Particle Bombardment Method Research Articles

Efficient transformation of the isolated microspores of Chinese cabbage (Brassica rapa L. ssp. pekinensis) by particle bombardment

BackgroundThe low efficiency of genetic transformation in Chinese cabbage (Brassica rapa L. ssp. pekinensis) is the key problem affecting functional verification. Particle bombardment is a widely used method along with the Agrobacterium-mediated method. As a physical means, it has almost no restrictions on the type of host and a wide range of receptor types, which largely avoids the restriction of explants. The bombardment parameters, which include the number of bombardments, the bombardment pressure, and the bombardment distance, may affect the microspores' genetic transformation efficiency.ResultsThe transformation efficiency was improved using the particle bombardment method under the combination of bombardment shot times (3, 4, 5) × bombardment pressure (900, 1100, 1350 psi) × bombardment distance (3, 6, 9 cm). The average viability of microspores in the treatment group ranged from 74.76 to 88.55%, while the control group was 88.09%. When the number of shot times was 4, the number of embryos incubated in the treatment group ranged from 16 to 236 per dish, and the control group had 117 embryos per dish. When the bombardment parameters of the biolistic method were 4 shot times—1350 psi—3 cm, 4 times—1100 psi—3 cm, and 4 times—900 psi—3 cm, they had high transient expression efficiency, and the average number of transformed microspores was 21.67, 11.67, and 11.67 per dish (3.5 mL), respectively. When the bombardment parameters were 4 times, 900 psi, and 6 cm, the highest genetically transformed embryos were obtained, and the transformation efficiency reached 10.82%.ConclusionA new genetic transformation system with proper parameters for Chinese cabbage microspores was established using particle bombardment. This proper transformation system could provide a useful tool for the improvement of cultivar quality and the investigation of functional genes in Chinese cabbage.

An efficient transformation method for genome editing of elite bread wheat cultivars.

An efficient genetic transformation protocol is necessary to edit genes for trait improvement directly in elite bread wheat cultivars. We used a protein fusion between a wheat growth-regulating factor 4 (GRF4) and its interacting factor (GIF1) to develop a reproducible genetic transformation and regeneration protocol, which we then used to successfully transform elite bread wheat cultivars Baj, Kachu, Morocco, Reedling, RL6077, and Sujata in addition to the experimental cultivar Fielder. Immature embryos were transformed with the vector using particle bombardment method. Transformation frequency increased nearly 60-fold with the GRF4-GIF1-containing vectors as compared to the control vector and ranged from ~5% in the cultivar Kachu to 13% in the cultivar RL6077. We then edited two genes that confer resistance against leaf rust and powdery mildew directly in the aforementioned elite cultivars. A wheat promoter, TaU3 or TaU6, to drive the expression of guide RNA was effective in gene editing whereas the OsU3 promoter failed to generate any edits. Editing efficiency was nearly perfect with the wheat promoters. Our protocol has made it possible to edit genes directly in elite wheat cultivars and would be useful for gene editing in other wheat varieties, which have been recalcitrant to transformation thus far.

Interspecific divergence of circadian properties in duckweed plants.

The circadian clock system is widely conserved in plants; however, divergence in circadian rhythm properties is poorly understood. We conducted a comparative analysis of the circadian properties of closely related duckweed species. Using a particle bombardment method, a circadian bioluminescent reporter was introduced into duckweed plants. We measured bioluminescence circadian rhythms of eight species of the genus Lemna and seven species of the genus Wolffiella at various temperatures (20, 25, and 30°C) and light conditions (constant light or constant dark). Wolffiella species inhabit relatively warm areas and lack some tissues/organs found in Lemna species. Lemna species tended to show robust bioluminescence circadian rhythms under all conditions, while Wolffiella species showed lower rhythm stability, especially at higher temperatures. For Lemna, two species (L. valdiviana and L. minuta) forming a clade showed relatively lower circadian stability. For Wolffiella, two species (W. hyalina and W. repanda) forming a clade showed extremely long period lengths. These analyses reveal that the circadian properties of species primarily reflect their phylogenetic positions. The relationships between geographical and morphological factors and circadian properties are also suggested.

