云烟 87 八倍体植株的诱导及其主要生殖特征

通过不同浓度秋水仙素溶液浸泡"云烟 87"四倍体无菌苗茎尖 72 h,经组织培养,成功获得八倍体植株,其中以 0.4%浓度秋水仙素处理检出率最高,为 28.57%。与"云烟 87"四倍体植株相比,八倍体植株花器官明显较大,叶片较厚、较小,节间较短。八倍体植株花粉量小,花粉大小不均一,可见超大花粉,花粉萌发率低,仅为 6.45%。八倍体自交不能结实,但与四倍体烟草品种杂交均可获得大量有活力的种子,发芽率均大于 65%,且与同"云烟 87"四倍体杂交不能获得种子的野生烟草Nicotiana plumbaginifolia杂交也可获得有活力的种子。可见,通过秋水仙素浸泡结合组织培养获得烟草八倍体是可行的。      

Nicotiana tabacum Lin.-N. plumbaginifolia V iv.杂种的鉴定及其育性和黑胫病抗性的初步分析

为明确一株云烟87八倍体(2n=8x=96)与N.plumbaginifolia Viv.(2n=2x=20)的杂交后代的部分生物学特性。对该后代进行了形态观测,细胞学遗传学分析,育性分析,并进行黑胫病抗性的离体鉴定。结果显示,该杂交后代(无性系)大部分外观形态指数介于云烟87四倍体与N.plumbaginifolia之间;花冠颜色与云烟87相近,花药颜色与N.plumbaginifolia相近。其染色体数目为58条,其中含有来自N.plumbaginifolia的10条染色体。花粉离体培养未见萌发;与云烟87四倍体杂交,做母本时坐果率为100%,平均每个蒴果获得102.60±25.12粒可萌发的种子,以其为父本及自交时未获得成熟蒴果。该杂交后代(无性系)对黑胫病抗性强于云烟87,与N.plumbaginifolia相近。综上所述,该杂交后代为云烟87与N.plumbaginifolia的真杂种(2n=5x=58);其雄性败育,但雌性可育;且具较强的黑胫病抗性。     

普通烟草不完全染色体组有性导入黄花烟草

本研究探讨两个杂交不亲和种间有性导入一个不完全染色体组的可能性。普通烟草(Ni-cotiana Tabacum)花粉经~(137)Cs γ-射线(5-40千伦)照射后,授到黄花烟草(N·rusti-ca)的花柱上。授粉后,第六天、第七天,将胚珠从胎座上分离出来,移植到固体培养基培养。培养结果,获得114株长成的杂种植株。根据其形态特征,可以分为下列三组:1、正常型杂种:与未辐射花粉杂交获得的杂种无区别。2、异常型杂种:与正常型杂种有明显区别。3、母本型杂种:与母本黄花烟草无区别,可育。在所有杂种植株中,50%以上是异常型杂杂种,其形态特征在母本单倍体至正常型杂种间变化,染色体变幅24-48条。这些结果表明,结合用辐射花粉杂交和杂种胚珠离体培养两种技术,可将普通烟草(n=24)的不完全染色体组导入黄花烟草(n=24)。     

花生种间杂种异源多倍化早期世代性状和SSR变化研究

以四倍体栽培种花生仲恺花4号为母本、二倍体野生种花生Arachis chacoensis为父本,对其种间杂种F1及人工加倍获得的异源六倍体S0及自交世代（S1～S3）植株的植物学性状和花粉育性进行观察,采用SSR标记研究各世代的基因组变化情况.结果显示随着自交的进行,F1～S3植株的总分枝数、第一对侧枝长和花旗瓣宽变异系数分别达到48.48％、34.56％和13.74％,表现出明显的不稳定性;F1～S3的花粉萌发率分别为0、11.03％、10.58％、16.44％和18.53％,花粉育性随着世代的增加逐渐增强;扩增出的微卫星条带在F1开始发生变化,主要包括为亲本片段的丢失、跳跃式继承和新片段的产生,随着世代的增加表现出稳定的趋势,表明微卫星及侧翼区域变化剧烈而快速,其生物学功能可能与多倍体进化过程有关.     

