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近年来,对于人参及其他药用植物的组织培养做了不少工作,但是对愈伤组织细胞的特点研究的较少。本工作是为配合人参细胞培养进行的。通过比较观察,研究人参愈伤组织细胞亚显微结构与愈伤组织形态特征之间的关系。材料与方法材料取自5年生人参鲜根和茎切段诱导的愈伤组织。每隔30—40天转移一次进行继代培养。根愈伤组织经16次继代培养,茎经6次继代培养。转移前取样。培养基 Ms,补加0.5 mg/1 2,4-D。将供试愈伤组织按其形态特征分为5种类型。1淡黄白色,新鲜, 相似文献
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苏云金杆菌内毒素蛋白基因转入葡萄胚性愈伤组织细胞及转基因植株再生的研究 总被引:5,自引:0,他引:5
以葡萄的胚性愈伤组织作为农杆菌介导,Ti质粒转化材料,利用共培养法将苏云金杆菌内毒素蛋白基因转入葡萄胚性愈伤组织细胞,通过胚状体发生途径再生转基因植株。实验发现:80μmol/L的乙酰丁香酮诱导处理农杆菌和葡萄愈伤组织后可将转化效率提高50倍。OD值为0.8的农杆菌菌液稀释8—10信后与在G培养基预培养10天的胚性愈伤组织共培养2—3夭,Ti质粒对葡萄愈伤组织细胞的转化效率可达50%左右。筛选得到的转基因植株在含Km 30 mg/L的选择培养基上继代存活6个月,生长正常;提取叶片染色体DNA做Southern blot,杂交结果为阳性。将转基因植株各部分切段置于含Km 50 mg/L的选择培养基上,能够脱分化产生抗性愈伤组织并能增殖。 相似文献
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麻栎茎段体胚发生和组织学观察初报 总被引:2,自引:0,他引:2
廖婧;方炎明;虞木奎 《植物研究》2011,31(5):575-578
分别于2010年4月和6月采集同一麻栎母株的成熟茎段,在培养基MS+6-BA 1 mg·L-1+IBA 1 mg·L-1+谷氨酰胺1 000 mg·L-1+脯氨酸500 mg·L-1上诱导体胚发生,选出较好的外植体采集时间。通过组织学和扫描电镜观察,研究麻栎体胚的起源和愈伤组织结构特征。结果表明,外植体较好的采集时间为4月。组织切片表明,麻栎体胚具有三种不同的起源方式,起源于愈伤组织内部、表皮或者初生胚性复合体表面;胚性愈伤组织的细胞较小,细胞质浓厚,染色较深,和非胚性组织细胞明显不同。扫描电镜(SEM)观察表明,胚性愈伤组织细胞呈球形,大小均一,多以细胞团形式存在;非胚性愈伤组织细胞无规则形状,细胞间隙较多,大多分散存在,这些可以作为区分两类愈伤组织的典型特征。 相似文献
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玉米未成熟胚乳愈伤组织和器官的发生及其倍性变化的初步研究 总被引:2,自引:1,他引:1
本试验在附加和不附加外源激素的MS培养基上,均得到了玉米未成熟胚乳愈伤组织。愈伤组织在附加外源激素的MS培养基上达到器官分化,获得了发育正常的和许多畸形的胚乳植株。所得到的愈伤组织细胞和植株根尖细胞染色体数目和倍性是不稳定的,二者有相同的趋向,其中有整倍体的细胞(2n=10,20,30,40,50),也有各种非整倍体的细胞(2n=5—49)。 相似文献
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通过研究接种量、激素配比、糖浓度、培养基种类对巫山淫羊藿悬浮培养细胞生长及其愈伤组织黄酮类含量的影响,建立了巫山淫羊藿细胞悬浮培养的技术体系.结果表明:巫山淫羊藿愈伤组织细胞悬浮培养在B5基本培养基中并附加1.0 mg·L-12,4-D和0.2 mg· L-1BA,蔗糖浓度40 g·L-1,接种量每30 mL为鲜重2 ... 相似文献
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丝裂霉素C诱发中国仓鼠体外培养细胞姐妹染色单体互换的研究 总被引:1,自引:0,他引:1
本文对不同年龄中国仓鼠各种组织的体外培养细胞姐妹染色单体互换(SCE)频率进行了比较研究。实验结果表明,体外培养细胞的自发SCE频率与动物年龄无关,相同组织细胞经MMC处理后,老龄仓鼠SCE频率比幼龄仓鼠SCE频率明显低。心脏和皮肤细胸的SCE频率高于肺和尾的SCE频率。 相似文献
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白皮松和油松雌配子体愈伤组织的诱导和分化 总被引:11,自引:0,他引:11
以白皮松(Pinus bungeana Zucc.)和油松(P.tabulaeform is Carr.)的未成熟胚乳,即雌配子体为外植体进行培养,将雌配子体分别接种在添加不同激素种类和不同浓度配比的改良MS培养基上诱导愈伤组织。经过20多天的培养,在含有1—6 m g/L 萘氧乙酸(NOA)和0.5m g/L6-BAP及3% 蔗糖浓度的培养基上诱导产生了愈伤组织,愈伤组织的诱导频率最高为25% 。经细胞学观察证明:愈伤组织细胞确为单倍性的,染色体数目为n= 12,正常的体细胞染色体数目为2n= 24,并在含有ABA 的原诱导愈伤组织培养基上分化出绿色小芽 相似文献
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Detection of bromodeoxyuridine incorporation in mammalian chromosomes by a bromodeoxyuridine antibody 总被引:1,自引:0,他引:1
A commercially available bromodeoxyuridine (BrdUrd) antibody was used to demonstrate sister chromatid differentiation (SCD) and to evaluate sister chromatid exchanges (SCEs) in V79 Chinese hamster cells. V79 cells were cultivated for one cell cycle in the presence of BrdUrd, followed by a second cell cycle in the absence of BrdUrd. Chromosome preparations were stained by a common immunologic staining technique. The staining pattern observed is similar to that after FPG (fluorescent plus Giemsa) staining, though with reverse staining specificity. The sensitivity of BrdUrd detection is enhanced by a factor of 20 compared to the FPG technique and thus allows the evaluation of SCEs at very low BrdUrd concentrations. The application of the antibody technique gives information about the origin and localization of SCEs and produces further evidence for the spontaneous occurrence of SCEs. 相似文献
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This report describes staining techniques for chromosome banding and sister chromatid exchanges (SCEs) suited to a method that allows simultaneous analysis of cell morphology and karyotype. Mitotic cells are first identified by either cytochemical staining or immunologic methods. The preparations are then destained and treated with acid fixative. For G- and C-banding, the cells are incubated overnight at room temperature in S?orensen buffer and then stained with Giemsa. To demonstrate SCEs, the cells are fluorescent stained before being stained with Giemsa. 相似文献
