Breeding maize lines for complete and partial resistance to maize streak virus (MSV)

Summary S₁ to S₅ inbred lines, derived from a maize population bred for its overall resistance to three tropical viruses, were screened for resistance to maize streak virus (MSV) by artificial plant infection using viruliferous leafhoppers. Symptoms were rated and intra-line frequency distributions studied for all pedigree inbred lines. Mortality due to MSV was very low among these inbreds. Symptoms appeared later, developed slower and were less severe than in the susceptible control hybrid. Results of a study of 500 S₁ and 93 S₂ lines suggested that resistance is under genetic control via a system involving loci with major genes (with dominance for resistance) controlling high to complete resistance, associated with a genetic system involving loci with minor genes controlling partial resistance. Lines expressing complete resistance to MSV were developed from 5 cycles of inbreeding and selection. The relevance of such complete and partial resistance is discussed.Abbreviations MRPS Mean Rating for Plants exhibiting Symptoms     

Effect of maize streak virus disease on the growth and yield of maize as influenced by varietal resistance levels and plant stage at time of challenge

Field experiments were conducted from 1989 to 1991 at Ibadan, Nigeria, to assess effects of maize streak virus (MSV) disease on growth and yield of maize varieties having different levels of disease resistance. MSV disease reduced yield and growth in all years, but varieties differed significantly in amount of loss, disease severity and incidence. MSV disease was negatively correlated with plant height and dry weight, grain weight per plot, 1000-grain weight, ear length and diameter. In 1989 MSV disease decreased yield of resistant variety TZB-SR by 1.5%, of resistant hybrid 8321-21 by 10%, and of moderately resistant hybrid 8329-15 by 17%. Yield of susceptible variety TZB Gusao was reduced significantly more, by 71%. Plant age at time of virus challenge had significant effects on yield and growth characters, with earlier infection resulting in greater disease severity and yield reduction. A significant interaction between variety × age at challenge was also detected, indicating that varieties were differentially affected by MSV in relation to the growth stage when challenged. Disease incidence after challenge was lower for the most resistant varieties. This property of lower disease incidence under equal challenge opportunities (tolremicity) is an important aspect of resistance. The resistant varieties discussed here were bred for tolerance - good yield performance when diseased -, but TZB-SR and 8321-21 also exhibited tolremicity. Tolremicity combined with tolerance constitutes the overall disease resistance of a variety to a systemic pathogen such as MSV. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transgenic barley by particle bombardment. Inheritance of the transferred gene and characteristics of transgenic barley plants

Summary Transgenic barley plants (Hordeum vulgare L. cv. Kymppi) were obtained by particle bombardment of various tissues. Immature embryos and microspore-derived cultures were bombarded with gold particles coated with plasmid DNA carrying the gene coding for neomycin phosphotransferase II (NPTII), together with plasmid DNA containing the gene for -glucuronidase (GUS).Bombarded immature embryos were grown to plants without selection and NPTII activity was screened in small plantlets. One plant proved to be transgenic (T₀). This chimeric plant passed the transferred nptII gene to its T₁ progeny. The presence of the nptII gene was demonstrated by the PCR technique and enzyme activity was analyzed by an NPTII gel assay. Four T₀ spikes and 15 T₁ offspring were transgenic. The integration and inheritance was confirmed by Southern blot hybridization. Transgenic T₂ and T₃ plants were produced by isolating embryos from green grains of transgenic T₁ and T₂ plants, respectively and growing them to plants. After selfing, the ratio of transgenic to non-transgenic T₂ offspring was shown to follow the rule of Mendelian inheritance. The general performance of transgenic plants was normal and no reduction in fertility was observed.Microspore-derived cultures were bombarded one and four weeks after microspore isolation. After bombardment, cultures were grown either with or without antibiotic selection (geneticin ^R or kanamycin). When cultures were grown without selection and regenerated plants were transferred to kanamycin selection in rooting phase, one out of a total of about 1500 plants survived. This plant both carried and expressed the transferred nptII gene. The integration was confirmed by Southern blot hybridization. This plant was not fertile.     

Maize streak virus: evidence for resistance againstCicadulina mbila Naude,the main vector species

Summary Settling, probing and oviposition behaviour ofCicadulina mbila, the main vector of maize streak virus (MSV) disease were studied on four maize genotypes, 100MSR, HASR, Reunion and H512. Of the four, only H512 was completely susceptible to MSV while the other three genotypes were all known to have some resistance to MSV. Generally,C. mbila settled in higher numbers on the MSV susceptible genotype compared to the three virus resistant ones. However, the number of probing marks left on H512 and 100MSR were similar while more marks were made on Reunion and HASR in increasing order. The oviposition patterns were similar to those observed for probing behaviour. The results suggest the existence of potentially useful resistance mechanisms against the vector. The possible implications of these results are discussed.     

