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1.
Ge Y  Norton T  Wang ZY 《Plant cell reports》2006,25(8):792-798
Zoysiagrass (Zoysia japonica Steud.) is an important turfgrass that spreads by stolons and rhizomes. By exploring the potential of direct shoot formation from stolons, we developed a straightforward and efficient transformation protocol without callus induction and propagation. Sterilized stolon nodes were infected and co-cultivated with Agrobacterium tumefaciens harboring pCAMBIA vectors. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Both green and albino shoots were directly regenerated from the infected stolon nodes 4–5 weeks after hygromycin selection. Greenhouse-grown plants were obtained 10–12 weeks after Agrobacterium-mediated transformation. Based on the number of transgenic plants obtained and the number of stolon nodes infected, a transformation frequency of 6.8% was achieved. Stable integration of the transgenes in the plant genome was demonstrated by PCR and Southern blot hybridization analyses. Expression of the transgenes was confirmed by RT-PCR analysis and GUS staining. The new transformation system opens up new opportunities for the functional characterization of genes and promoters and the development of novel germplasm in zoysiagrass.  相似文献   

2.
The present study reports the development of 30 polymorphic microsatellite markers for zoysiagrass Zoysia japonica Steud. The 30 markers produced a total of 125 alleles with an average of 4.2 alleles per locus. Values for observed and expected heterozygosities ranged from 0.10 to 0.95 and from 0.15 to 0.81, respectively. At significance threshold (P < 0.05), 11 loci deviated from Hardy–Weinberg equilibrium, whereas significant linkage disequilibrium values were observed between 16 pairs of loci. These markers may provide information needed to select genetically diverse parents for developing breeding and mapping populations of zoysiagrass.  相似文献   

3.
介绍了分别以日本结缕草根部、叶部、匍匐茎等为材料的总RNA提取的改良步骤,质量和浓度检测及其注意事项,并采用Promega公司的Poly ATract Systems Ⅲ(Z5300)试剂盒进行mRNA分离,尽管这一方法成熟可行,但仍存在洗脱体积较大,常需先沉淀浓缩后才能进行后续试验(如cDNA合成),在制备少量mRNA时回收率较低等问题。据此,对mRNA分离方法作了改良,介绍了mRNA分离的优化步骤,通过增加体系形成了一种更适合于日本结缕草mRNA分离的方法,从而为日本结缕草的分子生物学研究提供基础资料。结果证明所提取的总RNA和mRNA完整,质量较高,并应用到日本结缕草cD-NA文库的构建。  相似文献   

4.
Genetic transformation of arctic bramble (Rubus arcticus L.) was achieved utilizing a Ti-plasmid vector system of Agrobacterium tumefaciens. Internodal stem segments were inoculated with Agrobacterium strain EHA101 carrying a T-DNA with the CaMV 35 S promoter-gus-int marker gene from which β-glucuronidase (GUS) is expressed only in plants. Regenerants were produced on Murashige and Skoog medium. Growth of Agrobacterium was inhibited with cefotaxime. Kanamycin was used as the selective agent for the transformants. Regenerants were assayed by histochemical GUS staining, and by Southern analysis using a gus-int probe. Transgenic arctic bramble plants containing gus-int and expressing GUS were recovered. Expression has been stable for 3 years in micropropagation. Received: 22 October 1997 / Revision received: 7 January 1998 / Accepted: 2 February 1998  相似文献   

5.
The effect of dithiothreitol (DTT) on the expression of the β-glucuronidase (GUS) reporter gene under the control of the CaMV-35 S promoter has been investigated by radioactive labelling and immunoprecipitation of the enzyme in protoplasts from stably transformed tobacco plants and compared with that observed in protoplasts transiently expressing the same gene construct. An increase in net accumulation of GUS during the culture period in response to externally added DTT (2 mm) was observed both in protoplasts from transformed tobacco plants and in electroporated protoplasts. DTT had no effect on rate of degradation of the mature GUS protein, as shown in a pulse-chase experiment. Relevant aspects of protoplast physiology, such as viability, synthesis of 35S-labelled cellular proteins, or synthesis and export of pathogenesis-related proteins (one putative chitinase and two 1,3-β-glucanases) were not affected by the reducing reagent. Received: 15 December 1997 / Revision received: 14 April 1998 / Accepted: 1 May 1998  相似文献   

