Chlorsulfuron resistant transgenic tobacco as a tool for broomrape control

Broomrape (Orobanche ramosa L.) is the most important parasitic plant that infests tobacco (Nicotiana tabacum L.). Chemical treatment of the soil is not effective and crop rotation is not acceptable to solve this problem because of the long viability period of Orobanche seeds in the soil. Application of systemic herbicides in the field with herbicide resistant tobacco could be a successful tool for broomrape control. Several tobacco cultivars were transformed with a mutant ahas3R gene for resistance to the herbicide chlorsulfuron (Glean^®, DuPont). Transformed plants were selfed and the segregation of resistance was followed in the next generation. The efficiency of the herbicide was demonstrated in greenhouse and field trials. An Orobanche/tobacco growth system was used in order to prove the lethal effect of the herbicide to the attached broomrape plants.     

石防风原生质体遗传转化及抗除草剂植株的再生

以石防风（Ｐｅｕｃｅｄａｎｕｍｔｅｒｅｂｉｎｔｈａｃｅｕｍ（Ｆｉｓｃｈ．）Ｆｉｓｃｈ．ｅｘＴｕｒｃｚ．）叶柄愈伤组织为材料建立以致密细胞团为主的悬浮培养物,用酶解法获得原生质体。用ＰＥＧ法将拟南芥菜（Ａｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）抗除草剂基因导入原生质体后进行液体浅层培养。转化细胞经除草剂ｃｈｌｏｒｓｕｌｆｕｒｏｎ筛选,通过胚状体途径产生抗性小植株。转化处理１０６个原生质体,以加入４０～５０μｇ／ｍＬ质粒ＤＮＡ,ＰＥＧ终浓度为１０％,于１ｍＬ转化介质中,２６℃黑暗下处理２０ｍｉｎ的效果最好。ｃｔＤＮＡ的加入对转化结果无明显影响。分子杂交试验表明,突变的ａｌｓ基因已整合进转化植株的基因组中。转基因植株的细胞在脱分化过程中仍具有抗ｃｈｌｏｒｓｕｌｆｕｒｏｎ的能力;移栽成活的转基因小苗仍表现出抗除草剂特性,而未转化的植株在低浓度除草剂下即逐渐死亡。表明转入的ａｌｓ基因在石防风细胞内能够稳定表达。     

Analysis and regulation of legume inoculants in Canada: The need for an increase in standards

Herbicide residues may affect seedlings during early stages of their development. We studied this possibility by the use of light and electron microscopy after incubation of germinating seeds ofPisum sativum L. andZea mays L. with different concentrations of chlorsulfuron and metsulfuron-methyl. By in vitro experiments, we have shown that both herbicides caused growth reduction of the very young roots, and severe ultrastructural alterations and injuries of the root caps of both species. Chlorsulfuron caused increase of electron-dense material in the vacuoles, cytoplasmic degeneration even in the inner secretory cell layers of the cap, and disruption of the amyloplast envelopes with release of the statolithic starch grains. In the initial cell complex of the root cap, the herbicides caused the formation of large concentric aggregates of the rough ER and wall disformations in the cells adjacent to this complex. Scanning electron microscopic observations revealed a decrease of the slime layer ensheathing the root cap and the subapical root surface.We conclude that even in early stages of seed germination, both herbicides seriously affect the gravity perception centre (consisting of the statocytes), and the secretory tissue of the root caps, thus probably disturbing the processes of gravitropism and the protective slime secretion of the roots.     

The influence of chlorsulfuron and metsulfuron methyl on root growth and on the ultrastructure of root tips of germinating maize seeds

Seeds of Zea mays L., germinating in soil, were exposed to very low doses of the sulfonylurea herbicides chlorsulfuron and metsulfuron methyl. At a concentration of 0.012 mg L^–1, chlorsulfuron caused 72% and metsulfuron methyl 55% growth reduction of the young primary roots. Both herbicides also caused obvious injuries to the root tips. Scanning electron microscopic observations of the root tip surfaces indicated an inhibition of slime secretion at a herbicide concentration of 1.5 mg L^–1. Transmission electron microscopy revealed obvious changes to the nuclei and deformation of radial cell walls in the primary root cortex at 0.012 and 1.5 mg L^–1 for both herbicides. Moreover, the secretory cells of the root cap periphery showed partially irregular deposition of premature cell wall or slime material at a concentration of 0.012 mg L^–1 of both herbicides.From the results of our electron microscopic observations we conclude that the primary roots of maize seedlings are seriously affected by extremely low concentrations of even those herbicides which (as chlorsulfuron and metsulfuron methyl) have been developed to inhibit the growth of dicotyledonous weeds. Moreover, we suggest that the frequently observed growth retardation of crop seedlings is a consequence of early root tip injuries caused by herbicide residues in the soil. ei]H Lambers     

Mode of action of chlorsulfuron in a sensitive wheat (Triticum aestivum) cultivar: primary and secondary effects on nitrogen assimilation

