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.     

水稻基因枪法多基因转化研究

为探索多基因转化系统,利用基因枪法进行水稻3个质粒的共转化研究,结果从25个转基因植株中获得3个三转化植株N12、K4-39和K1-1-66,成功地将分别位于不同质粒载体上的GUS,hpt和RTBV CP基因同时导入到水稻中。3个三转化植株中N12、K4-39正常可育。对N12的后代遗传分析表明,该转基因植株中3个载体所携外源基因均呈孟德尔式分离(3:1),3个外源基因分别整合到水稻的两条染色体上。GUS基因与hpt连锁,RTBV CP位于另一条染色体上。     

Optimization of delivery of foreign DNA into higher-plant chloroplasts

We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of -glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.     

The ectopic expression of apple <Emphasis Type="Italic">MYB1</Emphasis> and <Emphasis Type="Italic">bHLH3</Emphasis> differentially activates anthocyanin biosynthesis in tobacco

Two direct DNA transfer methods, biolistic transformation and a protoplast transformation approach using the INRA-clone 717 1B4 (Populus tremula?×?P. alba), are applied to poplars and compared. Both the in vitro culture and the transformation parameters were optimized to receive a maximum quantity of transformed cells to achieve a stable transformation. For the first time, the stable integration of gfp and dsred in the poplar genome and their expression as visual reporter genes in regenerated plantlets can be shown. For biolistic transformation, stem segments cut lengthwise and incubated for 10 days on a callus induction medium revealed the highest number of transient Gfp- and dsRed signals. After optimization of the in vitro culture parameter, Gfp and dsRed-expressing transgenic poplars were regenerated, proven by PCR and Southern blot analysis. For protoplast transformation, the focus was initially on the development of a highly efficient protoplast isolation and plant regeneration system. Using an enzyme solution consisting of 1.0% cellulase R10 and 0.24% macerozyme, 1?×?10⁷ protoplasts were obtained from 1 g fresh weight leaves. Following incubation of the protoplasts in 600 mOsm culture medium, a high number of microcalli were obtained, from which plantlets were regenerated. The parameters for isolation and regeneration were then complemented by an efficient protoplast transformation protocol with 40% PEG₁₅₀₀. The results of this study confirm that both the biolistic and the protoplast transformation methods can be considered suitable for transferring cisgenes directly into poplar.     

Elite Indica Transgenic Rice Plants Expressing Modified Cry1Ac Endotoxin of Bacillus Thuringiensis Show Enhanced Resistance to Yellow Stem Borer (Scirpophaga Incertulas)

Bt-transgenics of elite indica rice breeding lines (IR-64, Pusa Basmati-1 and Karnal Local) were generated through biolistic of Agrobacterium-mediated approaches. A synthetic cry1Ac gene, codon optimised for rice and driven by the maize ubiquitin-1 promoter, was used. Over 200 putative transformants of IR-64 and Pusa Basmati-1 and 26 of the Karnal Local were regenerated following use of the hpt (hygromycin phosphotransferase) selection system. Initial transformation frequency was in the range of 1 to 2% for particle bombardment while it was comparatively higher ( 9%) for Agrobacterium. An improved selection procedure, involving longer selection on the antibiotic-supplemented medium, enhanced the frequency of Bt-transformants and reduced the number of escapes. Molecular evaluation revealed multiple transgene insertions in transformants, whether generated through biolistic or Agrobacterium. In the latter case, it was also observed that all genes on the T-DNA do not necessarily get transferred as an intact insert. Selected Bt-lines of IR-64 and Pusa Basmati-1, having Bt-titers of 0.1% (of total soluble protein) and above were evaluated for resistance against manual infestation of freshly hatched neonate larvae of yellow stem borers collected from a hot spot stem borer infested area in northern India. Several Bt-lines were identified showing 100% mortality of larvae, within 4-days of infestation, in cut-stem as well as vegetative stage whole plant assays. However, there was an occasional white head even among such plants when assayed at the reproductive stage. Results are discussed in the light of resistance management strategies for deployment of Bt-rice.     

Major improvements in biolistic transformation of suspension-cultured tobacco cells

Summary Suspension cultures of the NT1 line ofNicotiana tabacum L. were used as a model system to study plant biolistic transformation, because of their uniformity, rapid growth, and ease of handling. The β-glucuronidase gene and the neomycin phosphotransferase genes were used to assay transient and stable transformation. Numerous factors were studied and optimized, such that the frequency of transformation was increased roughly 60-fold for transient transformants and 20-fold for stable transformants. Both biological parameters (the promoter used to drive gene expression, osmotic preconditioning and posbombardment handling of the cells) and physical parameters of the bombardment process (particle acceleration device and accelerator parameters) were tested. The factors that increased transformation rates the most were promoter strength, use of a helium-driven particle accelerator, and osmotic preconditioning of the cells.     

