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1.
水稻原生质体产生细胞团的冰冻保存和冻后再生植株形成 总被引:4,自引:0,他引:4
水稻(Oryza sativa L.)原生质体产生的细胞团加上10-20%的二甲亚枫(DMSO)和10-20%的蔗糖,置于液氮中保存。冻后细胞生存率达到对照的40-50%。存活的细胞在附加2×10~(-5)mol/l 2,4-D 的Linsmier-Skoog(Ls)固体培养基上再生长,然后将形成的愈伤组织块转到附加10~(-6)mol/l NAA,4×10~(-6)mol/l 激动素和10~(-6)mol/l 2 IP 及8%的蔗糖的 LS培养基上分化出芽并形成植株。 相似文献
2.
O-Methylthreonine (OMT) inhibits the growth of plated Rosa cells (ID506·10-6M). Isoleucine is able to reverse efficiently and specifically this OMT toxicity. From OMT-resistant colonies occurring at a frequency of 1.58·10-7 variants per cell plated at 10-4M OMT, the variant strains OMTR-1 and OMTR-2 were isolated, cloned via protoplasts and characterized. Both variants were ten times more resistant to OMT than the wildtype and were cross-resistant to another isoleucine analog, dl-4-thiaisoleucine. The resistant variants retained their resistance after storage for three years in liquid nitrogen. Both resistant strains were stable for several months when subcultured in the absence of OMT although it was shown in a reconstitution experiment that wildtype cells overgrow OMTR-2 variant cells if co-cultivated for many passages in drug-free medium. One case of instability was observed upon long-term subculturing in drug-free medium: the strain OMTR-1D* partially lost phenotypic properties. Resistance to OMT was followed qualitatively by a new method based on inhibition-zone formation in cell suspensions plated in agar medium. The OMT-resistant variants showed a reduction in sensitivity of the enzyme l-threonine deaminase to feedback inhibition by isoleucine, a decreased stability of l-threonine deaminase when stored at-18°C or incubated at +55°C and a two- to threefold increase of the free isoleucine pool within the cells. The genetical events and the biochemical mechanisms which might lead to the observed stable and biochemically defined character are discussed with particular reference to the high ploidy level of the Rosa cell line.Abbreviations OMT
l-O-methylthreonine
- TD
l-threonine deaminase 相似文献
3.
M. M. H. Kristensen J. I. Find F. Floto J. D. MØller J. V. NØrgaard P. Krogstrup 《Protoplasma》1994,182(1-2):65-70
Summary The development of somatic embryos in an embryogenic suspension culture ofPicea sitchensis was followed every day for two weeks after thawing from liquid nitrogen (LN2). Only a few cells, primarily located at the periphery of the embryonic region of the embryos, survived cryopreservation in LN2. Surviving cells were classified into two groups: embryogenic cells (EC) and non-embryogenic cells (NEC), based on their morphology and embryogenic competence. The dense cytoplasmic EC underwent organized growth and differentiation with first divisions occurring after 24 h, and embryo formation 6–8 days after thawing from LN2. No evidence of asymmetrical divisions or free-nuclear stages was found during somatic embryo formation. NEC had less dense cytoplasm with numerous small vacuoles. One to five days after thawing the NEC became progressively more vacuolated and elongated. Histological examination revealed no mitotic activity in NEC, and six days after thawing NECs were seen as single cells or unorganized cell aggregates. Two weeks after thawing the appearance of the cryopreserved cultures was comparable to that of the untreated cultures.Abbreviations EC
embryogenic cells
- ECC
embryogenic cell clusters
- FDA
fluorescein diacetate
- GMA
glycol methacrylate
- LN2
liquid nitrogen (–196°C)
- NEC
non-embryogenic cells 相似文献
4.