Fluorescent Labeling and Confocal Microcopy of Plastids and Stromules.

While chlorophyll has served as an excellent label for plastids in green tissue, the development of fluorescent proteins has allowed their ready visualization in all tissues of the plants, revealing new features of their morphology and motility, including the presence of plastid extensions known as stromules. Gene regulatory sequences in nuclear transgenes that target proteins to plastids, as well as in transgenes introduced into plastid genomes, can be assessed or optimized through the use of fluorescent protein reporters. Fluorescent labeling of plastids simultaneously with other subcellular locations reveals dynamic interactions and mutant phenotypes. Transient expression of fluorescent protein fusions is particularly valuable to determine whether or not a protein of unknown function is targeted to the plastid. Fluorescent biosensors can assay molecules such as ATP, calcium, or reactive oxygen species. Particle bombardment and agroinfiltration methods described here are convenient for imaging fluorescent proteins in plant organelles. With proper selection of fluorophores for labeling the components of the plant cell, confocal microscopy and multiphoton microscopy can produce extremely informative images at high resolution at depths not feasible by standard epifluorescence microscopy.

Seed-specific expression of TaYUC10 significantly increases auxin and protein content in wheat seeds.

Present study revealed that specific expression of TaYUC10.3 in wheat young seeds could increase the content of auxin, and protein. Auxin is a vital endogenous hormone in plants, which is involved in the regulation of various physiological and biochemical processes in plants. The flavin-containing monooxygenase encoded by the YUCCA gene is a rate-limiting enzyme in the tryptophan-dependent pathway of auxin synthesis. TaYUC10.3 was identified, cloned and found that it was abundantly expressed in wheat young seeds. In this study, a seed-specific expression vector of TaYUC10.3 was constructed with the promoter of 1Bx17 glutenin subunit gene and transformed wheat using the particle bombardment method. The quantitative RT-PCR showed that TaYUC10.3 was expressed in a large amount in young seeds of the transgenic lines. Plant hormone-targeted metabolomics showed that the auxin content of the transgenic lines was significantly increased compared with controls. The GC / MS non-targeted metabolite multiple statistical analyses showed that the variable importance in projection (VIP) of tryptophan reduced in the transgenic lines. Simultaneously, the VIP of indole acetic acid increased. The precursor amino acids for synthesizing some proteins and carbohydrates were upregulated in the transgenic lines. Subsequently, it was found that the protein content of the seeds of the transgenic TaYUC10.3 wheat was significantly higher than that of the control. The wet gluten content and sedimentation value of the transgenic TaYUC10.3 wheat were also high. This result indicated that TaYUC10.3 might participate in auxin synthesis and affects the protein content of wheat seeds.

In planta particle bombardment (iPB): A new method for plant transformation and genome editing.

Transformation is a key step in modern breeding technology that involves genome editing. The requirement for in vitro tissue culture and regeneration hampers application of this technology to commercially important varieties of many crop species. To overcome this problem, we developed a simple and reproducible in planta transformation method in wheat (Tritticum aestivum L.). Our in planta particle bombardment (iPB) method utilizes the shoot apical meristem (SAM) as a target tissue. The SAM contains a subepidermal cell layer termed L2, from which germ cells later develop during floral organogenesis. The iPB method can also be used for genome editing through transient CRISPR/Cas9 expression or direct delivery of the CRISPR/Cas9 ribonucleoprotein. In this review, we describe the iPB technology and provide an overview of its current and future applications in plant transformation and genome editing.

Monitoring single-cell bioluminescence of Arabidopsis leaves to quantitatively evaluate the efficiency of a transiently introduced CRISPR/Cas9 system targeting the circadian clock gene ELF3.

