Improved frequency of transformation in rice and maize by treatment of immature embryos with centrifugation and heat prior to infection with Agrobacterium tumefaciens

The efficiency of transformation was improved by treating immature embryos with heat and centrifugation before infection with Agrobacterium tumefaciens in rice and maize. Because the effects were detected both in the levels of transgene expression after co-cultivation and in the number of independent transgenic plants obtained per embryo, conditions were first optimized based on the transgene expression, and then transformants were produced. The optimal conditions varied considerably depending on species and genotypes, but reasonably good parameters were identified for Japonica rice, Indica rice or maize. As a general tendency, the effect of centrifugation was greater than that of heat in Japonica rice, whereas that of heat was greater than that of centrifugation in Indica rice and maize A188, and the combination of the treatments was the most effective in all of the genotypes tested. The frequency of transformation was improved several fold in rice and maize. In addition, transformation of certain genotypes of maize, which were not transformable before, and transformation of maize with a less efficient vector, which could not transform maize before, became possible by these pre-treatments. In the highest case, 18 independent transgenic plants were obtained from a single immature embryo of Japonica rice. Although nothing is known about the mechanism, these pre-treatments seemed to render cells of rice and maize more competent for transformation mediated by A. tumefaciens.     

抗逆相关基因GmAREB转基因小麦的获得与鉴定

从大豆中克隆一个抗逆相关的bZIP类转录因子基因GmAREB,功能分析表明:GmAREB基因的过表达可以显著提高转基因拟南芥和烟草的抗旱、耐盐和耐寒性。为了获得抗逆转基因小麦,本研究利用玉米的Ubiqutin启动子控制GmAREB基因表达,构建了用于小麦转化的载体pSK-GmAREB。采用基因枪共转化法转化小麦品种郑147和济麦22。通过PCR检测共获得T0代的阳性植株70株,转化率为1.37%。其中,郑147阳性植株共31株,转化率为2.14%;济麦22阳性植株39株,转化率为1.08%。目前,已经获得T1代转基因株系18个,其中以郑147为受体的株系4个,以济麦22为受体的株系14个。对部分株系进行Southern blotting分析,进一步证实GmAREB基因已经整合到小麦基因组中。在低温胁迫条件下,3个以济麦22为受体的转基因株系体内脯氨酸的积累与受体小麦相比有显著增加,初步证明在小麦中过表达GmAREB基因,可以促进渗透调节物质脯氨酸的积累,可能有助于转基因小麦抗逆性的提高。本研究为进一步筛选抗逆转基因小麦新材料奠定了基础。     

转SAMS基因玉米自交系获得及抗旱性分析

以玉米自交系‘掖478’的茎尖为受体,采用农杆菌介导法将小麦抗旱SAMS基因转入玉米中,用PCR和RT-PCR法对转化玉米进行检测,并以18%PEG-6000模拟水分胁迫对T1代转基因玉米和非转基因玉米进行抗旱性分析。结果显示:(1)共转化518个玉米芽,得到453株转化苗。获得T0代阳性植株16株,转化率为3.53%,有14株自交结实,经RT-PCR检测,T1代转基因玉米有9个株系为稳定遗传阳性株系。(2)在同一水分胁迫时间下,转基因玉米的叶片相对含水量、叶绿素含量、脯氨酸含量和SOD活性均高于非转基因玉米,而转基因玉米叶片的电导率、MDA含量均低于非转基因玉米。在60 h水分胁迫处理下,转基因玉米叶片相对含水量、叶绿素含量、脯氨酸含量和SOD活性比非转基因玉米上升8.16%、20.02%、32.21%、22.77%,而转基因玉米叶片的电导率、MDA含量比非转基因玉米下降14.38%、29.41%。研究表明,通过导入SAMS基因,可以提高玉米的抗旱性。     

影响农杆菌介导玉米优良自交系遗传转化的因素

以我国玉米骨干自交系9046,齐319,414,Mo17的幼胚为材料,在已经建立的农杆菌介导的玉米幼胚转化体系的基础上,研究了影响农杆菌介导玉米优良自交系遗传转化的因素,建立了优化的玉米优良自交系的遗传转化体系。研究结果表明,1．0—2．0mm的玉米幼胚是最适宜的转化受体;在感染液和共培养基中都加入乙酰丁香酮(200μmol／L)和抗坏血酸(50mg／L),能显著提高农杆菌对玉米的侵染能力;而感染前将幼胚高渗透压预处理未能提高转化率;延迟筛选有利于提高抗性愈伤组织的存活率。应用优化后的转化体系,获得了这4个玉米优良自交系的转基因植株,PCR阳性植株率为1．71％-4．09％。转化植株叶片总DNA的PCR和Southern杂交分析表明,T-DNA上的外源基因已经整合进了玉米基因组,并且在大多数转基因植株(71．4％)中为单位点插入。这一体系的建立,为进一步将有用基因导入玉米优良自交系奠定了基础。     

