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。     

激光对香石竹愈伤组织蛋白质和同工酶谱的影响

用氦氖激光处理香石竹愈伤组织,可使材料中的34kD和28kD蛋白含量高于对照,并产生分子量约为22kD的新的蛋白带。而用氩离子激光处理后,则出现一条分子量约为45kD的新的蛋白带。此外,激光处理亦明显影响愈伤组织的过氧化物酶同工酶谱及其活性,其中氦氖激光处理后产生了Rf为0.17和0.26的新酶带,而用氩离子激光处理则只产生Rf为0.17的新酶带。激光对香石竹愈伤组织的酯酶同工酶无影响。     

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。     

耐盐突变体小麦后代耐盐稳定性分析研究

以卫星搭载小麦种子为原始材料,利用其幼穗、幼胚诱导的愈伤组织进行耐盐突变体的筛选,对耐盐愈伤组织再生植株后代进行耐盐稳定性生理生化特性分析。结果表明：(1)耐盐系后代在土壤高盐浓度条件下,游离脯氨酸含量稳定增加,且高于对照系;(2)耐盐系再生植株后代保持较高的K^ ／Na^ 比;(3)与对照相比,种子醇溶蛋白电泳带谱中的b2,b3,b5,b7带为耐盐系所特有,b8带消失;(4)耐盐系再生植株后代可溶蛋白电泳带为26条,而对照系为23条蛋白带。其中98kD、75kD、52kD、49kD和32kD为耐盐系的特有蛋白带。而38kD和35kD蛋白带为对照系所特有。     

苜蓿抗甲硫氨酸变异体的筛选

紫花苜蓿(Medicago sativa L.)下胚轴愈伤组织用NaN_3溶液诱变处理后,在含有全致死浓度甲硫氨酸的MS培养基上进行了6个月的连续筛选培养,获得了能抗100mmol/L甲硫氨酸的变异细胞系,并分化成再生植株。所获变异细胞系在脱离选择压力6个月后,对甲硫氨酸的抗性仍比对照高7.2倍,并表现出对乙硫氨酸的交叉抗性(为对照抗性的3.3倍)。抗性细胞系及其再生植株的甲硫氨酸、赖氨酸、苏氨酸和异亮氨酸含量均比对照有大幅度增加。抗性系的SDS-PAGE电泳图谱及过氧化物酶同工酶谱带均与对照有显著不同,并出现了新带,表明变异系已经产生变化了的基因产物。     

枸杞耐盐突变体的筛选及生理生化分析

用不同剂量60Coγ对以枸杞叶为外植体诱导出的愈伤组织进行辐射处理,并将恢复增殖的愈伤组织采用逐步提高盐浓度的方法,直到筛选出耐1%的愈伤组织变异体,通过对其生理生化的分析表明:变异体在不同盐浓度胁迫下干、鲜重增长均高于对照组;MDA含量和质膜透性、O-·2与H2O2含量都低于对照组;抗氧化酶SOD、POD、CAT活性均高于对照组;对可溶性蛋白SDS电泳结果表明:耐盐变异体有22条蛋白带,而对照组有21条蛋白带,其中31.3kD、21.0kD、18.5kD带为耐盐变异体所特有,而74.1kD、52.1kD带为对照组所特有,并且73.7kD、67.9kD的蛋白耐盐变异体含量高于对照组.     

苜蓿盐适应愈伤组织中蛋白质性质和组分的变化

通过一步筛选获得1.0％NaCl适应愈伤组织。在盐适应愈伤组织中水溶性蛋白和稀碱提取蛋白含量增加,而脂溶性蛋白含量没有明显变化。盐适应愈伤组织水溶性蛋白热稳定性下降;总蛋白抗脱水能力增强。盐适应愈伤组织水溶性蛋白在K~ 存在时,262nm吸收峰增高。盐适应愈伤组织蛋白氨基酸组成发生变化,其中最为明显的是Tyr摩尔百分数增加。Lys摩尔百分数减少。 盐适应愈伤组织中24kD蛋白含量明显增加。应用IEF-,NEPHGE-和native-PAGE-SDS-PAGE三种双向电泳方法同时证明在盐适应愈伤组织蛋白组分与非适应愈伤组织有明显差别。     

