Agrobacterium-mediated transient GUS gene expression in buffel grass (Cenchrus ciliaris L.)

The study was conducted to standardize a protocol for Agrobacterium-mediated genetic transformation of buffel grass (Cenchrus ciliaris L.). Embryogenic calli, produced from one-year-old mature seeds of buffel grass, were used as target cells for Agrobacterium-mediated transformation. A. tumefaciens strain LBA4404, harbouring pCAMBIA-1301 or pCAMBIA-2301, was used for co-cultivation with embryogenic calli from three genotypes (IG-3108, IG-9757 and IG-97101). Co-culturing of calli with Agrobacterium for 30 minutes, followed by co-cultivation with 0.1 mM acetosyringone for 3 days was found to be optimum for maximum transformation efficiency. Presence of acetosyringone during co-cultivation was found to be necessary for transformation. Transient GUS (beta-glucuronidase) gene expression was used to monitor T-DNA delivery into the target cells. Significant genotypic variations in response to transformation were observed among the tested genotypes. A very high frequency (63.3%) of GUS gene expression was obtained following Agrobacterium-mediated gene transfer into embryogenic calli. The standardized protocol would be useful for Agrobacterium-mediated genetic transformation of buffel grass with genes of agronomic importance.     

根癌农杆菌介导的苜蓿体胚转化

以苜蓿体细胞胚胎作为根癌农杆菌介导转化的受体,通过对GUS基因瞬时表达率的分析,研究该转化体系的最佳实验参数。实验结果显示,负压处理10min和共培养5d时表达率最高(可达17．4％)。以这一转化方法分别对带有3种不同启动于的表达载体进行比较,发现由CMV35S启动于驱动的GUS基因的瞬时表达率可达82．7％,Ubil启动于驱动的可达57．8％,而Actl启动于驱动的则未见表达。     

In vitro plant regeneration and genetic transformation of Dichanthium annulatum

Optimization of in vitro plant regeneration and genetic transformation of apomictic species such as Dichanthium annulatum would enable transfer of desirable genes. Seven genotypes of this grass species were screened through mature seed explant for embryogenic callus induction, callus growth and quality (color and texture), and shoot induction. Genotype IG-1999, which produced highly embryogenic, rapidly growing good-quality callus capable of regenerating at a high frequency, was selected for transformation experiments. Using a binary vector (pCAMBIA1305), frequency of GUS expression was compared between two methods of transformation. Bombardment of embryogenic calli with gold particles coated with pCAMBIA1305 at a distance of 11 cm, pressure of 4 bars, and vacuum of 27 Hg passing through 100 muM mesh produced maximum GUS expression (23%). Agrobacterium infection was maximum at an optical density of 2.0 when cocultured under vacuum for 15 min and cocultivated for 3 days at 28 degrees C in constant dark on MS medium of pH 5.8 with 3 mg/l 2,4-D, and 400 muM acetosyringone. Among two binary vectors used for Agrobacterium-mediated transformation, pCAMBIA1301 showed higher frequency of GUS expression while pCAMBIA1305 recorded more of the GUS spots per callus. Supplementation of acetosyringone in the cocultivation medium was found indispensable for Agrobacterium-mediated transformation. Injuring the calli through gold particle bombardment before their cocultivation with Agrobacterium improved the transformation efficiency. Several transgenic plants were developed using the PIG method, while stable GUS-expressing calli were multiplied during selection on MS medium containing 250 mg/l cefotaxime and 50 mg/l hygromycin, incubated in constant dark. A highly significant difference was observed between two methods of transformation for both frequency of GUS expression and GUS spots per callus. PIG-mediated transformation resulted in higher GUS expression compared to the Agrobacterium method. These results demonstrate that Dichanthium annulatum is amenable to Agrobacterium-mediated genetic transformation using a binary vector.     

