真空渗透法转化油菜及转化种子的筛选

真空渗透遗传转化法首先在拟南芥(Arabidopsis thaliana)中获得成功,是一种简便、快速且无需经过组织培养阶段即可获得大量转化植株的基因转化方法。该研究以不同遗传背景的3个甘蓝型油菜(Brassica napus)品种(系)陕3B、L45和Mg23为材料,对真空渗透遗传转化方法中真空渗透时间和Silwet L-77浓度与遗传转化效果的关系进行了比较,同时对转化种子的筛选方法进行了优化。结果表明,卡那霉素(Km)对油菜种子的萌发影响不显著,但对其生长发育有明显的抑制作用。不同油菜品种对卡那霉素的敏感性不同,各自的致死浓度也不一样。在0%-0.05%的浓度范围内,随着Silwet L-77浓度的增加,在相同的真空渗透时间内,3个油菜品种的转化率逐渐升高。当Silwet L-77浓度为0.05%时,10分钟的真空渗透时间可获得最高转化效率,此时陕3B、L45和Mg23的转化率分别达到1.97%、2.09%和2.30%。     

一种优化的大肠杆菌感受态细胞制备及转化方法

Silwet L-77是一种非离子型的表面活性剂,常用于植物的转化。本研究发现,Silwet L-77的加入也可以显著地提高大肠杆菌的转化效率。同时,我们比较了不同培养温度、不同培养浓度(OD_(600)值)及不同冷冻保护剂对感受态细胞转化效率的影响。我们发现,28℃培养E.coli至OD_(600)值为0.55~0.6之间时制备感受态细胞,利用9%的DMSO做为冷冻保护剂冷冻保存感受态细胞,转化时加入0.001 5%~0.002%的Silwet L-77,可以获得最高的转化效率。总之,该研究进一步优化了大肠杆菌感受态细胞的制备及转化方法。     

农杆菌介导巨桉Eg5高效遗传转化

以巨桉(Eucalyptus grandis)无性系Eg5叶片为外植体, 探讨了农杆菌(Agrobacterium tumefaciens)侵染时间、共培养pH值和共培养时间对瞬时转化效率的影响, 分析了不同筛选策略对遗传转化植株筛选效果的影响。结果表明, 外植体侵染45分钟, 共培养pH值为5.8, 共培养3天所得到的瞬时转化效率最高; 逐步提高卡那霉素(Km)浓度筛选转基因植株有效, 筛选率达到15%, 转化率达到0.26%。经过GUS染色分析和PCR检测, 证实为转基因植株。     

EuFPS基因表达载体构建及对杜仲遗传转化的研究

本实验用EcoRⅠ和BamHⅠ双酶切植物表达载体pSH737和含有目的基因的pUC-FPS,定向连接得到重组质粒pSH-FPS,将其导入农杆菌EHA105.采用农杆菌介导法对杜仲进行遗传转化,研究了卡那霉素(kanamycin,Km)浓度、预培养时间、菌液浓度及侵染时间、乙酰丁香酮(acetosyringone,AS)浓度、共培养时间等对杜仲遗传转化效率的影响.结果表明,选择无菌苗苗龄15 d的杜仲下胚轴,卡那霉素浓度50mg/L,农杆菌浓度OD600值0.3-0.6,侵染时间8 min,侵染时菌体重悬液中添加50 μmol/L乙酰丁香酮,共培养时间3 d,抗性芽的获得率最高.对再生植株进行GUS检测发现有45%的植株呈阳性.     

农杆菌介导的大豆子叶节非组织培养遗传转化体系优化

本研究以GUS基因在子叶节区的瞬时表达为依据,通过探讨影响农杆菌转化效率的因素,优化了大豆子叶节非组织培养遗传转化体系;利用该体系对冀豆16号进行Bar基因的遗传转化,并使用针刺法对转基因植株进行草铵膦筛选.结果表明,侵染液中附加3％蔗糖、OD600=0.6、以脱脂棉作为菌液附着介质同时不添加表面活性剂Silwet L-77、侵染1次的GUS阳性率最高达到62.13％.草铵膦抗性植株经PCR检测获得T0阳性植株10个,转化率为2.5％.经PCR和RT-PCR鉴定共获得3株T1阳性植株,初步证明目的基因已整合到大豆基因组中.     

