehmannia root, respectively. PCR and Southern blot analyses confirmed that rolB gene (564 bp) of TL-DNA was inserted in the genome of transformed hairy roots and their regenerated plantlets. RT-PCR analysis and opine paper electrophoresis detection revealed that TR-DNA containing opine synthetase gene was integrated and expressed in the genome of transformed hairy roots and their regenerated plantlets.

[Protoplast culture and plant regeneration of the methionine resistant variant of Astragalus cicer L].

A protoplast-to-plant system for the methionine resistant variant of Astragalus cicer L. has been developed. The friable calli induced from stem segments of variant plants were used as materials for protoplast isolation through enzyme digestion. The effects of different media and plating densities on protoplast divisions and plant regeneration were studied. Sustained cell divisions and colony formation from the protoplasts of the methionine resistant cell line of Astragalus cicer L. were obtained by a DPD medium containing 2.0 mg/L 2,4- dichlorophenoxyacetic acid (2,4-D), 0.2 mg/L 6 -benzylaminopurine(6-BA), 0.3 mol/L mannitol, 200 mg/L casein hydrolysate and 2% (W/V) sucrose at a plating density of 2x10(5) /ml. The division frequency was 38.3%. At the same time, different dividing types of protoplasts were found. Organogenesis and shoot formation from the protoplast-derived calli were induced on MS medium supplemented with 0.5 mg/L NAA, 10 mg/L KT and 2% (W/V) sucrose. The protoplast-derived calli still expressed resistance to methionine. The protoplast to plant regeneration protocol developed in this study might provide the foundation for the resistant cell line as a parent for somatic hybridization.

[Hairy root induction and plant regeneration of crownvetch (Coronilla varia L.) transformed by Agrobacterium rhizogenes].

An efficient system of genetic transformation and plant regeneration via somatic embryogenesis was established in crownvetch (Coronilla varia L.) by infecting the segments of cotyledons and hypocotyls of 15d-old seedlings with Agrobacterium rhizogenes strain 15834. Hairy roots were produced directly from the wounded surface of the explants or via calluses on hormone-free Murashige and Skoog (MS) medium after infection by A. rhizogenes. Transformed roots grew rapidly either on solid or liquid MS medium, and exhibited typical hairy root phenotypes. The highest transformation frequency (87.4%) was achieved by preculturing cotyledons for 2d and pre-treating the A. rhizogenes with suitable concentration of acetosyringone at logarithmic phase (OD600 = 0.8). The embryogenic calluses with 100% induction frequency were induced from hairy roots on MS medium containing 0.2mg/L 2,4-D, 0.5mg/L NAA and 0.5mg/L KT. Globular-, heart-, torpedo-, and cotyledon shaped somatic embryos were produced orderly and developed into plantlets when transferred the embryogenic calluses on MS medium supplemented with 0.5mg/L KT, 0.2mg/L IBA and 300mg/L proline. The transformed plants did not show differences in morphology except abundant lateral root branches compared to the non-transformed plants. However, the contents of 3-nitropropanic acid in hairy roots and leaves of one of 5 transformed clones were 57.68% and 58.17% in roots and leaves of untransformed plants, respectively. Opine paper electrophoresis revealed the integration and expression of TR-DNA. PCR analysis confirmed that the TL-DNA including 654 bp rol B sequence was inserted into the genome of transformed hairy roots and their regenerated plants.

Physiological and biochemical characteristics and molecular-biological identification of a gigantic tobacco mutant.