The rapid assessment of gene function is crucial in biological research. The CRISPR/Cas9 system is widely used as a tool for targeted gene editing in many organisms including plants. Previously, we established a transient gene expression system for investigating cellular circadian rhythms in duckweed. In this system, circadian reporters and clock gene effectors-such as overexpressors, RNA interference (RNAi), and CRISPR/Cas9-were introduced into duckweed cells using a particle bombardment method. In the present study, we applied the CRISPR/Cas9 system at a single cell level to Arabidopsis thaliana, a model organism in plant biology. To evaluate the mutation induction efficiency of the system, we monitored single-cell bioluminescence after application of the CRISPR/Cas9 system targeting the ELF3 gene, which is essential for robust circadian rhythmicity. We evaluated the mutation induction efficiency by determining the proportion of cells with impaired circadian rhythms. Three single guide RNAs (sgRNAs) were designed, and the proportion of arrhythmic cells following their use ranged from 32 to 91%. A comparison of the mutation induction efficiencies of diploid and tetraploid Arabidopsis suggested that endoreduplication had a slight effect on efficiency. Taken together, our results demonstrate that the transiently introduced CRISPR/Cas9 system is useful for rapidly assessing the physiological function of target genes in Arabidopsis cells.

Down-Regulation of CYP79A1 Gene Through Antisense Approach Reduced the Cyanogenic Glycoside Dhurrin in [Sorghum bicolor (L.) Moench] to Improve Fodder Quality.

A major limitation for the utilization of sorghum forage is the production of the cyanogenic glycoside dhurrin in its leaves and stem that may cause the death of cattle feeding on it at the pre-flowering stage. Therefore, we attempted to develop transgenic sorghum plants with reduced levels of hydrogen cyanide (HCN) by antisense mediated down-regulation of the expression of cytochrome P450 CYP79A1, the key enzyme of the dhurrin biosynthesis pathway. CYP79A1 cDNA was isolated and cloned in antisense orientation, driven by rice Act1 promoter. Shoot meristem explants of sorghum cultivar CSV 15 were transformed by the particle bombardment method and 27 transgenics showing the integration of transgene were developed. The biochemical assay for HCN in the transgenic sorghum plants confirmed significantly reduced HCN levels in transgenic plants and their progenies. The HCN content in the transgenics varied from 5.1 to 149.8 μg/g compared to 192.08 μg/g in the non-transformed control on dry weight basis. Progenies with reduced HCN content were advanced after each generation till T3. In T3 generation, progenies of two promising events were tested which produced highly reduced levels of HCN (mean of 62.9 and 76.2 μg/g, against the control mean of 221.4 μg/g). The reduction in the HCN levels of transgenics confirmed the usefulness of this approach for reducing HCN levels in forage sorghum plants. The study effectively demonstrated that the antisense CYP79A1 gene deployment was effective in producing sorghum plants with lower HCN content which are safer for cattle to feed on.

Testing the infectivity of a Begomovirus by particle bombardment method using a gene gun

This study was design to identify the causal agent of Horsegram yellow mosaic disease and to investigate the pathogenicity of Horsegram yellow mosaic viruses (HgYMV) infective clones. The samples were obtained using standard method from the two main horsegram growing areas of Bangalore, Karnataka State of India. The viral DNA from horsegram plants exhibiting severe symptoms was amplified by PCR. An isolate of HgYMV1 and HgYMV2 were associated with severe symptom phenotype from HgYMV. Full-length clones of DNA-A and DNA-B genomic components were constructed and attempts were made to introduce homologous (HgYMV1/HgYMV2) combinations of DNA-A and DNA-B genomic components into Nicotiana benthamiana plants. Inoculation of linearized constructs containing full-length clones or partial head-to-tail dimers of DNA-A and DNAB genomic components resulted in the introduction of DNA-A genomic components into the host plant. However, these combinations of genomic DNA component were not detected in the inoculated plants bombarded using the gene gun. Thus, this study was unable to confirm the pathogenicity of HgYMV infective clones using N. benthamiana as model plant.Keywords: Horsegram, Begomovirus, yellow mosaic viruses, Particle bombardment, Gene gun