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。     

红豆草抗甲硫氨酸变异体的筛选

用ＮａＮ３ 诱变过的红豆草（Ｏｎｏｂｒｙｃｈｉｓｖｉｃｉａｅｆｏｌｉａ Ｓｃｏｐ．）愈伤组织筛选得到了能抗８０ ｍ ｍ ｏｌ／Ｌ甲硫氨酸的变异细胞系——ＯＮｍ ｅｔｒ,并得到了再生植株。ＯＮｍ ｅｔｒ 脱离选择压力６ 个月后,其抗性仍比野生型的高５．６ 倍。同时还表现出对乙硫氨酸的交叉抗性,其抗性是野生型的６．５ 倍, 表明该抗性系抗性表达稳定。ＯＮｍ ｅｔｒ 愈伤组织中甲硫氨酸、赖氨酸、苏氨酸含量分别是野生型的４．００、１．０９、１．５０ 倍。ＯＮｍ ｅｔｒ再生植株中甲硫氨酸、赖氨酸、苏氨酸、异亮氨酸含量分别为野生型的２．０、３．５、３．５、２．５ 倍。在ＯＮｍ ｅｔｒ 愈伤组织可溶性蛋白ＳＤＳ－ＰＡＧＥ图谱中,出现了两条新多肽（３０ ｋＤ、２６ ｋＤ）。ＯＮｍ ｅｔｒ 细胞系过氧化物酶同工酶谱中亦出现了两条新酶带。这些变化表明该变异系已经产生变化了的基因产物     

白桦愈伤组织化学诱变

用不同浓度的甲基磺酸乙酯(ethyl methane sulfonate, EMS)对白桦(Betula platyphalla Sak.)愈伤组织进行化学诱变处理。结果表明: EMS诱变剂的浓度和处理时间对愈伤组织的存活率有很大影响。在高浓度EMS短时间处理和低浓度EMS长时间处理条件下得到叶柄、叶片愈伤组织的半致死剂量。通过观察半致死剂量下愈伤组织的染色体发现, 诱变后细胞中单倍体、非整倍体及多倍体比例均高于对照, 这说明EMS的诱变处理引起了愈伤组织细胞中染色体数量的变化。     

不同状态银杏愈伤组织蛋白质电泳分析及电镜观察

通过对同一来源3种银杏愈伤组织中蛋白质、黄酮含量测定,蛋白质SDS-PAGE电泳分析,细胞超微结构观察等研究银杏不同状态愈伤组织的生长情况。结果表明,银杏的这3种愈伤组织代表愈伤组织细胞生长的3个阶段。绿色愈伤组织处于分生组织状态,细胞生长旺盛,蛋白质较多,蛋白质条带宽,细胞内物质丰富,但黄酮的含量并不是最高;微黄色愈伤组织细胞处于初生代谢向次生代谢转化的阶段,蛋白质含量开始减少,45.7、38.4、33.4kD的蛋白质开始减少,核膜不完整,淀粉含量较高,其黄酮含量高于绿色愈伤,属次生代谢的阶段;褐色愈伤组织蛋白质含量很低,条带数减少,有5条蛋白质条带消失,且黄酮的含量最低,细胞结构发生质壁分离现象,内含物解体,细胞核解体,是衰亡的主要特征。微黄色愈伤组织是获取高黄酮含量的最好材料。     

The effects of DNA hypomethylating drugs on androgenesis in barley (Hordeum vulgare L.)

Summary The effects of DNA hypomethylating drugs (azacytidine and ethionine) on induction of microspore-derived calluses and embryos were studied in barley (Hordeum vulgare L.) ev. Igri. The results were as follows: (1) Yield of calluses and embryos pretreated with the different concentrations of azacytidine for 3 d was several-fold higher than that of the control. The highest yield of calluses and embryos in all treatments appeared at a concentration of 3 mg l⁻¹, which reached 11.03 per anther. It was 110-fold higher than the control. (2) There was a significant difference in yield of calluses and embryos between the different days of pretreatment. The highest yield was obtained at a 3-d pretreatment. If the period of pretreatment was shorter or longer than 3 d, yield of calluses and embryos was reduced sharply, and was similar to that of the control. (3) The data obtained with ethionine pretreatment were very similar to those obtained with azacytidine. (4) Tests on the different methods of pretreatment showed that yield of calluses and embryos pretreated with distilled H₂O, mannitol, azacytidine, and ethionine was much higher than other pretreatments and the control, and reached 6.53–11.39 per anther. The yield of calluses and embryos pretreated with DNA hypomethylating drugs was higher than with mannitol. However, pretreatment with hypomethylation drugs supplemented with induction medium was not effective.     

