苜蓿抗乙硫氨酸愈伤组织的筛选和特性

用乙硫氨酸为筛选剂,通过幼苗和组织培养筛选得到乙硫氨酸抗性愈伤组织。该愈伤组织在含乙硫氨酸的培养基上表现出较高的半抑制剂量和相对生长量。作为天门冬氨酸途径的产物,甲硫氨酸、异亮氨酸和赖氨酸在所筛选的愈伤组织中分别增加到对照的两倍多,但苏氨酸保持正常水平,另外酪氨酸、半胱氨酸和亮氨酸也有所增加,而在所筛选的愈伤组织中缬氨酸浓度却下降。说明在所筛选愈伤组织中存在一个以上与氨基酸合成相关的酶发生改变。同工酶分析表明,该愈伤组织中出现对照中没有的分子量为44kD的超氧化物歧化酶的分子量为45kD的酯酶谱带。     

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。     

南苜蓿组织和原生质体培养及转化试验

主要探讨南苜宿子叶和下胚轴外植体,子叶原生岳体培养中的器官发生及遗传转化。南苜蓿子叶和下胚细外植体培养在附加１ＡＡ０．５－１ｍｇ／Ｌ和细胞分裂素（ＢＡ或ＺＴ）０．５－２ｍｇ／Ｌ的ＭＳ培养基上,在有当照的条件下诱导形成不定芽,进而再生成完整植株。子叶原生质体培养在附加２,４－Ｄ０．５ｍｇ／Ｌ和ＫＴ０．２ｍｇ／Ｌ的Ｂ５液体培养基中,细胞分裂频率可达３０－４１％,原生质全来源的愈伤组织在ＭＳＢ培养基（Ｍ     

苜蓿愈伤组织中的盐胁迫蛋白

0.5％NaCl抑制愈伤组织生长,处理后期(第21天和28天检测)24kD蛋白含量明显增加。1.0％,2.0％和3.0％NaCl处理的愈伤组织不生长,未检出24kD蛋白增加,但36～42kD蛋白大量增加,并且有随处理的盐浓度增高而增加的趋势。 在1.0％NaCl适应愈伤组织(简称S-1)中24kD蛋白含量明显增加,而36～42 kD蛋白含量下降到对照水平。~(35)S-Met体内标记表明,增加的24kD蛋白是新合成的。S-1回到无盐5代后,仍保持提高的耐盐性和24kD蛋白含量。24kD蛋白含量的增加不受甘露醇胁迫的诱导。初步离心分离的细胞亚组分表明,24kD蛋白主要位于胞质和细胞器膜。在烟草S-1细胞中也发现24kD蛋白含量增加。     

大麦耐酸铝愈伤组织变异体的筛选及其特性研究

从对酸铝敏感的栽培大麦品种“早熟3号”的花药和幼胚培养物中,分别诱导了单倍体和二倍体愈伤组织。并筛选出生长快、质地松散的愈伤组织系,在不同的 pH、铝浓度培养基中观察其生长情况,建立耐酸铝愈伤组织变异体筛选条件(pH 4.5,Al~(+3)10 ppm)。并从经5000Rad γ-射线处理和未处理的材料中筛选到耐性变异体。比较结果表明,在酸铝条件下,变异体与原始型在愈伤组织生长速度、呼吸强度上存在显著差异。并且在 pH4.5,Al~(+3)10—20ppm时,变异体能基本正常生长,细胞呈圆形,荧光强度测定表明细胞活力大;而原始型在同样条件下基本停止生长,细胞呈长柱形和椭圆形,荧光强度弱、活力低。两者间过氧化物酶同工酶酶谱也存在差异。至今还未能从单倍体耐性变异系分化成植株,但在二倍体愈伤组织的筛选过程中直接成苗5株,3株在越夏和移栽中死亡。对存活结种子的2株后代鉴定结果为耐性,适应在酸铝红壤上生长,但农艺性状和品质性状还需进一步改良。最后,讨论了离体筛选技术在改良大麦耐酸铝性育种中的应用。     

渗透胁迫下苜蓿愈伤组织的生长和植株再生（简报）

通过三种途径获得的抗２０％ＰＥＧ的苜蓿愈伤组织获得的逐步选择法获得的抗２５％ＰＥＧ的愈伤组织以及抗％ＰＥＧ的芽愈伤组织,其抗性稳定,经过三代悬浮培养可以形成生长速度快,由均一细胞团组成的悬浮培养物,从抗２０％ＰＥＧ的愈伤组织和带芽愈伤组织均可再生成植株。     

