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.

Changes of gentiopicroside synthesis during somatic embryogenesis in Gentiana macrophylla.

IN VITRO plant regeneration of Gentiana macrophylla Pall. and determination of gentiopicroside content during somatic embryogenesis are described in the present work. The highest percentage of embryogenic callus formation was observed in Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg/L 6-benzylaminopurine (BA). Calli were subcultured on MS medium containing 1.0 mg/L 2,4-D, 1.0 mg/L BA and 500 mg/L lactalbumin hydrolysate (LH) at intervals of 25 days. A higher frequency of somatic embryo maturation was achieved on MS medium containing B5 vitamins (MB) supplemented with different concentrations of 1-naphthaleneacetic acid (NAA) and BA than with a combination of NAA and kinetin (KT). Addition of AgNO(3) improved maturation of somatic embryos while thidiazuron (TDZ) promoted vitrification. The gentiopicroside contents of embryogenic calli and globular-, heart-, torpedo-, and cotyledon-shaped embryoids were determined by high-performance liquid chromatography (HPLC). Gentiopicroside was not detectable in embryogenic calli, but in all types of somatic embryos. The highest gentiopicroside content was observed in cotyledon-shaped embryoids, reaching more than 12 mg/g dry weight.

[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.

[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.

[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.

[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.

[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.