Identification of a dicot infecting mastrevirus along with alpha- and betasatellite associated with leaf curl disease of spinach (Spinacia oleracea) in Pakistan

Spinach is a common vegetable crop and very little data is available about its virus infection. Symptomatic leaves of spinach were collected during field survey. Circular DNA molecules were amplified from symptomatic samples using rolling circle amplification (RCA). After restriction analysis, presumed bands of virus and satellites were cloned, sequenced and analyzed. Analysis of sequenced RCA product revealed the presence of chickpea chlorotic dwarf virus (CpCDV; Mastrevirus). Further analyses of the cloned virus showed that strain “C” of CpCDV was present in symptomatic samples of spinach collected from field associated with vein darkening, curling and enations on leaves. Amplification of alpha- and betasatellites with universal primers was performed. CpCDV showed association with cotton leaf curl Multan betasatellite (CLCuMB) and cotton leaf curl Multan alphasatellites (CLCuMA). Infectivity analysis of CpCDV and CpCDV/CLCuMB were done in N. benthamiana using particle bombardment method and the results showed that CpCDV was able to transreplicates CLCuMB in this host. To our knowledge, this is the first report of a dicot infecting mastrevirus (CpCDV) along with CLCuMB and CLCuMA associated with leaf curl disease of spinach in Pakistan. The significance of the results is discussed.

Biotechnology of oil palm: Current status of oil palm genetic transformation

Abstract Oil palm is the most productive vegetable oil in the world for human consumptions and industrial applications. Increasing demands of palm oil over the years have led the research interests to improve yield and oil quality through breeding approach. Concurrently, genetic transformation has been identified as the alternative method to accelerate the breeding and also to produce high-value traits that are almost impossible to obtain through breeding. During the last 25 years, significant progress has been made in oil palm genetic transformation, particularly in the development of transformation methods such as particle bombardment and Agrobacterium-mediated method. In parallel, various tissues have also been examined to identify the best target material for transformation, particularly tissue with high regenerability. Similarly, various selection systems have been developed for efficient selection of transformed cells. Transformation vectors for most target traits for oil palm improvements also have been constructed. This review provides the recent major advances and new directions that could be applied in oil palm genetic transformation.

Antifungal activity of chitinase II against Colletotrichum falcatum Went. causing red rot disease in transgenic sugarcane.

We evaluated transgenic lines of sugarcane modified with the barley chitinase class-II gene to create resistance against the red rot causative agent Colletotrichum falcatum Went. Local sugarcane cultivar SP93 was transformed with a 690-bp coding sequence of the chitinase-II gene under the influence of a polyubiquitin promoter. Transgenic sugarcane lines (T 0) overexpressing the chitinase gene were obtained through a particle bombardment method with 13.3% transformation efficiency. Four transgenic sugarcane lines, SCT-03, SCT-05, SCT-15, and SCT-20, were tested for resistance against red rot by in vitro antifungal assays. Crude protein extracts from transgenic sugarcane plants SCT-03, SCT-05, SCT-15, and SCT-20 inhibited the mycelial growth of C. falcatum by 49%, 40%, 56%, and 52%, respectively, in a quantitative in vitro assay. Our findings revealed that two transgenic lines, SCT-15 and SCT-20, exhibited the highest endochitinase activity of 0.72 and 0.58 U/mL, respectively. Furthermore, transgenic lines SCT-15 and SCT-20 exhibited strong resistance against inoculated C. falcatum in an in vitro bioassay, as they remained healthy and green in comparison with the control sugarcane plants, which turned yellow and eventually died 3 weeks after infection. The mRNA expression of the transgene in the C. falcatum-inoculated transgenic sugarcane lines increased gradually compared to the control plant. The mRNA expression was the highest at 72 h in both transgenic lines and remained almost stable in the subsequent hours.

