Transient and stable transformation of wheat with DNA preparations delivered by a biolistic method

An optimized procedure for transformation of wheat with the use of a Biolistic Particle Delivery System PDS 1000/He to deliver foreign DNA is described in detail. The bacterial uidA and bar genes (both driven by plant promoters) were utilized as the reporter and selectable marker genes, respectively. Moderately high gas pressure appeared to be most important to achieve the highest level of transient GUS expression in target tissues. There was, however, no apparent correlation between transient and stable GUS expression. The presence of telomeric DNA sequences in an uidA gene-containing vector did not influence transient GUS expression but, apparently, prevented its stable expression. Mechanical lesions caused by the bombardment (tungsten particles) seemed to be less severe when embryo- derived calli, instead of freshly excised immature embryos, were used as the target tissue. The limited ability of callus cells for regeneration, together with a restricted number of cells that receive the foreign DNA by particle bombardment, result in a low efficiency of wheat stable transformation.     

Genetic transformation of wheat: progress during the 1990s into the Millennium

Wheat transformation technology has progressed rapidly during the past decade. Initially, procedures developed for protoplast isolation and culture, electroporation- and polyethylene glycol (PEG)-induced DNA transfer enabled foreign genes to be introduced into wheat cells. The development of biolistic (microprojectile) bombardment procedures led to a more efficient approach for direct gene transfer. More recently, Agrobacterium-mediated gene delivery procedures, initially developed for the transformation of rice, have also been used to generate transgenic wheat plants. This review summarises the considerable progress in wheat transformation achieved during the last decade. An increase in food production is essential in order to sustain the increasing world population. This could be achieved by the development of higher yielding varieties with improved nutritional quality and tolerance to biotic and abiotic stresses. Although conventional breeding will continue to play a major role in increasing crop yield, laboratory-based techniques, such as genetic transformation to introduce novel genes into crop plants, will be essential in complementing existing breeding technologies. A decade ago, cereals were considered recalcitrant to transformation. Since then, a significant research effort has been focused on cereals because of their agronomic status, leading to improved genetic transformation procedures (Bommineni and Jauhar 1997). Initially, the genetic transformation of cereals relied on the introduction of DNA into protoplasts and the subsequent production of callus from which fertile plants were regenerated. More recently, major advances have been accomplished in the regeneration of fertile plants from a range of source tissues, providing an essential foundation for the generation of transgenic plants. This review summarises procedures, vectors and target tissues used for transformation, high-lights the limitations of current approaches and discusses future trends. The citation of references is limited, where possible, to the most relevant or recent reports.     

Gene transfer to inflorescence and flower meristems using ballistic micro-targeting

Direct gene transfer to floral meristems could contribute to cell-fate mapping, to the study of flower-specific genes and promoters, and to the production of transgenic gametes via the transformation of sporogenic tissues. Despite the wide potential of its applications, direct gene transfer to floral meristems has not been achieved so far because of the lack of suitable technology. We show in this paper that ballistic micro-targeting is the technique of choice for this purpose, and in this way, we were able to transfer genes efficiently into excised wheat immature spikes. Particle size was adjusted for optimal penetration into the L1 and L2 cell layers of the spikes with limited cell damage. Spikes at different developmental stages were shot either with a plasmid containing two genes involved in anthocyanin biosynthesis or with a plasmid bearing the uidA (-glucuronidase) gene. The transient expression of these marker genes was observed in the different developmental stages tested and in cells of both the L1 and the L2 layers. The transient expression of the uidA gene was significantly increased when the sucrose concentration in the culture medium was increased from 0.06 to 0.52 M. At the highest concentration, 100% of the targeted spikes expressed the uidA gene, with an average of 69 blue cells per spike. Twelve days after microtargeting, multicellular sectors showing transgene expression and containing up to 17 cells were found in 85% of the shot immature inflorescences. This indicated that targeted cells survived particle bombardment. Sectors were found in primordia of both vegetative and reproductive organs.     