转PvPGIP2基因小麦的获得与纹枯病抗性鉴定

多聚半乳糖醛酸酶抑制蛋白(PGIP)是一种植物防卫蛋白,可阻止一些病原真菌的侵害。本研究克隆出扁豆PvP-GIP2基因编码序列,构建了受玉米泛素(ubiquitin)启动子控制的PvPGIP2基因表达载体pA25-PvPGIP2;采用基因枪法将pA25-PvPGIP2转化小麦推广品种扬麦18幼胚愈伤组织4000块,获得了203株再生植株。PCR检测出阳性植株65株,转化率为1.625%。对转PvPGIP2基因小麦T1～T2植株,进行外源基因的PCR、RT-PCR、荧光定量RT-PCR(Q-RT-PCR)分析和小麦纹枯病抗性鉴定。结果表明,转入的PvPGIP2能够在转基因小麦中遗传、转录与表达;PvPGIP2基因的表达提高了转基因植株对小麦纹枯病的抗性。     

Production of transgenic maize from bombarded type II callus: Effect of gold particle size and callus morphology on transformation efficiency

Summary Here we present a routine and efficient protocol for year-round production of fertile transgenic maize plants. Type II callus derived from maize Hi II immature zygotic embryos was transformed using the PDS 1000/He biolistic gun and selected on bialaphos. In an effort to improve the transformation protocol, the effects of gold particle size and callus morphology on transformation efficiency were investigated. Reducing gold particle size from 1.0 μm or 0.6 μm resulted in a significant increase in the rate of recovery of bialaphos-resistant clones from Type II callus. The average transformation efficiency of pre-embryogenic, early embryogenic and late embryogenic callus did not vary significantly. Rates of transformation, regeneration and fertility achieved for Type II callus are summarized and compared to those achieved for greenhouse- and field-derived immature zygotic embryos.     

The ploidy level of transgenic plants in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of tomato cotyledons (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> L.Mill.) is genotype and procedure dependent

A protocol avoiding the feeder-layer cell system was optimized for Agrobacterium-mediated transformation of tomato cotyledonary explants. Over 500 transgenic plants from five tomato cultivars were regenerated in 15 independent experiments. Depending on both genotype and procedure, transformation frequencies ranged from 1.8% to 11.3%. The optimal transformation rate was obtained by inoculating explants with a bacterial suspension in exponential growth ( D(600) = 10(2)-10(3) cells/ml) and transferring cotyledon explants to fresh selective regeneration medium every 3 weeks. The ploidy level of both tomato genotypes used as explant source and primary transformants, was studied by flow cytometry. The inbred lines and cultivars were diploid but a polysomatic pattern in the cotyledon explant was confirmed. The rate of tetraploid transgenic plants ranged from 24.5% to 80% and depended on both the genotype and the transformation procedure. Surprisingly, the percentages of transformed plants with higher ploidy levels were not related to the proportion of 4C and 8C nuclei in the cotyledonary tissue. For some genotypes the optimisation of the transformation rate resulted in an increase of tetraploid transgenic plants. Results obtained in this work indicate the convenience of checking the ploidy level of the primary transformants before performing basic studies or introducing tomato transgenic material in a breeding program.     

农杆菌介导的大麦Mlo反义基因转化小麦获得抗白粉病后代

从大麦‘斯特林’幼叶总RNA中分离Mlo基因cDNA完整编码区,反向连接到植物双元载体（pBI-121.2）35S启动子下游,通过农杆菌介导的苗端转化法获得两种小麦基因型（‘烟优2801’和‘烟优361’）的转基因小麦。T0代405株中有55株PCR检测阳性,平均转化率达到13.58%,T0和T1基因组DNA Southern杂交可以证明大麦Mlo基因片段已整合到小麦基因组中并可传递到后代。两种基因型的转基因小麦T0和T1植株在温室及大田中均表现出对白粉病抗性的提高。农杆菌介导的苗端转化法可以简单、快速、高效地获得转基因株系;排除体细胞变异对转基因植株的影响;克服基因型对农杆菌转化的限制,是小麦遗传转化的一种实用方法。     