安徽省棉田马唐对草甘膦的抗药性研究

为明确安徽省不同生态区棉田马唐对草甘膦的抗药性水平,采用培养皿种子检测法、整株植株测定法和症状级别法测定池州市、安庆市、芜湖市、滁州市、蚌埠市、阜阳市等地共9个点马唐对草甘膦的抗药性水平。3种检测方法结果均表明:安徽安庆市大观区海口镇皖河农场马唐相对抗药性倍数最高,抗药性指数分别为3.23、3.52和4.91;培养皿种子检测法和整株植物测定法检测的结果基本一致,而使用症状级别法检测的马唐的相对抗药性倍数略高于其他2种方法。     

基因枪介导法转化苎麻获得转基因植株的研究

以苎麻品种中苎1号子叶愈伤组织为受体材料,利用基因枪法将外源双价抗虫基因(CryIA+CpTI)导入苎麻,以期建立基因枪转化苎麻的技术体系.结果表明,基因枪转化苎麻的最佳条件为轰击压力1 300psi、金粉+DNA用量500μg+1.0μg/枪、轰击距离9cm、轰击次数2次,共获得4株阳性转基因植株.苎麻子叶愈伤组织的基因枪转化法是苎麻遗传转化的新方法,为建立苎麻基因枪转化体系奠定了基础.     

Heavy-ion-induced mutants in sweet pepper isolated by M1 plant selection

We examined the effects of heavy-ion bombardment on mutagenesis in sweet pepper (Capsicum annuum L.). Dose–response studies indicated that 10 Gy irradiation of ¹²C or ²⁰Ne ions on dry seeds is suitable for inducing mutations in plants. From ²⁰Ne-irradiated M₁ plants, putative mutants included two dwarf plants and one plant whose pericarp was yellow were isolated. Phenotypes of their M₂ progeny were similar to those of the M₁ plants and did not segregate. F₁ plants resulting from reciprocal crosses between the mutants and wild-type plants showed the wild-type phenotype, but phenotypes of F₂ and BC₁F₁ segregated at 1:3 (mutant:wild) and 1:1, respectively. These crossing experiments indicate that the three mutants have monogenic recessive mutations in nuclear genes. In light of these data, we discuss the effectiveness of using heavy-ion bombardment to mutate sweet peppers.     

转AcAMP-sn基因抗全蚀病小麦新种质的创制与鉴定

抗菌肽是一类具有广谱抗菌性的小分子多肽,在植物防卫反应中起着重要作用。本研究人工合成了洋葱抗菌肽基因AcAMP-sn,利用基因重组技术构建了该基因的单子叶植物表达载体pAHC25::AcAMP-sn,使AcAMP-sn基因的表达受玉米Ubiqutin启动子控制。采用基因枪介导法,将AcAMP-sn基因导入小麦品种扬麦18,利用PCR、半定量RT-PCR及荧光实时定量RT-PCR检测、分析转基因小麦T0~T4代目的基因及其表达量,并对转基因植株进行全蚀病的抗性鉴定。PCR检测结果表明,导入的AcAMP-sn基因能够在转基因小麦中遗传。与受体扬麦18相比,在5个转基因小麦T4代株系中,AcAMP-sn基因的表达量及全蚀病抗性均显著提高,且全蚀病菌的相对含量明显下降,说明AcAMP-sn基因的过表达可以增强转基因小麦对全蚀病的抗性。     

广州市叶菜田杂草群落组成及其年动态变化

于2008年利用倒置“W”九点取样法对广州市叶菜田杂草群落年动态变化进行了调查。调查共计发现60种杂草,属23科,45属。春季杂草群落为马齿苋(Portulaca oleracea)+繁缕(stellaria media)+腋花蓼（Echinochloa colonum）为主;夏季以马齿苋+碎米莎草（Cyperus iria）+凹头苋（Amaranthus lividus）为主;秋季以马齿苋+凹头苋+光头稗（Echinochloa colonum）为主;冬季以腋花蓼+繁缕+酸模叶蓼（Polygonum lapathifolium）为主。通过计算杂草的优势度得出全年危害最严重的杂草为马齿苋、繁缕、腋花蓼、酸模叶蓼、凹头苋、碎米莎草、马唐（Digitaria sanguinalis）、牛筋草（Eleusine indica）、光头稗、小藜（Cardamine hirsuta）等10种。其中,马齿苋、凹头苋、碎米莎草在春夏秋季为优势杂草;马唐在夏秋冬为优势杂草;光头稗、牛筋草为春秋季优势杂草;繁缕、腋花蓼、酸模叶蓼、小藜为冬春季优势杂草。     