A new gigantic late-flowering tobacco mutant was isolated in tobacco field in Xunyang county, Shaanxi province, in 2001. It was rescued through tissue culture and a large amount of regenerated plantlets were obtained. The results of the comparison between the regenerated plants from this mutant and the wild type plant (K346) were as follows: (1) Morphological observation showed that the leaf number of the mutant was 3.3 times as many as that of the wild type, the height of mutant was 2.2 folds that of the wild type, and the mutant had the late-flowering character. (2) Cytological examination showed that the mutant was a normal diploid, and the number of chloroplasts in a guard cell of mutant was 1.3 times that of the wild type. (3) Both chlorophyll a and b content of the mutant were larger than that of the wild type; soluble protein content of the mutant was 1.18 times as much as that of the wild type. Peroxidase isozyme and cytochrome-oxidase isozyme electrophoresis analyses indicated differences between the mutant and the wild type. The soluble protein SDS-PAGE patterns showed the absence of four bands [P1 (114.6 kD), P2 (103 kD), P3 (66.2 kD), P4 (24 kD)] in the mutant. (4) RAPD analysis showed that the similarity index of the mutant and the wild type was 0.612. This suggested that there were changes at DNA level. The mRNA of mutant leaves at flowering stage was isolated; DDRT-PCR was carried out using 10 random primers, using OligodT(15)M (M=A/G/C) as anchor primer. It has been proved that gene expression at anthesis was different between the mutant and the wild type. (5) Genetic observation showed that the mutant was homozygotic and bred true. And the mutant was a late-flowering one.

[Genetic diversity of yam (Dioscorea opposita Thunb) detected by ISSR markers].

Genetic diversity of 28 cultivars of yam (Dioscorea opposita Thunb) was assessed by means of Inter-simple sequence repeat (ISSR) markers. The results showed that seven proper primers, with rich polymorphism, could be selected from a total of forty four ISSR ones; distinct differences appeared among 28 cultivars amplified bands, and the rate of polymorphic bands was 83.01%; Shannon's Information index was 0.3191; a Jaccard's genetic similarity matrix and a dendrogram for these cultivars were formed, in which they could be divided into four groups: Group 1 was composed of D. opposita. cv. Ribenbai, D. opposita. cv. Huashanyao and D. opposita. cv. Ribenyuan; Group2 contained D. opposita. cv. Xiaoye; Group 3 contained D. opposita. cv. No.1 Songye; other 23 cultivars were put into Group4. PCA(Principal component analysis) was employed to evaluate the resolving power of the markers to differentiate among them. This laid the foundation of the identification of yam cultivars and the efficient use of its germplasm resources.

Expression and significance of CD44s, CD44v6, and nm23 mRNA in human cancer.

AIM: To investigate the relationship between the expression levels of nm23 mRNA, CD44s, and CD44v6, and oncogenesis, development and metastasis of human gastric adenocarcinoma, colorectal adenocarcinoma, intraductal carcinoma of breast, and lung cancer. METHODS: Using tissue microarray by immuhistochemical (IHC) staining and in situ hybridization (ISH), we examined the expression levels of nm23 mRNA, CD44s, and CD44v6 in 62 specimens of human gastric adenocarcinoma and 62 specimens of colorectal adenocarcinoma; the expression of CD44s and CD44v6 in 120 specimens of intraductal carcinoma of breast and 20 specimens of normal breast tissue; the expression of nm23 mRNA in 72 specimens of human lung cancer and 23 specimens of normal tissue adjacent to cancer. RESULTS: The expression of nm23 mRNA in the tissues of gastric and colorectal adenocarcinoma was not significantly different from that in the normal tissues adjacent to cancer (P>0.05), and was not associated with the invasion of tumor and the pathology grade of adenocarcinoma (P>0.05). However, the expression of nm23 mRNA was correlated negatively to the lymph node metastasis of gastric and colorectal adenocarcinoma (r = -0.49, P<0.01; r = -4.93, P<0.01). The expression of CD44s in the tissues of gastric and colorectal adenocarcinoma was significantly different from that in the normal tissues adjacent to cancer (P<0.05; P<0.01). CD44v6 was expressed in the tissues of gastric and colorectal adenocarcinoma only, the expression of CD44v6 was significantly associated with the lymph node metastasis, invasion and pathological grade of the tumor (r = 0.47, P<0.01; r = 5.04, P<0.01). CD44s and CD44v6 were expressed in intraductal carcinoma of breast, the expression of CD44s and CD44v6 was significantly associated with lymph node metastases and invasion (P<0.01). However, neither of them was expressed in the normal breast tissue. In addition, the expression of CD44v6 was closely related to the degree of cell differentiation of intraductal carcinoma of breast (c2 = 5.68, P<0.05). The expressional level of nm23 mRNA was closely related to the degree of cell differentiation (P<0.05) and lymph node metastasis (P<0.01), but the expression of nm23 gene was not related to sex, age, and type of histological classification (P>0.05). CONCLUSION: Patients with overexpression of CD44s and CD44v6 and low expression of nm23 mRNA have a higher lymph node metastatic rate and invasion. In addition, overexpression of CD44v6 is closely related to the degree of cell differentiation. Detection of the three genes is able to provide a reliable index to evaluate the invasion and metastasis of tumor cells.