Production of transgenic diploid <Emphasis Type="Italic">Cucumis melo</Emphasis> plants

Melon (Cucumis melo L.) is considered to be a recalcitrant species for genetic transformation. Additionally, many studies have observed that regenerated transgenic plants are frequently polyploids. Here we have studied several aspects of melon transformation with the aim of improving transformation efficiency and producing diploid transformed plants. The protocol was based on using cotyledon explants from quiescent seeds that retain meristematic cells, which facilitated the regeneration of transformed diploid melon plants. In this study we evaluated the effect of using two different explant types from the proximal portion of melon seeds on the ploidy status (evaluated by flow cytometry) of regenerated plants. We also determined the transformation efficiencies obtained with these types of explants from four different genotypes. Regeneration was obtained from all explant types. Using quiescent seeds the percentage of diploid plants produced ranged from 85.2 to 94.1%, depending on the type of explant. On the other hand, only half of the plants regenerated from older-seed cotyledons (2- or 3-day-old) were diploids. Transgenic plants were produced with variable transformation efficiencies depending on the explant and which of the four melon genotypes was used. The explants with the best behavior produced transgenic plants with the highest efficiencies ever published both, in terms of plants expressing the visual marker transgenes (ranging from 4.5 to 15.4%) and the number of rooted plants in selective medium (ranging from 1.3 to 3.8%). Although the transformation efficiencies were still relatively low, they were consistent for the four very different melon genotypes tested. Furthermore, at least 85% of plants produced were diploid.     

Novel synthetic Bacillus thuringiensiscry1B gene and the cry1B-cry1Ab translational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in transgenic tropical maize

In order to develop a resistance management strategy to control tropical pests based on the co-expression of different toxins, a fully modified Bacillus thuringiensis cry1B gene and the translational fusion cry1B-cry1Ab gene have been developed. Both constructs were cloned under the control of a maize ubiquitin-1 or a rice actin-1 promoter and linked to the bar gene driven by the CaMV 35S promoter. Immature embryos from the tropical lines CML72, CML216, and their hybrids, were used as the target for transformation by microprojectile bombardment. Twenty five percent of the transformed maize plants with cry1B expressed a protein that is active against southwestern corn borer and sugarcane borer. Ten percent of the transgenic maize expressed single fusion proteins from the translational fusion gene cry1B-1Ab and showed resistance to these two pests as well as to the fall armyworm. Transgenic maize plants that carried the cry1B gene in T1 to T3 progenies transmitted trangenes with expected Mendelian segregation and conferred resistance to the two target insects. Molecular analyses confirmed the cry genes integration, the copy number, the size of protein(s) expressed in maize plants, the transmission, and the inheritance of the introduced cry gene. These new transgenic products will provide another recourse for reducing the build-up of resistance in pest populations. Received: 25 September 2000 / Accepted: 15 December 2000     

Successful <Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Genetic Transformation of Maize Elite Inbred lines

An efficient transformation system was developed for maize (Zea mays L.) elite inbred lines using Agrobacterium-mediated gene transfer by identifying important factors that affected transformation efficiency. The hypervirulent Agrobacterium tumefaciens strain EHA105 proved to be better than octopine LBA4404 and nopaline GV3101. Improved transformation efficiencies were obtained when immature embryos were inocubated with Agrobacterium suspension cells (A₆₀₀ = 0.8) for 20 min in the presence of 0.1% (v/v) of a surfactant (Tween20) in the infection medium. Optimized cocultivation was performed in the acidic medium (pH5.4) at 22 °C in the dark for 3 days. Using the optimized system, we obtained 42 morphologically normal, independent transgenic plants in four maize elite inbred lines representing different genetic backgrounds. Most of them (about 85%) are fertile. The transformation frequency (the number of independent, PCR-positive transgenic plants per 100 embryos infected) ranged from 2.35 to 5.26%. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to three copies of the transgene were integrated into the maize nuclear genome. About 70% of the transgenic plants received a single insertion of the transgenes based on Southern analysis of 10 transformed events. T₁ plants were analyzed and transmission of transgenes to the T₁ generation in a Mendelian fashion was verified. This system should facilitate the introduction of agronomically important genes into commercial genotypes.     