Culturing shoot-tip clumps of pearl millet [Pennisetum glaucum (L.) R. Br.] and optimal microprojectile bombardment parameters for transient expression

Microprojectile bombardment and transient expression of the reporter gene, β-glucuronidase (GUS) in a novel target tissue, multiple-shoot-tip clumps of pearl millet [Pennisetum glaucum (L.) R. Br.] is reported here. The multiple-shoot-tip clumps were developed in vitro from shoot-apices of seedlings. Using this method, the apical meristems along with the germline cells were easily exposed for bombardment without loss of viability. Further growth of the multiple-shoot-tip clumps was not substantially affected by microprojectile bombardment. Transient expression of β-glucuronidase gene was detected in the form of blue transformed cell sectors in the bombarded tissue by an in situ enzyme assay. The blue sectors were used as convenient criteria to study several factors affecting gene transfer efficiency. Optimal conditions for efficient transient expression of the GUS gene have been defined to aid future strategies of genetic engineering in pearl millet with agronomically important genes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transgenic fertile soybean plants derived from somatic embryos transformed via the combined DNA-free particle bombardment and <Emphasis Type="Italic">Agrobacterium</Emphasis> system

An Agrobacterium-mediated transformation procedure for soybean [Glycine max L. Merrill] proliferating somatic embryos is here described. The Agrobacterium tumefaciens LBA4404 strain harboring pTOK233, pCAMBIA1390-olp or pH7WG2Dwrky plasmids was used to mediate gene transfer into the plant genome. Prior to Agrobacterium inoculation, proliferative soybean embryogenic clusters were microwounded by DNA-free tungsten particle bombardment. Three independent transformation experiments were performed. In Experiment I, 26 transgenic plants were obtained from a unique clone of cv Bragg, while 580 plants were recovered from 105 clones of cv IAS5. In Experiment II, a single hygromycin-resistant clone of cv BRSMG68 Vencedora was recovered and gave rise to five plants. In Experiment III, 19 plants of cv Bragg and 48 plants of IAS5 were recovered, representing five and 14 independent transformation events, respectively. PCR and Southern analyses confirmed the transgenes’ integration into plant genomes. Transgenic plants were fertile. They flowered, set pods and seeds. Transgene segregation in two T₁ progenies fits the Mendelian pattern (3:1 transgenic:non-transgenic plants). This is the first report of transgenic fertile soybean plants obtained from somatic embryogenic tissues transformed by the system that combines DNA-free particle bombardment and Agrobacterium.     

转Rs-AFP2基因小麦的分子分析及其纹枯病抗性

Rs-AFP2属于r-硫堇类抗菌肽,主要通过形成离子通道直接破坏细胞来杀灭病原菌。本研究通过基因枪介导法结合对目标基因的分子检测,证明已将外源Rs-AFP2基因转入小麦推广品种扬麦12中。通过逐株抗纹枯病接种鉴定、PCR、PCR-Southern blot、Southern blot和 RT-PCR/荧光定量RT-PCR(Q-RT-PCR)分析,对转Rs-AFP2基因小麦T₁至T₄代植株跟踪检测。结果表明,Rs-AFP2在转基因小麦中能够稳定遗传,以单拷贝整合到小麦基因组中,遗传方式符合孟德尔遗传规律,并能在转录水平上表达。对转Rs-AFP2基因小麦的抗病性、主要农艺性状以及Rs-AFP2表达活性分析结果表明,与受体扬麦12相比,Rs-AFP2表达活性高的转基因小麦植株对纹枯病抗性有明显提高,其抗病性可以遗传,而主要农艺性状没有明显差异,证明可以利用Rs-AFP2基因和基因工程途径创制抗纹枯病小麦新种质。     

大豆DNA导入引起大麦籽粒蛋白含量及氨基酸含量变异的遗传稳定性分析

采用花粉管通道法和两种不同压力基因枪法将大豆总DNA导入大麦，提高了大麦后代籽粒的蛋白质含量和必需氨基酸含量。四代大麦籽粒蛋白质含量追踪分析及第四代籽粒氨基酸含量分析结果表明，部分大麦籽粒的高蛋白含量及质量变异性状能够稳定遗传。本试验还分析比较了两种DNA导入法，采用基因枪法比花粉管通道法在保持后代高蛋白遗传稳定性方面效果更好。在两种不同压力基因枪处理中，1300psi压力处理比1500psi压力处理更适合大豆总DNA的导入。在本试验中我们还获得了两个高蛋白性状较稳定的穗行，它们的蛋白质平均含量分别比对照提高20．67％和17．99％。     

