Selection and characterization of a feedback-insensitive tissue culture of maize

Tissue culture selection techniques were used to isolate a maize (Zea mays L.) variant D33, in which the aspartate family pathway was less sensitive to feedback inhibition by lysine. D33 was recovered by successively subculturing cultures originally derived from immature embryos on MS medium containing growth-inhibitory levels of lysine+threonine. The ability of D33 to grow vigorously on lysine+ threonine medium was retained after growth for 12 months on nonselection medium. New cultures initiated from shoot tissues of plants regenerated from D33 also were resistant to lysine+threonine inhibition. The K_i of aspartokinase for its feedback inhibitor, lysine, was about 9-fold higher in D33 than for the enzyme from unselected cultures. The free pools of lysine, threonine, isoleucine and methionine were increased 2–9-fold in D33 cultures. This was consistent with the observed change in feedback regulation of aspartokinase, the first enzyme common to the biosynthesis of these amino acids in the aspartate pathway. The accumulated evidence including the stability of resistance in the cultures, the resistance of cultures initiated from regenerated plants, the altered feedback regulation, and the increased free amino acids, indicates a mutational origin for these traits in line D33.Abbreviation LT lysine+threonine in equimolar concentration Paper No. 10880, Scientific Journal Series, Minnesota Agricultural Expertment Station     

通过组织培养筛选小麦抗赤霉病突变体的研究

选用中抗赤霉病的春小麦品种和品系的花药进行离体培养,以小麦赤霉病29号菌株产生的致病培养滤液为选择剂,结合物理诱变处理,进行抗病鉴定,用赤霉病菌分生孢子直接接种在愈伤组织和再生植株筛选抗病突变体。在83块愈伤中有53块抗病。在11株的再生植株中有9株均比未经培养滤液处理的对照提高了抗病性,从中选出4株抗病性接近或超过“苏麦3号”品种。     

Advances in somatic cell genetics of higher plants — the protoplast approach in basic studies on mutagenesis and isolation of biochemical mutants

Summary Selection strategies developed in microbial genetics were successfully extrapolated to in vitro cell culture systems of higher plants and are having a major impact in the elucidation of regulatory mechanisms of basic cellular processes in eukaryotes. Although an increasing number and wide spectrum of biochemical variants have been isolated in such cell culture systems, their routine selection, characterization, and manipulation have not yet been achieved. Methodological limitations are considered to be one of the major reasons. Suspension or callus cultures, so extensively employed during the last decade in mutation-selection experiments and so useful in demonstrating the potentialities of in vitro screening techniques in obtaining various biochemical markers, have inherent drawbacks which limit in our opinion their further contribution in this field. Protoplast cultures represent an ideal tool for mutation and selection experiments. It is the purpose of this review to show how, due to recent methodological advances in the manipulation of some model protoplast culture systems, essential aspects of mutagenesis and selection of biochemical mutants can be reconsidered. These systems are simple and efficient, and lend themselves to statistical interpretation. Genetic analysis of selected variants should help us to understand and define better the new set of problems and concepts revealed by the somatic cell genetics of higher plants; combined with biochemical analyses it should elucidate the basic relationship between control of biological processes at cellular and whole organism level.     

唐菖蒲花色突变株开花后子房组织培养与植株再生的研究

以唐菖蒲花色突变株开花后的子房为外植体,接种于MS+1.0mg/L BA+0.01-0.1mg/L NAA或MS+1.0mg/LBA+1.0mg/L KT+0.01-0.1mg/LNAA的培养基中,从膨大的组织表面直接诱导不定芽.膨大的组织或带不定芽的组织每隔周转入1/2MS_1.0mg/L BA+0.01-0.1mg/L ANN培养基中,不断地诱导产生不定芽,分化的不定芽转入不含激素的1/2MS培养基中,形成幼苗、然后生根,最后可形成小球茎.幼苗转入1/2MS+0.1mg/L NAA培养基中,幼苗生根,再可形成小球茎.1个唐葛蒲突变株子房,经6—7个月培养,获得了上千株试管苗.     

