Ammonium nitrate improves direct somatic embryogenesis and biolistic transformation of Triticum aestivum

Triticum aestivum is of major importance both nutritionally and economically. Introduction of new genes has been difficult to apply to elite wheat varieties mainly as a result of their recalcitrance to prerequisite tissue culture. We attempted to improve the frequency of wheat transformation by exposing plants to high level of ammonium nitrate. Our experiments showed that modification of the ammonium nitrate content in the direct somatic embryogenesis induction medium can increase the number of primary embryos produced over twofold in the elite hard red wheat cultivar Superb. The number of primary embryos that were capable of transitioning into shoot development also increased twofold. Biolistic transformation efficiency improved as much as sevenfold when targeted scutellar tissue was exposed to elevated ammonium nitrate levels. This simple approach could become extremely useful for increasing transformation efficiency in wheat.     

A protocol for Agrobacterium-mediated transformation in rice

Agrobacterium-mediated transformation of rice is an important method that has been widely adopted by many laboratories. However, because current approaches rely on culture systems, routine protocols have been established only in japonica rice, especially those varieties with higher regeneration potential. Some very efficient methods have been developed for japonica varieties that enable high-throughput functional analysis in rice; however, many elite japonica, and most indica, varieties are difficult to regenerate, leading to low transformation efficiencies. Much effort has been devoted to improving transformation efficiency for all rice genotypes. Here, we describe an Agrobacterium-mediated rice transformation method that is applicable to easily cultured varieties in addition to elite japonica varieties that are more difficult to culture. Using this method, transgenic rice plants can be obtained in about 2-3 months with a transformation frequency of 30-50%, both in easily cultured varieties and recalcitrant elite japonica rice.     

Efficient production of haploid wheat (Triticum aestivum) through crosses between Japanese wheat and maize (Zea mays)

Summary Four Japanese wheat varieties, three crossable and one non-crossable with Hordeum bulbosum, were pollinated with maize pollen of 5 genotypes. By the application of 2,4-dichlorophenoxyacetic acid after pollination, embryos kept developing on wheat plants until 14 days after pollination. The frequency of embryo formation was significantly different among the maize genotypes, varying from 18.0% to 31.9%, but not among the wheat varieties. By bagging spikes with flag leaves the frequency of embryo formation was increased by about 7%. Ten- to twelve-day-old embryos gave higher frequencies of plant formation (83.6%) than 14-day-old embryos(50.0%). All 6 regenerated plants investigated cytologically were found to be haploid. Twelve of the 14 colchicine-treated plants produced florets setting seeds. The overall efficiency of our procedure is considered to be higher than that reported by Laurie and Bennett (1988).     

Efficient Agrobacterium transformation of elite wheat germplasm without selection

A previously reported Agrobacterium tumefaciens transformation system that transformed wheat cultivar Fielder at high efficiency was shown to also transform eight out of nine Triticum aestivum (hexaploid wheat) cultivars tested and two Triticum turgidum (durum wheat) cultivars. Transformation efficiencies of these wheat lines ranged from 1.5 to 51 %. Included amongst this germplasm were elite Australian hexaploid wheat cultivars that are currently in commercial cultivation and two of these cultivars, Gladius and Westonia, were transformed at 32 and 45 % efficiency, respectively. Similar high transformation efficiencies were observed for durum wheat cultivars Kronos (51 %) and Stewart (26 %). This highly efficient transformation system was used to generate transgenic plants in the absence of selection and high heritability of unselected transgenes was observed. Selectable marker free transgenic wheat plants were produced at 3 % efficiency. These data demonstrate highly efficient Agrobacterium transformation of diverse wheat germplasm, including elite cultivars, which enables routine production of selectable marker free transgenics.     