基因枪转化小麦主要轰击参数的优化

基因枪转化是目前小麦遗传转化的主要方法。影响基因枪转化效率的主要参数有轰击距离、轰击压力、DNA总量及浓度、金粉颗粒大小及用量等。为改善目前小麦遗传转化效率偏低的现状, 以普通小麦品种科农199为受体材料, 设金粉大小(0.6 μm和1.0 μm)、轰击距离(5.5 cm和8.5 cm)及轰击压力(650、900和1100 psi)三因素共12个处理, 利用pAHC25质粒转化小麦幼胚, 以确定最佳轰击参数, 建立稳定高效的普通小麦基因枪转化体系。结果表明, 3个因素的不同组合对幼胚的愈伤诱导效率没有明显影响, 但对再生率影响较大, 以金粉0.6 μm、轰击距离5.5 cm的再生率最高。3个因素组合的不同处理对GUS瞬时表达有明显影响, 以金粉0.6 μm、轰击距离5.5 cm和轰击压力650 psi处理的GUS瞬时表达量最高。本试验最终获得28株转基因植株, 其中12株来自金粉0.6 μm、轰击距离5.5 cm和轰击压力650 psi的处理, 该处理的平均转化率达2.66%。     

长江中下游棉田马唐(Digitaria sanguinalis)对草甘膦的抗药性初步研究

采用培养皿种子检测法和整株植物测定法的测定了从长江中下游3个省13个县棉田采集的18个马唐(Digitaria sanguinalis)种群,其草甘膦抗药性水平,并采用分光光度计测定了1个相对敏感种群和3个相对抗性种群在施用草甘膦后莽草酸积累量和谷胱甘肽S￣转移酶(glutathione S-transferase,GST)活力的变化情况。整株植物测定法结果表明:湖南常德安障乡地区马唐种群相对抗性水平最高,抗性指数为7.637,培养皿种子检测法与整株植物测定法测定的结果基本一致。测定的4个种群莽草酸积累量均随时间的变化而上升,其中3个抗性马唐种群的莽草酸积累量在9 d内始终低于相对敏感种群。4个种群GST活力变化无显著性差异,表明GST的代谢活力与马唐对草甘膦产生抗药性不相关。     

Genetic transformation of Eustoma grandiflorum Griseb. by microprojectile bombardment

Summary Foreign DNA was introduced into cell suspension cultures and leaf tissue of Eustoma grandiflorum Griseb. (lisianthus) by microprojectile bombardment. For this purpose a low-cost bombardment device that uses a helium flux to accelerate microprojectiles was built. When cell suspensions were used, an average of 4.1 Kan resistant calli were recovered per shot after 4 months' cultivation on selective medium. Most of the Kan resistant plants regenerated from calli were positive to GUS assay. Both the nptII and gus genes were successfully amplified from alkali-treated leaves of putative transgenic plants by PCR analysis. Transgenic plants were not recovered from bombarded leaves. Considering the host range specificity of Agrobacterium, and the response of the species to plant regeneration from suspension culture, microprojectile bombardment is, at present, the most efficient procedure for genetic transformation of lisianthus.Abbreviations BA 6-benzyladenine - Cx cefotaxime - 2,4 D (2,4-dichlorophenoxy) acetic acid - FDA fluorescein diacetate - gus -glucuronidase - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2iP (2-isopentenyl) adenine - Kan kanamycin - nptII neomycin phosphotransferase II - PCR polymerase chain reaction     

Parameters influencing the regeneration capacity of calluses derived from mature indica and japonica rice seeds after microprojectile bombardment

The microprojectile bombardment procedure has allowed the stable transformation of indica and japonica rice varieties, although at different frequencies of transformation depending mainly on their regeneration capacity and on the specific parameters of the transformation protocol. A study of the process of regeneration to whole plants from primary calli derived from mature indica and japonica rice seeds, via embryogenesis, has shown that somatic embryos are produced by division and differentiation of the external cell layers of callus tissues. Adjusting the bombardment conditions to optimize gene delivery to those regenerable cells, we have evaluated the influence of parameters such as the target distance, particle penetration and the effect of osmotic treatment on the regeneration capacity of bombarded cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