烟草18S rDNA染色体原位杂交技术优化研究

荧光原位杂交（Fluorescence in situ hybridization,FISH）在细胞遗传学中有较多应用,其中重复序列常被作为探针来研究物种进化、分类。但现常用的探针序列均较长（200 bp-500 bp）,杂交耗时较多。本研究以烟草（Nictiana tabacum）为材料,克隆并标记18S rDNA保守序列（254 bp）,以其为探针对烟草染色体进行FISH,经18 h杂交获得了清晰的信号。在此基础上,利用荧光素直接标记引物序列（寡聚核苷酸）,经较短时间（2 h）的杂交,也获得了清晰的信号。以寡聚核苷酸为探针对云烟87四倍体、N.tabacum-N.plumbaginifolia五倍体、烟草六倍体、云烟87八倍体以及N.plumbaginifolia的染色体进行杂交,均获得了良好效果。且杂交液可简化配制,洗脱过程也可简化。18S rDNA寡聚核苷酸探针与5S rDNA寡聚核苷酸探针结合,实现了N.plumbaginifolia的18S rDNA和5S rDNA双色FISH。所以,该寡聚核苷酸序列可以应用于烟草属植物18S rDNA（45S rDNA）位点的分析,且较大程度缩短了操作时间并简化了操作过程。      

抗PVY云烟87定向改良新品种“云烟301”的选育及特征特性

抗PVY云烟87新品种“云烟301”是云南省烟草农业科学研究院、国家烟草基因工程中心在克隆烟草隐性抗PVY基因eif4e1（又称为感PVY基因eIF4E1）、开发功能性分子标记、分析国内外抗PVY品种和种质资源基因型过程中,以云烟87为母本、Y85×RY2/F₂为父本（携带eif4e1）杂交,以定向改良云烟87的PVY抗性为主要目标,通过连续回交、分子标记辅助选择、全基因组基因芯片背景检测等技术培育而成（非转基因）。云烟301的遗传背景恢复为云烟87的比率为99.43%,云烟301的PVY抗性显著提高,保留了云烟87的栽培烘烤特性、原烟风格特征和感官质量。在打顶后PVY发病率达到1%的云烟87种植区,种植云烟301可挽回病害损失,具有良好的推广价值。      

烟草雄性不育系的转育及其利用的研究

烟草杂种优势现象比较普遍,一般两个亲本杂交以后,杂种 F_1在生长势、生产量和抗逆性等方面,都比双亲平均值有明显提高。但是对于自花传粉的两性花的烟草来说,在未解决人工去雄的较大劳力耗费以前,生产上大量推广利用的实际可能性受到限制。因此,积极选育烟草雄性不育系,对于节省劳力和繁育纯度高的杂交种种子,开辟烟草杂种优势利用的新前景,有其重要的实际意义。一、雄性不育系的转育烟草细胞质雄性不育系的转育,是把某优良品种为父本与胞质雄性不育系杂交,再以前者为父本进行连续多代的回交,使前者的细胞     

白菜型油菜和诸葛菜远缘杂交的子房培养及杂种鉴定

对白菜型油菜和诸葛菜正反属间杂交及杂交子房培养进行了研究。结果表明,以授粉后7d取子房进行离体培养其子房存活率和结籽率均较高。对于所使用的4种培养基,培养基A（MS＋2．0mg·L^-16-BA＋0．2mg·L^-1NAA＋500mg·L^-1水解酪蛋白＋0．5％活性炭）显示较好的效果,在杂交组合澧临白菜×诸葛菜中,其最高结籽率达2．56％。适宜的母本结合授粉后恰当的子房离体时间和合适的培养基成分有利于提高杂种的获得频率。通过子房培养,从杂交组合澧临白菜×诸葛菜获得的1株杂种植株,形态上显示出双亲的中间型。细胞学研究显示,该杂种小孢子和花粉发育呈现异常现象,花粉育性低。形态学和SSR分子鉴定表明,该杂种植株为真杂种。因此,通过有性杂交结合子房培养能够将诸葛菜的有益性状转入白菜型油菜中。     