Effect of promoter driving selectable marker on corn transformation

Identification of an appropriate selection agent and its corresponding selectable marker gene is one of the first steps in establishing a transformation protocol for a given plant species. As the promoter controls expression level of the genes, the promoter driving the selectable marker gene can affect transformation. However, investigations into the direct effect of promoters driving selectable marker on transformation are lacking in the literature though many reports of relative strengths of promoters driving reporter genes like GUS or CAT or GFP are available. In the present study, we have compared rice Actin1 and CaMV.35S (commonly used promoters in monocotyledonous plant transformation) promoters driving nptII for their effectiveness in paromomycin selection of transgenic corn events. To enable statistically meaningful analysis of the results, a large sample size of nearly 5,000 immature embryos (explants) was employed producing approximately 1,250 independent events from each of the two constructs in four independent experiments. The rate of appearance of resistant calli and percentage of resistant calli recovered was higher with P-Os.Actin1/nptII/nos3' as compared to P-CaMV.35S/nptII/nos3' in all four experiments. There was no appreciable difference either in the frequency of plant regeneration or in the morphological characteristics of plants recovered from the two constructs. Although the escape rate trended lower with P-Os.Actin1 as compared to P-CaMV.35S, the recovery of low copy events was significantly higher with P-CaMV.35S. The higher transformation frequency with P-Os.Actin1 could be related to the strength of this promoter as compared to P-CaMV.35S in the explants and/or calli. Based on these results, we infer that the promoter driving the selectable marker is an important factor to be considered while establishing a high throughput transformation protocol as it could not only influence the transformation frequency but also the copy number of the transgene in the recovered transgenics.     

Early events in microprojectile bombardment: cell viability and particle location

Tungsten and gold particles, coated with plasmid DNA harboring the β-glucuronidase (GUS) and neomycin phosphotransferase II (npt-II) genes, were delivered into tobacco primary leaves and suspension-cultured cells of maize using the helium particle inflow gun. Cell viability and particle localization were determined 1 and 2 days after bombardment. Of the counted particles, 7–10% penetrated into or through the epidermis. Blue spots on tobacco leaves appeared as a blue area around a single, densely stained particle-containing central cell. DNA-coated gold particles provoked smaller spots with less diffusion and gave rise to more individual events than tungsten particles. In more than 90% of the GUS-positive epidermal and mesophyll cells, a particle was detectable within their nucleus. Two days after bombardment, viability had decreased to 1–2% in particle-containing cells. Penetration of a cell by a particle was accompanied by callose formation in the wound area. Dead suspension culture cells of maize without callose formation but containing particles were detected just 1 h post-bombardment. Living cells with callose spots appeared more frequently after bombardment with tungsten than gold. As in tobacco, GUS expression was limited to those cells containing a particle in their nucleus, and the number of particle-containing, viable cells was low after 48 h. The frequency of stable expression events was compared to the number of surviving tobacco leaf cells. On average, four kanamycin-resistant calli or plantlets were recovered per bombarded dish, of which approximately 50% were also GUS-positive. This corresponds to a stable-to-transient ratio of approximately 0.8%, and is similar to the number of particle-containing cells surviving after 48 h.     

Factors affecting gene delivery by particle bombardment ofDendrobium orchids

Summary Five parameters were examined for their effect on transformation ofDendrobium tissues by microprojectile bombardment. The superpromoter in pBI426 produced at least 1.5 times as many transient transformants as the single cauliflower mosaic virus 35S promoter in pBI121 (37 to 69% vs. 0 to 44%) with dark and frequent GUS (β-glucuronidase) staining. Tissue, genotype, and type of microparticle significantly affected transient GUS activity. Higher expression was seen in protocormlike bodies and in hybrid UH44 compared to etiolated shoots and protocorms and to hybrids M61 and K1329-39. Microparticles of 1.6-μm Bio-Rad gold were more effective than 1.0-μm ASI gold. Transient GUS activity did not differ among protocormlike bodies bombarded using helium propellant pressures of 650, 900, or 1100 psi. Transgenic plants were recovered fromDendrobium UH800 protocormlike bodies bombarded with pBI426-coated, 1.1-μm tungsten particles using an early-model gunpowder-driven apparatus with an estimated stable transformation rate of 11.7%. One transgenic plant ofDendrobium UH44 was recovered from etiolated shoot explants bombarded with pBI121-coated, 1.1-μm tungsten particles using the Dupont PDS-1000 with a stable transformation rate of 0.17%. Positive selection results showed 100 to 200 mg·liter⁻¹ kanamycin to be appropriate for regeneration of transgenic plants from protocormlike bodies, protocorms, and etiolated shoot explants over a 3- to 9.5-mo. period.     