Influence of exogenous proline on embryogenic and organogenic maize callus subjected to salt stress

The effect of exogenous proline (6 mM) and increasing NaCl doses (from 0.4 to 1.2% w/v) on the maintenance of organogenic and embryogenic callus lines derived from the salt-sensitive maize inbred W64Ao2 were studied. To this end, total protein, free amino acid and polyamine content were analyzed. The demand of exogenous nitrogen and especially of proline, even in the presence of salt, differed in the two types of morphogenic calluses. The total protein content of embryogenic calluses was higher in the presence of proline than in its absence, in all the cases studied. An opposite effect of proline was observed in organogenic calluses: the presence of proline and salt decreased significantly their protein content. With respect to amino acid and polyamine contents, the organogenic calluses showed physiological characteristics of salt-adaptation, whereas the embryogenic calluses were more sensitive to NaCl. Although endogenous proline increased in the organogenic calluses cultured in the presence of salt, in embryogenic calluses it only rose at the lowest salt concentration. Furthermore, the endogenous arginine content under saline conditions was higher in organogenic calluses. A compensatory effect between proline and polyamine metabolism related to the endogenous arginine content in response to salt stress was also observed. This effect differed in the two types of calluses.     

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.     

Influence of exogenous proline on embryogenic and organogenic maize callus subjected to salt stress

The effect of exogenous proline (6 mM) and increasing NaCl doses (from 0.4 to 1.2% w/v) on the maintenance of organogenic and embryogenic callus lines derived from the salt-sensitive maize inbred W64Ao2 were studied. To this end, total protein, free amino acid and polyamine content were analyzed. The demand of exogenous nitrogen and especially of proline, even in the presence of salt, differed in the two types of morphogenic calluses. The total protein content of embryogenic calluses was higher in the presence of proline than in its absence, in all the cases studied. An opposite effect of proline was observed in organogenic calluses: the presence of proline and salt decreased significantly their protein content. With respect to amino acid and polyamine contents, the organogenic calluses showed physiological characteristics of salt-adaptation, whereas the embryogenic calluses were more sensitive to NaCl. Although endogenous proline increased in the organogenic calluses cultured in the presence of salt, in embryogenic calluses it only rose at the lowest salt concentration. Furthermore, the endogenous arginine content under saline conditions was higher in organogenic calluses. A compensatory effect between proline and polyamine metabolism related to the endogenous arginine content in response to salt stress was also observed. This effect differed in the two types of calluses.     

卫星搭载红豆草后代中耐盐细胞系的筛选及鉴定

将红豆草种子搭载于940703返地卫星,经田间繁殖得后代种子,先将种子在1.5%NaCl上筛选、并在该盐浓度下诱导愈伤组织和筛选,在无盐培养基上恢复生长后再在1.2%NaCl上筛选得到耐盐变异系.变异系具有正常的分化能力并表现出对PEG胁迫的交叉抗性.变异系在无胁迫条件下脯氨酸含量较低但在有盐胁迫时具有高效积累脯氨酸的能力.后者可能对红豆草耐盐系更为重要.变异系中脯氨酸的这种合成机理可能是由于一些基因在调控中对水的敏感性改变引起.梯度聚丙烯酰胺凝胶电泳表明耐盐系的SOD和酯酶分别出现175kD和75kD的新形式.说明空间诱变和组织培养相结合可以筛选耐盐变异系.     

卫星搭载亚麻后代中PEG和NaCl抗性系的初步筛选

把空间生物学和细胞工程相结合,通过组织培养技术对其离体筛选,得到抗１．２％ＮａＣｌ和３５％ＰＥＧ的愈伤组织,将所得抗性系愈伤组织在２．０ｍｇ／Ｌ６－苄基氨基嘌呤、０．５ｍｇ／Ｌ吲哚乙酸的ＭＳ培养基上分化得到完整的植株。抗性系能在胁迫条件下保持高的生长速度和高效的脯氨酸合成能力,表明空间诱变与组织培养相结合有望可成为筛选抗胁迫变异系的有效途径。     

卫星搭载亚麻后代中PEG和NaCl

把空间生物学和细胞工程相结合,通过组织培养技术对其离体筛选,得到抗1.2% NaCl和35% PEG的愈伤组织。将所得抗性系愈伤组织在2.0 mg/L 6-苄基氨基嘌呤、0.5 mg/L吲哚乙酸的MS培养基上分化得到完整的植株。抗性系能在胁迫条件下保持高的生长速度和高效的脯氨酸合成能力。表明空间诱变与组织培养相结合有望可成为筛选抗胁迫变异系的有效途径。