Use of the firefly luciferase gene in a barley (Hordeum vulgare) transformation system

Transgenic lines of the spring barley variety Golden Promise containing the firefly luciferase gene were produced by particle bombardment of immature embryos. Non-destructive analysis of luciferase gene expression was used to monitor the transformation process. This revealed that transformation efficiency, in terms of the percentage of bombarded immature embryos giving rise to transformed callus lines, was very high, up to 40%. Following the expression of the luciferase gene provided a method for the sensitive, non-destructive, real-time monitoring of gene expression throughout the transformation process. Luciferase expression could also be used to easily identify transgenic plants and to identify homozygous transgenic plants at an early stage. The production of transgenic barley by selecting for luciferase-positive material, without an additional selection system, was possible but technically difficult.     

基因枪转化小麦主要影响因素细述

基因枪转化是目前小麦遗传转化的主要技术之一,高效的基因枪转化系统对于转基因小麦新品种培育、候选基因功能鉴定和功能基因组学研究具有重要意义。本文综述了影响基因枪转化小麦效率的主要因素,包括基因型、外植体、植物生长调节剂、轰击参数、筛选体系等,以期为进一步改进小麦基因枪转化技术,提高基因枪转化小麦的效率提供参考。     

Biolistic transformation of animal tissue

Summary The biolistic technique transforms cells by bombardment with DNA-coated microprojectiles. It has been used to transform plants, microbes, and organelles. We adapted a standard Biolistic PDS-1000 device for use with animals and have successfully transformed tissues in live mice. The firefly luciferase gene was introduced into mouse skin and ear tissue. One day after transformation 344±74 and 1648±254 pg of luciferase were detected in skin and ear samples, respectively. Expression of the gene product was transient but detectable up to 7 days after bombardment. A further modification of the device allowed transient transformation of liver tissue in vivo. Liver contained 293±122 pg of luciferase 1 day postransformation. Expression of the gene in liver tissue was unchanged at Day 3 but declined to low levels by Day 5. This new device allowed a fourfold increase in gene expression in ear tissue extending a minimum of 14 days. This technology is applicable to a broad range of tissues and organs in situ and makes it possible to test numerous reporters and the tissue specificity of promoters. It may also be useful in protocols for somatic cell therapy. Presented in the Session-in-Depth Genetic Transformation and Genetic Analysis Using Microprojectile Bombardment at the 41st Annual Meeting of the Tissue Culture Association, Houston, Texas, June 10–13, 1990.     

Construction and testing of an intron-containing luciferase reporter gene from<Emphasis Type="Italic">Renilla reniformis</Emphasis>

We describe a newRenilla reniformis luciferase reporter gene,RiLUC, which was designed to allow detection of luciferase activity in studies involvingAgrobacterium-based transient expression studies. TheRLUC gene was altered to contain a modified intron from the castor bean catalase gene while maintaining consensus eukaryotic splicing sites recognized by the plant spliceosome.RLUC andRiLUC reporter genes were fused to the synthetic plant SUPER promoter. Luciferase activity within agrobacteria containing the SUPER-RLUC construct increased during growth in culture. In contrast, agrobacteria harboring the SUPER-RiLUC gene fusion showed no detectable luciferase activity. Agrobacteria containing these gene fusions were cotransformed with a compatible normalization plasmid containing a cauliflower mosaic virus 35S promoter (CaMV) joined to the firefly luciferase coding region (FiLUC) and infused into tobacco leaf tissues through stomatal openings. The kinetics of luciferase production from theRLUC orRiLUC reporters were consistent, with expression of theRiLUC gene being limited to transiently transformed plant cells.RiLUC activity from the reporter gene fusions was measured transiently and within stably transformed tobacco leaf tissues. Analysis of stably transformed tobacco plants harboring either reporter gene fusion showed that the intron altered neither the levels of luciferase activity nor tissue-specific expression patterns driven by the SUPER promoter. These results demonstrate that theRiLUC reporter gene can be used to monitor luciferase expression in transient and stable transformation experiments without interference from contaminating agrobacteria.     

Optimization of sorghum transformation parameters using genes for green fluorescent protein and beta-glucuronidase as visual markers

Early and reliable detection of plant transformation events is essential for establishing efficient transformation protocols. We have compared the effectiveness of using the gene encoding a green fluorescent protein (GFP) and a beta-glucuronidase (gus) as reporter genes for early detection of transgene expression in explants subjected to biolistic bombardment and Agrobacterium-mediated transformation. The results indicate that gfp gene is superior to gus gene in following transgene expression in transiently transformed materials in both methods of transformation. Using GFP as the screenable marker, we have optimized sorghum transformation with respect to the conditions for transformation, type of explants, promoters, and inbreds. These optimized conditions have been used to obtain stably transformed explants for subsequent regeneration.     