烟草体细胞杂交优质抗病株系的选育

为了选育抗花叶病的烟草新品种 ,用G 140与抗花叶病的野生波叶烟草 (N .undulata)进行了体细胞杂交 (即两种烟草的体细胞原生质体融合 ) ,其后代经过形态学鉴定 ,过氧化物酶同功酶酶谱鉴定、细胞学染色体数目检测 ,确定为体细胞杂种植株。采用定向筛选和回交手段选育体细胞杂种植株 ,经过 3年的田间筛选鉴定 ,得到了体细胞杂交单株自交系 ,从中再经筛选和攻毒试验获得了几个综合农艺性状好 ,抗或耐花叶病的优良单株和株系     

烟草单倍体基因编辑系统的建立

  背景和目的  单倍体材料作为转化受体具有不受同源染色体联会配对影响、转化效率较高、外源基因在后代基因组中稳定性较好、加倍后即可获得纯合转化植株等优点,结合基因编辑CRISPR/Cas9系统,可大大加快烟草品种选育进程。  方法  通过花粉离体培育获得单倍体,利用CRISPR/Cas9系统编辑八氢番茄红素脱氢酶基因位点,产生白化单倍体植株。  结果  （1）经过4℃处理4~6天的实验组,花药愈伤组织形成率最高可达11%;（2）获得转化后单倍体植株120株,其中白化86株（白化率71.67%）,红花大金元51株,其中白化24株（白化率47.06%）。  结论  （1）适当低温处理可提高花药培育单倍体效率;（2）以单倍体为受体的基因编辑效率有所提高。      

不同pH赤霉素引发对烟草种子萌发和幼苗素质的影响

以MS云烟87、MSK326和红花大金元种子为材料,研究了不同pH的赤霉素（GA3）引发对种子萌发和幼苗素质的影响。结果表明,MS云烟87经pH3．0的50mg／LGA3引发,幼苗茎高和苗高分别提高0．59和0．40cm;对于MSK326,用pH3．0的50mg／LGA3引发效果优于其他处理,种子平均发芽时间较对照缩短0．43d,发芽指数提高12．98％,幼苗根长、茎高及苗高分别提高0．28、0．33和O．61cm;红花大金元经pH3．0的50mg／LGA3引发,种子平均发芽时间较对照缩短0．36d,发芽指数提高10．82％,幼苗茎高和苗高分别提高0．18和0．43cm。此研究结果说明,pH影响引发效果,用GA3引发时应选择适宜的pH。     

施钾量对烤烟钾积累与分配的影响

研究四川省凉山州主栽烤烟品种云烟85对钾的积累和分配特征,可为当地合理施用钾肥提供理论依据。以云烟85为材料进行土培盆栽和大田试验,分析了不同施钾量下云烟85的产量、含钾量、钾积累量和钾分配率,探讨施钾量对云烟85钾积累与分配的影响。结果表明,云烟85在不同钾肥用量下的钾积累高峰均出现在旺长后期和中部叶成熟期。随着钾肥用量的增加,烟叶产量、含钾量、钾积累量和钾分配率先增大后减小,含钾量和钾积累量差异显著。本试验条件下,种植云烟85的最佳钾肥用量为158kg／hm2。在凉山烟区种植云烟85时,可适当降低钾肥用量,并增加后期钾肥追施比例,在保证产量的同时,有望提高烟叶含钾量。     

茉莉酸浸种对云烟87斜纹夜蛾抗性的影响

为明确茉莉酸（Jasmonic acid,JA）浸种对烟草抗虫性的影响,测定了JA浸种的云烟87对斜纹夜蛾（Spodoptera litura）的抗性及植株中JA和茉莉酸-异亮氨酸（JA-Ile）的含量。结果表明,以JA浸种的云烟87为食物的斜纹夜蛾幼虫重量比未浸种的降低了15%。在模拟斜纹夜蛾取食后,JA浸种的云烟87幼苗诱导出的JA含量比未浸种的增加41%,JA-Ile含量增加42%。斜纹夜蛾取食的JA浸种的云烟87幼苗,其抗虫次生代谢物如咖啡酰丁二胺、二咖啡酰亚精胺、尼古丁和二萜糖苷的含量分别增加60%,79%,19%和29%,胰蛋白酶抑制剂活性增强80%。同时,JA浸种后6周,云烟87植株干质量与对照植株差异不显著。JA浸种可以提高云烟87对斜纹夜蛾的抗性,同时不影响其产量。     

