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1.
The attempts of this investigation were to develop a system for plant regeneration from explants of adult plants and its use for genetic transformation of important commercial Pelargonium zonale hybrid and P. peltatum hybrid cultivars. To this aim, leaf blade and petiole explants of eight cultivars were cultured on modified MS (Murashige and Skoog, 1962) medium with two concentrations of TDZ, BA, and zeatin (5 and 20 M). Petiole explants showed a higher regeneration response than leaf blade explants and TDZ was the most effective cytokinin. Petioles of 16 cultivars were incubated on medium containing 5, 10, 15, and 20 M TDZ, respectively, in order to identify the most effective TDZ concentration. For the majority of genotypes 10 M TDZ resulted in the best regeneration response. Cefotaxim at 500 mg l –1 had no effect on shoot regeneration and did not show interaction with glufosinate. For selection of transgenic cells, a concentration of 2.5 M glufosinate was shown to be appropriate. LBA4404 and EHA101 Agrobacterium strains carrying pIBGUS vector with pat gene as selectable marker gene and GUS gene as reporter gene were compared in transformation studies. With regard to GUS expression in petiole explants 16 days after coculture, better results were obtained with EHA 101 than with LBA 4404.  相似文献   

2.
Baroux C  Fransz P  Grossniklaus U 《Planta》2004,220(1):38-46
Somatic polyploidization is recognized as a means to increase gene expression levels in highly active metabolic cells. The most common mechanisms are endoreplication, endomitosis and cell fusion. In animals and plants the nuclei of multinucleate cells are usually prevented from fusing. Here, we report that the nuclei from the syncytial cyst of the chalazal endosperm of Arabidopsis thaliana (L.) Heynh. are polyploid with some intermediate ploidy levels that cannot be attributed to endoreplication, suggesting nuclear fusion. Analysis of isolated nuclei, together with fluorescent in situ hybridization (FISH), revealed that nuclei from the chalazal endosperm are two or three times bigger than the nuclei from the peripheral endosperm and have a corresponding increase in ploidy. Together with the consistent observation of adjoined nuclei, we propose that nuclear fusion contributes, at least in part, to the process of polyploidization in the chalazal endosperm. Confocal analysis of intact seeds further suggested that free nuclei from the peripheral endosperm get incorporated into the chalazal cyst and likely participate in nuclear fusions.Abbreviations BAC Bacterial artificial chromosome - CZE Chalazal endosperm - DAPI 4,6-Diamino-2-phenylindole - FISH Fluorescent in situ hybridization - NOR Nucleolar organizing region - NCD Nuclear cytoplasmic domain - PEN Peripheral endosperm  相似文献   

3.
Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm2 sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.  相似文献   

4.
Superoxide dismutases (SODs) are ubiquitous metalloenzymes that catalyze the dismutation of superoxide radicals (O2-) to molecular oxygen (O2) and hydrogen peroxide (H2O2). In this study we characterized an Arabidopsis thaliana CuZnSOD (CSD1), a close ortholog of a previously identified Brassica juncea CuZnSOD (MSOD1). CSD1 and other two homologs CSD2 and CSD3 were spatially regulated in Arabidopsis, and CSD1 exhibited distinct expression patterns in response to different stress treatments. To investigate the in vivo function of SOD, transgenic Arabidopsis plants, expressing sense and antisense MSOD1 RNAs, were generated and those with altered SOD activity were selected for further characterization. Although SOD transgenic plants exhibited normal phenotypes, the shoot regeneration response in transgenic explants was significantly affected by the modulated SOD activity and the corresponding H2O2 levels. Transgenic explants with downregulated SOD activity were poorly regenerative, whereas those with upregulated SOD activity were highly regenerative. These results suggest that shoot regeneration in vitro is regulated by the SOD activity.  相似文献   