用floral dip法对黄瓜遗传转化的初步研究

用floral dip方法进行遗传转化已经在模式植物拟南芥、部分十字花科及豆科植物苜蓿中取得成功,但在黄瓜转化中未见报道.本研究首次采用floral dip法转化黄瓜,结果表明,采用0.05%Silwet-77和含质粒pX6-GAD-GLP-1的工程农杆菌EHA105转化黄瓜自交系P2及2M1,分别收获种子544和1022粒:将收获种子经60 mg/L的卡那霉素筛选,其抗性植株移栽成活后分别进行PCR及斑点杂交检测,在2个自交系中均获得了阳性转化植株,转化率分别为0.55%和0.68%.本研究首次将floral dip法用于黄瓜遗传转化并获得成功,为扩展floral dip法的应用领域、研究floral dip法在葫芦科植物上的应用奠定了基础,同时也进一步完善了黄瓜转基因手段.     

蔗糖转运蛋白基因VvSUC11和VvSUC12双价植物表达载体的构建及在甜菜中的遗传转化

将葡萄Vitis vinifera L.的蔗糖转运蛋白基因VvSUC11和VvSUC12与甘薯Ipomoea batatas L. Lam.的甘薯贮藏蛋白 (Sporamin) 基因的根部特异性启动子命名为SP1和SP2重组。以pCAMBIA2301为起始载体,构建了pCAMBIA2301- SP1-VvSUC11-SP2-VvSUC12用农杆菌介导法转化了甜菜Beta vulgaris L.品种KWS-9103,发现预培养4 d,侵染时农杆菌的浓度OD600值为0.5,附加0.005％表面活性剂Silwet L-77,延迟筛选4 d,转化效率最高,可达42％。对在卡那霉素中分化并生根的甜菜植株进行PCR和RT-PCR检测,证明目的基因已整合到甜菜中并表达,为进一步研究该基因在甜菜Beta vulgaris中的功能奠定了基础。     

不同抗生素对金发草愈伤组织生长分化的影响

该研究以金发草愈伤组织为材料,通过分析比较不同抗生素种类(卡那霉素、潮霉素、头孢噻呋钠和氨苄青霉素)和浓度对金发草愈伤组织生长分化的影响,来确定适用于金发草遗传转化体系中的抗性筛选剂和抑菌剂。结果表明：(1)金发草愈伤组织对卡那霉素很敏感,且其分化率随着卡那霉素浓度的增加显著减少( P＝0.01)。当卡那霉素浓度为10 mg·L-1时,金发草愈伤组织的生长分化受到明显抑制,且有大量的白化苗形成,但分化率仍有36.56％;当卡那霉素浓度为15 mg·L-1时,金发草愈伤组织的分化率为11.94％,只有很少部分的愈伤分化出绿色的丛生苗;当卡那霉素浓度为20 mg·L-1时,金发草愈伤组织基本褐化死亡,分化率仅为2.26％。因此,浓度为15 mg·L-1的卡那霉素适合作为金发草遗传转化体系中的抗性筛选剂。(2)金发草愈伤组织对潮霉素的敏感性要比卡那霉素弱,且潮霉素对金发草愈伤组织分化率的影响小,但毒害作用大。因此,潮霉素不适合作为金发草遗传转化体系中的抗性筛选剂。(3)300 mg·L-1的头孢霉素和氨苄青霉素对金发草愈伤组织生长分化影响很小且能有效抑制杂菌的生长,较高浓度的氨苄青霉素对金发草愈伤组织的抑制作用不太明显。因此,300 mg·L-1的头孢霉素和较高浓度的氨苄青霉素均可作为金发草遗传转化体系中的抑菌剂。该研究确定了适用于农杆菌介导的金发草遗传转化体系中的抗性筛选剂和抑菌剂,为金发草的遗传改良及功能性基因的研究奠定了基础。     

根癌农杆菌介导GUS基因转化玉米芽尖生长点的研究

本试验以玉米(Zea mays)杂交种‘郑单958’的芽尖生长点作为农杆菌侵染受体,以β-葡萄糖醛酸苷酶基因(GUS)作为转化基因,吸水链霉菌草丁膦乙酰转移酶基因(bar)作为筛选标记基因,通过GUS颜色反应,分别从侵染液浓度、侵染时间和真空处理时间三个方面分析了影响GUS基因转化率的因素,初步得出结论:当侵染液浓度(OD600)为0.6、侵染时间2 h、真空处理时间为10 min时,选用杂交种‘郑单958’作为受体材料将会达到较高的遗传转化率。     

影响花椰菜农杆菌介导转化因素的研究

以花椰菜赛雪的带柄子叶为外植体,以MS为基本培养基,GUS基因为报告基因,分析了遗传转化过程中的影响因子,如预培养时间、农杆菌菌液浓度、侵染时间、共培养时间、乙酰丁香酮浓度、延迟筛选时间等对外植体瞬间表达和稳定表达的影响。结果显示,以花椰菜的带柄子叶为外植体,预培养2d,农杆菌菌液为OD6000.3～0.4,侵染8min,共培养2d,乙酰丁香酮浓度为100μmol/L,延迟筛选7d,卡那霉素筛选压为5mg/L为最优的遗传转化方案,转化率最高可达35.7%。另外,GUS瞬间表达率和转化率并不存在绝对的相关性,但瞬间表达分析仍然可以作为外源基因进入受体细胞的指示。花椰菜农杆菌介导转化方案的优化研究为芸薹属蔬菜高效遗传转化提供了技术保障,有利于芸薹属蔬菜遗传育种与种质创新研究。     

