红豆草抗羟脯氨酸变异系原生质体培养后愈伤组织的抗性特征

通过ＮａＮ３诱变得到的红豆草抗羟脯氨酸变异系,在酶液中游离原生质体进行培养,获得再生植株。在含不同浓度ＮａＣｌ,羟脯氨酸或ＰＥＧ的ＭＳ培养基上,原生质体来源抗性愈伤组织中的游离脯氨酸含量在１周之内均急剧增加,随后开始下降,３周后接近正常水平。随着胁迫程度的提高,抗性愈伤组织中游离脯氨酸含量呈递增趋势,生长速度呈递减趋势。     

红豆草抗羟脯氨酸变异系的筛选及其特性研究

通过选择-NaN_3诱变处理-再选择的程序从红豆草下胚轴愈伤组织获得了抗脯氨酸类似物L-羟脯氨酸的变异细胞系。抗性变异细胞系的游离脯氨酸含量比正常细胞系高4.6倍,并且抗性变异系亦表现出了很强的耐NaCl、PEG 能力。这可能为筛选抗逆境植物提供一条有效途径。     

大麦籽粒及花药愈伤组织的游离氨基酸含量分析

对同一基因型大麦花药愈伤组织与籽粒中的游离氨基酸含量尤其是赖氨酸含量之间的关系进行分析,结果表明,对同一基因型而言,愈伤组织及籽粒中的同一氨基酸含量高低具有相同趋势。同时着重分析了愈伤组织中游离脯氨酸的含量与绿苗分化的关系,结果表明游离脯氨酸含量高的愈伤组织,其绿苗分化率较高,说明脯氨酸对绿苗分化具有重要作用。966259的花药愈伤组织及籽粒中的游离氨基酸总含量及游离赖氨酸含量均最高。     

细胞壁羟脯氨酸和游离脯氨酸与棉花对枯萎病抗性的关系

氟乐灵处理能推迟棉苗枯萎病症状的出现、降低发病株率和病情指数,说明氟乐灵处理能提高棉酋对枯萎病的抗性。在健康棉苗中,抗病品种（中棉１２）细胞壁结合的羟脯氨酸含量显著高于感病品种（６０３７）,但游离脯氨酸含量无明显差异。在氟乐灵处理和未处理而接种棉枯萎病菌的两个处理中,两个品种的细胞壁结合的羟脯氨酸和游离脯氨酸含量均高于各自的健康株对照,而且抗病品种中的含量都高于感病品种,说明细胞壁结合的羟脯氨酸和游禹脯氨酸的积累是氟乐灵诱发的抗病性表现及棉苗受枯萎病菌侵染后抗性反应的生化机制之一。     

单倍性烟草Hyp抗性愈伤组织变异系的选择及其生理生化特性的研究Ⅱ．Hyp抗性变异系内游离脯氨酸的积累

在正常条件下,烟草Hyp抗性变异系愈伤组织内的游离脯氨酸含量是其野生型的4倍多。这种积累游离脯氨酸的特性在愈伤组织的转代、再生及次生过程中均十分稳定。虽然其他多种氨基酸含量也有所改变,但变异系内游离脯氨酸的积累具有特异性。由于脯氨酸对Hyp抑制效应具有部分的逆转作用,所以游离脯氨酸的积累是变异系抗Hyp抑制作用的重要原因。然而,变异系内蛋白质组成中的脯氨酸并没有特异性地增加。在Hyp存在条件下,变异系内的硝酸还原酶、转化酶及过氧化氢酶活性均显著地高于野生型。表明变异系对培养基中的碳源和氮源具有较高的利用能力。     

抗羟脯氨酸水稻变异系的筛选及其特性的研究

利用Hyp反复筛选经EMS处理的水稻愈伤组织,得到4个抗性细胞系。它们的游离脯氨酸含量高于对照2—25倍。Hyp(2 mmol/L)和NaCl(1%)胁迫对M_1、M_2变异系γ-谷氨酰磷酸合成活力及游离脯氨酸积累的影响呈一定的正相关性。变异系在0.8%,15NaCl 培养基中分化出植株。与对照相比,各变异系在愈伤组织、植株水平上均表现较强的耐盐性,且与游离脯氨酸的积累量呈正相关。     