Deletion analysis of pollen-expressed promoters

Summary We have used microprojectile bombardment of tobacco pollen to study the DNA sequences involved in the expression of pollen-expressed genes. Promoter-reporter gene fusions constructed with the promoters of three different pollen-expressed genes from tomato (LAT52, LAT56, and LAT59) and either theβ-glucuronidase or luciferase reporter genes were assayed by bombarding hydrated tobacco pollen with the gene constructs precipitated onto tungsten microprojectiles. Reporter gene expression can be assayed within 30 min, with the maximal level of expression between 6 and 12 h after bombardment. By constructing and assaying promoter deletion derivatives, we have been able to delimit regions of the promoters that are necessary for high level expression in pollen. We also demonstrate that results with this transient expression system parallel the expression levels seen in pollen from stably transformed transgenic plants. The microprojectile bombardment assay can be used to rapidly test constructs for pollen expression beforeAgrobacterium-mediated plant transformation. Furthermore, it may be possible to adapt the microprojectile bombardment technique to achieve stable transformation of pollen. Presented in the Session-in-Depth Genetic Transformation and Genetic Analysis Using Microprojectile Bombardment at the 41st Annual Meeting of the Tissue Culture Association, Houston, Texas, June 10–13, 1990. This work was supported by the U.S. Department of Agriculture, Washington, DC, ARS CRIS 5335-22230-002-00D, and by the NSF Center for Plant Developmental Biology, UC-Berkeley, DIR-8719933.     

High-efficiency,microprojectile-mediated cotransformation of sugarcane,using visible or selectable markers

Transient expression of the maize anthocyanin regulatory elements,R andC1, was used to optimise parameters for microprojectile-mediated delivery of DNA into sugarcane embryogenic callus. Osmotic treatment of target tissues and particle acceleration in a high-pressure helium pulse increased the frequency of transient expression to 5–8×10³ cells per bombardment, with minimal tissue damage. An average of 0.34% of transiently expressing cells developed into stably transformed, anthocyanin-pigmented proembryoids which subsequently regenerated into plantlets. However, constitutive expression ofR andC1 proved deleterious, and no anthocyanin-pigmented plant survived beyond 3 cm in height. We also compared selective subculture of callus portions showing luciferase activity with antibiotic selection on medium containing G418 or phosphinothricin, upon bombardment of callus with constructs driving strong expression ofluc, aphA orbar genes. Selective subculture based on luciferase activity enabled recovery of 1.4±0.5 independent transgenic plants per bombardment, compared to 19.8±3.7 independent transgenic plants per bombardment from an optimised G418 selection regimen, and no transformed plants from phosphinothricin selection. Whenluc andaphA on separate plasmids were coprecipitated onto microprojectiles before bombardment, 67–79% of callus lines selected for G418 resistance also showed luciferase activity detectable under a low-light camera. Southern analysis confirmed a very high cotransformation frequency, with variable copy numbers of introduced genes. The high efficiencies of gene transfer, selection and cotransformation in the optimised system, coupled with the simple initiation and regeneration of embryogenic callus, provide an effective tool for practical genetic transformation of sugarcane.     

A Rapid,Highly Efficient and Economical Method of Agrobacterium-Mediated In planta Transient Transformation in Living Onion Epidermis

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.     

利用花色素苷合成调节基因C1-R作为基因枪转化效果指示的研究

报道了玉米花色素苷合成调节基因Ｃ１－Ｒ在小麦幼胚、玉米愈伤组织、水稻愈伤组织、烟草叶片中的瞬时表达情况。由于调节基因Ｃ１－Ｒ激活了植物体细胞内花色素苷的合成,因此不需任何生色底物,即可活体观察到花色素苷的表达。结果表明,对于目前仍主要通过基因枪法转化的几类主要粮食作物──小麦、玉米、水稻、枪击４８ｈ后,放大２倍便清晰可见红色斑点,且其表达强度远高于ＧＵＳ的表达,证明Ｃ１－Ｒ可作为一个很好的衡量打枪效果的指示,同时还证明其在双子叶植物──烟草叶片的基因枪转化瞬时表达体系中也起同样的作用。     

基因枪法转化小麦的金粉用量及转基因植株表型特征分析

研究了不同金粉用量对小麦幼胚瞬间及稳定转化频率的影响,结果表明此实验系统的金粉用量以每枪500μg金粉为佳。对获得的T     

质粒DNA物理形态和其它因素对获得可育转基因小麦的影响

本工作分析了不同形态质粒DNA和未成熟胚高渗透压处理对基因枪小麦转化体系的适用性.高渗处理对瞬间表达和转基因小麦的再生均有明显的促进作用.轰击之前对质粒DNA进行变性处理导致瞬间表达反应大幅度下降,但稳定转化频率(指从100个轰击未成熟胚得到的再生可育转基因植株数)与双链DNA相差不大.使用单链质粒DNA、线性双链质粒DNA和环状双链质粒DNA均可以得到转基因小麦植株.迄今已得到26个不同的转基因冬小麦株系和4个不同的转基因春小麦株系.这些转基因小麦大多数已产生种子,几个春小麦株系已获第二代种子.     