5.
Gaj MD  Zhang S  Harada JJ  Lemaux PG 《Planta》2005,222(6):977-988
The capacity for somatic embryogenesis was studied in lec1, lec2 and fus3 mutants of Arabidopsis thaliana (L.) Heynh. It was found that contrary to the response of wild-type cultures, which produced somatic embryos via an efficient, direct process (65–94% of responding explants), lec mutants were strongly impaired in their embryogenic response. Cultures of the mutants formed somatic embryos at a low frequency, ranging from 0.0 to 3.9%. Moreover, somatic embryos were formed from callus tissue through an indirect route in the lec mutants. Total repression of embryogenic potential was observed in double (lec1 lec2, lec1 fus3, lec2 fus3) and triple (fus3 lec1 lec2) mutants. Additionally, mutants were found to exhibit efficient shoot regenerability via organogenesis from root explants. These results provide evidence that, besides their key role in controlling many different aspects of Arabidopsis zygotic embryogenesis, LEC/FUS genes are also essential for in vitro somatic embryogenesis induction. Furthermore, temporal and spatial patterns of auxin distribution during somatic embryogenesis induction were analyzed using transgenic Arabidopsis plants expressing GUS driven by the DR5 promoter. Analysis of data indicated auxin accumulation was rapid in all tissues of the explants of both wild type and the lec2-1 mutant, cultured on somatic embryogenesis induction medium containing 2,4-D. This observation suggests that loss of embryogenic potential in the lec2 mutant in vitro is not related to the distribution of exogenously applied auxin and LEC genes likely function downstream in auxin-induced somatic embryogenesis.  相似文献   

6.
Shoot organogenesis in Arabidopsis thaliana wasstudied with regard to the timing of key developmental phases and expression ofthe SHOOTMERISTEMLESS (STM) gene.Shoot regeneration in the highly organogenic ecotype C24 was affected byexplanttype and age. The percentage of C24 cotyledon explants producing shootsdecreased from 90% to 26% when donor seedlings were more than 6 dold, but 96% of root explants produced shoots regardless of the age of thedonorplant. Using explant transfer experiments, it was shown that C24 cotyledonexplants required about 2 days to become competent and another 8-10 days tobecome determined for shoot organogenesis. A C24 line containing the promoterofthe SHOOTMERISTEMLESS (STM) genelinked to the -glucuronidase(GUS) gene was used as a tool for determining the timingofde novo shoot apical meristem (SAM) development incotyledon and root explants. Cotyledon and root explants from anSTM:GUS transgenic C24 line were placed on shoot inductionmedium and GUS expression was examined after 6-16 days ofculture. GUS expression could be found in localizedregionsof callus cells on root and cotyledon explants after 12 days indicating thatthese groups of cells were expressing the STM gene, hadreached the key time point of determination, and were producing an organizedSAM. This was consistent with the timing of determination as indicated byexplant transfer experiments. Root explants from anSTM:GUStransgenic Landsberg erecta line and a two-step tissue culture method revealedasimilar pattern of localized GUS expression duringde novo shoot organogenesis. This is the first studydocumenting the timing and pattern of expression of theSTMgene during de novo shoot organogenesis.  相似文献   

7.
The species Solanum surattense Burm.f. has importance in ayurvedic medicine and also as vegetable. Streptomycin-resistant plantlets were induced showing chloroplast encoded mutants in S. surattense from mutagenised (ethyl methane sulphonate and gamma-rays) cotyledon explants. Chloroplast encoded – streptomycin resistant – shoots were developed from green (unbleached) sectors of the cotyledons. The streptomycin-resistant plants were similar to parental plants in morphology and ploidy level (2n=2x=24). Reciprocal crosses between streptomycin-resistant and the original streptomycin sensitive plants have shown the non-Mendelian transmission under the control of chloroplast – DNA. These antibiotic resistant plants are useful in designing biochemical selection schemes aimed at somatic hybrid/cybrid recovery in S. surattense.  相似文献   