Chlorsulfuron (15 g a.i. ha^-1) inhibited growth of wheat (Triticurn aestivum L. cv. Rongotea) especially on high nitrate (NO₃) supply. Decreased growth at high NO^-₃ was associated with higher concentrations of reduced nitrogen (N) and NO^-₃ in the shoots. Seven days after spraying (DAS), shoot dry weight (dry wt) of sprayed plants was similar with NO^-₃ or branched chain amino acids as main N supply but 28 DAS, shoot dry wt was greater with the amino acid treatment. One DAS, chlorsulfuron caused substantial decreases in extension of the youngest leaf and acetolactate synthase activity and valine content of shoots of plants supplied with NO^-₃ or branched chain amino acids. Total amino acid content of shoots was greater in sprayed plants than in unsprayed plants 1 DAS. Acetolactate synthase activity of sprayed plants supplied low NO^-₃ returned to normal 14–21 DAS. For sprayed plants transferred from low to high NO^-₃ supply 7, 14 or 21 DAS, shoot dry wt 50 DAS increased with increased time of transfer to high NO^-₃ while shoot NO^-₃ content decreased. Shoot NO₃ content of sprayed plants transferred to high NO^-₃ supply 7 or 14 DAS was similar to that in unsprayed plants at applied NO^-₃ concentrations which inhibited growth. It is concluded that inhibition of acetolactate synthase is likely to be the primary mode of action of chlorsulfuron in this wheat cultivar; data are consistent with the proposal that subsequent NO^-₃ accumulation can also inhibit growth.     

Crop responses to applications of both chlorsulfuron and monoammonium phosphate

Field studies were conducted at two locations of different soil pH in Saskatchewan using preemergence chlorsulfuron and granular monoammonium phosphate. In year one, barley was not adversely affected by chlorsulfuron applied in the spring at 11.25 and 22.5 g a.i. ha^–1 (Kernen location, soil pH 7.5) or 22.5 and 45.0 g a.i. ha^–1 (Scott location, soil pH 5.5). In year two, the residual chlorsulfuron did not affect flax seed yield on the acid soil, but caused substantial crop damage to flax and lentil on the neutral soil. In general, flax and lentil were more injured by chlorsulfuron residues where lower rates of MAP had been applied the previous year, suggesting that the presence of a MAP band in the soil at the time of herbicide application may have slowed the rate of chlorsulfuron degradation. A corn-root bioassay procedure was adapted to estimate residue levels in soil samples taken from the field trials.     

Chlorsulfuron reduces rates of zinc uptake by wheat seedlings from solution culture

Wheat plants differing in zinc efficiency (Excalibur; Zn-efficient, Gatcher and Durati; Zn-inefficient) were grown in HEDTA chelate-buffered nutrient solution in controlled conditions and supplied with 0 or 40 g chlorsulfuron L^-1 . Zinc uptake rates of 12-d-old plants were measured over 80 or 90 minutes using⁶⁵ Zn added to nutrient solutions. Increasing the zinc concentration of the solution increased the rate of zinc uptake, while the percentage of zinc transported to shoots was decreased. Addition of chlorsulfuron to uptake solutions for 90 minutes did not influence rate of zinc uptake or transport of zinc to shoots. Pretreating plants with chlorsulfuron for 5 days decreased zinc uptake rates, but transport to shoots was proportionally increased. Three-day pretreatment with chlorsulfuron was the minimum required for significant differences in uptake and transport of zinc to occur. Plants exposed to chlorsulfuron for 3 days required a further 5 days of growth in chlorsulfuron-free solutions before uptake rates recovered to control plant rates. It is concluded that chlorsulfuron deleteriously but reversibly affects uptake of zinc across the plasma membrane after prolonged exposure.     

寡聚核苷酸介导的基因突变技术创制抗除草剂烟草新种质北大核心CSCD

为进一步优化烟草(Nicotiana tabacum)品种‘K326’的种质,该研究采用寡聚核苷酸介导的基因突变(oligonucleotide-mediated mutagenesis,OMM)技术,利用植物中支链氨基酸合成途径中第一个关键酶——乙酰乳酸合成酶(acetolactate synthase,ALS)突变后烟草对氯磺隆除草剂不敏感且产生抗性的特征,以及NCBI报道的ALS基因序列同源克隆了烟草品种‘K326’中的ALS基因,并根据ALS基因序列设计用于定点突变的RNA/DNA嵌合体,导入烟草品种‘K326’创制对氯磺隆除草剂具有抗性的烟草新种质。结果表明:(1)烟草品种‘K326’具有2条ALS基因,即ALS SuRA和ALS SuRB,大小分别为2004 bp和2010 bp。(2)根据2个基因的保守位点ALS SuRA 588脯氨酸位点和ALS SuRB 1719色氨酸位点设计用于ALS基因核苷酸第588位点的Chl-588嵌合体和第1719位点的Chl-1719嵌合体。(3)利用基因枪成功将这2个片段导入烟草愈伤组织,愈伤组织依次经抗性芽分化和生根,共获得氯磺隆抗性植株22株。(4)抗性植株ALS酶活性测定显示,8株抗性植株具有较强的活性,进一步对抗性植株中跨突变位点保守扩增、测序,最终确定有2株(f11和b18)分别在588、1719位点产生定点突变。综上认为,该研究在获得烟草品种‘K326’抗氯磺隆新种质同时,也为培育抗性烟草新种质提供了理想的亲本材料。     

APHIDICOLIN INHIBITS DNA REPLICATION IN THE MICRONUCLEUS AND BLOCKS CYTOKINESIS IN PARAMECIUM CAUDATUM

Aphidicolin, a selective inhibitor of DNA polymerase, totally blocks DNA replication in the micronucleus but not in the macronucleus of Paramecium caudatum. The ciliates no longer divide and after 4 days the DNA content of the macronucleus has increased by 64%. Concomitantly the cell volume has increased by 53%.