Evaluation of parameters for high efficiency gene transfer via particle bombardment in Indian mulberry

Particle bombardment is a popular method of direct gene delivery into cell, tissue and organs since it requires minimum pre- and post-bombardment manipulation. In addition, this technique is much easier and fast to perform with intact tissue/organ and reduces the period of in vitro culture. Genetic transformation of mulberry, Morus indica cv. K2 was attempted by particle bombardment using hypocotyl, cotyledon, leaf and leaf callus explants. The effect of various physical and biological parameters during bombardment were studied by the histochemical localization of GUS reporter gene following two days of bombardment and by assessing the number of blue spots per explant. p35SGUSINT was used for optimization of different parameters. The percentage of GUS positive explants was very low with tungsten (20%) as compared to gold particles (36%) indicating tungsten toxicity to the tissue. Maximum GUS activity was observed at 1100 psi helium pressure and 9 cm target distance for hypocotyl, cotyledon and leaf. Double bombardment of explants with 10 microg of DNA loaded on macrocarriers clearly yielded a better (up to 56%) result as compared to a single bombardment (30%). Amongst the various plasmids tested, pBI221 gave the highest (100%) GUS positive explants in the leaf callus.     

The effect of the parameters of biolistic transformation of spring barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) on the level of transient expression of <Emphasis Type="Italic">GFP</Emphasis> reporter gene

The effect of the parameters of biolistic transformation (rupture disk pressure of helium, vacuum pressure, stopping screen to target tissue distance, material (gold or tungsten) and size of particles, and duration of explant culturing before bombardment) on the level of transient expression of GFP reporter gene was studied in barley embryos. The highest transient expression was observed after explant preincubation for 12–14 days and bombardment with 1 μm gold particles at the helium pressure of 61.24–74.85 atm, vacuum pressure of 0.064 atm, and distance to target of 9 cm.     

Screening promoters for Anthurium transformation using transient expression

Key message

There are multiple publications on Anthurium transformation, yet a commercial product has not been achieved. This may be due to use of non-optimum promoters here we address this problem.

Abstract

Different promoters and tissue types were evaluated for transient β-glucuronidase (GUS) expression in Anthurium andraeanum Hort. ‘Marian Seefurth’ following microprojectile bombardment. Plasmids containing the Ubiquitin 2, Actin 1, Cytochrome C1 from rice, Ubiquitin 1 from maize and 35S promoter from Cauliflower Mosaic Virus fused to a GUS reporter gene were bombarded into in vitro grown anthurium lamina, somatic embryos and roots. The number of GUS foci and the intensity of GUS expression were evaluated for each construct. Ubiquitin promoters from rice and maize resulted in the highest number of expressing cells in all tissues examined. Due to the slow growth of anthurium plants, development of transgenic anthurium plants takes years. This research has rapidly identified multiple promoters that express in various anthurium tissues facilitating the development of transformation vectors for the expression of desirable traits in anthurium plants.     

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.     

基因枪法将GAI基因导入巴西橡胶的研究

为了探讨利用基因工程技术进行橡胶树品质改良的可行性,用基因枪轰击巴西橡胶(Hevea brasiliensis)愈伤组织,将GAI矮化基因导入橡胶受体中,通过50mg L-1卡那霉素筛选鉴定,优化了基因枪法转化橡胶的各种参数。结果表明,DNA金弹与靶细胞的距离为9cm,每皿轰击一次时,胚状体诱导率可以达到1.87%。经过GUS组织染色和PCR扩增鉴定,初步确定GAI基因已经整合到橡胶基因组中。     

Development of an efficient transient transformation protocol for avocado (Persea americana Mill.) embryogenic callus

An efficient protocol for transient transformation of avocado embryogenic callus has been established, using the PDS-1000/He system and the reporter gus gene driven by the sunflower polyubiquitin promoter. Best physical parameters for transient transformation were 900 psi helium pressure and 6 cm target distance. The level of transient gus expression was slightly higher when the amount of DNA per shot was increased from 0.6 to 1.8 μg, but it was not significantly modified by the type of microprojectile used (tungsten vs. gold particles). The transient transformation assay developed in this research was used to test the strength of different promoters and the expression of fluorescent reporter genes. Four constitutive promoters, sunflower polyubiquitin, CaMV35S, CaMV35S with enhancer, and rice actin 1, as well as a trichome-specific promoter, ATP, were analyzed. Polyubiquitin and ATP promoters yielded the highest number of gus expressing foci, while no expression was detected with the Act1 promoter from rice. Embryogenic callus was also bombarded with plasmids pXK7S*NF2 and pXK7RNR2, harboring the enhanced green fluorescent gene, EGFP, and the red fluorescent gene DsRed, respectively. Both fluorescent proteins were detected 24 and 72 h after bombardment, but the observed transformation efficiency was slightly higher in GFP bombarded cells. The transient transformation system described here can be used as a fast way to select suitable promoters and/or fluorescent genes needed to undertake stable transformation studies in avocado using currently available protocols.     