8.
Studies on chromosome numbers and karyotypes in Orchid taxa from Apulia (Italy) revealed triploid complements inOphrys tenthredinifera andOrchis italica. InO. tenthredinifera there is no significant difference between the diploid and the triploid karyotypes. The tetraploid cytotype ofAnacamptis pyramidalis forms 36 bivalents during metaphase I in embryo sac mother cells. Aneuploidy was noticed inOphrys bertolonii ×O. tarentina with chromosome numbers n = 19 and 2n = 38. There were diploid (2n = 2x = 36), tetraploid (2n = 4x = 72), hexaploid (2n = 6x = 108) and octoploid (2n = 8x = 144) cells in the ovary wall of the diploid hybridOphrys apulica ×O. bombyliflora. Evolutionary trends inOphrys andOrchis chromosomes are discussed.  相似文献   

9.
Polyploidy, which is thought to have played an important role in plant evolution and speciation, is prevalent in Chrysanthemum (x = 9). In fact, polyploid series are known in C. zawadskii (2x, 4x, 6x, 8x, and 10x) and C. indicum (2x, 4x, and 6x), but the mechanism by which polyploidization occurs is unknown. Here we show that in diploid individuals of both C. zawadskii and C. indicum, the fusion between two adjacent pollen mother cells (PMCs) occurs at a frequency of 1.1–1.3% early in the first meiotic division. While possessing the chromosomes of both PMCs, the fused cell or syncyte undertakes subsequent meiotic division processes as a single large PMC, producing four 2n pollen grains that are able to germinate. Despite their low frequency, syncyte formation may have played a major role in the production of infraspecific polyploids in Chrysanthemum.  相似文献   

10.
Summary The occurrence of 2n pollen-producing plants was investigated in 187 plant introductions (PIs) of 38 wild species of tuber-bearing Solanum. These 2x, 4x, and 6x species are from Mexico, and Central and South America. The determination of 2n pollen-producing plants was conducted using acetocarmine glycerol. Plants with more than 1% large-size pollen were regarded as 2n pollen-producing plants. 2n pollen-producing plants were identified in the following species: 10 out of 12 Mexican 2x species, seven of nine South American 2x species, seven of seven Mexican and Central American 4x species, five of five South American 4x species, and five of five Mexican 6x species. The frequency of 2n pollen-producing plants varied among species at the same ploidy level, but the range of frequency, generally between 2 and 10% among species, was similar over different ploidy levels. The general occurrence of 2n pollen in both 2x and polyploid species, which are evolutionarily related, is evidence that the mode of polyploidization in tuber-bearing Solanums is sexual polyploidization. Furthermore, the frequencies of 2n pollen-producing plants in autogamous disomic polyploid species were not markably different from those of their related diploid species. It is thought that the frequent occurrence of 2n gametes with autogamy tends to disturb the fertility and consequently reduce fitness of polyploids. Thus, we propose that the breeding behavior of polyploids and the occurrence of 2n gametes may be genetically balanced in order to conserve high fitness in polyploid species in tuberbearing Solanum.Paper No. 3114 from the Laboratory of Genetics. Research supported by the College of Agriculture and Life Sciences; International Potato Center; USDA, SEA, CGRO 84-CRCR-1-1389; and Frito Lay, Inc.  相似文献   

11.
Castasterone (CS) and brassinolide (BL) were administered to mung bean (Vigna radiata) explants, Arabidopsis thaliana seedlings, and cultured Catharanthus roseus cells, and the glucosylated metabolites were analyzed using LC/MS/MS. In mung bean and C. roseus, CS-2-O-glucoside (CS-2G), -3-O-glucoside (CS-3G), -22-O-glucoside (CS-22G), and -23-O-glucoside (CS-23G) were identified as metabolites of CS, whereas BL-2G, BL-3G, and BL-23G were identified as metabolites of BL. In A. thaliana, CS and BL were converted into their respective 2-O- and 23-O-glucosides. Of the metabolites identified with BL and CS administration, BL-23G was the predominant metabolite in mung bean and A. thaliana, whereas the 3-O-glucoside of BL was abundant in C. roseus. This is the first report of the metabolic conversion of CS into CS-2G, CS-3G, CS-22G, and CS-23G, and of BL into BL-2G and BL-3G. Our results indicate that the glucosylation profiles of BL and CS vary with plant species, and that the glucosylation of CS is rather limited quantitatively, compared with that of BL.  相似文献   