Jie Su Caiyun Wang Yongli Song Yanyan Yang Guifang Cao 《Asian-Australasian Journal of Animal Sciences》2022,35(9):1351
ObjectiveThe objective of this study was to analyse the differentially abundant proteins caused by freeze–thawing of ram sperm and explore candidate proteins of interest for their ability to improve ram sperm cryopreservation outcomes in vitro.MethodsSperm were from three mature Dorper. Fresh and frozen sperm proteins were extracted, and the differentially abundant proteins were analysed by mass spectrometry. Among these proteins, lactoferrin (LTF) was selected to be added before cryopreservation. Next, sperm samples were diluted in Tris extender, with the addition of 0, 10, 100, 500, and 1,000 μg/mL of LTF. After thawing, sperm quality was evaluated by motility, plasma membrane integrity, mitochondrial activity and reactive oxygen species (ROS).ResultsCryopreservation significantly altered the abundance of 40 proteins; the abundance of 16 proteins was increased, while that of 24 proteins was decreased. Next, LTF was added to Tris extender applied to ram sperm. The results showed that sperm motility and plasma membrane integrity were significantly improved (p<0.05) by supplementation with 10 μg/mL LTF compared to those in the control group. There was no significant difference in mitochondrial activity between the 0 μg/mL group and other groups (p>0.05). Supplementation of the cryoprotective extender with 10 μg/mL LTF led to decreased ROS levels compared with those in the control and other groups (p<0.05).ConclusionThe LTF is an important protein during cryopreservation, and the addition of 10 μg/mL LTF to a cryoprotective extender can significantly improve the function of frozen ram sperm. 相似文献
5.
Suk Young Oh Chun Hua Wu Elena Popova Eun Joo Hahn Kee Yoeup Paek 《Journal of Plant Biology》2009,52(4):348-354
We tested desiccation and/or vitrification procedures to cryopreserve the adventitious roots of Panax ginseng, the source of commercially produced ginsenosides. When only desiccation was applied, the post-freeze survival of 3- to 4-mm
root tips was <14% regardless of the composition of the preculture medium or the explant origin. Callus formation was frequently
observed after cryopreservation. In contrast, 90% survival and 32.5% root formation efficiency were achieved after cryopreservation
when a vitrification protocol was followed. Adventitious root cultures in flasks and bioreactors were reestablished from root
tips cryopreserved by vitrification. A prolonged lag-phase and lower biomass production were recorded in post-freeze-regenerated
cultures compared with control roots that were subcultured four times in flasks. However, biomass accumulations did not differ
between control and regenerated roots at the end of the sixth subculturing period. After 40 days of culture in bioreactors,
a mean value of 12.5 g dw L−1 was recorded for post-freeze-regenerated cultures versus 9.1 g dw L−1 for the control roots. Production of triol and diol ginsenosides in our bioreactor cultures also was enhanced after cryopreservation,
by 41.0% and 89.8%, respectively. These results suggest that the vitrification method is successful for cryopreservation of
P. ginseng adventitious roots. 相似文献
6.
以君迁子(Diospyros lotus L.)和柿(D.kaki Thunb.)组培苗茎尖为试材,对影响超低温保存效果的主要因素,如低温锻炼方式、预培养条件、PVS:(30%甘油+15%乙二醇+15%二甲基亚砜+0.4mol/L蔗糖)处理时间等进行了研究。建立了2种柿属植物的超低温保存程序:(1)切取1cm左右试管苗梢段继代到1/2MS(KNO3和NH4NO3减半)培养基中,交替低温[昼(25±1)℃、夜(4±1)℃]锻炼6周;在含0.5mol/L蔗糖的1/2MS培养基上预培养5d,20℃下装载液(2.0mol/L甘油+0.4mol/L蔗糖)过渡10min,0℃下PVS2处理1.5h;(2)投入液氮保存;(3)40℃水浴化冻,洗涤5~6次后接种于含1.0mg/LTDZ、0.6g/L可溶性PVP、30g/L蔗糖和7.0g/L琼脂的培养基(作者在优化柿属植物离体培养体系试验中获得)上暗培养1周,转入25℃,1500lx培养室。按照上述程序培养,‘鄂柿1号’、‘湘西甜柿’和君迁子的成活率分别为79.6%、67.4%和60.9%。 相似文献
7.
Dovgan Barbara Miklavčič Damijan Knežević Miomir Zupan Janja Barlič Ariana 《Cytotechnology》2021,73(3):391-411
Cytotechnology - Trehalose is a nontoxic disaccharide and a promising cryoprotection agent for medically applicable cells. In this study, the efficiency of combining trehalose with reversible... 相似文献
8.