用核基质结合区(SAR)序列提高小麦最小表达框转基因表达的稳定性

采用最小表达框技术转化植物可以规避由骨架序列引起的安全风险。核基质结合区序列SAR (scaffold attachment region)可作为边界元件与核基质结合阻挡转基因片段邻近染色质区的作用与影响, 提高外源基因稳定性。本研究在最小表达框序列两端添加SAR序列, 提高小麦最小表达框转基因表达的稳定性, 提高转化基因的表达效率。首先, 以GUS为目的基因构建带有SAR序列的最小表达框, 以科农199为受体进行基因枪转化, 同时以不加SAR序列的最小表达框片段为对照。带有SAR序列的最小表达框片段共轰击857个幼胚, T₀代获得40株再生植株, PCR检测到16株阳性植株, 转化效率为1.87%; 对这16个阳性单株进行GUS染色, 15株显色; 从来自4个T₀阳性植株的18个T₁代植株中随机选取18株进行PCR和GUS染色检测, 有15株表现为阳性。不带SAR序列的对照片段轰击1012个幼胚, 获得31株再生植株, 其中5株PCR阳性, 转化效率0.49%, 这5个阳性植株中仅2株为GUS染色阳性; 来自于5个T₀代PCR阳性株系的10个T₁代单株中没有发现PCR和GUS染色阳性株。表明SAR序列可以提高基因枪转基因效率和目的基因表达稳定性。为了创制抗旱转基因小麦, 以来自大豆的抗旱相关转录因子基因GmDREB3为目的基因, Bar基因为筛选标记基因, 转化受体小麦济麦22, 共轰击6045个幼胚, 获得再生植株130株, PCR检测阳性植株30株, 转化效率为0.50%; 随机选取6株PCR阳性植株进行RT-PCR分析, 其中5株可检测到外源基因的转录。进一步对这5株RT-PCR阳性植株插入片段完整性进行分析, 其中4株插入片段基本完整。通过real-time PCR分析, 发现T₀代6个RT-PCR阳性植株的外源GmDREB3的拷贝数为1~3个。以上结果证明, 在最小表达框两端加上SAR序列后可以提高小麦最小表达框转基因表达的稳定性。     

导入外源玉米C_4型NADP-ME基因对小麦光合效能的影响

为研究玉米C_4型NADP-ME基因对小麦光合特性的影响,以T_3代转NADP-ME基因小麦株系10T(9)-1-1和10T(9)-225-4及其对照周麦23为试材,进行了分子特征鉴定、光合生理特性分析和单株产量性状调查,并对其作用机制进行了初步解析。结果表明,外源基因已整合到转基因小麦的基因组中,并能正确转录和翻译;与对照周麦23相比,两个转基因株系在4个测定时期旗叶NADP-ME酶活性均明显提高,而旗叶净光合速率(P_n)则明显下降;尤其在花后第7天,两个转基因株系的酶活性较对照分别提高1.33倍和1.13倍,P_n分别较对照下降17.26%和10.35%;千粒重和籽粒产量较对照均显著下降。与对照周麦23相比,转基因株系10T(9)-225-4利用强光和同化CO_2能力下降,光合效率下降;旗叶气孔张开率和气孔导度均显著下降;苹果酸含量降低了5.6%,丙酮酸含量则提高了17.1%;外施苹果酸可恢复其光合速率。以上研究结果表明,玉米C_4型NADP-ME基因的导入降低了小麦的光合特性,苹果酸含量降低引起的气孔导度下降可能是导致转基因小麦光合效能降低的原因。     

The Introduction of Transgenes to Control Blackheart in Pineapple (Ananas Comosus L.) cv. Smooth Cayenne by Microprojectile Bombardment

Summary A transformation technique for the introduction of transgenes to control blackheart by particle bombardment has been developed for pineapple cv. Smooth Cayenne. Leaf callus cultures capable of high frequency organogenesis with a short regeneration time were used as explant material. Gus and gfp reporter genes were used to observe and determine transient and stable expression. The ppo gene, isolated from pineapple, was introduced to control blackheart. Co-transformation occurred with constructs containing the nptII gene conferring geneticin resistance. We have recovered 15 independent transgenic gus and gfp lines each from 8 separate experiments and 22 ppo lines from 11 experiments. Gus, gfp, ppo and nptII positive plants have been regenerated, which have been shown by Southern blot analysis to be stable transgenics containing multiple copies of the introduced genes. These results show that biolistic gene delivery in pineapple can be successfully achieved at an acceptable efficiency of 0.21–1.5% for genetic improvement of ’Smooth Cayenne’, the industry standard throughout the world.