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Presence of a Base Line Level of Sister Chromatid Exchanges in Somatic Cells of DROSOPHILA MELANOGASTER IN VIVO and IN VITRO
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Hideo Tsuji 《Genetics》1982,100(2):259-278
Sister chromatid exchanges (SCEs) under in vivo and in vitro conditions were examined in ganglion cells of third-instar larvae of Drosophila melanogaster (Oregon-R). In the in vivo experiment, third-instar larvae were fed on synthetic media containing 5-bromo-2'-deoxyuridine (BrdUrd). After two cell cycles, ganglia were dissected and treated with colchicine. In the in vitro experiment, the ganglia were also incubated in media containing BrdUrd for two cell cycles, and treated with colchicine. SCEs were scored in metaphase stained with Hoechst 33258 plus Giemsa. The frequencies of SCEs stayed constant in the range of 25-150 micrograms/ml and 0.25-2.5 micrograms/ml of BrdUrd in vivo and in vitro, respectively. SCEs gradually increased at higher concentrations, strongly suggesting that at least a fraction of the detected SCEs are spontaneous. The constant levels of SCE frequency were estimated, on the average, at 0.103 per cell per two cell cycles for females and 0.101 for males in vivo and at 0.096 for females and 0.091 for males in vitro. No difference was found in the SCE frequency between sexes at any of the BrdUrd concentrations. The analysis for the distribution of SCEs within chromosomes revealed an extraordinarily high proportion of the SCEs at the junctions between euchromatin and heterochromatin; the remaining SCEs were preferentially localized in the euchromatic regions of the chromosomes and in the heterochromatic Y chromosome. These results were largely inconsistent with those of Gatti et al. (1979). 相似文献
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Germinated seeds ofVicia faba were continuously irradiated at low dose rate of gamma rays (0.05 Gy h-1) up to a total accumulated dose of 2 Gy. The FPG (fluorescence plus Giemsa) technique of differential chromatid staining
was used to monitor the frequency of sister chromatid exchanges (SCEs) in irradiated root tip meristem cells. The results
of the experiments have demonstrated that SCE frequency is raised by continuous gamma irradiation only in plant cells containing
BrdU in the chromosomal DNA. No effect concerning SCE formation was recorded at continuous irradiation of meristematic cells
of Vicia faba with native, i. e. BrdU-nonsubstituted, DNA. In contrast to SCEs, a significant increase was found in the yield
of chromosomal aberrations in all variants of irradiation. 相似文献
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The DNA lesions responsible for the formation of sister chromatid exchanges (SCEs) have been the object of research for a long time. SCEs can be visualized by growing cells for either two rounds of replication in the presence of 5-bromo-2'-deoxyuridine (BrdU) or for one round with BrdU and the next without. If BrdU is added after cells were treated with a DNA-damaging agent, the effect on SCEs can only be analyzed in the second post-treatment mitosis. If one wishes to analyze the first post-treatment mitosis, cells unifilarily labeled with BrdU must be treated. Due to the highly reactive bromine atom, BrdU interacts with such agents like ionizing and UV radiation enhancing the frequency of SCEs. However, its precise role in this process was difficult to assess for a long time, because no alternative technique existed that allowed differential staining of chromatids. We have recently developed a method to differentially label sister chromatids with biotin-16-2'-deoxyuridine-5'-triphosphate (biotin-dUTP) circumventing the disadvantage of BrdU. This technique was applied to study the SCEs induced by ionizing and UV radiation as well as by mitomycin C, DNaseI and AluI. This article is a review of the results and conclusions of our previous studies. 相似文献
