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101.
Three methods of increasing the productivity of somatic embryogenesis in Medicago sativa L. were investigated. In the basic procedure, somatic embryos were initiated from young petioles and carried through several phases: callus formation, suspension culture, selection of the embryogenic fraction by sieving, development, maturation, desiccation and storage. The suspensions were normally separated into three fractions by sieving. Fraction I (<200 m) containing nonembryogenic cells or cell clusters was discarded. Fraction II (200–500 m) consisting of embryogenic cell clusters was collected for embryo development and maturation. Fraction III (over 500 M) containing the mixture of petiole residues with large pieces of calli and globular somatic embryos was usually discarded. Several methods to scale-up the suspension phase were unsuccessful. Direct subculture of the entire suspension by the addition of fresh liquid medium resulted in the loss of embryogenic capacity by the third subculture. Subculture of fraction II decreased embryogenic cell mass, and hence reduced total productivity. The recycling of fraction III back to fresh B5g liquid medium resulted in high productivity in the first culture but further subculture of this fraction resulted in a rapid decline in the embryogenic capacity.As an alternative, somatic embryos from the first tissue culture cycle were also used as explants for the initiation of secondary embryogenic callus. The embryogenic capacity of these somatic embryo explants declined rapidly as they matured. More than 100 secondary somatic embryos could be induced from embryo explants removed from development medium at 10 days after sieving the suspension, but only 40 somatic embryos were produced from each mature somatic embryo explant, and 13 from desiccated embryos. The secondary somatic embryos were comparable to the primary embryos in quality according to germination tests. The implications of the results to the efficiency of somatic embryo production of Medicago are discussed.Abbreviations ABA
abscisic acid
- 2,4-d
2,4-dichlorophenoxyacetic acid
- DAS
days after sieving
- PPF
photosynthetic photon flux density
- SE
somatic embryo 相似文献
102.
Stephanie W. Watts Marlene L. Cohen† Patrick Q. Mooney† Bryan G. Johnson† Darryle D. Schoepp† Melvyn Baez† 《Journal of neurochemistry》1994,62(3):934-943
Abstract: Heterogeneity of the 5-hydroxytryptamine2 (5-HT2) receptor across species has been implicated in several pharmacological and physiological studies. Although 5-HT2 receptors in the rat have been linked to increases in Phosphoinositide (PI) hydrolysis, little evidence exists to support the association of guinea pig 5-HT2 receptors with Pl hydrolysis, the second messenger generally linked with 5-HT2receptors. In the present study, we have taken a molecular and biochemical approach to determining whether species differences in brain 5-HT2 receptors exist between rat and guinea pig. First, we isolated partial cortical 5-HTa receptor cDNA clones that encompassed the third intracellular loop, a receptor area putatively important in receptor-effector coupling. The amino acid sequences deduced from the cDNA clones for rat and guinea pig brain 5-HT2 receptor were 97% homologous. However, the guinea pig 5-HT2 receptor had two tandem substitutions that disrupted a potential alpha helix in the region of the third cytoplasmic loop, which theoretically could alter the intracellular coupling of the guinea pig cortical 5-HT2 receptor. Because of these molecular differences, we examined further the pharmacological activation of the brain 5-HT2 receptor from guinea pig. 5-HT and the 5-HT2 receptor agonist α-methyl-5-HT increased PI hydrolysis in guinea pig cortical slices whereas the 5-HT1c receptor agonist 5-methyltryptamine was significantly less potent. In addition, the 5-HT2 receptor antagonists LY53857, ketanserin, and spiperone blocked 5-HT-stimulated Pl hydrolysis. These pharmacological data suggested that activation of the 5-HT2 receptor in guinea pig cortical slices was associated with PI hydrolysis. Thus, although areas of the guinea pig brain 5-HT2 receptor that influence receptor-effector coupling were different from the rat, such differences were not critical to receptor-effector coupling because, as in the rat, guinea pig brain 5-HT2 receptors were also coupled to PI hydrolysis. 相似文献
103.
A protocol was developed combining non-radioactive in situ hybridization histochemistry with enzyme based immunohistochemistry, detect the expression of mRNA in phenotypically defined neurons. Freefloating brain sections were hybridized with the oligonucleotide probes which have been 3-end labelled with biotin-11-dUTP. The hybridized probe was visualized by a combined avidin-biotin bridge method, anti-avidin immunohistochemistry, and horseradish peroxidase detection using diaminobenzidine as a substrate. The in situ hybridization step yielded a very stable reaction product enabling subsequent immunohistochemical reactions using horseradish peroxidase and benzidine dihydrochloride as a chromogen. Magnocellular neurons of the hypothalamo-neurophypophysial system synthesize either vasopressin or oxytocin; water deprivation and chronic saline ingestion are potent stimuli for the expression of both of the genes encoding these neuropeptides. A number of other neuropeptides with putative transmitter action are synthesized in magnocellular neurons during such stimulation. Experiments were performed to explore whether neuropeptide Y immunoreactivity is present within magnocellular vasopressin mRNA-expressing neurons of the hypothalamo-neurophypophysial system. The results clearly demonstrated that neuropeptide Y-immunoreactive elements were present within a number of magnocellular vasopressin mRNA-containing cells. In addition, immunohistochemical detection of the neuropeptides ocytocin and cholecystokinin was carried out on sections hybridized non-radioactively for vasopressin; as expected vasopressin mRNA did not co-exist with cholecystokinin, whereas a few oxytocin immunoreactive neurons in osmotically stimulated animals also contained vasopressin mRNA. The developed method makes possible the immunohistochemical detection of intracellular antigens with concomitant detection of intracellular mRNA. 相似文献
104.