12.
Summary The gene frequency for parallel spindles (ps) was estimated from the frequency of plants producing 2n pollen in three cultivated groups: 2x Phureja (phu), 2x Stenotomum (stn), and 4x Andigena (adg), as well as in four related wild taxa: 2x Solanum brevicaule (brc), 2x S. sparsipilum (spl), 4x S. gourlayi (grl) and 4x S. gourlayi-S. infundibuliforme hybrids (grl-ifd). Plants with more than 1% large pollen were considered as 2n pollen producers. Observations of meiosis in a sample of 2n pollen-producing plants indicated that parallel spindles is the mechanism of 2n pollen formation. The number of plants with 2n pollen among the total examined was 228 plants (15.5%) of 1,473 in 2x spl, 31 (26.7%) of 116 in 2x brc, 92 (17.4%) of 528 in 2x stn, 665 (22.1%) of 3,008 in 2x phu, 731 (51.4%) of 1,421 in 4x adg, 591 (41.2%) of 1,436 in 4x grl, and 36 (64.3%) out of 56 in 4x grl-ifd. The ps gene frequencies assuming Hardy-Weinberg equilibrium were: 0.393 for 2x spl, 0.462 for 2x brc, 0.417 for 2x stn, 0.470 for 2x phu, 0.847 for 4x adg, 0.801 for 4x grl, and 0.895 for 4x grl-ifd. Twenty-five adg clones were randomly selected from a large population and were crossed with 2x clone W5295.7, which produces 2n pollen by parallel spindles (ps). The 4x progenies from 4x×2x crosses were used to determine the genotypes at the ps locus by screening 10–20 plants in each family for 2n pollen. Based on chromosome segregation at the ps locus, 9, 14, 1, and 1 clones were nulliplex, simplex, simplex or duplex, and duplex, respectively. The frequency of the ps gene in the adg population was estimated to be 0.825 and 0.815 for chromosome and chromatid segregation, respectively. The high frequencies of 2n pollen and the ps gene in cultivated 2x and 4x groups, and in wild taxa closely related to them, provide evidence for sexual polyploidization in the tuber-bearing Solanums.Paper No. 3032 from the Laboratory of Genetics. Research supported by the College of Agriculture and Life Sciences; International Potato Center; USDA, SEA, CGRO 84-CRCR-1-1389; and Frito Lay, Inc.  相似文献   

13.
Summary More than 28,000 pollinations were carried out between 5 Ipomoea batatas and 41 diploid I. trifida accessions of diverse origins to obtain 4x interspecific hybrids. From the resultant 730 seeds, 248 plants were finally obtained. Ploidy level determination of the progeny showed unexpected results: 52 individuals were hexaploid, 5 were pentaploid, 190 were tetraploid, as expected, and one was not determined. The existence of 5x and 6x progenies from 6x x 2x crosses not only confirmed the presence of 2n gametes but also their successful function in gene flow between ploidy levels and polyploidization within this genus. The progeny and their cultivated parents were planted in an observation field. The cultivated parents produced 0.49 kg/plant or less. Most 4x progenies did not produce storage roots or had very poor yields; nonetheless, and despite their cultivated parents' poor yields, 8 genotypes yielded between 0.81 and 1.50 kg/plant.A new scheme, using the 4x interspecific hybrids, is proposed for evaluating 2x and 4x wild accessions of the section Batatas to which the sweet potato belongs. Other possible uses of the 4x hybrids in breeding and genetics of the sweet potato are also discussed.  相似文献   