6.
7.
Recurrent selection for high seed oil content was carried out with 2,008 progeny of 28 plants regenerated via embryogenesis, 95 via organogenesis and 25 from protoplasts via organogenesis from five different soybean cultivars. Two lines derived from plants regenerated from the cultivar Jack with small increases in seed oil content emerged after three selection cycles in the field but in both cases the protein content was decreased and the seed yield of one of the lines was also decreased. Apparently somaclonal variation for seed oil content can arise, but on the basis of the decreases in protein and yield found in this study, this small change is not useful for soybean improvement. Received: 22 July 2000 / Accepted: 28 July 2000  相似文献   

8.
A protocol was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. The bacterial strain ABI containing the binary vector pMON977 with the nptII gene as selectable marker and an intron-interrupted uidA gene (encoding β-glucuronidase) as visible marker was used for the experiments. Selection of transformed tissue with paromomycin resulted in the establishment of antibiotic-resistant, β-glucuronidase-expressing lines of friable embryogenic callus, from which embryos and subsequently plants were regenerated. Southern blot analysis demonstrated stable integration of the uidA gene into the cassava genome in five lines of transformed embryogenic suspension cultures and in two plant lines.  相似文献   

9.
A procedure for culturing protoplasts from slowly growing embryogenic calli of wheat was developed. The procedure was dependent on the ability to isolate large numbers of culturable protoplasts from slowly growing embryogenic callus. Approximately 68% of the isolated protoplasts divided, and 22% formed colonies; of the latter, 67% continued to proliferate. Plating efficiency was reduced when protoplasts were transformed by polythylene glycol, electroporation, and/or Agrobacterium. Intact cells were also directly transformed by electroporation. Direct electroporation of the Agrobacterium binary vector into intact cells resulted in a significant increase of GUS activity over the control.  相似文献   

10.
A cytological and molecular analysis was performed to assess the genetic uniformity and true-to-type character of plants regenerated from 20 week-old embryogenic suspension cultures of meadow fescue (Festuca pratensis Huds.), and compared to protoplastderived plants obtained from the same cell suspension. Cytological variation was not observed in a representative sample of plants regenerated directly from the embryogenic suspensions and from protoplasts isolated therefrom. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in the plants analyzed. Randomly amplified polymorphic DNA markers (RAPDs) have been used to characterise molecularly a set of mature meadow fescue plants regenerated from these in vitro cultures. RAPD markers using 18 different short oligonucleotide primers of arbitrary nucleotide sequence in combination with polymerase chain reaction (PCR) allowed the detection of pre-existing polymorphisms in the donor genotypes, but failed to reveal newly generated variation in the protoplast-derived plants compared to their equivalent suspensionculture regenerated materials.The genetic stability of meadow fescue plants regenerated from suspension cultures and protoplasts isolated therefrom and its implications on gene transfer technology for this species are discussed.Abbreviations PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism.  相似文献   

11.
ABSTRACT

(2-Nitroethyl)benzene, methyl 4-methoxybenzoate and 4-methoxybenzaldehyde have been known as major scent components in flowers of the Japanese loquat Eriobotrya japonica [Rosales: rosaceae], together with 13 related benzenoids, including Z- and E-2-phenylacetaldoxime and benzyl alcohol. The scents air-trapped from a flowering panicle during 24 h incubation with d8-L-phenylalanine were composed of 15 deuterium labeled compounds {d6-styrene, d5-benzaldehyde, d7-2-phenylacetaldehyde, methyl d5-benzoate, d7 ?2-phenylethanol, d7-2-phenylacetonitrile, d4-1,4-dimethoxybenzene, d7-Z-2-phenylacetaldoxime, d4-4-methoxybenzaldehyde, d7-E-2-phenylacetaldoxime, d4-4-methoxybenzyl alcohol, d7-(2-nitroethyl)benzene, methyl d4-4-methoxybenzoate, methyl d6-cinnamate and ethyl d4-4-methoxybenzoate}. On the other hand, hexane extracts of the flower petal incubate with a mixture of d5-Z- and d5-E-2-phenylacetaldoxime after 24 h indicated generation of six d5-labeld components {d5-benzaldehyde, d5-benzyl alcohol, d5-2-phenylacetaldehyde, methyl d5-benzoate, d5-2-phenylethanol, and d5-(2-nitroethyl)benzene}. By comparing those results, (2-nitroethyl)benzene was concluded as a product directly generated from a mixture of Z- and E-2-phenylacetaldoxime together with six minor benzenoids, while two major compounds (4-methoxybenzaldehyde and methyl 4-methoxybenzoate) together with three minors from L-phenylalanine, presumably via L-tyrosine. The other two minor components were derived from L-phenylalanine.  相似文献   