Parameters Influencing Stable Transformation of Rice Immature Embryos and Recovery of Transgenic Plants using Electric Discharge Particle Acceleration

Recovery of transgenic rice plants from elite varieties wasreported from the author's laboratory. By using electric dischargeparticle acceleration, recombinant technology was extended tocommercially important japonica and indica rice cultivars notamenable to conventional transformation methodologies. Criticalparameters influencing the recovery of transformed embryogeniccallus from which transgenic plants were recovered have beendefined. Such parameters included the physiological conditionof explants prior to bombardment, DNA and gold particle loadingrates, accelerating voltage, and depth of particle penetration.Selection for recovery of stable transformants was not criticalin this transformation/regeneration system; both selected andnon-selected tissues yielded transformed embryogenic callusand plants at approximately similar frequencies.Copyright 1995,1999 Academic Press Oryza sativa L., transformation parameters, transgenic indica and japonica plants, particle bombardment, ß-glucuronidase     

Optimization of wheat co-transformation procedure with gene cassettes resulted in an improvement in transformation frequency

Genetic manipulation using gene cassettes was applied to the elite wheat variety EM12 via particle bombardment, which allows an improvement in transformation frequency. We simultaneously transferred to wheat immature embryos with two non-linked genes, gus and bar, on either separate gene cassettes or one plasmid. The linear gene cassettes were excised and purified by restriction digestion of the plasmid, and consisted of promoters, open reading frames and terminators. No difference was observed in GUS transient expression of between gene cassettes and single whole plasmid. However, the stable transformation frequency was significantly increased to 1.1% using gene cassettes, compared with 0.4% when using single plasmid. Procedures of the efficient co-transformation with gene cassettes were developed. Factors influencing on the transformation frequency were also studied in order to optimize the procedure. These were acceleration pressure, target distance, gold particle size, the quantity ratio of gene cassettes and the age of target explants. Based on the transient and stable expression of the gus gene cassettes, optimization of transformation parameters improved the reproducibility of transformation in the elite wheat variety.     

不同磷效率小麦品种对缺磷胁迫反应的比较

在营养液培养条件下,以根据相对产量为指标筛选出的6个不同磷效率的小麦(Triticum aestivum L.)品种为材料,对其苗期在缺磷条件下生长、根冠磷含量及其分配,以及叶片韧皮部汁液中磷浓度等进行了比较研究.结果表明,缺磷抑制植株地上部生长,但刺激根系生长,导致植株根/冠比增加.无论在供磷或缺磷条件下,磷高效品种的根冠生长速率都低于磷低效品种.缺磷导致植株体内的磷含量下降与根系相比,地上部磷含量的下降速率更快.但在缺磷条件下,不同磷效率的小麦品种根冠间的磷分配变化没有差异.研究发现,在正常供磷条件下,磷高效小麦品种的叶片韧皮部汁液中磷浓度较低,而磷低效品种的叶片韧皮部汁液中磷浓度较高.但开始缺磷后,磷高效品种的叶片韧皮部汁液中的磷浓度下降较慢,使其相对磷浓度较高.缺磷后1 0天,磷低效品种叶片韧皮部汁液中的磷浓度为供磷对照的35.9%,而磷高效品种叶片韧皮部汁液中的磷浓度为供磷对照的59%.     

不同磷效率小麦品种对缺磷胁迫反应的比较

在营养液培养条件下,以根据相对产量为指标筛选出的6个不同磷效率的小麦(Triticum aestivum L.)品种为材料,对其苗期在缺磷条件下生长、根冠磷含量及其分配,以及叶片韧皮部汁液中磷浓度等进行了比较研究。结果表明,缺磷抑制植株地上部生长,但刺激根系生长,导致植株根／冠比增加。无论在供磷或缺磷条件下,磷高效品种的根冠生长速率都低于磷低效品种。缺磷导致植株体内的磷含量下降与根系相比,地上部磷含量的下降速率更快。但在缺磷条件下,不同磷效率的小麦品种根冠间的磷分配变化没有差异。研究发现,在正常供磷条件下,磷高效小麦品种的叶片韧皮部汁液中磷浓度较低,而磷低效品种的叶片韧皮部汁液中磷浓度较高。但开始缺磷后,磷高效品种的叶片韧皮部汁液中的磷浓度下降较慢,使其相对磷浓度较高。缺磷后10天,磷低效品种叶片韧皮部汁液中的磷浓度为供磷对照的35．9％,而磷高效品种叶片韧皮部汁液中的磷浓度为供磷对照的59％。     