Induction,regeneration, and biolistic sensitivities of different genotypes of common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> 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°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.     

Comparison of Biolistic and Agrobacterium -mediated Transformation Methods on Transgene Copy Number and Rearrangement Frequency in Rice

Transferring foreign DNA into plant cells by biolistic and Agrobacterium -mediated methods may result in random integration of different copy numbers of the transgene, and different proportions of intact vs. rearranged copies of the transgene. This may, in turn, affect transgene expression levels. To test the above hypothesis, we first introduced the same plasmid, pAc1PG-CAM, into rice (BX)Oryza sativa L.) calli separately by the biolistic method and by the Agrobacterium -mediated method. To show whether different plasmids may affect the results, we also introduced pTOK233 by the Agrobacterium -mediated method and pJPM44 by the biolistic method. Transgene expression of R0 plants was monitored by histochemical analysis of GUS activity. Transgene copy number was determined by Southern blot analysis after digesting genomic DNA with an enzyme that has a unique cutting site within the input plasmid. The total genomic DNA was also digested by a two-cut enzyme (the cuts are located at two sides of a given transgene expression cassette), followed by Southern blotting analysis, for determining the number of intact transgene expression cassettes. Our data showed that Agrobacterium -mediated transformation resulted in lower transgene copy number (average between 2.1 and 2.3) in transgenic rice plants, compared with those plants obtained by the biolistic method (average between 4.2 and 5.6). The frequency of DNA rearrangement in expression cassettes is lower in transgenic rice plants obtained by the Agrobacterium -mediated method than those obtained by the biolistic method. The average rearrangement frequency is 0.07 to 0.106 for the Agrobacterium -mediated method, and 0.57 to 0.66 for the biolistic method. Our results suggest that it is better to compare the number of intact expression cassettes instead of the total copy number of the transgene in demonstrating their influence on the level of transgene expression. This is the first report on the frequency of expression cassette rearrangement in transgenic plants transformed with the same plasmid by two different transformation methods.     

根癌农杆菌介导转化水稻成熟胚及转Metr基因植株的获得

以粳稻品种广陵香糯为材料,用携带有双元载体pBB的根癌农杆菌EHA105为载体,研究了根癌农杆菌介导转化粳稻成熟胚愈伤组织的几个影响因素,建立了合适的粳稻成熟胚愈伤组织转化系统。一种基于MS为基本培养基的商品培养基(HRM)较适合于作为粳稻成熟胚愈伤组织诱导培养基,合适的愈伤组织诱导培养天数为7~8 d,合适的筛选培养基为CC培养基。在此基础上,将Metr基因导入粳稻品种广陵香糯中,获得了一批转基因水稻植株,PCR分析表明外源基因已经整合进了水稻的基因组中。部分转基因植株后代遗传分析表明外源基因的分离符合3∶1的理论比例。     

双管基因枪介导的基因瞬时表达技术在拟南芥中的应用

基因瞬时表达是植物中研究目标基因功能的常用手段。在模式植物拟南芥(Arabidopsis thaliana)中, 相比原生质体和农杆菌介导的基因异源表达技术, 利用粒子轰击进行基因瞬时表达一直鲜有报道。其主要原因是拟南芥叶型相对较小、基因枪操作相对烦琐以及基因表达效率差异较大。该研究通过优化双管基因枪系统, 在营养生长旺盛的拟南芥莲座叶中实现GFP和GUS基因高效表达。同时, 通过GUS报告基因明确了坏死诱导因子BAX、Avh238和ATR13/Rpp13激发拟南芥细胞坏死的表型。但在本氏烟(Nicotiana benthamiana)中明显诱导细胞坏死的Avrblb1/RB基因对, 在拟南芥中却丧失了诱导细胞坏死的活性。由于双管基因枪系统每次轰击时设置平行对照, 可有效降低转化实验中的样本变异度, 为拟南芥及其突变体研究中准确评价基因功能和高通量筛选目标基因提供新的技术参考。     

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。     

Analysis of a large number of independent transgenic rice plants produced by the biolistic method

Summary Over 500 independent transgenic rice plants have been obtained by the biolistic method with an average transformation frequency of 9.7% for japonica variety Taipei 309. A tight selection procedure using 50 mg/l of hygromycin B successfully prevented the growth of nontransformed tissues. Analysis of the T0 transgenic rice plants revealed that more than 97% of the transgenic plants were morphologically normal and more than 80% were at least partially fertile. The hyg^r trait was inherited as a dominant trait in a Mendelian manner in 8 out of 11 transgenic events assayed. Thirty-seven out of fifty transgenic plants were estimated to contain no more than five copies of the transgenes. In six out of seven transformation events, unlinked, co-transformed genes co-segregated in the T1 generation. The hyg^r trait has been stably inherited to the T4 generation. No chimerical transgenic plant has been found in an intensive search. Novel phenomena observed in transgenic rice plants are also reported.     

Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.)

Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene. The transformation yielded on the average 5 callus lines per bombardment (1.4×10⁶ cells). Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines. The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass. Long-term GUS expression was observed in ca. 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture.     

Development of transformation vectors based upon a modified plant alpha-tubulin gene as the selectable marker

A plant transformation and selection system has been developed utilizing a modified tubulin gene as a selectable marker. The vector constructs carrying a mutant alpha-tubulin gene from goosegrass conferring resistance to dinitroaniline herbicides were created for transformation of monocotyledonous and dicotyledonous plants. These constructs contained beta- and/or mutant alpha-tubulin genes driven either by ubiquitin or CaMV 35S promoter. The constructs were used for biolistic transformation of finger millet and soybean or for Agrobacterium-mediated transformation of flax and tobacco. Trifluralin, the main representative of dinitroaniline herbicides, was used as a selective agent in experiments to select transgenic cells, tissues and plantlets. Selective concentrations of trifluralin estimated for each species were as follows: 10 microM for Eleusine coracana, Glycine max, Nicotiana plumbaginifolia and Nicotiana sylvestris; 3 microM for Linum usitatissimum. PCR and Southern blotting analyses of transformed lines with a specific probe to nptII, alpha-tubulin or beta-tubulin genes were performed to confirm the transgenic nature of regenerated plants. Band specific for the mutant alpha-tubulin gene was identified in transformed plant lines. Results confirmed the stable integration of the mutant tubulin gene into the plant genomes. The present study clearly demonstrates the use of a plant mutant tubulin as a selective gene for plant transformation.     

Biolistic plant transformation

The biolistic process represents a completely new approach to the problem of how to deliver DNA into intact cells and tissues. High velocity microprojectiles are used to carry DNA or other substances past cell walls and membranes. Because DNA is being 'shot' into cells, it represents a type of biological ballistics, hence the term "biolistics".
There are several fundamental advantages to the biolistic process over other plant transformation techniques. The biolistic process appears to be effective regardless of species or tissue type, it is a rapid and very simple procedure, and it should facilitate the direct transformation of totipotent tissues such as pollen, embryos, meristems and morphogenic cell cultures. In addition, the biolistic process appears to be uniquely suitable for organelle transformation.
The disadvantages of the biolistic process are that it requires special instrumentation, and is still in the early stages of its development. Consequently, delivery efficiencies are still not as high as can be achieved in highly optimized transformation systems such as electroporation or agrobacterial-infection of tobacco. Furthermore, potential users should be prepared to spend some time adapting existing protocols to their specific species or tissue of interest.     

Development of a non-lethal selection system by using the aadA marker gene for efficient recovery of transgenic rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The application of aminoglycoside-3-adenyltransferase (aadA) gene-mediated streptomycin resistance for non-lethal selection of transgenic rice resulted in plant regeneration frequencies under selection pressure as high as those in non-transformed controls without selection. Since streptomycin does not kill non-transgenic cells, and allows plant regeneration from them, a selection procedure was developed that made the visual identification of transgenic calli and regenerants possible. For callus-level selection, a vital pH indicator—Chlorophenol Red—was applied together with streptomycin, making use of the phenomenon that fast-growing cell lines lower the pH in the culture medium. Transgenic plants were selected according to their main distinctive features; their green colour (photomixotrophic assimilation), and more intense growth. At the same time, non-transgenic regenerants were bleached (heterotrophic assimilation), and growth was retarded in the presence of streptomycin and sucrose. The final efficiency of genetic transformation based on streptomycin resistance was found to be double that of transformations where the selective agent was l-phosphinothricin, and nearly three times more compared to transformations resulting in hygromycin-resistant regenerants. To the best of our knowledge, this is the first report on producing nuclear transformed rice plants by using a non-lethal selection strategy based on the chimaeric aadA gene.Communicated by D. Dudits     

Multiple copies of virG enhance the transient transformation of celery,carrot and rice tissues by Agrobacterium tumefaciens