Specificity of Agrobacterium-mediated delivery of maize streak virus DNA to members of the Gramineae

Parameters affecting the efficiency of agroinfection of maize streak virus (MSV) in maize have been determined. Monomeric units, cloned at a number of sites in the MSV genome were not infectious but multimeric units containing partial duplications were equally as infectious as complete tandem dimeric clones. Inoculation of tandem dimeric units conjugated into different strains of Agrobacterium showed that both A. tumefaciens and A. rhizogenes were able to transfer DNA to maize and this ability was Ti (or Ri) plasmid-specific. Nopaline strains of A. tumefaciens and both agropine and mannopine A. rhizogenes strains efficiently transferred MSV DNA to maize. A number of strains were capable of MSV DNA transfer to other members of the Gramineae, providing information which may be essential for Agrobacterium-mediated transformation of monocotyledonous plants.     

Hydroxamic acids affecting barley yellow dwarf virus transmission by the aphid Rhopalosiphum padi

2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), a hydroxamic acid (Hx) occurring in wheat, was shown to deter feeding by the aphid Rhopalosiphum padi (L.), and to reduce BYDV transmission to the plant. Dual choice tests with wheat leaves showed the preferential settlement of aphids on leaves with lower levels of DIMBOA. Electric monitoring of aphid feeding behaviour showed that in seedlings with higher DIMBOA levels fewer aphids reached the phloem and they needed longer times to contact a phloem vessel than in those with lower levels. When aphids carrying BYDV were allowed to feed on wheat cultivars with different DIMBOA levels, fewer plants were infected with BYDV in the higher DIMBOA cultivars than in the lower ones. Preliminary field experiments showed a tendency for wheat cultivars with higher Hx levels to be more tolerant to infection by BYDV than lower Hx level ones.     

Accelerated production of transgenic wheat (Triticum aestivum L.) plants

We have developed a method for the accelerated production of fertile transgenic wheat (Triticum aestivum L.) that yields rooted plants ready for transfer to soil in 8–9 weeks (56–66 days) after the initiation of cultures. This was made possible by improvements in the procedures used for culture, bombardment, and selection. Cultured immature embryos were given a 4–6 h pre-and 16 h post-bombardment osmotic treatment. The most consistent and satisfactory results were obtained with 30 g of gold particles/bombardment. No clear correlation was found between the frequencies of transient expression and stable transformation. The highest rates of regeneration and transformation were obtained when callus formation after bombardment was limited to two weeks in the dark, with or without selection, followed by selection during regeneration under light. Selection with bialaphos, and not phosphinothricin, yielded more vigorously growing transformed plantlets. The elongation of dark green plantlets in the presence of 4–5 mg/l bialaphos was found to be reliable for identifying transformed plants. Eighty independent transgenic wheat lines were produced in this study. Under optimum conditions, 32 transformed wheat plants were obtained from 2100 immature embryos in 56–66 days, making it possible to obtain R3 homozygous plants in less than a year.     

小麦穗发芽鉴定方法的比较与分析

穗发芽是小麦生产中较为严重的灾害之一,易受外界环境的影响,一旦发生不仅会影响产量,而且还会严重影响小麦的品质,因此培育抗穗发芽的小麦品种至关重要。该研究通过对65份小麦材料进行穗发芽试验,比较分析了小麦穗发芽抗性的常用方法,即籽粒发芽法、整穗发芽法和大田穗发芽法。结果表明:三种方法之间均呈极显著正相关关系,而且在1%水平上均存在极显著性差异;发芽指数与籽粒发芽率的相关性最高,能够更好地评价小麦材料的休眠特性,但不能得出材料的总体抗性;籽粒发芽法和整穗发芽法的变异程度相对较小,试验条件更易控制,可作为小麦穗发芽抗性评价的简易方法;多数参试材料的平均籽粒发芽率平均整穗发芽率平均大田穗发芽率,且三者差异程度均达到极显著水平,这说明麦穗的外部结构及外部环境对小麦穗发芽的影响显著。因此,籽粒发芽法可以从休眠性方面,对小麦种子资源进行初步筛选;整穗发芽法可用于穗发芽抗性的进一步鉴定和验证,评价小麦材料穗发芽的综合抗性;大田穗发芽法较易受自然条件的影响、变异程度较大,其结果可以作为室内发芽试验的参考数据。     