Mutation induced by ethylmethanesulphonate (EMS), in vitro screening for salt tolerance and plant regeneration of sweet potato (<Emphasis Type=Italic>Ipomoea</Emphasis><Emphasis Type=Italic>batatas</Emphasis> L.)

Salt tolerant cultivars of sweet potato (Ipomoea batatas L.) can be obtained from induced mutation. The objective of the present study was to induce mutation for salt tolerance using ethylmethanesulphonate (EMS) in calli of sweet potato, followed by cell line selection and subsequent plant regeneration. Calli initiated from leaf explants were treated with 0.5% EMS for 0, 1, 1.5, 2, 2.5 and 3 h, followed by rinsing with sterile distilled water for four times. Preliminary experiments showed that 200 mM NaCl could be used as selection pressure. Salt tolerant calli were sub-cultured on medium supplemented with 200 mM NaCl for selection of mutant cell lines and this process repeated 5 times (20 days each). The selected calli were transferred onto somatic embryo formation medium, which was Murashige and Skoog (MS) medium supplemented with 4 mg l⁻¹ abscisic acid (ABA), 10 mg l⁻¹ gibberellic acid (GA). After 15 days, somatic embryos were transferred onto MS medium supplemented with 0.05 mg l⁻¹ ABA, 0.2 mg l⁻¹ zeatin (ZT) for regeneration. Plants designated as ML1, ML2 and ML3 were regenerated from the somatic embryos formed by calli treated with 0.5% EMS for 2 and 2.5 h. After propagation, salt tolerance of these mutants was investigated. Data suggested the mutants were more salt tolerant than control plants.     

Engineering Thrombin for Selective Specificity toward Protein C and PAR1

Thrombin elicits functional responses critical to blood homeostasis by interacting with diverse physiological substrates. Ala-scanning mutagenesis of 97 residues covering 53% of the solvent accessible surface area of the enzyme identifies Trp²¹⁵ as the single most important determinant of thrombin specificity. Saturation mutagenesis of Trp²¹⁵ produces constructs featuring k_cat/K_m values for the hydrolysis of fibrinogen, protease-activated receptor PAR1, and protein C that span five orders of magnitude. Importantly, the effect of Trp²¹⁵ replacement is context dependent. Mutant W215E is 10-fold more specific for protein C than fibrinogen and PAR1, which represents a striking shift in specificity relative to wild-type that is 100-fold more specific for fibrinogen and PAR1 than protein C. However, when the W215E mutation is combined with deletion of nine residues in the autolysis loop, which by itself shifts the specificity of the enzyme from fibrinogen and PAR1 to protein C, the resulting construct features significant activity only toward PAR1. These findings demonstrate that thrombin can be re-engineered for selective specificity toward protein C and PAR1. Mutations of Trp²¹⁵ provide important reagents for dissecting the multiple functional roles of thrombin in the blood and for clinical applications.     

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。     

稻瘟病菌AVR-pita等位基因的遗传多样性研究(简报)

由真菌Magnaporthe grisea引起的稻瘟病是我国水稻三大病害之一．也是遍及世界各水稻产区的重要病害．每年均有不同程度的发生．流行年份一般减产10％-20％．严重的达40％-50％．局部田块甚至颗粒无收。稻瘟病菌在进化过程中形成了遗传多样性和毒性易变的特性．是水稻品种抗病性容易丧失的主要原因之一。对稻瘟病系统研究的证据表明．水稻与稻瘟病菌之间的互作．符合“基因对基因”假说。也就是说．水稻有一抗病基因,稻瘟病菌中就会有相对应的无毒基因．     