In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect of multiple copies of virG genes contained in Agrobacterium tumefaciens strains upon the transient transformation of celery, carrot and rice tissues. Four days after A. tumefaciens infection, we performed histochemical -glucuronidase (GUS) assays to determine the frequency of transient transformation of calli from celery and carrot, and explants from rice and celery. Additional copies of octopine- and agropine-type virG genes in A. tumefaciens strains containing an agropine-type Ti-plasmid enhanced the frequency of transient transformation of celery and rice. This enhancement ranged from 25% to five-fold, depending upon the source of the virG gene and the plant tissues inoculated. For both rice and celery, we observed a greater enhancement of transformation using A. tumefaciens strains containing additional copies of an octopine-type virG gene than with strains harboring additional copies of an agropine-type virG gene. Multiple copies of virG genes contained in A. tumefaciens strains harboring a nopaline-type Ti-plasmid had a smaller enhancing effect upon the transformation of celery tissues, and no enhancing effect upon the transformation of rice. In contrast, we obtained a three-fold increase in the transient transformation frequency of carrot calli using an A. tumefaciens strain harboring a nopaline-type Ti-plasmid and additional copies of an octopine-type virG gene. Our results show that multiple copies of virG in A. tumefaciens can greatly enhance the transient transformation frequency of celery, carrot and rice tissues, and that this enhancement is influenced by both the type of Ti-plasmid harbored by A. tumefaciens and by the infected plant species.Current address: Department of Agronomy, Purdue University     

High-efficiency biolistic co-transformation and regeneration of 'Chardonnay' (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) containing<Emphasis Type="Italic"> npt-II</Emphasis> and antimicrobial peptide genes

A reliable and efficient system for transformation and regeneration of 'Chardonnay' (Vitis vinifera L.) plants via microprojectile bombardment was developed. Improvements over the previous biolistic transformation system included: (1) the use of gold particles for bombardment; (2) step-wise selection at 10 then 15 mg/l kanamycin; and (3) embryo induction at 27°C. Embryogenic cell cultures were either bombarded with pBI426, which contains the reporter gene gus (uidA) coding for -glucuronidase (GUS), or were co-bombarded with pSAN237 carrying the npt-II (neomycin phosphotransferase II) selectable marker gene, and a second plasmid with an antimicrobial peptide gene. A large number of transient (7,883±1,928) and stable (46±32) blue spots per plate at 2 and 95 days after bombardment, respectively, were obtained according to GUS expression analyses. A total of 447 putative transgenic embryos was harvested from 84 bombarded plates. From these embryos, 242 (54%) were regenerated into plants within the first year of the experiment. Southern blot analyses confirmed integration of the transgenes into the grape genome. Co-transformation was tested with four separate antimicrobial constructs. The co-transformation frequency of unlinked genes was 48% as measured by polymerase chain reaction (PCR), and 56% as estimated by dot blot hybridization. Expression of the gus gene, and PCR and Southern blot analyses of npt-II and antimicrobial genes from regenerated plants document stable transformation of 'Chardonnay' and establish the parameters for highly-efficient biolistic transformation in V. vinifera.Abbreviations AMP Antimicrobial peptide - DBH Dot blot hybridization - GM+NOA Glycerol and maltose liquid medium with -naphthoxyacetic acid - gus -Glucuronidase gene - GUS -Glucuronidase - Km Kanamycin - Km^R Kanamycin resistant - mag2 Magainin-2 - MS Murashige and Skoog medium - MS/2 Half-strength Murashige and Skoog medium - nos Nopaline synthase - npt-II Neomycin phosphotransferase II gene - PCR Polymerase chain reaction - PGL Peptidyl-glycine-leucine - Pubq3 Arabidopsis ubiquitin-3 promoter - Pubq10-L Arabidopsis ubiquitin-10L promoter - Pubq11 Arabidopsis ubiquitin-11 promoter - SP Signal peptide - Tnos Nopaline synthase terminator - WPM Woody plant mediumCommunicated by E.D. Earle     

Use of a feedback-insensitive α subunit of anthranilate synthase as a selectable marker for transformation of rice and potato

A selection system based on a mutant rice gene for a feedback-insensitive subunit of anthranilate synthase (OASA1D) was developed for the transformation of rice and potato. Expression of OASA1D conferred resistance to the tryptophan analog 5-methyltryptophan (5MT) in transformed cells of rice and potato. The selection system based on OASA1D and 5MT was associated with a high transformation efficiency, a short time frame for the generation of transgenic plants, simple culture procedures, and it was as effective as hygromycin B selection in rice (monocotyledon) and kanamycin selection in potato (dicotyledon). Transgenic rice and potato plants established by 5MT selection had normal morphology and accumulated tryptophan when OASA1D was expressed under the control of a constitutive promoter. These results demonstrate the efficacy of OASA1D as a selectable marker and they suggest that the 5MT selection system based on this gene will prove applicable to a wide range of plant species and culture procedures.