Generation of DNA double-strand breaks and inhibition of somatic embryogenesis by tungsten microparticles in wheat

Particles of metallic tungsten, known also as tungsten microprojectiles, are routinely used to deliver foreign DNA into target cells and tissues. Some side effects of biolistic transformation have been observed but never studied in detail. Here we present evidence that intact tungsten particles can promote a breakage of phosphodiester bonds in native DNA, at a limited number of sites. A single, double-strand break appeared within almost each of the circular pUC119 molecules after a short incubation of plasmid DNA with a suspension of tungsten particles. No further DNA cutting could be induced even if the reaction rate was accelerated by increasing the concentration of tungsten in the incubation mixture. Indirect evidence indicates that similar lesions may be generated in cellular DNA of bombarded tissues. These lesions are rapidly repaired, as evidenced by increasing incorporation of labelled DNA precursors in bombarded wheat embryos. The rate of repair is, however, not high enough to restore all the genome functions. Neither germination of mature embryos nor initiation of callus tissues from immature embryos was inhibited by biolistic bombardment. Nevertheless, the frequency of formation of somatic embryos in calli derived from bombarded embryos was markedly lower than in calli derived from control embryos. Both immediate (generation of a limited number of double-strand breaks) and remote (selective inhibition of somatic embryogenesis) side effects of the biolistic process strongly suggest that biological activity of tungsten deserves special attention. This revised version was published online in June 2006 with corrections to the Cover Date.     

Screening methods for salinity tolerance: a case study with tetraploid wheat

Fast and effective glasshouse screening techniques that could identify genetic variation in salinity tolerance were tested. The objective was to produce screening techniques for selecting salt-tolerant progeny in breeding programs in which genes for salinity tolerance have been introduced by either conventional breeding or genetic engineering. A set of previously unexplored tetraploid wheat genotypes, from five subspecies of Triticum turgidum, were used in a case study for developing and validating glasshouse screening techniques for selecting for physiologically based traits that confer salinity tolerance. Salinity tolerance was defined as genotypic differences in biomass production in saline versus non-saline conditions over prolonged periods, of 3–4 weeks. Short-term experiments (1 week) measuring either biomass or leaf elongation rates revealed large decreases in growth rate due to the osmotic effect of the salt, but little genotypic differences, although there were genotypic differences in long-term experiments. Specific traits were assessed. Na⁺ exclusion correlated well with salinity tolerance in the durum subspecies, and K⁺/Na⁺ discrimination correlated to a lesser degree. Both traits were environmentally robust, being independent of root temperature and factors that might influence transpiration rates such as light level. In the other four T. turgidum subspecies there was no correlation between salinity tolerance and Na⁺ accumulation or K⁺/Na⁺ discrimination, so other traits were examined. The trait of tolerance of high internal Na⁺ was assessed indirectly, by measuring chlorophyll retention. Five landraces were selected as maintaining green healthy leaves despite high levels of Na⁺ accumulation. Factors affecting field performance of genotypes selected by trait-based techniques are discussed.     

Genetic transformation through the use of hyperhydric tobacco meristems

Exposed shoot meristems from normal and hyperhydric (vitrified) tobacco, Nicotiana tabacum, were bombarded with gold particles either coated with plasmid DNA containing neomycin phosphotransferase (NPTII), rolC and -glucuronidase (GUS) genes (plasmid pGA-GUSGFrolC) or left uncoated. Meristems bombarded with uncoated particles were co-cultivated with Agrobacterium tumefaciens strain EHA 101 harboring the binary vector pGA-GUSGFrolC. Whole-plant transformants were produced from 4 of 40 hyperhydric meristems bombarded with uncoated particles followed by co-cultivation with A. tumefaciens. One transgenic plant was obtained from 40 normal, non-hyperhydric meristems treated. Transformation was verified by growth on kanamycin-containing medium, GUS assays, PCR, and Southern analysis. The plants tested through Southern analysis appeared to have 2 or more copies of the transgene insert. Seeds obtained from self-pollination of these transgenic plants segregated 3:1 or 15:1 (kanamycin resistant:sensitive) when germinated on medium containing 100 mg/l kanamycin, indicating transfer of foreign genes through the sexual cycle. Whole-plant transformants were not produced from 50 normal tobacco meristems bombarded with plasmid-coated gold particles and not exposed to engineered A. tumefaciens, but 1 plant of 60 bombarded hyperhydric meristems produced transgenic roots, the result of a chimera. We suggest that hyperhydric meristems are more readily transformed.     