Gametic embryos of maize as a target for biolistic transformation: comparison to immature zygotic embryos

The aim of the present study was to determine the suitability of maize gametic embryos of three ETH genotypes as a target for biolistic transformation. We studied parameters considered essential for a successful transformation, such as the frequency of secondary embryo formation, their regeneration ability and the transient transgene expression. Transformable zygotic embryos of one of the ETH genotypes were used as positive control. Our results indicate that gametic embryos can potentially be transformed by particle bombardment, since they responded positively to all the studied parameters, although with lower efficiencies than the zygotic embryos. In particular, differences were found in the rate of secondary embryogenesis and the density of transformed cells.     

Evaluation of peanut (Arachis hypogaea L.) leaflets from mature zygotic embryos as recipient tissue for biolostic gene transfer

Leaflets from mature peanut embryos are a useful recipient tissue for biolistic DNA transfer. Fertile plants were regenerated from leaflets from genotypes representing all botanical types of peanut. Regeneration frequency was strongly influenced by genotype. NPT II and GUS chimaeric gene fusions, driven by the CaMV 35S promoter, were expressed transiently following biolistic delivery to unexpanded leaflets. Bombardment conditions affecting transient expression frequency were determined using a prototype of the Bio Rad PDS 1000/He helium-powered particle acceleration apparatus. Stably transformed calli were derived routinely from leaflet tissue bombarded with the NPT II gene and subsequently cultured on kanamycin. Several plants have been regenerated from treated explants under kanamycin selection. Thus far, none of these has been stably transformed. The occurrence of escapes suggests that kanamycin is an inefficient selective agent for the recovery of transgenic peanuts from this explant. Experiments designed to regenerate plants using published regeneration protocols from stably transformed calli, devoid of primary explant tissue, have been unsuccessful.     

质粒DNA物理形态和其它因素对获得可育转基因小麦的影响

The applicability of hyperosmotic treatment and different configurations of plasmid DNA for stable transformation of wheat mediated by particle bombardment was investigated. Hyperosmotic treatment increased the frequency of transient expression and had also a positive effect on stable transformation. Denaturation of plasmid DNA prior to bombardment led to dramatic reduction of transient expression. However, there were no marked differences between single-stranded and double-stranded DNA in stable transformation. Single-stranded plasmid DNA, double-stranded plasmid DNA in linear state and double-stranded plasmid DNA in circular state could all be used to produce transgenic wheat plants. A total of 26 independent transgenic plants of winter wheat genotype Florida and 4 independent transgenic plants of spring wheat genotype Veery were obtained. Most transgenic plants have set seeds. T2 seeds of some spring wheat transgenic plants have also been harvested.     

Optimization of biolistic method for transient gene expression and production of agronomically useful transgenic Basmati rice plants

We have developed a reproducible biolistic procedure for the efficient transformation of embryogenic suspension cells of an improved aromatic Indica rice variety, Pusa Basmati 1. The -glucuronidase gene was used to assay transient transformation; other plasmids carrying either a potato protease inhibitor 2 (Pin2) gene, or a late embryogenesis-abundant protein (LEA3) gene from barley, were used for the optimization of biolistic process and transgenic plant production. After optimization of the procedure, over 600 transient transformants and at least five fertile plants showing integrative transformation were obtained per bombarded filter. At least 30% of the plants were derived from independent transformation events. The new improved procedure involves the use of a reporter gene or other useful genes driven by the strong rice actin 1 gene (Act1) promoter, osmotic pre-conditioning of cells for 24 h on medium supplemented with 0.25 M mannitol prior to bombardment, use of gold particles for DNA delivery, and use of plant regeneration medium with high (1.0%) agarose concentration.     

Transient gene expression assays in rose tissues using a Bio-Rad Helios® hand-held gene gun

Rose tissues of different varieties were transformed using a Bio-Rad Helios® hand-held biolistic gun. Parameters for optimum transient expression were optimized and included rose variety, flower age, tissue, gold particle size and DNA Loading ratio. Smooth flowers without thick waxy layers and young unopened actively growing flowers were found to be better suited for the transient expression assays. The DNA amounts, gold particle amounts and size etc. were not found to influence the efficiency of the transient transformation in these tissues. These studies indicate that biolistic transformation using hand-held guns can be used for successful transient expression assays in rose flower tissues. This is especially useful for a quick and easy analysis of genes and their expression before attempting stable transformation.     