荞麦愈伤组织分化与过氧化物酶活性及其同工酶关系的研究

在附加不同外源激素的培养基中,龙山荞的子叶愈伤组织表现出不同的分化状态：（１）不分化;（２）分化根;（３）分化芽;（４）分化全苗。四种愈伤组织的过氧化物酶活性的相对幽会为１：１．４７：５．９５：７．７８,并且过氧化物酶同工酶谱也存在明显差异。酶带４’为分化愈伤组织所特有;酶带２’为未分化和分化愈伤组织所共有;在分化根和未分化愈伤组织中缺少酶带１’;未分化愈伤组织比分化愈伤组织多出酶带３’。     

酵母菌高产蛋氨酸机理的研究

乙硫氨酸是蛋氨酸的结构类似物,１５０μｇ／ｍｌ浓度的乙硫氨酸可抑制丝孢酵母（Ｔｒｉｃｈｏｓｐｏｒｏｎｃｕ－ｔａｎｅｕｍ）ＳＴ８５１生长,又能被蛋氨酸回复。经紫外线处理后,得到抗性突变株ＳＴ８５１－１０,菌体蛋氨酸含量提高了４３．５％。分析合成机理表明：该突变株ＳＴ８５１－１０的Ｓ－腺苷蛋氨酸合成酶活性降低,增大了细胞内蛋氨酸库（Ｍｅｔｈｉｏｎｉｎｅｐｏｏｌ）。     

杂花和紫花苜蓿原生质体分离培养条件的筛选

以杂花苜蓿‘甘农1号’和紫花苜蓿‘甘农4号’、‘阿尔冈金’及‘清水’4个适宜西北内陆黄土高原地区栽培的苜蓿愈伤组织为材料,研究酶解时间、酶液组合、酶液渗透压、愈伤组织继代培养时间、预处理措施及不同培养方法等对原生质体分离和培养效果的影响,并对培养条件进行优化。结果表明:(1)适宜4个苜蓿品种愈伤组织酶解的最佳预处理措施为0.55mol/L蔗糖或CPW溶液中预质壁分离1h,最佳继代时间均为12d。(2)‘甘农1号’、‘甘农4号’和‘清水’的最佳酶液组合均为2%纤维素酶+0.5%果胶酶+0.3%崩溃酶;‘阿尔冈金’的最佳酶液组合为2%纤维素酶+0.5%果胶酶+0.3%半纤维素酶+0.3%离析酶+0.3%崩溃酶;‘甘农1号’和‘阿尔冈金’的最佳酶解时间为12h,‘甘农4号’和‘清水’分别为14h和10h。(3)适宜4个品种酶解的甘露醇浓度分别为‘甘农1号’0.75mol/L,‘甘农4号’0.65mol/L,‘阿尔冈金’0.6mol/L,‘清水’0.55~0.6mol/L。(4)经液体浅层培养和固液培养方式均可获得4个苜蓿品种的再生愈伤组织,且固液培养法较液体浅层培养法更有利于苜蓿原生质体早期的培养和再生。     

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。...     

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.     

The Response of Sugarcane (Saccharum officinarum) Cultured Cells to Anoxia and the Selection of a Tolerant Cell Line

The effect of anoxia on the sugarcane (Saccharum officinarum L.) cultured cells was studied in order to elaborate a technique for in vitro selection of cell lines, which would be tolerant to anaerobic stress. Inhibitory and lethal doses of anaerobic incubation were established from the state of the mitochondrial ultrastructure during the anaerobic incubation of cells either with or without exogenous glucose, as well as from the pattern of the post-anaerobic callus growth. An intact state of the mitochondrial ultrastructure and the viability of some cells in the presence of 3% glucose were shown to be maintained for at least 14 days of anaerobic incubation, while the index of post-anaerobic growth decreased by almost 50% even after 72-hour-long anaerobiosis. In the absence of exogenous glucose, a marked destruction of mitochondria and a twofold decrease in the callus growth index were observed as early as after six-hour-long anaerobic stress. A 48-hour-long incubation under these conditions resulted in the maintenance of the intact ultrastructure only in 7–10% of cells, while a 96-hour-long anaerobiosis brought about the complete degradation of the subcellular structure and cell death. A 48-hour-long anaerobiosis without exogenous glucose was chosen for selecting the anoxia-tolerant cell lines. After three cycles of selection, the anoxia tolerance of the selected cell line exceeded the respective index of the initial callus several-fold. In selected line, about 50% of cells retained viability and could resume growth even after 96-hour-long anaerobic incubation. The experimental results obtained were used to determine the possible causes of the heterogeneity of callus cells as regards their anoxia resistance.     

Resistant Host Responses to Ten California Populations of Meloidogyne incognita

Resistant and susceptible cultivars of tomato, lima beans, cotton, and alfalfa were tested with 10 populations of Meloidogyne incognita from different California locations. Nine of the populations differed in aggressiveness on the nine cultivars tested. Two populations were especially aggressive toward resistant tomato cultivars.     