A model wheat cultivar for transformation to improve resistance to Fusarium Head Blight

Fusarium head blight (FHB), caused primarily by Fusarium graminearum, is a major disease problem in wheat (Triticum aestivum). Genetic engineering holds significant potential to enhance FHB resistance in wheat. Due to the requirement of screening for FHB resistance on flowers at anthesis, the number of screens carried out in a year is limited. Our objective was to evaluate the feasibility of using the rapid-maturing dwarf wheat cultivar Apogee as an alternative genotype for transgenic FHB resistance research. Our transformation efficiency (number of transgenic plants/number of embryos) for Apogee was 1.33%. Apogee was also found to exhibit high FHB susceptibility and reached anthesis within 4 weeks. Interestingly, microsatellite marker haplotype analysis of the chromosome 3BS FHB resistant quantitative trait locus (QTL) region indicated that this region maybe deleted in Apogee. Our results indicate that Apogee is particularly well suited for accelerating transgenic FHB resistance research and transgenic wheat research in general. C.A. Mackintosh and D.F. Garvin contributed equally to the article and should be considered co-first authors     

甜菜碱醛脱氢酶(BADH)基因转化小麦及其表达

采用基因枪法将山菠菜甜菜碱醛脱氢酶 (BADH)基因导入小麦 (TriticumaestivumL .)品种 ,并且得以表达。该基因由玉米Ubi1启动子控制。在盐胁迫条件下 ,多数转基因植株叶片的BADH活性比受体亲本提高 1～ 3倍 ,部分植株相对电导率比亲本明显低 ,表明转基因植株的细胞膜在胁迫时有受损较轻倾向。PCR和Southern杂交分析证实外源BADH基因已插入小麦基因组 ,平均转化频率为 4.1%。     

Effect of copper on stem melanosis and yield of wheat: Sources,rates and methods of application

Park wheat (Triticum aestivum cv. Park) is susceptible to stem melanosis when grown on Cu-deficient soils. Three field experiments were conducted to evaluate the effectiveness of various Cu sources, using different methods and rates of application for reducing the incidence of stem melanosis and increasing the grain yield of Park wheat on a Cu-deficient Black Chernozemic soil in central Alberta. A fourth experiment determined the residual effect of Cu over 4 years. In the first experiment, Park wheat had less disease and higher grain yield when Cu-chelate solution at 2 and 4 kg Cu ha⁻¹ or Cu-sulfate solution at 10 and 20 kg Cu ha⁻¹ was applied to the soil surface and incorporated to a depth of 8 cm or was applied at these rates as a foliar spray. The foliar application, however, was phytotoxic and delayed maturity by approximately two weeks. Sidebanding Cu (4 cm to the side and 4 cm below the seed row) was least effective. In the second experiment, Cu-sulfate solution incorporated into the soil was more effective than the soil incorporated granular Cu-sulfate in reducing disease incidence and increasing grain yield in the year of application. In the following year the granular Cu was as effective as solution Cu. The third experiment showed that Cu seed dressings did not have any effect on disease incidence or grain yield of Park wheat. In the fourth experiment, the residual effect of Cu-chelate was evident four years after application. The grain yield in the fourth year was about four times that of the control. Contribution No. 564     

A quick method to isolate RNA from wheat and other carbohydrate-rich seeds

No reports on isolating RNA from carbohydrate-rich wheat seeds have been published. Because of the presence of carbohydrates, published protocols yield small amounts of poor quality RNA. Extracting seeds in a buffer (pH 9, 150 mM NaCl, 1% sarcosyl) ensured maximum RNA solubility and the removal of most interfering substances. Extracted RNA was purified using a guanidine hydrochloride-based buffer system. This protocol yields up to 148 μg of RNA from 100 mg of tissue in 3.5 h. An A₂₆₀/A₂₈₀ ratio of 1.85 indicates RNA purity. Isolated RNA was amenable to downstream applications such as differential display. The developed method was extended to other carbohydrate-rich seeds, such as barley and maize, with success.     