14.
The thale cress, Arabidopsis thaliana, is considered to be an important model species in studying a suite of evolutionary processes. However, the species has been criticized on the basis of its comparatively small size at maturity (and consequent limitations in the amount of available biomass for herbivores) and on the duration and timing of its life cycle in nature. In the laboratory, we studied interactions between A. thaliana and the cabbage butterfly, Pieris rapae, in order to determine if plants are able to support the complete development of the herbivore. Plants were grown in pots from seedlings in densities of one, two, or four per pot. In each treatment, one, two, or five newly hatched larvae of P. rapae were placed on fully developed rosettes of A. thaliana. In a separate experiment, the same densities of P. rapae larvae were reared from hatching on single mature cabbage (Brassica oleracea) plants. Pupal fresh mass and survival of P. rapae declined with larval density when reared on A. thaliana but not on B. oleracea. However, irrespective of larval density and plant number, some P. rapae were always able to complete development on A. thaliana plants. A comparison of the dry mass of plants in different treatments with controls (= no larvae) revealed that A. thaliana partially compensated for plant damage when larval densities of P. rapae were low. By contrast, single cress plants with 5 larvae generally suffered extensive damage, whereas damage to B. oleracea plants was negligible. Rosettes of plants that were monitored in spring, when A. thaliana naturally grows, were not attacked by any insect herbivores, but there was often extensive damage from pulmonates (slugs and snails). Heavily damaged plants flowered less successfully than lightly damaged plants. Small numbers of generalist plant-parasitic nematodes were also recovered in roots and root soil. By contrast, plants monitored in a sewn summer plot were heavily attacked by insect herbivores, primarily flea beetles (Phyllotreta spp.). These results reveal that, in natural populations of A. thaliana, there is a strong phenological mismatch between the plant and most of its potential specialist insect herbivores (and their natural enemies). However, as the plant is clearly susceptible to attack from non-insect generalist invertebrate herbivores early in the season, these may be much more suitable for studies on direct defense strategies in A. thaliana.  相似文献   

15.
Yang X  Chen H  Xu W  He Z  Ma M 《Plant cell reports》2007,26(10):1889-1897
The callus of Pteris vittata was induced from gametophytes generated from spores in vitro, and grew rapidly with periodical medium change. Arsenic tolerance and accumulation of P. vittata callus were compared with those of Arabidopsis thaliana callus. Cell death was not detected in P. vittata callus even at arsenate concentrations up to 2 mM; however, A. thaliana callus died at low (0.2 mM) arsenate concentrations. Meanwhile, P. vittata callus accumulated almost three times more As than A. thaliana callus when exposed to 0.2 mM arsenate. About 60% of the total As was removed when 7.5 g of P. vittata callus was cultured on 150 ml of half-strength MS liquid medium containing 450 μg As for 2 days. Furthermore, P. vittata callus, sporophytes, and gametophytes all grew well under 1 mM of arsenate and accumulated 1,250; 1,150 and 2,180 mg kg−1 dry weight As when grown on 2 mM arsenate for 15 or 30 days. The characteristics of non-differentiated cells, large biomass, ease of culture, good synchronization, and excellent As sequestering, make the callus of P. vittata a new ideal system to study the mechanisms of As hyperaccumulation and phytoremediation in As-contaminated groundwater.  相似文献   

16.
High frequency transformation of Arabidopsis thaliana leaf explants has been obtained using a disarmed Ti plasmid containing the coding region of a neomycin phosphotransferase gene (NPT II) as a selectable marker. The rate of transformation ranged from 55 to 63 percent when acetosyringone (AS), a natural wound response molecule, was added to an Agrobacterium tumefaciens culture prior to incubation with leaf segments. Without acetosyringone, the transformation rate was approximately 2 to 3 percent. Calli resistant to G418 were regenerated into mature flowering plants in the presence of 10 g/ml G418. Southern analysis and neomycin phosphotransferase assays confirmed the insertion and expression of the NPT II gene in regenerated Arabidopsis plants.  相似文献   