Transgenic basmati rice transformed with the Xa21 gene shows resistance against bacterial leaf blight

Rice (Oryza sativa L.) cultivar Super Basmati was transformed with a cloned gene, Xa21, through particle bombardment method. Twenty-two plants were recovered from the selected calli. Initial screening through PCR confirmed the presence of the transgene in 21 plants. Southern analysis further confirmed the integration of the Xa21 gene into the genome of these transgenic plants. Analysis of the T1 generation revealed that both the hpt and Xa21 genes were successfully transmitted from the primary transformants to the progeny. Bioassays with two local isolates of bacterial leaf blight pathogen, Xanthomonas oryzae pv. oryzae (Xoo), showed that 14 transgenic lines were resistant to both Xoo isolates. No significant differences were observed in yield and grain characteristics between transgenic lines and the control; however, the transgenic lines took 8-12 days longer to flower and showed reduced plant height compared to the control plants. In conclusion, we have developed bacterial blight-resistant transgenic lines that can be further exploited by breeders for rice improvement.

Transformation of the Diatom Phaeodactylum tricornutum with its Endogenous (E)-4-Hydroxy-3-methylbut-2-enyl Diphosphate Reductase Gene

Phaeodactylum tricornutum is a model diatom that its genomic information and biological tools are well established. In this study, a gene encoding (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (PtHDR), a terminal enzyme of the methylerythritol phosphatepathway regulating chlorophyll and carotenoid biosynthesis, was isolated from P. tricornutum. The isolated gene was cloned into pPha-T1 vector containing fcpA promoter to prepare pPha-T1-HDR plasmid. As a positive control, pPha-T1-eGFP plasmid was constructed with egfp gene. Stable nuclear transformation was carried out with these plasmids by particle bombardment method and zeocin resistant colonies of P. tricornutum were selected on f/2 agar plate. In result, transformation efficiency was evaluated according to the amount of plasmid DNA coated with gold particles. Integration of introduced plasmids was confirmed with genomic DNA of each transformant by polymerase chain reaction. The eGFP fluorescence was visible in the cytoplasm, indicating that eGFP was successively expressed in P. tricornutum system. The transcript level of exogenous Pthdr gene was evaluated with the obtained transformants. The results presented here demonstrated that introduction of Pthdr gene into P. tricornutum chromosome succeeded and expression of PtHDR was enhanced under the fcpA promoter.

Lignin down-regulation of Zea mays via dsRNAi and klason lignin analysis.

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Characterisation of circadian rhythms of various duckweeds.

The plant circadian clock controls various physiological phenomena that are important for adaptation to natural day-night cycles. Many components of the circadian clock have been identified in Arabidopsis thaliana, the model plant for molecular genetic studies. Recent studies revealed evolutionary conservation of clock components in green plants. Homologues of clock-related genes have been isolated from Lemna gibba and Lemna aequinoctialis, and it has been demonstrated that these homologues function in the clock system in a manner similar to their functioning in Arabidopsis. While clock components are widely conserved, circadian phenomena display diversity even within the Lemna genus. In order to survey the full extent of diversity in circadian rhythms among duckweed plants, we characterised the circadian rhythms of duckweed by employing a semi-transient bioluminescent reporter system. Using a particle bombardment method, circadian bioluminescent reporters were introduced into nine strains representing five duckweed species: Spirodela polyrhiza, Landoltia punctata, Lemna gibba, L. aequinoctialis and Wolffia columbiana. We then monitored luciferase (luc+) reporter activities driven by AtCCA1, ZmUBQ1 or CaMV35S promoters under entrainment and free-running conditions. Under entrainment, AtCCA1::luc+ showed similar diurnal rhythms in all strains. This suggests that the mechanism of biological timing under day-night cycles is conserved throughout the evolution of duckweeds. Under free-running conditions, we observed circadian rhythms of AtCCA1::luc+, ZmUBQ1::luc+ and CaMV35S::luc+. These circadian rhythms showed diversity in period length and sustainability, suggesting that circadian clock mechanisms are somewhat diversified among duckweeds.