单倍性烟草HyP抗性愈伤组织变异系的选择及其生理生化特性的研究Ⅲ．Hyp抗性变异系在逆境条件下的反应

在水分胁迫、盐分胁迫、抑制剂胁迫和⁶⁰C0 γ-射线等逆境条件下,烟草愈伤组织系中的游离脯氨酸含量随着逆境胁迫程度的增加而幅度不同地提高。在一定范围内,二者之间存在着特定的正相关。在正常条件下积累游离脯氨酸的烟草Hyp抗性愈伤组织变异系C^r-2虽在耐寒能力方面并不比其野生型C^w高,但对盐分逆境和模拟干旱处理的抗性均是其野生型的两倍多。烟草Hyp抗性愈伤组织变异系在研究抗旱和抗盐中具有潜在的应用价值。     

菜心耐羟脯氨酸初选系的耐热性

分析了离体筛选出的一个菜心耐羟脯氨酸选择系的脯氨酸含量和耐热性。Hypr01比原品种六十天特青有较高的游离脯氨酸含量：35℃热胁迫下Hypr01中有显著的游离脯氨酸含量增加,为六十天特青的4倍和耐热品种四九菜心的近2倍。与对照相比,在35℃下连续培养的Hypr01愈伤组织表现出更强的活力和根的分化能力,鲜重增量明显加大：相对电解质渗出率、愈伤组织褐变程度和MDA含量低：SOD、CAT活性较高。在人工气候箱模拟栽培高温逆境下,Hypr01再生苗生长优于未经选择的六十天特青,并具较低的电解质渗出率和较高的鲜重增长,说明有较强的耐热性。     

羊草细胞变异系HR20—8的氨基酸组成水平与脯氨酸合成代谢变化的…

离体培养,筛选获得羊草抗羟脯氨酸细胞变异系ＨＲ２０－８,其愈伤组织中游离氨基酸和蛋白质氨基酸含量均发生了较大的变化,与供体对照比较,分别提高２．３５倍和１．４０倍,其中的脯氨酸组分在变异系体内表现异常增加。     

枸杞抗羟脯氨酸细胞变异系筛选及其耐盐特性的研究

试验以枸杞(Lycium barbarum L.)成熟胚诱导的胚性愈伤组织为材料研究了：(1)^60Co—γ射线诱变处理对愈伤组织的影响;(2)耐羟脯氨酸(HYP)愈伤组织变异系的筛选;(3)耐HYP愈伤组织变异系的耐盐性及稳定性分析;(4)耐HYP愈伤组织变异系生理生化特性;(5)变异系的分化和再生苗的生理生化特性。初究结果表明：由胚性愈伤组织经30Gy的^60Co—γ辐射处理,在含16mmol／L HYP选择培养基和无HYP培养基上交替培养4代,分离出耐HYP的愈伤组织变异体;该变异体游离脯氨酸含量比对照高3．3倍,其再生植株叶片游离脯氨酸比对照高13．5倍。     

Selection and Characterization of Maize Variants Resistant to S-(2-Aminoethyl)-L-Cysteine and 5-Methyltryptophan

Using tissue culture selection techniques, variants resistant to S-(2-aminoethyl)-L-cysteine (AEC) and 5-methyltryptophan(5MT) were, respectively, isolated from Opaque-2 maize inbred line “Zhongxi 037/02” and “Zhongxi 091/02”. After growing 5 months on AEC free medium, the AEC-resistant cell line (Raec) still showed high level AEC resistance which was 4 times. higher than that of its wild type, “Zhongxi 037/02”. The resistance was expressed at the plant level. New cultures initiated from shoot tissue of plants regenerated from Raec was also resistant to AEC inhibition. The free pool of lysine, threonine, isoleucine, methionine and arginine increased 0.5–3.4 fold in Raec culture. The aspartokinase from both AEC-resistant and -sen- sitive lines exhibited similar sensitivity to lysine and AEC inhibition. But the aspartokinase activity in the resistant line was 2.3 times of that in sensitive line. Seed were obtained from the plants resistant to AEC when crossed with pollen of sensitive plants. The resistance of 5MT-resistant cell line, tested after growth for 11 months on nonselection medium, was 3.5 times higher than that of its wild type, “Zhongxi 091/02”. The 5MT-resistance was possibly due to the accumulation of free tryptophan (from 0 to 61.6 nmol/g fr. wt) in the resistant cells. There was also an increase in free phenylalanine (14.5 fold) and tyrosine (28.8 fold).     

Isolation and Characterization of a Chinese Aneurolepidium Cell Line Resistant to Hydroxyproline

A hydroxyproline (HYP) resistant cell line of Chinese aneurolepidium (Aneurolepidium chinense (Trin.) Kitag. ) was isolated under selection pressure of 20 mmol/L HYP. Comparison of the free amino acid pool levels in the cell line with that of donor showed substantial accumulation of proline (6.6 × ). Enzyme examination indicated that γ-glutamyl kinase controlling proline biosynthesis in HR20-8 cell line had 2.5 times as much activity as that of the donor. Exogenous L-proline inhibited the enzyme activities both in the HR20-8 and the donor by the same rate of 30% at 100 mmol/L. The responses of HR20-8 cell line to NaC1, PEG and cold temperature (5 ℃) were also compared with those of donor and the former exhibited remarkably increased tolerance to the tested stress condition. The results showed that changes of γ-glutamyl kinase property exhibited the phenomenon of extra accumulation of proline which might favor the increased tolerance to NaC1, PEG and cold temperature in the resistant cell line.     