17.
Summary We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.Abbreviations bar bialaphos resistance gene - CIM callus-inducing medium - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - GM germination medium - HPT hygromycin phosphotransferase - MS Murashige and Skoog salts - NPTII neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin - SIM shoot-inducing medium  相似文献   

18.
Summary Electrofusion was carried out between mesophyll protoplasts from the transformed diploid S. tuberosum clone 413 (2n=2x=24) which contains various genetic markers (hormone autotrophy, opine synthesis, kanamycin resistance, -glucuronidase activity) and mesophyll protoplasts of a diploid wild-type clone of N. plumbaginifolia (2n=2x=20). Hybrid calli were obtained after continuous culture on selection medium containing kanamycin. Parental chromosome numbers, determined at 2 months after fusion, revealed hybrid-specific differences between the individual calli. On the basis of these differences three categories of hybrids were distinguished. Category I hybrids contained between 8 and 24 potato chromosomes and more than 20 N. plumbaginifolia chromosomes; category II hybrids had between 1 and 20 N. plumbaginifolia chromosomes and more than 24 potato chromosomes; category III hybrids contained diploid or subdiploid numbers of chromosomes from both parents. The hybrids were evenly distributed over the three categories. After a 1-year culture of 24 representative hybrid callus lines on selection medium the karyotype of 10 hybrids remained stable, whereas 8 hybrids showed polyploidization of the genome of one parent, together with no or minor changes of the chromosome numbers of the other parent. Six hybrids showed slight changes in the hybrid karyotype. The elimination of chromosomes of a particular parent was not correlated to their metaphase location. The processes of spontaneous biparental chromosome elimination leading to the production of asymmetric hybrids of different categories are discussed.  相似文献   

19.
Both diploid and tetraploid experimental interspecific hybrids betweenRanunculus silerifolius (2x) andR. chinensis (2x) exhibit normal bivalent pairing. However, microspores of diploid hybrids do not undergo mitosis and their pollen grains are highly sterile, whereas tetraploid hybrids form good pollen grains after microspore division. Evidence is forwarded for the assumption thatR. cantoniensis (4x) has originated by hybridization between these two diploid parental species and by polyploidization of the diploid hybrids. Parallelisms between the different karyotypes ofR. cantoniensis (4x) andR. silerifolius (2x) suggest that the former is a species of polyphyletic origin.  相似文献   

20.
Polyploidy is known to be common in plants and recent work has focused on the rapid changes in genome structure and expression that occur upon polyploidization. In Arabidopsis, much of this work has been done on a synthetic allotetraploid obtained by crossing a tetraploid Arabidopsis thaliana (2= 4= 20) with A. arenosa (2= 4= 32). To explore an alternative route to polyploidy in this model species, we have developed a synthetic allopolyploid by crossing two diploid species: A. thaliana (2= 2= 10) and Arabidopsis lyrata subsp. petraea (2= 2= 16). F1 hybrids were easy to obtain and phenotypically more similar to A. lyrata. Spontaneous chromosome doubling events occurred in about 25% of the F1s, thus restoring fertility. The resulting allotetraploids (2= 26) exhibited many genomic changes typically reported upon polyploidization. Nucleolar dominance was observed as only the A. lyrata rDNA loci were expressed in the F1 and allotetraploids. Changes in the degree of methylation were observed at almost 25% of the loci examined by MSAP analysis. Finally, structural genomic alterations did occur as a large deletion covering a significant portion of the upper arm of chromosome II was detected but no evidence of increased mobility of transposons was obtained. Such allotetraploids derived from two parents with sequenced (or soon to be sequenced) genomes offer much promise in elucidating the various changes that occur in newly synthesized polyploids.  相似文献   

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