Development of an airgun device for particle bombardment

Construction and operation of an airgun device for transient gene-expression studies in monocots is described. Compressed air in a cylinder of an airgun was used as the source of propulsion for DNA-coated gold or tungsten particles. Under a partial vacuum of 700 mm Hg, velocity of the macrocarrier was measured at 520 m sec^?1 and 432 m sec^?1 at atmospheric pressure. Optimum distance from the stopping plate to different target cells during bombardment ranged from 4 to 7 cm. Mean transformation efficiency of the GUS-gene marker was estimated at 350 transformants per 65 mg fresh weight of the maize cultured cells. Up to 200 GUS transformed cells were detected per 100 mg of embryogenic rice callus. Use of gold flakes or tungsten powder as microcarriers resulted in similar transformation rates. No transformation was observed in any cells when DNA constructs contained prokaryotic translation initiation sequences for the GUS gene. Based on transient GUS assays, further modification of the airgun device will likely be necessary to obtain high stable transformation rates.     

Factors affecting transient gene expression in cultured radiata pine cotyledons following particle bombardment

Transfer and expression of the β–glucuronidase gene ( gusA ) in cultured cotyledons of radiata pine ( Pinus radiata D. Don ) were obtained by particle bombardment. Conditions for optimum transient expression were established by using plasmid pB[/12], delivered by gold particles, 1.6 μm in diameter, into 8-day-old cultured cotyledons. Helium pressure of 7.6 MPa, bombardment distance between the stopping screen and the target tissues of 6 cm, and 0.8 μg of plasmid DNA per bombardment proved to be the best parameters for transient expression; using these parameters 79% of bombarded cotyledons showed GUS activity, with 4.3 blue spots per cotyledon. This system was used for studying the expression of several gus-driven promoters the expression of the sunflower ubiquitin gene promoter was higher (99% of positive cotyledons, with 14.2 blue spots per cotyledon) than that of the CaMV 35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower gusA expression, as determined histochemically. These results were confirmed by using the gus fluorometric assay. Use of the sunflower ubiquitin gene promoter resulted in gusA expression up to 20 days after bombardment, with a significant level of gus expressing loci per bombarded cotyledon, whereas with the CaMV 35S promoter gusA expression was lost 12 days after bombardment.     

Transient expression of the uidA gene in Pinus pinea cotyledons: A study of heterologous promoter sequences

Transfer and expression of the β-glucuronidase gene (uidA) in cultured cotyledons of stone pine (Pinus pinea L.) was obtained by microprojectile bombardment. Conditions for optimum transient expression were established by using plasmid pBI121 delivered by 1.0 μm-diameter gold particles, into 1-day-old cultured cotyledons. Helium pressure of 6.2 MPa, microcarrier travel distance of 6 cm, and 0.8 μg of plasmid DNA per bombardment, were the best parameters for high levels of transient uidA expression. By using these parameters, 98% of bombarded cotyledons showed β-glucuronidase activity, with a mean of 63 Gus foci per cotyledon. This system was used to study the expression of uidA gene driven by several heterologous promoters. The expression under the control of the sunflower polyubiquitin gene (UbB1) promoter (Δ1 deletion) was higher (99% of GUS positive cotyledons) than under the control of the CaMV35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower uidA expression, as determined histochemically. These results were confirmed by using the GUS fluorometric assay. Use of a deletion of the sunflower polyubiquitin promoter resulted in GUS activity detectable 35 days after bombardment, and significant levels of GUS activity were confirmed at the end of that period. The results will be useful to design protocols for stable transformation and high levels of transgene expression in P. pinea. This revised version was published online in June 2006 with corrections to the Cover Date.     