12.
Three partial S-RNase genes, MSRN-1, MSRN-2, and MSRN-3, in the Japanese apricot (Prunus mume Sieb. et Zucc.) were isolated from the three cultivars Nankou, Gyokuei, and Kairyouuchidaume, respectively. The structural characteristics revealed that S-RNase genes from the Japanese apricot were in the T2/SRNase-type S-RNase family with five conserved regions (C1, C2, C3, RC4, and C5) and one variable region (RHV) as reported in the other rosaceous plants. In the phylogenetic tree of T2/S SRNase-type RNases, three S-RNase genes of the Japanese apricot did not form a species-specific subgroup but the Prunus subfamily did. At least seven S-allelic genes were present in the Japanese apricot, and S-genotypes of six representative cultivars, including Nankou, Gyokuei, Kairyouuchidaume, Baigou, Kagajizou, and Oushuku were first established in this study as S 1 S 7, S 2 S 6, S 3 S 4, S 3 S 6, S 3 S 6 and S 1 S 5, respectively. An extended elucidation of the S-genotype would contribute to a more efficient breeding program of the Japanese apricot. Received: 5 September 2000 / Revision accepted: 22 December 2000  相似文献   

13.
It is desirable that the expression of transgenes in genetically modified crops is restricted to the tissues requiring the encoded activity. To this end, we have studied the ability of the heterologous ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small-subunit (SSU) gene promoters, RBCS3CP (0.8 kbp) from tomato (hycopersion esculentum Mill.) and SRS1P (1.5 kbp) from soybean (Glycine max [h.] Mers.), to drive expression of the β-glucuronidase (gusA) marker gene in apple (Malus pumila Mill.). Transgenic lines of cultivar Greensleeves were produced by Agrobacterium-mediated transformation and the level of gusA expression in the vegetative tissues of young plants was compared with that produced using the cauliflower mosaic virus (CaMV) 35S promoter. These quantitative GUS data were assessed for their relationship to the copy number of transgene loci. The precise location of GUS activity in leaves was identified histochemically. The heterologous SSU promoters were active primarily in the green vegetative tissues of apple, although activity in the roots was noticeably higher with the RBCS3C promoter than with the SRS1 promoter. The mean GUS activity in leaf tissue of the SSU promoter transgenics was approximately half that of plants containing the CaMV 35S promoter. Histochemical analysis demonstrated that GUS activity was localised to the mesophyll and palisade cells of the leaf. The influence of light on expression was also determined. The activity of the SRS1 promoter was strictly dependent on light, whereas that of the RBCS3C promoter appeared not to be. Both SSU promoters would be suitable for the expression of transgenes in green photosynthetic tissues of apple. Received: 15 June 1999 / Accepted: 12 August 1999  相似文献   

14.
Phylogenetic analyses based on the mitochondrial ND5 gene comparisons and the geohistory of the Japanese Islands suggest that each Japanese species belonging to the subtribe Carabina has its own history for the establishment of its present habitat in the Japanese Islands. It can be roughly classified into two categories: (1) species which were derived from the ancestry that inhabited ancient Japan at the time of its split from the Eurasian Continent [ca. 15 million years ago (MYA)], followed by diversification within the Japanese Islands; and (2) species which invaded Hokkaido from the Eurasian Continent through land-bridges from Sakhalin and/or the Kuriles or from western Japan from the Korean Peninsula during the glacial era (<2 MYA). Received: 28 September 1999 / Accepted: 25 February 2000  相似文献   

15.
Dioscorea zingiberensis Wright has been cultivated as a pharmaceutical crop for production of diosgenin, a precursor for synthesis of various important steroid drugs. Because breeding of D. zingiberensis through sexual hybridization is difficult due to its unstable sexuality and differences in timing of flowering in male and female plants, gene transfer approaches may play a vital role in its genetic improvement. In this study, the Agrobacterium tumefaciens-mediated transformation of D. zingiberensis was investigated with leaves and calli as explants. The results showed that both leaf segments and callus pieces were sensitive to 30 mg/l hygromycin and 50–60 mg/l kanamycin, and using calli as explants and addition of acetosyringone (AS) in cocultivation medium were crucial for successful transformation. We first immersed callus explants in A. tumefaciens cells for 30 min and then transferred the explants onto a co-cultivation medium supplemented with 200 μM AS for 3 days. Three days after, we cultured the infected explants on a selective medium containing 50 mg/l kanamycin and 100 mg/l timentin for formation of kanamycin-resistant calli. After the kanamycin-resistant calli were produced, we transferred them onto fresh selective medium for shoot induction. Finally, the kanamycin resistant shoots were rooted and the stable incorporation of the transgene into the genome of D. zingiberensis plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The method reported here can be used to produce transgenic D. zingiberensis plants in 5 months and the transformation frequency is 24.8% based on the numbers of independent transgenic plants regenerated from initial infected callus explants.  相似文献   