Interrelationship of Meloidogyne hapla and Ditylenchus dipsaci on Resistant and Susceptible Alfalfa

Simultaneous inoculations of alfalfa with Meloidogyne hapla larvae and Ditylenchus dipsaci at 16, 20, 24, and 28 C did not depress penetration of either nematode in ''Nev Syn XX'' -a selection resistant to M. hapla and D. dipsaci, ''Vernal 298'' -a selection resistant to M. hapla and susceptible to D. dipsaci, ''Lahontan'' -a cultivar resistant to D. dipsaci and susceptible to M. hapla, and ''Ranger'' -a cultivar susceptible to both M. hapla and D, dipsaci. Infection with D. dipsaci depressed growth of susceptible ''Vernal 298'' and ''Ranger'' at all soil temperatures, except for ''Vernal 298'' at 16 C. Infection with M. hapla alone did not depress growth of any of the alfalfas. A combination of M. hapla and D. dipsaci resulted in a synergistic weight depression on ''Ranger'' at all soil temperatures. Inoculation of the four alfalfas with D. dipsaci 2, 4, 6, and 8 wk before inoculation with M. hapla at 16, 20, 24, and 28 C did not influence the resistance or susceptibility of ''Nev Syn XX,'' ''Lahontan,'' or ''Ranger.'' However, galling of ''Vernal 298'' by M. hapla was affected by soil temperature, plant age, and inoculation with D. dipsaci.     

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).     

Early Root Response to Meloidogyne incognita in Resistant and Susceptible Alfalfa Cultivars

The early events of Meloidogyne incognita behavior and associated host responses following root penetration were studied in resistant (cv. Moapa 69) and susceptible (cv. Lahontan) alfalfa. Ten-day-old seedlings of alfalfa cultivars were inoculated with second-stage juveniles (J2) and harvested 12, 24, 48, and 72 hours and 7, 14, and 21 days later. Both cultivars supported similar root penetration and initial J2 migration. By 72 hours after inoculation the majority of J2 were amassed inside the vascular cylinder in roots of susceptible Lahontan, while J2 had not entered the vascular cylinder of resistant Moapa 69 and remained clumped at the root apex. Nematode development progressed normally in Lahontan, but J2 were not observed in Moapa 69 after day 7. The greatest differences between RNA translation products isolated from inoculated and uninoculated roots of Lahanton occurred 72 hours after inoculation. Only minor differences in gene expression were observed between inoculated and uninoculated Moapa 69 roots at 72 hours. Comparison of translation products from inoculated versus mechanically wounded Lahontan roots revealed products that were specific to or enhanced in nematode-infected plants. Moapa 69 appears to possess a type of resistance to M. incognita that does not depend on a conventional hypersensitive response.     

耐盐药蒲公英(Taraxacum officinale Weber)愈伤组织筛选及生理生化特性分析

为获得耐1.5% NaCl的药蒲公英(Taraxacum officinale Weber)愈伤组织, 以药蒲公英叶片外植体为材料诱导愈伤组织。以NaCl为选择因子, 从愈伤组织直接筛选。在选择培养基上, 大部分愈伤组织褐化死亡, 个别褐化死亡的愈伤组织周围有少量新的细胞团长出, 将其转接到新鲜的选择培养基上, 每3周继代一次, 经3个月继代筛选获得了耐1.5% NaCl的药蒲公英细胞团。以普通愈伤组织为对照, 发现随着NaCl浓度升高, 耐盐愈伤组织的相对生长率下降但显著高于对照; 且随着盐胁迫处理时间延长持续升高, 而普通愈伤组织对照几乎停止生长, 说明耐盐愈伤组织具有相对稳定的耐盐性。在蛋白水平上, 耐盐愈伤组织与对照愈伤组织差异明显, SDS-PAGE分析显示: 耐盐愈伤组织比对照多出一条34 kD大小的蛋白带, 且30 kD、18 kD左右的蛋白带明显上调。相同处理条件下耐盐愈伤组织脯氨酸的增加幅度高于对照。盐胁迫条件下, 耐盐愈伤组织的超氧化物歧化酶(Super oxidase dimutase, SOD)、过氧化物酶(Peroxidase, POD)和过氧化氢酶(Catalase, CAT)活性明显高于对照,且随着处理时间的延长和盐浓度的增加呈现升高的趋势, 而对照则呈现先升高后下降的趋势。结果说明耐盐愈伤组织一方面通过小分子有机溶质如脯氨酸的方式调节其渗透平衡, 另一方面还可通过提高抗氧化能力降低盐分造成的次级伤害。积累蛋白也可能是耐盐愈伤组织调节渗透平衡的一种方式。通过生理生化分析确定我们获得的耐盐愈伤组织为耐盐变异体。     

Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.