Investigating Pollen and Gene Flow of WYMV-Resistant Transgenic Wheat N12-1 Using a Dwarf Male-Sterile Line as the Pollen Receptor

Pollen-mediated gene flow (PMGF) is the main mode of transgene flow in flowering plants. The study of pollen and gene flow of transgenic wheat can help to establish the corresponding strategy for preventing transgene escape and contamination between compatible genotypes in wheat. To investigate the pollen dispersal and gene flow frequency in various directions and distances around the pollen source and detect the association between frequency of transgene flow and pollen density from transgenic wheat, a concentric circle design was adopted to conduct a field experiment using transgenic wheat with resistance to wheat yellow mosaic virus (WYMV) as the pollen donor and dwarf male-sterile wheat as the pollen receptor. The results showed that the pollen and gene flow of transgenic wheat varied significantly among the different compass sectors. A higher pollen density and gene flow frequency was observed in the downwind SW and W sectors, with average frequencies of transgene flow of 26.37 and 23.69% respectively. The pollen and gene flow of transgenic wheat declined dramatically with increasing distance from its source. Most of the pollen grains concentrated within 5 m and only a few pollen grains were detected beyond 30 m. The percentage of transgene flow was the highest where adjacent to the pollen source, with an average of 48.24% for all eight compass directions at 0 m distance. Transgene flow was reduced to 50% and 95% between 1.61 to 3.15 m, and 10.71 to 20.93 m, respectively. Our results suggest that climate conditions, especially wind direction, may significantly affect pollen dispersal and gene flow of wheat. The isolation-by-distance model is one of the most effective methods for achieving stringent transgene confinement in wheat. The frequency of transgene flow is directly correlated with the relative density of GM pollen grains in air currents, and pollen competition may be a major factor influencing transgene flow.     

农杆菌介导抗储粮害虫转基因小麦(Triticum aestivum L.)的获得及分析

以本实验室选育的小麦优良品系的胚性愈伤组织为材料,采用农杆菌介导将抗虫基因豇豆胰蛋白酶抑制剂基因CpTI转入小麦培养细胞,经筛选获得抗卡那霉素的愈伤组织并再生植株。经PCR和实时PCR检测、PCR-Southern和Southernblot验证,确定了3株独立再生植株为含有CpTI的转基因植株。农杆菌菌浓度、侵染时间及转化处理方式对小麦转化率均有明显影响。3株转基因植株正常可育并结籽,形成转基因株系。外源基因在转基因植株T1代中的分离呈多样性,部分株系(转基因株系T-Ⅰ、T-Ⅲ)表现出孟德尔遗传规律。抗虫试验表明,3株转基因植株T2代籽粒对储粮害虫麦蛾具有一定的抗性,转基因株系T-Ⅰ、T-Ⅱ、T-Ⅲ及非转基因植株的T2代籽粒虫蛀率分别为19·8%、21·9%、32·9%和58·3%。转基因植株T1代群体农艺性状调查显示,3个株系具有良好的农艺性状,为小麦的遗传改良提供了新的种质抗虫材料。     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium

Immature embryo-derived calli of spring wheat (Triticum aestivum L.) cv Veery5 were transformed using Agrobacterium tumefaciens strain LBA4404 carrying either binary vector pHK22 or superbinary vector pHK21, the latter carrying an extra set of vir genes--vir B, -C and -G. In both cases, transient beta-glucuronidase ( GUS) expression ranging from 35-63% was observed 3 days after co-cultivation, but 587 calli infected with pHK22/LBA4404 failed to produce a single stably transformed plant, whereas 658 calli infected with pHK21/LBA4404 gave rise to 17 transformants carrying both the GUS and bar genes. Regeneration media supplemented with 0.1 M spermidine improved the recovery of transformants from pHK21/LBA4404-infected calli from 7% to 24.2%, resulting in an increase in the overall transformation frequency from 1.2% to 3.9%. The results suggest that two important factors that could lead to an improvement in transformation frequencies of cereals like wheat are (1) the use of superbinary vectors and (2) modification of the polyamine ratio in the regeneration medium. Stable expression and inheritance of the transgenes was confirmed by both genetic and molecular analyses. T1 progeny showed segregation of the transgenes in a typical Mendelian fashion in most of the plants. Of the transformed plants, 35% showed single-copy insertion of the transgene as shown by both Southern analysis and the segregation ratios.     