[Intergeneric somatic hybridization between Astragalus adsurgens Pall and Medicago sativa L].

The protoplast fusion was induced between Astragalus adsurgens Pall and Agrobacterium rhizogenes-transformed cell line of Medicago Sativa L. by use of PEG method. Putative somatic hybrid cells were selected based on the observation that only intergeneric hybrid cells continued to divide on the DPD medium without any phytohormone while the cells and clusters derived from parental cells did not survive further. After being cultured, the somatic hybrid calli were obtained. Although both of the parental calli were not capable of regenerating , one of the hydrid cell lines recovered the differentiation ability and produced small shoots. Characterizations of the hybrid calli were carried out by examination of chromsome number, opine synthetase assay, isoenzyme and RAPD analyses.

[Gene delivery system based on low molecular weight polyethylenimine and its transfection activity in the skin tissue].

Low molecular weight polyethylenimine (LMW-PEI) was linked to an expressing plasmid contains a green fluorescence protein (GFP) reporter gene and effective gene transfer was observed in CM7221 cell line tested. We examined the relationship among the molecular weight, structure of PEI and their transfection activity and cytotoxicity on CM7221 cell line. We also examined the position and continuance time of the GFP reporter gene expressed in the skin tissue of mouse. Results showed that LMW-PEI/DNA complexes led to high levels of expression in the CM7221 cell line (about 55%). However, with the increasing of PEI molecular weight, the transfection activity of PEI was decreasing. There was an increasing cytotoxicity with the larger PEI molecules. Further research showed that LMW-PEI induced a significant and long-lasting (7 days) expression of the GFP reporter gene in the hair vesicle, sweat, gland, sebaceous gland in the mouse skin tissues. The LMW-PEI described here is a new, highly efficient and non-cytotoxic vector. It would be a useful non-viral vector for gene delivery technology, particular useful as simple skin-specific vehicles of therapeutic genes.

[Protoplast culture and plant regeneration of the methionine resistant variant of Astragalus melilotoides Pall].

An efficient protocol for plant regeneration from protoplasts of the methionine resistant variant of Astragalus melilotoides was established. The friable calli induced from internode segments of variant plants were used for protoplast preparation. The protoplasts were isolated through enzyme digestion. Calli were formed after sustained divisions of protoplasts. High frequency of shoot differentiation was obtained from the protocalli on differentiated medium. The effects of different media, culturing methods and plating densities on protoplast divisions and plant regeneration were studied. The results show that agarose-beads culture method, KM8p medium supplemented with 1.0 mg/L 2,4-D, 0.5mg/L 6BA, 0.3 mol/L mannitol, 2% (W/V) sucrose and 500 mg/L casein hydrolysate at a plating density of 3 x 10(5)/mL are the appropriate conditions for protoplast division of the methionine resistant cell line. The division frequency is over 38%. The protoplast-regenerated plants still preserve resistance to methionine and ethionine.This research builds up the foundation for the resistant cell line as a parent of somatic hybridization.

[Assessment of genetic diversity of Rehmannia glutinosa germplasm detected by RAPDs and ISSRs].

RAPD and ISSR markers were used to assess the germplasm genetic diversity among 10 individuals of Rehmannia glutinosa, including 8 cultivars and 2 virus-free lines micropropagated by tip tissue culture. 17 RAPD primers and 10 ISSR primers, with polymorphic and informative patterns, were selected from a total of 80 RAPD ones and 44 ISSR ones to determine these individuals' genetic diversity. The 17RAPD primers and 10 ISSR primers generated 177RAPDfragments and 110 fragments, respectively. The number of effective loci, the percentage of polymorphic loci, Shannon's Information index (I) and effective number of alleles (Ne) is in turn109, 61.58%, 0.3135, 1.3641 for RAPD makers, and 79, 71.82 %, 0.3577 and 1.4037 for ISSR markers; Jaccard's genetic similarity matrice and dendrograms for the 10 individuals were formed based on RAPD and ISSR-generated polymorphic bands. In dendrograms, they could be divided into two groups: one group containing six individuals such as Zupei 85.5, Datian 85.5, jinzhuangyuan, Jinbai, Zupei 9302 and Datian9302; the other composed of 4 ones such as Beijing No.1, Dahongpao, Dihuang 9104 and wild dihuang; the correlation coefficient of 0.649 between RAPD and ISSR markers GSs indicated that these two markers were significantly correlated. The results revealed that RAPD and ISSR markers were suitable for assessment of germplasm genetic diversity of Rehmannia glutinosa, and ISSR marker was superior to RAPD marker.