芦笋抗S—（2—氨乙基）—L—半胱氨酸（AEC）变异体的筛选

S-(2-氨乙基)-L-半胱氨酸(AEC)可抑制芦笋愈伤组织的生长,此抑制作用可被赖氨酸或甲硫氨酸部分解除。用0.5mmol/L的AEC进行筛选,得到抗性愈伤组织AR10并再生植株。AR10愈伤组织经一年多的继代培养,在离开选择剂组培继代两代后仍保持对AEC的抗性。抗性系愈伤组织还表现出对2mmol/L的半胱氨酸具交叉抗性,对1mmol/L的赖氨酸加苏氨酸表现部分交叉抗性。AR10再生植株一部分保持对AEC的抗性,而一部分则无抗性。对抗性愈伤组织及其再生植株的氨基酸分析表明,愈伤组织内游离赖氨酸、苏氨酸、甲硫氨酸都有增加,而在再生植株内却发现半胱氨酸和赖氨酸的特异性增加,分别是对照植株的5.4和4.6倍。     

Expression of 5-Methyltryptophan Resistance in Plants Regenerated from Resistant Cell Lines of Datura innoxia

Seventy-nine 5-methyltryptophan-resistant cell lines have been selected from haploid Datura innoxia Mill. cell cultures by plating suspensions in agar medium containing a growth inhibitory concentration of 5-methyltryptophan. Mutagen treatment increased the frequency of resistance. The eleven variants tested posses an altered anthranilate synthase less sensitive to feedback inhibition by tryptophan. All five of the variants which were analyzed for free amino acids contained elevated levels of free tryptophan (8 to 30 times the wild type level). None of the selected cell lines were auxin-autotrophic. Resistance to 5-methyltryptophan, altered anthranilate synthase, and high free tryptophan (4 to 44 times) were also expressed in leaves of plants regenerated from the variant lines and in cultures reinitiated from the resistant plants. These results show that the amino acid overproduction phenotype can be selected at the cellular level of organization and be expressed identically in whole plants regenerated from the selected cells.     

Utilization of putrescine in tobacco cell lines resistant to inhibitors of polyamine synthesis

Three tobacco cell lines have been analyzed which are resistant to lethal inhibitors of either putrescine production or conversion of putrescine into polyamines. Free and conjugated putrescine pools, the enzymic activities (arginine, ornithine, and S-adenosylmethionine decarboxylases), and the growth characteristics during acidic stress were measured in suspension cultures of each cell line. One cell line, resistant to difluoromethylornithine (Dfr1) had a very low level of ornithine decarboxylase activity which was half insensitive to the inhibitor in vitro. Intracellular free putrescine in Dfr1 was elevated 10-fold which was apparently due to a 20-fold increase in the arginine decarboxylase activity. The increased free putrescine titer was not reflected in an increased level of spermidine, spermine, or putrescine conjugation. Dfr1 cultures survived acidic stress at molarities which were lethal to wild type cultures. Two other mutants, resistant to methylglyoxal bis(guanylhydrazone) (Mgr3, Mgr12), had near normal levels of the three decarboxylases and normal titers of free putrescine, spermidine, and spermine. Both mutants however had elevated levels of conjugated putrescine. Mgr12 had an increased sensitivity to acidic medium. These results suggest that increased levels of free putrescine production may enhance the ability of tobacco cells to survive acid stress. This was supported by the observation that cytotoxic effects of inhibiting arginine decarboxylase in wild type cell lines were dependent on the acidity of the medium.     