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Differential staining of sister chromatids with Giemsa after BrdU incorporation into DNA was performed in Allium cepa L. chromosomes. A treatment solution containing 10–7 M FdU, 10–4 M BrdU and 10–6 M Urd was found to ensure BrdU incorporation without affecting cell cycle duration. After several procedures before staining the slides with Giemsa had been tested, treatment with the fluorochrome compound 33258 Hoechst, exposure to UV light and heating at 55° C in 0.5×SSC, were found to be essential for good differentiation. The distribution of SCEs per chromosome agrees with the expected Poisson distribution. The mean value of SCEs per chromosome occurring when cells were exposed to the treatment solution for two consecutive rounds of replication (=5.5) was double the mean value observed when cells were exposed to the same treatment for only one round of replication (=2.8). SCEs were found to occur more frequently in those chromosome regions corresponding neither to C-bands nor to late replicating DNA-rich regions. Finally, the occurrence of SCEs involving less than the width of a chromatid is discussed. 相似文献
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The cell killing and induction of sister-chromatid exchanges (SCEs) by X-rays and short-wave ultraviolet (UV) irradiation in combination with inhibitors of DNA repair, 3-aminobenzamide (3AB), cytosine arabinoside (ara-C) or aphidicolin (APC) were studied in wild-type CHO-K1 and two X-ray-sensitive mutants, xrs 5 and xrs 6 cells. The spontaneous frequency of SCEs was similar in the mutants and the wild-type CHO-K1 cells (8.4-10.3 SCEs/cell). Though X-rays are known to be poor inducers of SCEs, a dose-dependent increase in the frequency of SCEs in xrs 6 cells (doubling at 150 rad) was found in comparison to a small increase in xrs 5 and no increase in wild-type CHO-K1 cells. 3AB, an inhibitor of poly(ADP-ribose) synthetase increased the spontaneous frequency of SCEs in all the cell types. 3AB did not potentiate the X-ray-induced frequency of SCEs in any of the cell lines. Ara-C, an inhibitor of DNA polymerase alpha, increased the frequency of SCEs in all the cell lines. In combined treatment with X-rays, ara-C had no synergistic effect in xrs 5 and xrs 6 cells, but the frequency of SCEs increased in X-irradiated wild-type CHO-K1 cells post-treated with ara-C. For the induced frequency of SCEs, CHO-K1 cells treated with X-rays plus ara-C behaved like xrs 6 cells treated with X-rays alone, suggesting a possible defect in DNA base damage repair in xrs 6 cells, in addition to the known defective repair of DNA double-strand breaks (DSBs). Survival experiments revealed higher sensitivity of xrs 5 and xrs 6 mutant cells to the cell killing effect of X-rays in S-phase when compared to wild-type CHO-K1 cells. The mutants responded with lesser sensitivity to cell killing effect of ara-C and APC than CHO-K1 cells, the relative sensitivity to ara-C or APC being CHO-K1 greater than xrs 5 greater than xrs 6 cells. When X-irradiation was coupled with ara-C, the results obtained for survival were similar to those of the SCE test, i.e., unlike wild-type CHO-K1, no synergistic effect was observed in xrs 5 or xrs 6 cells. After UV-irradiation, the frequency of SCEs increased similarly in wild-type CHO-K1 and xrs 6 cells, but xrs 5 cells responded with lower frequency of SCEs.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