Transformation of pasta wheat (Triticum turgidum L. var. durum) with high-molecular-weight glutenin subunit genes and modification of dough functionality

Particle bombardment has been used to transform three cultivars (L35, Ofanto, Svevo) and one breeding line (Latino × Lira) of durum wheat (Triticum turgidum L. var. durum). These varieties were co-transformed with plasmids containing selectable and scorable marker genes (bar and uidA) and plasmids containing one of two high-molecular-weight (HMW) glutenin subunit genes (encoding subunits 1Ax1 or 1Dx5). Ten independent transgenic lines were recovered from 1683 bombarded scutella (transformation efficiency thus 0.6%). Five lines expressed either subunit 1Dx5 or 1Ax1 at levels similar to those of endogenous subunits encoded on chromosome 1B. To identify the effects of the transgenes on the functional properties of grain, three lines showing segregation for transgene expression were used to isolate sibling T₂ plants which were null or positive for the transgene product. Analysis of these plants using a small-scale mixograph showed that expression of the additional subunits resulted in increased dough strength and stability, demonstrating that transformation can be used to modify the quality of durum wheat for bread and pasta making.     

Induction, regeneration, and biolistic sensitivity of different genotypes of common wheat (Triticum aestivum L.)

The induction, regeneration, and biolistic sensitivities of different genotypes of common wheat (Triticum aestivum L.) have been determined in order to develop an efficient system for transformation of Russian cultivars of spring wheat. Short-term (two days) cold treatment (4 degrees C) has been demonstrated to distinctly increase the frequency of morphogenetic callus induction. The optimal phytohormonal composition of the nutrient medium ensuring an in vitro regeneration rate of the common wheat cultivar Lada as high as 90% has been determined. The optimal temporal parameters of genetic transformation of wheat plants (10-14 days of culturing after initiation of a morphogenetic callus) have been determined for two transformation methods: biolistic without precipitated DNA and transformation with the plasmid psGFP-BAR. Analysis of the transient expression of the gfp gene has confirmed that 14 days of culturing is the optimal duration.     

Biolistic transformation of elite genotypes of switchgrass (Panicum virgatum L.)

Key message

With a novel elite genotype, SA37, and an improved transformation protocol, it is now possible to routinely and efficiently engineer switchgrass using biolistic transformation.

Abstract

Transformation of elite switchgrass (Panicum virgatum L.) genotypes would facilitate the characterization of genes related to cell wall recalcitrance to saccharification. However, transformation of explants from switchgrass plants has remained difficult. Therefore, the objective of this study was to develop a biolistic transformation protocol for elite genotypes. Three switchgrass genotypes (ST1, ST2, and AL2) were previously selected for tissue culture responsiveness. One genotype, SA37, was selected for further use due to its improved formation of callus amenable to transformation. Various medium sets were compared and a previously published medium set provided cultures with >96 % embryogenic callus, and data on transient and stable gene expression of RFP were used to optimize biolistic parameters, and further validate the switchgrass (PvUbi1) promoter. SA37 proved to be the most transformable, whereas eight transgenic calli on average were recovered per bombardment of 20 calli (40 % efficiency) when using a three-day day preculture step, 0.6 M osmotic adjustment medium, 4,482 kPa rupture disks and 0.4 μm gold particles which traveled 9 cm before hitting the target callus tissue. Regenerability was high, especially for ST2, for which it is possible to recover on average over 400 plants per half-gram callus tissue. It is now possible to routinely and efficiently engineer elite switchgrass genotypes using biolistic transformation.     

Improvement of plant regeneration and GUS expression in scutellar wheat calli by optimization of culture conditions and DNA-microprojectile delivery procedures

Summary Genetic transformation of cereals by direct DNA delivery via microprojectile bombardment has become an established procedure in recent years. But the derivation of functional transgenic plants, especially in wheat, is still problematic, mainly due to low efficiency of DNA delivery and the reduced regeneration capability of microprojectile-bombarded tissue. We focussed on these two aspects and found that the regeneration of scutellar calli of wheat can be rendered highly efficient and considerably accelerated by a liquid culture phase in screen rafts. We also found that the expression of a reporter gene following DNA delivery by microprojectile can be improved by maintaining the scutellar calli in 0.25 M mannitol before and after bombardment, by bombardment in the presence of silver thiosulfate and Ca(NO₃)₂ (rather than CaCl₂) and by the elimination of spermidine from the DNA/microprojectile mixture. A protocol that includes all these features leads to several-fold higher transient expression of the reporter gene than have previously published procedures.