Microprojectile particle effect on stable transformation of black spruce via bombardment

Three types of microprojectile particles, 1.0-μm gold, 1.3-μm tungsten, and 1.6-μm gold, were studied for their effectiveness on genetic transformation of black spruce via bombardment with somatic embryos as the target tissue. Different particles resulted in different levels of transient expression of theGUS reporter gene; 1.0-μm gold particles produced the highest level of expression, and 1.6-μm gold particles produced the lowest level. Particle type also affected stable transformation; 1.0-μm gold particles had a 10-fold higher stable transformation efficiency than did 1.6-μm gold particles and a 2-fold higher efficiency than did 1.3-μm tungsten particles. This study indicates that microprojectile particle type and size are important in bombardment-mediated plant transformation.     

Comparison of promoters and selectable marker genes for use in Indica rice transformation

The effectiveness of different promoters for use in Indica rice transformation was compared. Plasmids encoding the Escherichia coli uidA (gus) gene under the control of CaMV 35S, Emu, Act1 or Ubi1 promoters were delivered into cell suspension cultures by particle bombardment. Transient gene expression, 48 h after delivery, was greatest from plasmids utilising the constitutive promoters, Act1 and Ubi1. Gene expression in stably transformed tissue was examined by bombarding embryogenic Indica rice calli with a pUbi1-gas plasmid and a plasmid containing either the selectable marker gene, hph, which confers hygromycin resistance, or bar, which confers resistance to the herbicide phosphinothricin (BASTA) each under the control of the CaMV 35S, Emu, Act1 or the Ubi1 promoters. The bombarded calli were placed on the appropriate selection media and stained for GUS activity at 1 day, 3 weeks and 5 weeks after shooting. Callus bombarded with the pUbi1-hph or the pEmu-hph constructs gave a dramatic increase in the size of the GUS staining areas with time. No such increase in the size of GUS staining areas was observed in calli co-bombarded with pUbi1-gus and any of the bar containing constructs.Co-bombardment of calli with either the pEmu-hph or pUbi1-hph construct and a virus minor coat protein (cp) gene construct resulted in many fertile transgenic Indica rice plants, containing one to eight copies of both the hph and cp genes. These genes were stably inherited by the T1 generation.     

High efficiency of stable genetic transformation in <Emphasis Type="Italic">Dendrobium via</Emphasis> microprojectile bombardment

Several parameters affecting transient β-glucuronidase (GUS) expression in protocorms of Dendrobium cv. Jaquelyn Thomas were examined using bombardment technique with pActin-1D plasmid. The GUS activity in Dendrobium protocorm was not significantly affected by size of the target, type of particles, and helium gas pressure. However, the numbers of surviving tissues after bombardment were different. Transgenic orchids were established by bombardment of actively dividing protocorms with gold particles coated with pMNK1005 plasmid containing hygromycin phosphotransferase (hgh) and green fluorescent protein (gfp) genes driven by a ubiquitin promoter. A high efficiency of orchid transformation was established using three selection steps. The bombarded protocorms were screened on medium supplemented with 5 mg dm⁻³ and 25 mg dm⁻³ hygromycin and surviving protocorms were stringently selected on medium containing 30 mg dm⁻³ hygromycin. The transformation efficiency was 19.87 % and GFP expressing protocorms were not chimeras. The integration of the transgene into genomic DNA of transgenic plantlets was confirmed by PCR and Southern blot hybridization. All but one of the transgenic lines contained multiple copies of the transgene.     

Stable transformation of tomato cell cultures after bombardment with plasmid and YAC DNA

Summary Stable transformants were obtained after microprojectile particle bombardment of tomato cell suspensions (Lycopersicon esculentum cv VFNT Cherry and L. pennellii). The suspensions were bombarded with tungsten particles coated with either plasmid (6.3 kb) or yeast artificial chromosome (YAC) (80 kb) DNA containing the ß-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. The YAC DNA contained an insert of approximately 50 kb of DNA from VFNT Cherry. L. pennellii suspensions were more amenable to transformation than VFNT Cherry; more kanamycin-resistant calli were recovered from L. pennelli after bombardment with plasmid DNA, and only L. pennellii cells produced transformants after bombardment with YAC DNA. DNA gel blot analysis confirmed the presence of the nptll and GUS genes. This analysis also confirmed the integration of YAC DNA into the genome of the kanamycin-resistant calli and suggested that the level of intactness of the integrated YAC DNA was fairly high in four of the five transformants examined. Microprojectile bombardment of regenerable cultures with YACs may ultimately aid in map-based cloning of agriculturally-important genes.Abbreviations YAC yeast artificial chromosome - MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indole-3-acetic acid - GUS ß-glucuronidase - nptII neomycin phosphotransferase II