γ-聚谷氨酸基因工程研究进展与展望

γ-聚谷氨酸(γ-PGA)是一种天然高分子可降解、环境友好型的新型阴离子聚合物。目前发现多种芽孢杆菌、古细菌和一种真核生物均可合成γ-PGA。根据其合成是否需要外加谷氨酸分为谷氨酸依赖性和谷氨酸非依赖型。γ-PGA的合成基因分为结合型的cap系和游离型的pgs系,其表达产物组成的γ-PGA合成酶复合体调节着γ-PGA的合成和转运。由于γ-PGA具有水溶性好、保湿性好、吸水性好、良好的生物兼容性和生物可降解性、可食用、对环境无污染等优点,在医药、农业、食品、环境、化妆品等领域具有广泛的应用前景。主要对γ-PGA的结构特点、微生物合成、相关基因、合成机理、应用、诱变处理进行综述。以期通过物理和化学等技术的诱变,获得γ-PGA的高产菌株,为提高γ-PGA产量提供依据。     

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     

Induced mutants from dihaploid potatoes after pollen mother cell treatment

Summary Microspore mother cells of dihaploid Solanum tuberosum plants were mutagenically treated during the stage of meiosis. Mutagenesis was performed either by irradiation with x- or -rays or by the application of nitrosomethylurethane or methylnitronitrosoguanidine. Then, by use of the anther culture technique, 913 functional plants and 442 untreated control plants were regenerated. From the exposed plants seven distinct mutants could be isolated, predominantly chlorophyll deficient lines, while from the controls no clear-cut mutants arose. One mutant turned out to be photomorphogenetic in addition to having a chlorophyll defect. In addition to the production of mutants the treatments significantly increased the frequency of multicellular structure formation from microspores.     

小麦×冰草属间杂种F_1的植株再生及其变异

在对0.5—40.cm长幼穗培养4周后诱导出愈伤组织的基础上,获得了88株普通小麦(Triticum aestivum cv.Chinese Spring,2n=6x=42,AABBDD)×沙生冰草(Agropyron desertorum Fisch.> Schult.,2n=4x=28,PPPP)杂种F_1的再生植株。不同长度的幼穗在培养时,其愈伤组织发生的部位及其增殖速度不同。再生植株的产生主要是通过直接器官发生途径。所有的再生植株染色体数目全与杂种F_1相同,为2n=35。与杂种F_1相比,再生植株的减数分裂行为是相当复杂的,证明有染色体结构变异的发生。每茎穗数、叶片失绿斑等形态上的变异是由环境效应引起的;而株高、每穗轴节上小穗数和育性是由遗传效应决定的。特别值得注意的是,再生植株的自交结实率高达5.49%,共获得自交种子484粒,这对利用P染色体组中的期望基因有着极为重要的意义。     

大肠杆菌乙酸代谢突变株的选育和特性研究

在大肠杆菌高密度培养中 ,因代谢副产物乙酸积累 ,导致抑制菌体的生长和产物表达的下降。为减小乙酸的抑制作用 ,采用60 Co诱变处理大肠杆菌JM1 0 1 ,结合连续培养 (含乙酸钠选择压力 )定向富集方法 ,选育到一株乙酸耐受性增强的菌株JL3。该菌株表现出明显的乙酸耐受性的提高 ,在含有 1 0 g/L乙酸钠的MA培养基中 ,菌体生长和葡萄糖消耗速率都有较大程度提高 ,并且具有良好的遗传稳定性     

Bioelectric activity is required for regional specificity of sensory ganglion projections to spinal cord explants cultured in vitro

Summary Chronic blockade of spontaneous nerve impulses by means of tetrodotoxin leads to abnormally diffuse afferent projections into spinal cord cross-sections cultured for two to six weeks in vitro. In addition, even untreated explants which show a low level of spontaneous cord discharges failed to develop the normal degree of dorsal pathway selectivity. It is therefore concluded that centrally generated neuronal activity may play an important role in eliminating exuberant connections which, during early development, are transiently present in this part of the nervous system.     

Simplified high-throughput screening of AOX1-expressed laccase enzyme in Pichia pastoris

The heterologous protein expression in Pichia pastoris under the control of alcohol oxidase (AOX1)promoter comprises two steps, the growth and induction phases, which are time-consuming and technically demanding. Here, we describe an alternate method where expression is carried out directly in the methanol-containing medium. Using this method, we were successful in screening high-activity laccase clones from a library of laccase mutants generated by random mutagenesis. This simplified method not only saves time but also is highly efficient and can be used for screening a large number of clones.