Action of Cyclodextrins on Germinating Seeds and on Micropropagated Plants

Effects of cyclodextrins on germinating barley seeds and on the growth of micropropagated strawberry plants were investigated. Treatment of barley seeds with α- or β-cyclodextrins resulted in a similar delay of germination as reported earlier [1]. The delay of germination occurred with agarose-immobilized as well as with free cyclodextrins. Since penetration of immobilized cyclodextrins into the seeds is limited, it was concluded that the delay resulted from complexation of growth factors. Soluble cyclodextrins caused a growth retardation with micropropagated strawberry plants and certain morphological changes resembling the ones observed when the action of auxins is enhanced in the respect to cytokinins.     

Amino acid analyses of germinating seeds of some members of the leguminosae

The free and bound amino acid contents of soaked seeds and 7- and 14-day old seedlings of Pisum sativum, Phaseolus vulgaris and Canavalia eniformis were determined. It was found that on germination the free amino acid content of the plants increased, while the total amount of amino acid decreased. Most of the essential amino acids were shown to be present in the plants.     

叶片衰老诱导FLP重组酶删除转基因烟草外源基因的研究

  目的   “Gene-deletor”系统可实现转基因植物中外源基因的清除。为了研究该系统在转基因烟草中对清除外源基因的作用并最终创制不含外源基因的烟草新种质。  方法   以含有“Gene-deletor”系统的转基因烟株为材料,分析外源筛合报告选融基因Bar::GUS、重组酶基因FLP在不同叶龄叶片中的表达水平,同时通过观察转基因烟草花粉中GUS蛋白的表达活性,分析外源基因GUS在花粉中的删除情况。  结果   （1）相同叶龄不同转基因烟株叶片均表现出GUS活性和对除草剂草铵膦抗性,且3个转基因烟草株系中GUS活性和对除草剂抗性都表现出随着叶龄增大而降低的特点。（2）Real-time PCR和RT-PCR结果进一步证明外源Bar::GUS基因表达水平随叶片发育成熟持续下降,而外源FLP基因的表达水平则出现先增后降的变化趋势,在测定后期两者的表达量均达到最低,（3）花粉GUS组织染色结果表明,“明,色结果降的变化趋势,特系统诱导各转基因植株中花粉外源基因清除效率不同,平均清除率分别78.3%,54.7%和75.2%。  结论   在转基因烟草中,叶片衰老特异表达基因SAG12基因启动子驱动“动启动子驱动达基因,平均清系统（LoxP/FRT）的表达不仅可引起转基因植株叶片中外源基因的特异性清除,还能诱导花粉中外源基因的删除,该技术可为进一步创制培育不含外源基因的烟草新种质提供参考。      

The effect of calcinated calcium and chlorine treatments on Escherichia coli O157:H7 87-23 population reduction in radish sprouts

Abstract: The effect of calcinated calcium spray on Escherichia coli O157:H7 87–23 population reduction during radish sprout production was studied. Artificially inoculated radish seeds were soaked in sodium hypochlorite (NaOCl) solutions (200 and 20000 ppm), rinsed in distilled water, and sprayed with water or a calcinated calcium solution during sprouting. Microbial plate count was obtained at each step of the process and germination rate was determined after 72 h of sprouting. Scanning electron microscopy (SEM) was done on treated seeds and sprouts to locate which parts were populated by the E. coli cells. The results showed that the active compound in the calcinated calcium was calcium oxide. The treatment of 200 ppm NaOCl soaking followed by 0.04% calcinated calcium spray resulted in no microbial growth after a 72‐h sprouting, while maintaining a high germination rate. The 0.4% calcinated calcium spray significantly reduced the germination rate and is therefore not recommended. Soaking the seeds in a 20000 ppm chlorine solution achieved the highest E. coli count reduction (1.65 log CFU/g). However, the E. coli cells that survived the 20000 ppm chlorine soak grew to 6 log CFU/g sprouts after a 72‐h sprouting, significantly higher than the initial count on the seeds. The SEM microimages showed that the bacteria were mostly located in the roots of the radish sprouts and all across the seed surface. The E. coli O157:H7 87–23 cells appeared to be located in biofilms or embedded into the radish sprout tissues during sprouting. Practical Application: The seed sanitation treatment with 20000 ppm chlorine solution that is currently used by the sprout industry was once again found to be ineffective in eliminating inoculated pathogenic cells. More importantly, the remaining cells that have survived the chlorine wash would grow during sprouting to reach an alarmingly high cell concentration. The new observation of E. coli cells and sprout tissue interaction manifested as embedding of the cells in sprout tissues, if confirmed, will have a significant impact on the microbial safety intervention strategies used in the sprout industry. This research demonstrated the importance of eliminating all pathogens on the seeds before germination and sprouting.     