An efficient mannose selection protocol for tomato that has no adverse effect on the ploidy level of transgenic plants

A protocol for Agrobacterium-mediated transformation with mannose selection was developed for cotyledon petiole, hypocotyl and leaf explants of tomato (Lycopersicon esculentum L. Mill). More than 400 transgenic plants from three tomato varieties were selected with 1% mannose in combination with 0.1–0.5% glucose. Average transformation frequencies ranged from 2.0 to 15.5% depending on the construct, genotype and type of tissue used for transformation. The highest transformation rate was obtained for hypocotyl explants from tomato variety SG048. The ploidy levels of 264 independent transgenic events and 233 non-transgenic plants regenerated from tissue culture were assessed by flow cytometry. The incidence of polyploids within the total population of transgenic plants varied from 10 to 78% and was not significantly different from the non-transgenic population. The greatest variation in the proportion of polyploids was observed in plants derived from different explant types, both in transgenic and non-transgenic regenerants, across three studied genotypes. Transgenic and non-transgenic plants regenerated from leaves included the highest number of normal diploid plants (82–100%), followed by cotyledon petiole-derived plants (63–78%). Transgenic plants produced from hypocotyls contained 22–58% diploids depending on the genotype used in transformation. Results described in this study demonstrate that, although transformation frequencies for leaf tissue are still lower under current protocols, the high percentage of diploids obtained make leaf tissue an attractive transformation target.Abbreviations BAP Benzylaminopurine - MS Murashige-Skoog - MsCHI Medicago sativa chalcone isomerase - PMI Phosphomannose isomerase     

Molecular and cellular evidence of chimaeric tissues in primary transgenics and elimination of chimaerism through improved selection protocols

Transgenic plants of strawberry cultivar Totem were developed by Agrobacterium-mediated transformation using a plasmid vector containing gus and nptII genes. Parallel experiments were carried out with and without repeated subculturing (iterative cultures) for generation of transgenic shoots on selection medium. The selection levels in the non-iterative pathway were kept constant, while in the iterative protocol, stepwise increase of selection pressure was applied at different stages of tissue growth. Rooted transgenic plants obtained via both protocols were outplanted in soil. Random leaf samples of greenhouse-grown transgenics were analysed for the presence of gus gene sequences by Southern hybridization as well as gus expression on leaf and petiole tissues by X-Gluc histological assay. Random leaf samples analysed from individual transgenic events developed under iterative culture were positive for the gus insert as verified by Southern analysis confirming the presence of transgenes and lack of chimaeras. Leaf samples of the transgenic events from the non-iterative protocol were either positive or negative on Southern analysis indicating the chimaeric nature of the transgenic plants. The absence of gus sequences in the transgenic plants grown under the non-iterative protocol reinforced the necessity of iterative cultures along with stepwise increase in selection levels for generating non-chimaeric transgenics in strawberry. The gus expression was highly variable, irrespective of the iterative or non-iterative protocol used for transformation. We conclude that strawberry is highly prone to develop chimaeric transgenics if derived from primary regenerants and that the iterative culture technique effectively converts chimaeras to pure line transgenic plants     

Particle inflow gun-mediated transformation of multiple-shoot clumps in rhodes grass (Chloris gayana)

Rhodes grass (Chloris gayana) is one of the most important warm-season forage grasses. It is cultivated in tropical and subtropical parts of the world and is mostly used for grazing and hay production. We have established a particle-bombardment transformation protocol for rhodes grass using multiple-shoot clumps (MSCs) as the target tissue. A vector pAHC25 containing a herbicide-resistance gene (bar) together with the beta-glucuronidase (GUS) gene was used in transformation experiments. The most efficient recovery of bialaphos-resistant tissue was achieved when the bombarded MSCs were first cultured for 15 d on bialaphos-free medium before being subjected to selection pressure. The resistant tissues regenerated transgenic plants that displayed GUS gene expression. Under optimized conditions, 251 target pieces yielded 46 transgenic plants from 4 independent transgenic lines.     

转AtNHX1基因玉米的产生及其耐盐性分析

以玉米(ZeamaysL.)骨干自交系DH4866、齐319和鲁原16106的胚性愈伤组织为材料,采用农杆菌介导法将AtNHX1和hpt基因转入玉米培养细胞,经筛选获得了抗潮霉素的愈伤组织并再生植株。经PCR检测和Southernblot验证,确定了22.8%的再生植株为转基因植株。农杆菌液浓度、愈伤组织基因型及共培养时间对转化率均有明显影响。外源基因在转基因植株后代中的分离呈多样性,在部分株系中表现出孟德尔遗传规律。耐盐筛选表明,一些转基因植株及其后代具有很好的耐盐性,部分株系可在0.8%-1.0%NaCl溶液浇灌下萌发和生长。Northern杂交表明,植株耐盐性提高与AtNHX1基因的转录水平相一致。     

Agrobacterium-mediated transformation of cotyledons of mature seeds of multiple genotypes of sunflower (Helianthus annuus L.)