Efficacy of Bauveria bassiana for red flour beetle when applied with plant essential oils or in mineral oil and organosilicone carriers

The carriers mineral oil and Silwet L-77 and the botanical insecticides Neemix 4.5 and Hexacide were evaluated for their impacts on the efficacy of Beauveria bassiana (Balsamo) Vuillemin conidia against red flour beetle, Tribolium castaneum (Herbst), larvae. The dosages of liquid treatments were quantified by both conidia concentration in the spray volume and conidia deposition on the target surface. The latter approach allowed comparison with dry, unformulated conidia. The median lethal concentrations of B. bassiana in 0.05% Silwet L-77 solution or without a carrier were approximately double that for conidia in mineral oil. Carriers had highly significant effects on the efficacy of B. bassiana. The lower efficacy of conidia in aqueous Silwet L-77 may have been the result of conidia loss from the larval surface because of the siloxane's spreading properties. Neemix 4.5 (4.5% azadirachtin) delayed pupation and did not reduce the germination rate of B. bassiana conidia, but it significantly reduced T. castaneum mortality at two of four tested fungus doses. Hexacide (5% rosemary oil) caused significant mortality when applied without B. bassiana, but it did not affect pupation, the germination rate of conidia, or T. castaneum mortality when used in combination with the fungus.     

Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana

The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.     

Efficacy of Silwet L-77 against several arthropod pests of table grape

Silwet L-77, an organosilicone surfactant, was applied to several arthropod pests of California table grapes. Eggs of grape mealybug, Pseudococcus maritimus (Ehrhorn), and omnivorous leafroller, Platynota stultana Walsingham, were tolerant to 0.1, 0.25, and 0.5% treatment solutions; however, eggs of Pacific spider mite, Tetranychus pacificus McGregor, were highly susceptible with mortality >99.4% (0.1% Silwet L-77). Mortality of immature and adult stages of cotton aphid (Aphis gossypii Glover), Western flower thrips (Frankliniella occidentalis Pergande), and Pacific spider mite (Tetranychus pacificus McGregor) was > or = 93.8, > or = 98.5, and > or = 99.4% for 0.1, 0.25, and 0.5% Silwet L-77, respectively. Grape mealybug crawlers had 100% mortality when treated with 0.5 and 1.0% Silwet L-77 solutions; however, mortality was only 6.7% when 0.1% Silwet L-77 was applied. 'Thompson Seedless' table grapes were not damaged when treated with up to 1% Silwet L-77; however, grapes treated with the 0.5 and 1.0% solutions appeared wet after removal from cold storage because of the effect of the surfactant spreading the water condensation. Grapes dried with the normal bloom on the berries when they reached room temperature.     

The effect of varying rates of glyphosate and an organosilicone surfactant on the control of gorse

The herbicidal effect of glyphosate applied to gorse (Ulex europaeus L.) was improved by the addition of increasing amounts (0.5–20 g/litre) of Silwet L-77, an organosilicone surfactant. Increasing the rate of herbicide also enhanced control. There was a highly significant interaction between surfactant rate and herbicide dosage; as the amount of Silwet L-77 was increased the rate of glyphosate could be reduced without loss of herbicide efficacy. However, without any added organosilicone surfactant, glyphosate did not provide more than 73% control of gorse at any rate up to 6.5 kg a.i./ha. With the addition of Silwet L-77, complete mortality of all plants could be achieved with 2.2 kg glyphosate/ ha.     

Improvement of Agrobacterium-mediated transformation of embryogenic calluses from maize elite inbred lines

Summary This study was carried out to evaluate the effects of purine synthesis inhibitor mizoribine, purine and pyrimidine synthesis inhibitors azaserine and acivicin, and surfactant Silwet L-77 on Agrobacterium-mediated transformation efficiency of embryogenic calluses from maize elite inbred lines Qi 319 and Ye 515. After transformation and three rounds of selection on 2.8 μM chlorsulfuron, resistant calluses were obtained subsequently, and morphologically normal plantlets were regenerated from 80 to 90% of the resistant calluses treated with the compounds. There were no obvious discrepancies between the frequencies of plantlet regeneration and the ratio of PCR positive plantlets of calluses treated with different compounds. Results of PCR assay with primers for betA showed that 40.2% (103/256) of the regenerated plantlets were positive. The percentage of resistant calluses was 2–3-fold higher than the control after being treated with 0.19–0.27 mM mizoribine. The most suitable concentration of azaserin was 0.36 mM, which gave a 4-fold increase in the percentage of resistant calluses. Acivicin at 0.28–0.84 mM yielded a 3–5-fold increase in the percentage of resistant calluses, which is significantly better than the control. When the calluses were treated with 0.01 or 0.02% Silwet L-77, the percentages of resistant calluses were 34.89 and 25.60%, respectively. We concluded that purine synthesis inhibitors, purine and pyrimidine synthesis inhibitor and the surfactant Silwet L-77 at optimal concentrations significantly improved the Agrobacterium-mediated transformation of maize calluses.     