[Tissue culture and plant regeneration of Helianthemum songaricum Schrenk].

The cotyledonary segments of sterile seedlings of Helianthemum Songaricum Schrenk were cultured on different media containing different phytohormons. It was found that the calli could be induced efficiently on MS basal medium supplemented with 10.0 mg/L NAA and 0.2 mg/L 6-BA. When calli were transferred on MS medium with 2.0 mg/L 6-BA and 0.2 mg/L NAA, shoots were produced. The frequency of shoot differentiation reached about 85%. The regenerated shoots were rooted on 1/2MS medium added with 0.5 mg/L NAA. The rooting rate was about 76%. Regenerated plantlets were successfully transplanted in soil, with a success rate of 67%.

[In vitro selection and characterization of methionine-resistant variant in Astragalus melilotoides Pall].

A variant cell line of Astragalus melilotoides Pall. resistant to 14 mmol/L methionine was selected from calluses via mutagenesis with sodium azide. The plantlets were induced from the variant calluses. The regenerated variant plants were transplanted in soil and had set seeds. After the variant calluses had been cultured for 25 days in the medium containing 15 mmol/L methionine, the relative growth rate was 10.2 folds of the control's. Free amino acids of the aspartate family in the variant cell line increased obviously in comparison with the control. Analyses of SDS-PAGE pattern and isozyme pattern of peroxidase revealed the differences between the variant plants and the control's.

[Sequence variation of the chloroplast gene ndh D region in cytoplasmic male sterile sorghum].

A fragment SAAU-02(700) was amplified specifically from total DNA of seven sorghun varieties with male-fertile cytoplasm (N-cytoplasm). PCR assays indicated that it was amplified from chloroplast (cp) DNA. Sequence analysis revealed this newly cloned fragment contained a portion of chloroplast gene psa C (88 bp) and part of ndh D gene (192 bp). Total DNA, mitochondrial (mt) DNA, and cpDNA were digested with EcoR I + Hind III and probed with fragment SAAU-02(700). The Southern hybridization patterns displayed a 0.74 kb band both in total DNA and cpDNA, but an additional faint band 0.45 kb in size was found only in the latter. No polymorphic hybridization signal between the N-cytoplasm and male-sterile cytoplasm (S-cytoplasm) was observed. Southern hybridization of total DNA of CMS line A1 Tx623 and fertile line Tx623 digested with Hae III gave a band 4.9 kb in size in the former and a 4.45 kb band in the latter. This revealed that the sequence of ndh D from CMS line was likely altered. Further studies designed to determine whether or not the variation has some effect on the metabolism of mitochondria and chloroplast, even on the occurrence of male sterility in sorghum are underway.

[Expression activity of maize Ubi-1 promoter in fertile transgenic maize plants].

The encoding sequence of gus gene from Escherichia coli was fused with maize Ubi-1 promoter and was introduced into maize genome via particle bombardment. Fertile transgenic maize plants were regenerated from bombarded type-I calluses which were derived from scutellar tissue of immature embryos based on PPT selection. Expression activity of gus gene under the control of Ubi-1 promoter was analysed using histochemical method, and the results showed that gus gene expressed in most tissues except anther. Ubi-GUS expression in pollen, egg cell and T1 immature embryos revealed that this promoter was active in early stages of plant development. Histochemically stained pollen grains of T0 plants showed a 1:1 segregation of the gus gene, which suggested that the foreign gene was inherited in Mendelian model in these plants. In addition, maize Ubi-1 promoter could reduce the copy number of foreign genes in transgenic maize plants, which might be useful in avoidance of gene silencing. T1 seeds have been harvested.