16.
 PCR-based DNA profiling of coconut palms indigenous to Sri Lanka was conducted using amplified fragment length polymorphism (AFLPs). A total of 322 amplification products were generated from the 42 genotypes with eight pairs of primers (EcoRI and MseI). Overall most variation was detected in the tall (Typica) rather than the intermediate (Aurantiaca) and dwarf (Nana) forms. A hierarchical analysis of molecular variance (AMOVA) was used to quantify and partition levels of variability into between- and within-form components. This revealed that for the inbreeding dwarf and intermediate forms most variation was observed between, rather than within, forms. In contrast, the outbreeding tall forms exhibited as much variation within as between forms. These observations have important implications for the maintenance and collection of coconut germplasm. This study also provided insights into the genetic (as opposed to phenotypic) relatedness of coconut accessions. Morphologically the Aurantiaca group of accessions are considered to be intermediate between the tall and dwarf accessions. Estimation of genetic relatedness based on AFLP analysis identified the Aurantiaca group as being more similar to the dwarf rather than the tall group. In addition, putative duplicate accessions were identified in the Aurantiaca group. Information emerging from this study will facilitate the management of coconut germplasm and optimise the choice of genetically divergent parents for crossing. Received: 16 June 1997 / Accepted: 14 October 1997  相似文献   

17.
Arabinogalactan proteins (AGPs) are abundant plant cell surface proteoglycans widely distributed in plant species. Since high concentrations of β-glucosyl Yariv reagent (βglcY), which binds selectively to AGPs, inhibited cell division of protoplast-regenerated cells of the liverwort Marchantia polymorpha L. (Shibaya and Sugawara in Physiol Plant 130:271–279, 2007), we investigated the mechanism underlying the inability of the cells to divide normally by staining nuclei, cell walls and β-1,3-glucan. Microscopic observation showed that the diameter of regenerated cells cultured with βglcY was about 2.8-fold larger than that of cells cultured without βglcY. The cells cultured with βglcY were remarkably multinucleated. These results indicated that βglcY did not inhibit mitosis but induced multinucleation. In the regenerated cells cultured with low concentrations of βglcY (5 and 1 μg ml−1), the cell plate was stained strongly by βglcY, suggesting abundant AGPs in the forming cell plate. In these cell plates, β-1,3-glucan was barely detectable or not detected. In multinucleated cells, cell plate-like fragments, which could not reach the cell wall, were frequently observed and they were also stained strongly by βglcY. Our results indicated that AGPs might have an important role in cell plate formation, and perturbation of AGPs with βglcY might result in remarkable multinucleation in protoplast-regenerated cells of M. polymorpha. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

18.
19.
Agrobacterium-mediated and direct gene transfer into protoplasts using PEG were both successfully used to produce stable, transformed peppermint plants (Mentha×piperita L. cultivar Black Mitcham) with the limonene synthase gene. Stem internode explants found to possess a high level of organogenesis through adventitious shoot formation were subjected to Agrobacterium tumefaciens disarmed strain GV3101 (pMP90). Following the development of an efficient protoplast-to-plant cycle from stem-isolated protoplasts, they were used in direct gene transformations. In both cases the binary vector pGA643 carrying the nptII/GUS genes, both driven by the CaMV35S promoter, was used in preliminary plant-transformation studies. Later, GUS was replaced with the limonene synthase gene. Kanamycin was used as a selective agent in all transformation experiments to obtain both transformed protoplast-derived calli as well as putative transgenic shoots regenerated from internode explants. Both types of transformation resulted in transgenic plants which were detected using PCR and confirmed by Southern-blot hybridizations. Southern analysis revealed that the method of Agrobacterium-mediated transformation is superior to the direct DNA uptake into protoplasts with regard to the stability of the insert during the transformation event. Single transgenic plants were grown to 10% flowering in a greenhouse and the plants derived both by the Agrobacterium and the protoplast-derived methods were generally observed to have essential oil profiles characterized by a high-menthone, low-menthol, high-menthofuran and –pulegone content in comparison to a typical mid-west peppermint. Limonene varied only slightly, around 1.2%, in transgenic plants produced by both methods. Received: 22 November 1998 / Accepted: 4 Januar 1999  相似文献   

20.
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