Lack of a genotype-independent transformation protocol for sunflower is a major bottleneck in improving this important oilseed crop. An efficient Agrobacterium-mediated transformation protocol is described, which was adaptable across a broad range of sunflower genotypes. The improved transformation approach used cotyledons from mature seeds vertically split through the embryo axis. The LBA 4404 Agrobacterium strain was used, which carried pCAMBIA 2301 plasmid containing UidA as the reporter gene and nptII as the selectable marker for transformants on kanamycin. Bacterial titer, cotyledon type, acetosyringone concentration and vacuum application enhanced the transformation efficiency. Wounding, enzyme pretreatment and sonication significantly reduced the transformation frequency. Putative transgenic shoots were obtained through both axillary proliferation and adventitious shoot regeneration. Following two and three cycles of selection on kanamycin for axillary and adventitious shoots, respectively, putative transformed shoots were obtained at an average frequency of 3.0?%. Reporter gene histochemical assay and molecular characterization of primary and T₁ transgenic plants revealed stable transgene integration, expression and monogenic inheritance. The standardized procedure was tested on 28 genotypes comprising sets of inbred, maintainer, restorer and hybrid lines. Transformation was successful in all genotypes albeit with variable frequency in all except the hybrid lines wherein it was stable around 4.0?%. The procedure opens possibilities of directly improving any commercial genotypes of sunflower.     

Detection of vector- and selectable marker-free transgenic maize with a linear GFP cassette transformation via the pollen-tube pathway

The pollen-tube pathway is feasible to transform vector- and selectable marker-free linear gene cassettes into plants to address the biosafety issues. However, its transformation frequency is low and the screening of selectable marker-free transformants by PCR analysis is time-consuming and expensive. In this study, a linear GFP cassette (Ubi-GFP-nos) flanked by 25bp T-DNA borders was transformed into maize via the pollen-tube pathway. The forepart of each maize ear was divided into five segments (segments I-V) at an interval of two rows of kernels. The segments that were most likely to contain transgenic kernels were identified by monitoring GFP expression in the immature embryos. A total of 21 ears were transformed with the linear GFP cassette. Seven out of 19 ears exhibited positive GFP expression in the immature embryos. Transgenic kernels were primarily identified in segments III and IV. A total of 121 plants derived from kernels located within segments III and IV of the remaining two ears were screened by PCR analysis. Six plants (4.96%) showed the presence of the GFP cassette. Southern blot analysis showed that the transgenic plants had simple integration patterns. The identification of transgenic kernels would facilitate PCR screening for marker-free transgenic plants.     

玉米Ubi－1启动子在可育转基因玉米植株中的表达活性

本工作将玉米泛素基因－1启动子（Ubi-1)与大肠杆菌β－葡萄糖苷酸酶基因（gus,uidA)的编码区融合,通过基因枪粒子轰击方法转化来自水成熟胚盾片组织的I－型愈伤组织,经PPT选择获得可育的玉米转基因植株,并采用组织化学方法分析了Ubi-1启动子驱动的gus基因在不同组织,细胞中的表达活性,发现gus基因在除花药壁以外的其它所试组织中均可以有效表达。Ubi：GUS在花粉,卵细胞中T1代转基因植株未成熟胚中的表达显示该启动子在植株发育的早期阶段即具有活性。对T0代转基因植株的花粉进行GUS组织化学染色,gus基因呈1：1分离,显示外源基因在转基因植株中以孟德尔方式遗传。同时发现,使用玉米本身的启动子Ubi-1可以降低外源基因在转基因玉米中的拷贝数,进而避免基因沉默现象的发生。目前已得到第二代转基因种子。     

单子叶植物表达载体的构建及农杆菌介导的玉米遗传转化的研究

构建了单子叶植物表达载体pCUA-tr-cat-als,其中含有豌豆过氧化氢酶基因cat和突变的乙酰乳酸合成酶基因als,分别由玉米ubi启动子和花椰菜花叶病毒35S启动子启动。以玉米昌7-2种子苗的茎尖分生组织为受体,用农杆菌介导法首次将目的基因定向转入玉米叶绿体中。以als基因作为选择标记,以氯磺隆为选择剂进行筛选获得一定数量的转基因植株。经PCR分析,可初步确定目的基因已经整合到玉米基因组中。