High frequncies of fertilization and embryo formation in hexaploid wheat x Tripsacum dactyloides crosses

The Hexaploid wheat variety Fukuho was crossed with Tripsacum dactyloides (2n=4x=72). The total fertilization frequencies for the egg cell, polar nuclei, and both, were 58.3%, 26.8% and 58.9% of the 168 ovaries examined. However, the fertilization frequency of single polar nuclei was much lower at only 0.6%. The total frequency of fertilization was higher than that in wheat x maize crosses. A total of 49 hexaploid wheat varieties, including Hope carrying the dominant genes Kr1 and Kr2, were crossed with T. dactyloides, and most gave embryos. The embryoformation frequencies ranged from 0.5% to 59.0%. A higher frequency of 32.0% embryo formation was obtained following pollination of the variety Hope. In comparison with embryo formation in wheat x maize crosses the difference of embryo-formation frequencies between the two crosses was significant. The results of high frequencies of fertilization and embryo formation in wheat x T. dactyloides crosses indicated that the Kr genes are as inactive in wheat x T. dactyloides, as they are in wheat x maize crosses, and also that the efficiency of fertilization and embryo formation is higher in wheat x T. dactyloides than in what x maize crosses. The potential of wheat x T. dactyloides crosses for wheat haploid production and wheat improvement is discussed.     

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     

Factors influencing successful<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated genetic transformation of wheat

The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like bread wheat requires the identification and optimisation of the factors affecting T-DNA delivery and plant regeneration. We have used immature embryos from range of wheat varieties and the Agrobacterium strain AGL1 harbouring the pGreen-based plasmid pAL156, which contains a T-DNA incorporating the bar gene and a modified uidA (beta-glucuronidase) gene, to investigate and optimise major T-DNA delivery and tissue culture variables. Factors that produced significant differences in T-DNA delivery and regeneration included embryo size, duration of pre-culture, inoculation and co-cultivation, and the presence of acetosyringone and Silwet-L77 in the media. We fully describe a protocol that allowed efficient T-DNA delivery and gave rise to 44 morphologically normal, and fully fertile, stable transgenic plants in two wheat varieties. The transformation frequency ranged from 0.3% to 3.3%. Marker-gene expression and molecular analysis demonstrated that transgenes were integrated into the wheat genome and subsequently transmitted into progeny at Mendelian ratios.     

Frequency — dependent advantage in wheat

Summary The effect of frequency-dependent advantage in wheat was investigated by growing F₁. hybrid seeds of the crosses (Warimek X Halberd) and (Wariquam x Halberd) in a stand of Halberd at 5 frequencies: 4 %, 6.25 %, 11.11 %, 25 % and 50 %. A reduction of 35 % to 40 % in grain yield of individual plants was observed with both hybrids as their frequencies changed from 4% to 50 %. A similar trend with frequency was noted for several other plant characteristics, including total grain number, particularly with those measured towards the end of the growing season. Halberd plants did not show a corresponding increase as their frequency declined from 96 % to 50%. — The following season, 76 F₄ lines from the cross (Warimek X Halberd) and 70 F₄ lines from (Wariquam x Gabo) were grown at frequencies of 6.25% and 18.75% in machine sown stands of Wariquam and Halberd, respectively. Again grain yield decreased as genotypic frequency increased. Furthermore, there was a positive correlation between frequency-dependent advantage and relative grain yield, suggesting that high yielding genotypes show a greater advantage at low frequencies than lower yielding ones.     