FITC示踪在优化小麦花粉管通道转化方法中的应用

利用FITC(fluorescein is othiocyanate,异硫氰酸荧光素)标记的外源DNA对小麦进行了花粉管通道法转化,在荧光显微镜下观察了外源DNA进入小麦胚囊的情况,并初步研究了转化时间及转化溶液组成对DNA进入胚囊效率的影响。结果表明,外源DNA沿着花粉管生长过程中形成的花粉管通道进入胚囊;授粉45 min和60min进行转化外源DNA进入胚囊的几率较大,因此在此段时间开始转化较为合适;相对于TE缓冲液,将0.05%Silwet L-77和5%蔗糖作为转化溶液可以缩短DNA进入胚囊的时间,但不能提高外源DNA进入胚囊的几率。研究表明,使用FITC标记观察外源DNA进入胚囊效率的方法,可简便有效地应用于花粉管通道法转化条件的初步优化。     

"Inert" formulation ingredients with activity: toxicity of trisiloxane surfactant solutions to twospotted spider mites (Acari: Tetranychidae)

Organosilicone molecules are important surfactant ingredients used in formulating pesticides. These methylated silicones are considered inert ingredients, but their superior surfactant properties allow them to wet, and either suffocate or disrupt important physiological processes in mites and insects. Aqueous solutions of the tri-siloxane surfactants Silwet L-77, Silwet 408, and Silwet 806 were bioassayed against adult female two-spotted spider mites, Tetranychus urticae Koch, with leaf dip methods to compare their toxicity with organosilicone molecules containing bulkier hydrophobic components. All three tri-siloxanes in aqueous solutions were equivalently toxic (LC50 = 5.5-8.9 ppm), whereas Silwet L-7607 solutions were less toxic (LC50 = 4,800 ppm) and Silwet L-7200 was nontoxic to mites. In another experiment, the toxicity of Silwet L-77 was affected by the wettability of leaf surfaces. The LC50 shifted from 22 to 84 ppm when mites were tested on bean and strawberry leaf disks, respectively. Droplet spreading on paraffin and surface tension were both related to the toxicity of surfactant solutions. Surface tensions of solutions below 23 mN/m caused > 90% mite mortality in leaf dip bioassays. A field test of Conserve SC and its formulation blank, with and without Dyne-Amic adjuvant (a vegetable oil-organosilicone surfactant mixture) revealed that Dyne-Amic had the greatest miticidal contribution, reducing mite populations by 70%, followed by formulation inactive ingredients. Spinosad, the listed active ingredient in Conserve, only contributed miticidal activity when synergized by Dyne-Amic. Researchers should include appropriate surfactant or formulation blank controls when testing insecticides or miticides, especially when using high spray volumes.     

Flower bud dipping or vacuum infiltration—two methods of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> transformation

The purpose of this work was to evaluate two methods (floral dip and vacuum infiltration) of in planta transformation of Arabidopsis thaliana. The key issue of this work is the identification of the developmental stages of A. thaliana flower buds subjected to agroinfection, optimal for the successful transformation. Histological tests performed after agroinfection made it possible to establish the patterns of a GUSPlus reporter gene expression in the examined plants and thus precisely define the range of flower developmental stages most appropriate for efficient transformation. Two plasmids, CAMBIA 1305.1 and CAMBIA 2301, were used. Verification of the transgenic nature of plants was carried out by detection of CaMV::gusA and CaMV::GUSPlus transgenes and their expression in transgenic plants by appropriate molecular and histochemical methods. For the flower dip transformation, three concentrations of Silwet L-77 surfactant and two inoculation times were tested. The most efficient treatment appeared to be 2-min-long flower bud inoculation and 400 μl/l surfactant (pCAMBIA 1305.1 − 1.73%; pCAMBIA 2301 − 2.01%). In the case of vacuum infiltration method, the highest efficiency of the transformation occurred when the inoculation time was 4 min (pCAMBIA 2301 − 1.55%; pCAMBIA 1305.1 − 1.37%). Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 619–628. This text was submitted by the authors in English.