Studies on the relationship between antioxidative activity of rice hull and germination ability of rice seeds

An investigation of the germination potentials of seeds of Indica and Japonica rice varieties revealed that while the long-life rice seeds retained the germination ability even on storage for a year at 30°C, the germination ability of the short-life rice seeds tended to decrease when stored beyond 6 months. The role of rice hull constituents in the preservation of the germination property during ageing of rice seeds was investigated with special emphasis on rice antioxidants. Antioxidative assay with chloroform-methanol and methanol-water extracts of hull, bran-germ and grain fractions of two Indica and five Japonica rice varieties indicated that all of the seed varieties belonging to Indica and Japonica possessed a strong antioxidative activity in the hull fraction. Hull extracts of long-life rice seeds showed relatively higher activities than those of short-life rice seeds. In contrast, hull extracts of long-life rice seeds had higher inhibitory effects on lipoxygenase activity than did those of short-life varieties. Quantification of the natural antioxidants indicated that ^α-tocopherol in rice oil to be present in the range of 318.3–450.1 μg g^?1 oil while the amount of oryzanol in the hull, bran-germ and grain fractions of rice was found to be 1.0–5.8, 95.4–279.8 and 3.0–9.4 μg g^?1 sample respectively. The results, however, did not show any positive correlation between the amount of natural antioxidants in rice hull and germination potentials of rice seeds. The amount of total phenolic constituents in rice hull was found to range from 190–390 μg g^?1 sample, the levels being far higher in long-life rice seeds in comparison with the short-life rice seeds. These values seemed to agree well with the germination data of the corresponding rice variety. The investigation revealed a strong influence of the level of phenolic constituents in rice hull on the storability of rice seeds.     

不同品种（系）烤烟西柏三烯二醇代谢差异及其对香气品质的影响

摘要:研究了不同烤烟品种（系）叶面分泌物组分以及主要成分西柏三烯二醇的代谢差异,并对烟叶感官质量进行了评价,以期初步阐明烟草西柏三烯二醇代谢机理并为寻找和选育烟草中具有特异香气的种质资源提供有效的研究方法。本研究采用气相色谱-质谱联用技术（GC-MS）分别测定了8306、12451、云烟87、Y892×8306、豫烟11号、NX232和中烟100,7个烤烟品种（系）中部叶龄60d叶面分泌物组分,并对其中差异较大的8306、12451和云烟87的西柏三烯二醇合成关键酶基因CYC-1和CYP71D16进行了荧光定量PCR测定,对成熟期叶面分泌物的变化、调制后烟叶茄酮含量和感官质量进行了分析评价。结果表明,7个烤烟品种（系）中部叶叶龄60d时烟叶的叶面分泌物中,共测定出13种非挥发性化合物,主要成分为西柏三烯二醇,材料间含量存在差异;8306具有最高的叶面分泌物和西柏三烯二醇含量,豫烟11号次之,12451最低;CYC-1和CYP71D16在8306中的相对表达量显著高于12451和云烟87,说明这两个基因的上调表达促进了西柏三烯二醇的合成。成熟期具有较大西柏三烯二醇降解量的8306和豫烟11号在调制后拥有更高的茄酮含量;与云烟87相比,豫烟11号获得了较高的感官评吸分数和工业评价。