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The influence of low doses of 5-bromodeoxyuridine (BrdU) on the occurrence of sister chromatid exchanges (SCEs) during the first cell cycle, when unsubstituted DNA templates replicate in the presence of the halogenated nucleoside (SCE1) has been assessed in third mitosis (M3) Chinese hamster ovary (CHO) cells showing three-way differential (TWD) staining. In addition, lower concentrations of BrdU, not detectable by Giemsa staining, have been tested by a high resolution immunoperoxidase method (anti-BrdU monoclonal antibody) and SCEs were scored in second mitosis (M2) cells. Our findings was a dose-response curve for SCE1 that allows an estimated mean spontaneous yield of 1.32/cell per cell cycle by extrapolation to zero concentration of BrdU. On the other hand, when the total SCE frequency corresponding to the first and second rounds of replication (SCE1+SCE2) found in M3 chromosomes was compared with the yield of SCEs scored in M2 cells grown in BrdU at doses lower than 1 M no further reduction was achieved. This seems to indicate that SCEs can occur spontaneously in this cell line, though the estimated frequency is higher than that reported in vivo.by S. Wolff 相似文献
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Chinese hamster lung embryonic cells (CL1) were treated with colchicine in order to induce endoreduplication and subsequently
with mitomycin-C (MMC) to induce exchanges within the diplochromosome. The use of chromosomal differential staining through
incorporation of 5-bromodeoxyuridine, resulting in only one stained chromatid, has allowed the analysis of all classes of
exchanges among the four chromatids of the diplochromosome. Three classes of exchanges may occur: intradiplochromatid exchanges
(ICEs) between the two inner chromatids, cousin chromatid exchanges (CCEs) between one inner and one outer chromatid, and
sister chromatid exchanges (SCEs) between the two sister chromatids of the diplochromosome. The results show that MMC treatment,
in the last cell cycle of endoreduplication, as expected, significantly increases only the frequency of SCEs, whereas the
frequency of ICEs and CCEs remains unchanged. This result supports replication models of formation of SCEs. Furthermore the
fact that the number of ICEs does not increase means that the molecular mechanism of somatic crossing over is not related
to that of SCE formation, or very rarely. The results also indicate a statistically significant lower induction of SCEs in
endoreduplicated metaphases as compared with diploid ones both in control and MMC-treated cells. Such a result may be due
to structural restrictions within the diplochromosome.
Received: 29 December 1995; in revised form; 4 March 1996 / Accepted: 24 March 1996 相似文献
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The present study was carried out in order to analyze how persistent the lesions in DNA are which elicit sister-chromatid exchanges (SCEs), induced by three different chemical agents, mitomycin C (MMC), 4-nitroquinoline-1-oxide (4NQO) and ethyl methanesulfonate (EMS), in proliferating human lymphocytes. Cells were exposed to the mutagens for 1 h just before starting bromodeoxyuridine substitution and SCEs were examined in third-cycle metaphases showing three-way-differential staining, by means of our previously standardized method. The results show that, in spite of the fact that these three compounds have different modes of action, the lesions induced by all of them seem to be capable of persisting in DNA and eliciting SCEs for at least three successive cell cycles. 相似文献