Alejandro Córdova Rocío Hernández-Gil Cristian Alejandro Córdova-Jiménez Diogo José Cardilli Kellen de Sousa Oliveira José Félix Pérez-Gutiérrez 《Reproductive biology》2013,13(2):166-168
The effect of post-thaw incubation (0 vs. 5 h at 15 °C) and straw size (5 vs. 0.5 ml) on motility, acrosomal integrity and in vitro fertilizing (IVF) capacity of cryopreserved boar spermatozoa was studied. In samples assessed immediately after thawing, no differences were found between the two straw sizes. After 5 h post-thaw incubation, all parameters, except polyspermy, decreased and, spermatozoa packaged in 5 ml straws showed better functional and IVF parameters than these in 0.5 ml straws. 相似文献
9.
《Cryobiology》2020
Improving aspects of platelet cryopreservation would help ease logistical challenges and potentially expand the utility of frozen platelets. Current cryopreservation procedures damage platelets, which may be caused by ice recrystallization. We hypothesized that the addition of a small molecule ice recrystallization inhibitor (IRI) to platelets prior to freezing may reduce cryopreservation-induced damage and/or improve the logistics of freezing and storage. Platelets were frozen using standard conditions of 5–6% dimethyl sulfoxide (Me2SO) or with supplementation of an IRI, N-(2-fluorophenyl)-d-gluconamide (2FA), prior to storage at −80 °C. Alternatively, platelets were frozen with 5–6% Me2SO at −30 °C or with 3% Me2SO at −80 °C with or without 2FA supplementation. Supplementation of platelets with 2FA improved platelet recovery following storage under standard conditions (p = 0.0017) and with 3% Me2SO (p = 0.0461) but not at −30 °C (p = 0.0835). 2FA supplementation was protective for GPVI expression under standard conditions (p = 0.0011) and with 3% Me2SO (p = 0.0042). Markers of platelet activation, such as phosphatidylserine externalization and microparticle release, were increased following storage at −30 °C or with 3% Me2SO, and 2FA showed no protective effect. Platelet function remained similar regardless of 2FA, although functionality was reduced following storage at −30 °C or with 3% Me2SO compared to standard cryopreserved platelets. While the addition of 2FA to platelets provided a small level of protection for some quality parameters, it was unable to prevent alterations to the majority of in vitro parameters. Therefore, it is unlikely that ice recrystallization is the major cause of cryopreservation-induced damage. 相似文献
10.
《Cryobiology》2020
Mesenchymal stromal/stem cells (MSCs) derived from bone marrow, umbilical cord and especially adipose tissue are increasingly being explored for their therapeutic potential to treat a wide variety of diseases. A prerequisite for most allogeneic off-the-shelf and some autologous MSC therapies is the ability to safely and efficiently cryopreserve cells during production or for storage prior to treatment. Dimethyl sulfoxide (Me2SO) is still the commonly used gold standard cryoprotectant (CPA). However, undesirable cellular impacts and side effects of Me2SO have led to an increasing demand for the development of safe and effective alternatives.This study investigated the effect of pentaisomaltose as a CPA for cryopreservation of adipose-derived stromal/stem cells (ASCs). We compared pentaisomaltose-based freezing media containing 1% Me2SO (PIM1) or 2% Me2SO (PIM2) to our in-house freezing media formulation containing 10% Me2SO (STD10) and to CryoStor freezing media containing 2% or 10% Me2SO (CS2 and CS10). We assessed the recovery of viable ASCs, their phenotype, differentiation potential, proliferation potential, and migratory potential. Further, their immunomodulatory potential was assessed by measuring their ability to suppress T cell proliferation and express immunomodulatory markers.The results showed that the post-thaw viability of ASCs cryopreserved with STD10, CS10 and PIM2 was improved compared to that of CS2. The recovery of ASCs with PIM1 and PIM2 was also improved compared to that of CS2. Proliferation and migration were comparable among the tested freezing media. The results showed no difference in the induction of PDL1, PDL2 or IDO1 expression. Nevertheless, the potential of cryopreserved ASCs to suppress T cell proliferation was reduced when the Me2SO concentration was reduced (CS10>STD10>CS2 and PIM2>PIM1).Altogether, the migratory and immunomodulatory potential combined with improved recovery indicate that the addition of pentaisomaltose in the freezing media may allow for the reduction of the Me2SO concentration to 2% while retaining a more potent cell product that what is recovered using comparable freezing media. With the desire to reduce the amount of Me2SO, these results suggest that 2% and potentially even 1% Me2SO in combination with 10% pentaisomaltose could be an effective and less toxic alternative to comparable freezing media. 相似文献