A comparative evaluation of two methods of selecting locations used for testing spring wheat cultivars

Summary One of the considerations of regional cultivar evaluation programs is to optimize the number of locations used for testing. Although optimization of numbers of locations using cluster analysis has been previously attempted, no objective comparison of methods has yet been made. A new clustering method that uses the pairwise contribution of locations to the cultivar x location mean square as the distance measure (LB) was compared to another method that employs diallel correlations as the distance measure (CL). Data from six spring wheat (Triticum aestivum L. em Thell) cultivars grown at 13 locations for five years were used in the initial cluster analysis. Another set of data, from a separate year, consisting of yields of the original 6 cultivars and a set of 12 independent cultivars was then used to check the validity of the original groupings and to compare the two methods. When the 6 original cultivars were considered, the LB technique was found to be superior to the CL. When the 12 independent cultivars were used, neither method was considered to be superior. Because of the lack of flexibility on the part of the LB method, neither technique could be deemed as fully adequate.Contribution Number 842 of the Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada     

Generation of transgenic wheat plants producing high levels of the osmoprotectant proline

Plasmid DNA (pBI-P5CS), containing the selectable neomycin phosphotransferase-II `npt II' gene for kanamycin resistance and the reporter -glucuronidase `gus' gene as well as the Vigna aconitifolia ¹-pyrroline-5-carboxylate synthetase `P5CS' cDNA that encodes enzymes required for the biosynthesis of proline, was delivered into wheat plants using Agrobacterium-mediated gene transfer via indirect pollen system. Southern, northern and western blot analysis demonstrated that the foreign gene had been transferred, expressed and integrated into wheat chromosomal DNA. Salinity test indicated that proline acts as an osmoprotectant and its overproduction in transgenic wheat plants results in the increased tolerance to salt.     

甜菜碱醛脱氢酶(BADH)基因转化小麦及其表达

Betaine aldehyde dehydrogenase (BADH) cDNA cloned from Atriplex hortensis L. in the plasmid pABH9 containing maize ubiquitin promoter and bar gene was transferred into wheat (Triticum aestivum L.) by microprojectile bombardment with 4.1% of average frequency of transformation. From 300 young embryo calli bombarded with the plasmid, 24 transgenic plants were obtained showing BADH gene integration by both PCR and Southern blotting analysis. Among the 24 transgenic plants, 13 exhibited higher BADH activity than the control. Some transgenic plants grew normally with healthy roots on the medium containing 0.7% NaCl while the control plants had very poor roots and finally died.     

The contribution of different regions of the seminal roots of wheat to uptake of nitrate from soil

A technique was developed to determine the physiological activity of defined sections of seminal roots of wheat grown in sand. Wheat plants were grown for 2 weeks in narrow columns of N-deficient sand to which all other nutrients had been added. The columns were split longitudinally and ¹⁵N-labelled nitrate, in an agar medium, supplied to 2 cm sections of root. Shoots and roots were analysed after 24 h to determine the uptake of ¹⁵N. Three sections were examined on either the secondary or tertiary seminal root: 1 cm from the seed (basal segment), 35 cm from the seed (middle segment) and 4 cm from the root apex (apical segment). Total uptake was greatest from the basal and middle segments, declining by 50% from the apical segment. However, uptake per unit root length, including exposed sections of lateral roots, was not significantly different along the root.     

A moderately repeated DNA sequence of wheat and rye genomes

A 371 base pair segment (bordered by Hind III and Eco RI cutting sites) of wheat embryo nuclear DNA has been cloned and sequenced. It is AT-rich (68%), shares some sequence features with autonomously replicating sequence (ARS) elements, and occurs in approximately 7600 copies per haploid genome. When used as probe for blot hybridization to Hind III-digested wheat DNA, it gives an irregular series of hybridization bands. Essentially the same hybridization pattern was observed for rye DNA. It is concluded that this segment is distributed irregularly but, apparently, according to the same rule in both wheat and rye genomes.