In vitro selection of endosulfan-tolerant strains of Brassica compestris (cv. Brown Sarson)

Endosulfan tolerant lines of mustard (Brassica campestris cv. Brown Sarson) have been developed through tissue culture methods. Cotyledonary expiants excised from eight day old in vitro grown seedlings were used for inducing callus. Fast growing friable callus was then transferred to MS medium containing (0.1–2.0 ugl^–1) endosulfan for selection. Five alternating exposures with and without endosulfan containing medium yielded an endosulfan tolerant cell line (ETL). The plants regenerated from ETL were found to tolerate three fold higher concentrations of endosulfan. Callus induced from randomly selected endosulfan tolerant regenerated plants were also tolerant to 3.0 ugl endosulfan, thereby, suggesting that tolerance has been acquired at the gene level.Biochemical investigation revealed higher levels of total free sugar, free amino acids, protein and activity of peroxidase in the tolerant cell line.Abbreviations MS Murashige and Skoog medium (1962) - NSM non-selective medium - SM selective medium - BAP 6-Benzylaminopurine - NAA -naphthaleneacetic acid - Z zeatin     

Selection of ethionine-resistant variants with increased accumulation of methionine from embryogenic protoplasts of the forage legume <Emphasis Type="Italic">Astragalus adsurgens</Emphasis>

In vitro selection was carried out to obtain ethionine-resistant plants with increased contents of free methionine in the vegetative tissues of the forage legume Astragalus adsurgens Pall. Three-week-old cell colonies were derived from protoplasts mutagenized with N-methyl-N-nitrosoguanidine from embryogenic callus and were selected with 0.6mM ethionine. Four colony lines were isolated and their resistance to ethionine was 7–8 times that of the wild-type callus. No plant regeneration occurred on these colony lines in the differentiation medium containing ethionine. Only one colony line (R-1) regenerated plants through somatic embryogenesis in the absence of ethionine. Stem and leaf segments from the regenerated plants showed the same potential to produce callus in the presence of ethionine as in the absence of ethionine. The formed callus kept continuously growing in ethionine-containing medium. Free amino acid analysis revealed that colony line R-1, its regenerated plants and callus from the regenerated plants accumulated methionine at levels at 5–9 times higher than in wild-type. These results suggested that ethionine resistance and methionine over-accumulation were also expressed at plant level. Thus, the obtained resistant colony line that could regenerate plants with over-accumulation of methionine might provide an alternative approach to improve the nutritional quality of this forage.     

利用组织培养技术选育玉米抗小叶斑病突变体

大叶斑病和小叶斑病是玉米最严重的病害,选育抗大、小叶斑病优良自交系及单交种是提高玉米产量的重要措施。Ｇｅｎｇｅｎｂａｃｈ等以玉米Ａ６１９ｃｍｓＴ愈伤组织为材料,筛选出对小叶斑病毒素具有抗性的愈伤组织与再生植株［１,２］,开辟了玉米抗病育种的新途径。...     

P-糖蛋白在多药耐药的K562细胞转运苯妥英纳与卡马西平中的作用

研究证实,多药转运体与难治性癫痫耐药机制密切相关,P-糖蛋白在其中起重要作用．主要研究P-糖蛋白拮抗剂维拉帕米对P-糖蛋白过表达的K562细胞耐药性及细胞内苯妥英纳与卡马西平浓度的影响．首先建立了P-糖蛋白高表达的K562/Dox(阿霉素诱导)耐药细胞株,比较耐药细胞株和P-糖蛋白表达阴性的K562细胞株对苯妥英纳和卡马西平的耐药性,并观察给予维拉帕米后,耐药细胞内抗癫痫药物的浓度变化．结果发现,苯妥英纳和卡马西平对K562/Dox细胞株的半数抑制浓度(IC₅₀)明显高于K562细胞株,加入维拉帕米后,苯妥英纳和卡马西平对K562/Dox 细胞的IC₅₀明显下降,逆转倍数分别为2.5和1.5．进一步研究发现,K562/Dox细胞内苯妥英纳和卡马西平的浓度均显著少于其药敏K562细胞,仅分别为正常K562细胞的23.6%和32.2%．当加入维拉帕米后,K562/Dox细胞内抗癫痫药物浓度明显升高(P < 0.05)．由此证明,高表达的P-糖蛋白参与了细胞的药物转运,在难治性癫痫的耐药机制中扮演重要角色．     

Selection and Characterization of a Daucus carota L. Cell Line Resistant to Four Amino Acid Analogues

Carrot suspension cultures resistant to growth inhibition byp-fluorophenylalanine, ethionine. aminoethylcysteine, and 5-methyltryptophanwere obtained by sequential selection for resistance to eachamino acid analogue. Resistance was increased at least 100-foldfor each analogue and the resistance was retained after growthaway from the inhibitors for 40 cell doublings. After each selection,the corresponding free natural amino acid was increased andthe line resistant to all four analogues showed levels of phenylalanine,methionine, lysine, and tryptophan which were 7, 6, 5, and 32times that of the parental wild type line, respectively. Thetotal free amino acid level was doubled in this line. Only afterselection for 5-methyltryptophan resistance did the anthranilatesynthetase show altered feedback sensitivity to tryptophan.