Wheat genetic diversity trends during domestication and breeding

It has been claimed that plant breeding reduces genetic diversity in elite germplasm which could seriously jeopardize the continued ability to improve crops. The main objective of this study was to examine the loss of genetic diversity in spring bread wheat during (1) its domestication, (2) the change from traditional landrace cultivars (LCs) to modern breeding varieties, and (3) 50 years of international breeding. We studied 253 CIMMYT or CIMMYT-related modern wheat cultivars, LCs, and Triticum tauschii accessions, the D-genome donor of wheat, with 90 simple sequence repeat (SSR) markers dispersed across the wheat genome. A loss of genetic diversity was observed from T. tauschii to the LCs, and from the LCs to the elite breeding germplasm. Wheats genetic diversity was narrowed from 1950 to 1989, but was enhanced from 1990 to 1997. Our results indicate that breeders averted the narrowing of the wheat germplasm base and subsequently increased the genetic diversity through the introgression of novel materials. The LCs and T. tauschii contain numerous unique alleles that were absent in modern spring bread wheat cultivars. Consequently, both the LCs and T. tauschii represent useful sources for broadening the genetic base of elite wheat breeding germplasm.     

Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation

The visual marker GUS has been utilized in this study to understand the Arabidopsis thaliana vacuum infiltration transformation process by Agrobacterium tumefaciens. High transformation frequencies of up to 394 transgenic seeds per infiltrated plant were achieved. The results showed that the majority of the transgenic seeds from single infiltrated plants were from independent transformation events based on Southern analysis, progeny segregation, distribution of transgenic seeds throughout the infiltrated plants and the microscopic analysis of GUS expression in ovules of infiltrated plants. GUS expression in mature pollen and anthers was monitored daily from 0 to 12 days post-infiltration. In addition, all ovules from a single infiltrated plant were examined every other day. GUS expression frequencies of up to 1% of pollen were observed 3-5 days post-infiltration, whereas frequencies of up to 6% were detected with ovules of unopened flowers 5-11 days post-infiltration. Most importantly, transgenic seeds were obtained only from genetic crosses using infiltrated plants as the pollen recipient but not the pollen donor, demonstrating Agrobacterium transformation through the ovule pathway.     

利用花粉管通道法获得转雪花莲凝集素基因（sgna）小麦

利用适用于禾谷类作物的表达载体pGU4AGBar和pGBIU4AGBar,采用花粉管通道法, 将人工合成的雪花莲凝集素基因sgna导入了优良冬小麦品系西农2208和西农132,经PCR 和Southern blot鉴定,证明获得了20株导入了sgna基因的转基因植株,转化率约为0.2 8%～0.84%,并通过Western blot鉴定检测到了目的蛋白的表达。     

Callus formation and plant regeneration from immature and mature embryos of rye (Secale cereale L.)

Summary An efficient protocol was developed to regenerate entire plants from immature embryos of elite genotypes of rye as a prerequisite to plant transformation. Three winter genotypes and one spring genotype were tested using both immature and mature embryos as explants. Four types of callus initiation media and five kinds of regeneration media were tested in all possible combinations. Immature embryos gave much higher levels of plant regeneration than mature embryos, but mature embryos could be induced to regenerate plants for all genotypes and media tested, although at low levels. A minimum stage of embryo development must be reached before embryos can be cultured successfully. Genotypic effects were less pronounced than those reported for inbred cereal species such as wheat and barley, but there was an effect of genotype on percentage of callus formation. There was a significant interaction between genotype and initiation media. Composition of the initiation media affected both the percentage of callus formation from embryos and subsequent frequencies of plant regeneration. Composition of the regeneration media had no effect on level of plant regeneration. Immature embryos of all genotypes tested could be induced to produce 90–100% callus on appropriate initiation media and all regenerated shoots from approximately one-half to three-quarters of the calluses produced.     

Effect of Combined Changes in Culture Medium and Incubation Conditions on the Regeneration from Immature Embryos of Elite Varieties of Winter Wheat

In this study, tissue culture method for plant regeneration from immature embryos of elite Hungarian winter wheat varieties was established. The influence of the growth regulators and the concentration of macroelements in the regeneration medium and of the incubation temperature and light intensity on regeneration frequency were investigated. The most noticeable effect on regeneration frequency was achieved by simultaneously reducing both the incubation temperature to 23 °C and the concentration of macroelements in the regeneration medium to half-strength. This modification increased the average regeneration frequency from about 10–78%. Changes in the light intensity and temperature gave an average plant regeneration frequency of 83%.