A comparison of three packaging techniques using two extenders for the cryopreservation of canine semen 总被引:1,自引:0,他引:1
The progressive motility of frozen-thawed canine semen was used as a criterion to compare methods of semen cryopreservation. Twenty-one ejaculates from 7 dogs were frozen in 2 extenders, Tris-citrate (TC) and BES-lactose (BL), in each of 3 packaging techniques (pellets, 0.5-ml, and 2.5-ml straws). Duplicate samples were frozen on dry ice (pellets) or in liquid nitrogen vapor (straws). Least squares means for the percentage of post-thaw progressive motility (PTPM) were greater for TC (33.2 ± 1.7) than for BL (20.9 ± 1.7; P<0.0001). Freezing in pellets (PTPM = 34 ± 2.3) resulted in greater PTPM than freezing in either 0.5-ml (24.7 ± 1.6) or 2.5-ml (22.1 ± 2.3) straws (P<0.001). The percentage of PTPM of spermatozoa frozen in TC pellets was greater than that in TC 2.5-ml straws or in BL in any packaging method (P<0.05). The percentage of PTPM of spermatozoa from semen extended in BL was greater in pellets than in 0.5 or 2.5-ml straws (P<0.05). 相似文献
105.
Kai Larsen 《Nordic Journal of Botany》1993,13(4):403-404
As a precursor for the treatment of Caesalpiniaceae in Flora Malesiana the following new combinations are proposed: Cassia javanica ssp. pubiflora stat. nov., C. javanica ssp. agnes stat. nov., C. javanica ssp. microcalyx stat. nov. C. javanica ssp. renigera stat. nov., C. javanica ssp. bartonii stat. nov., Senna divaricata comb. nov., Chamae-christa pumila comb. nov. and Chamaechrista mindanaensis comb. nov. 相似文献
106.
Gregory M. L. Patterson Kathleen K. Baker Cynthia L. Baldwin Christine M. Bolis Faith R. Caplan Linda K. Larsen Ira A. Levine Richard E. Moore E. Moore Carrie S. Nelson Kathryn D. Tschappat Grace D. Tuang Michael R. Boyd John H. Cardellina Ralph P. Collins Kirk R. Gustafson Kenneth M. Snader Owen S. Weislow Ralph A. Lewin 《Journal of phycology》1993,29(1):125-130
Lipophilic and hydrophilic extracts from approximately 600 strains of cultured cyanophytes, representing some 300 species, were examined for antiviral activity against three pathogenic viruses. Approximately 10% of the cultures produced substances that caused significant reduction in cytopathic effect normally associated with viral infection. The screening program identified the order Chroococcales as commonly producing antiviral agents. 相似文献
107.
108.
The effective molecular weight cut-off values of dialysis membranes for carrageenan and alginate oligosaccharides were evaluated by gel permeation chromatography and nuclear magnetic resonance spectroscopy. For the different membranes tested, i.e. Medicell, Spectra Por 1000D and 3500D, the porous sizes are analogous to tri- and tetrasaccharides. A simple dialysis can be used to recover the majority of the oligosaccharides produced by a carrageenase or an alginate lyase digestion. 相似文献
109.
Biosynthesis of alginate in algae may be studied by following the cell wall regeneration of brown seaweed protoplasts in culture. The enzyme mannuronan C-5 epimerase will control the composition of the alginate being synthetized.Freshly isolated protoplasts from the thallus of young Laminaria digitata plants showed only low expression of this enzyme. However, after prolonged periods in culture, this activity increased 15-fold. The synthesis of C-5 epimerase by the protoplasts is probably essential for the formation of a new cell wall.After cellular disruption by osmotic shock and centrifugation, most of the epimerase activity resided in the pellet fraction. This may indicate that the enzyme is membrane associated. 相似文献
110.
Summary We have improved zygote recovery 11–1,000 fold by optimizing the physiology of gamete release and mating inAcetabularia acetabulum. Gamete release was affected by agar purity, concentration, and volume/gametangial pair. Cold pre-treatment of gametangia (14–30 d at 10°C in the dark) synchronized subsequent gamete release at 21°C in the light. Cold pre-treatment was nearly twice as effective in synchronizing subsequent gamete release when intact, gametangia-bearing caps rather than isolated gametangia were pretreated. Synchronizing gamete release doubled mating efficiency. In a wild-type laboratory strain ofA. acetabulum, there were 1,561±207 gametes/gametangium which had half-lives of 14.5 d in 0.1% seawater-agar. We recovered 48–93% of the expected numbers of zygotes from a mass mating of 8 to 1,226 gametangia and 11–128% of the expected numbers of zygotes from mating single gametangial pairs: the large range in the calculated mating efficiency may be attributable to the variation in the numbers of gametes made per gametangium. Zygote recovery from single gametangial pairs was highly dependent on the volume of mating matrix. In addition, most zygotes recovered were unattached to any other zygotes in the subsequent generation (> 95% single cells from matings of 1–500 gametangial pairs). Our improvements in mating conditions and zygote recovery (1) have facilitated cell manipulation and culture ofA. acetabulum in the laboratory; and (2) have made controlled crosses for selection and genetic analysis of mutants feasible. These advances have removed a major barrier to genetic analysis of development inAcetabularia.Abbreviations LB
Luria-Bertani bacteriological broth
- SE
standard error of the mean
- Tg
agar gelling temperatures
- DAPI
4,6-diamidino-2-phenylindole 相似文献