Genetic engineering of the green alga Chlorella zofingiensis: a modified norflurazon-resistant phytoene desaturase gene as a dominant selectable marker

The unicellular green alga Chlorella zofingiensis has been proposed as a promising producer of natural astaxanthin, a commercially important ketocarotenoid. But the genetic toolbox for this alga is not available. In the present study, an efficient transformation system was established for C. zofingiensis. The transformation system utilized a modified norflurazon-resistant phytoene desaturase (PDS-L516F, with an leucine-phenylalanine change at position 516) as the selectable marker. Three promoters from endogenous PDS, nitrate reductase (NIT), and ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RBCS) genes were tested, with the RBCS promoter demonstrating the highest transformation efficiency. Inclusion of the first intron of the PDS gene further enhanced the efficiency by 91%. Both particle bombardment and electroporation methods were examined, and the latter gave a fourfold higher transformation efficiency. The introduction of PDS-L516F, which exhibited a 33% higher desaturation activity than the unaltered enzyme, enabled C. zofingiensis to produce 32.1% more total carotenoids (TCs) and 54.1% more astaxanthin. The enhanced accumulation of astaxanthin in transformants was revealed to be related to the increase in the transcripts of PDS, β-carotenoid ketolase (BKT), and hydroxylase (CHYb) genes. Our study clearly shows that the modified PDS gene is a dominant selectable marker for the transformation of C. zofingiensis and possibly for the genetic engineering of the carotenoid biosynthetic pathway. In addition, the engineered C. zofingiensis might serve as an improved source of natural astaxanthin.

Production and evaluation of transgenic sorghum for resistance to stem borer

Transgenic sorghum plants were produced through particle bombardment and Agrobacterium methods in two elite, but recalcitrant genotypes of Sorghum bicolor L. Moench. Use of target cells from developing tissues (immature embryos and multiple shoot buds), pre-culture of target tissue, small size of target tissue (2–3 mm), and regular subculture improved the selection and regeneration efficiencies. Addition of amino acid l-cysteine during co-cultivation and blotting sheet interface was helpful for complete decontamination of Agrobacterium tumefaciens and regeneration of transgenic plants. We demonstrated production of transgenic sorghum plants expressing a Bacillus thuringiensis lepidopteran toxin, through tailored in vitro protocols. Our results showed that decontamination of agrobacteria employing subtle treatments aided recovery of transgenic plants in recalcitrant genotypes. We generated 14 independent transgenic lines carrying different classes of B. thuringiensis toxin genes, cry1Aa and cry1B. Many single copy events were generated in two elite parental lines, CS3541 and 296B. Accumulation of the B. thuringiensis protein in leaves during the susceptible period of plant growth ranged from 35 to 500 ng/g fresh leaf tissue. Comprehensive insect bioassays for tolerance to spotted stem borer (Chilo partellus) were conducted through leaf disk and whole plant assays. Transgenic progeny plants showed 20–30% of damage as compared to 70–80% in non-transformed controls.

Expression analysis and subcellular localization of an ERF transcription factor from Salvia miltiorrhiza

A SmERF1 gene was isolated from Salvia miltiorrhiza, and expression patterns to different stress condition were analysed in the root tissues of S. miltiorrhiza. The cDNA of SmERF1 gene from S. miltiorrhiza was isolated by RTPCR, and the phylogenetic tree using the neighbour-joining tree method in Mega 5 was obtained. To confirm the protein is likely to localize in the nucleus, the SmERF1 coding region was fused to the N-terminus of the GFP gene under the control of the CaMV 35S promoter and transferred into onion epidermal cells using the particle bombardment method. Semi-quantitative RT-PCR analysis revealed different expression pattern of SmERF1 gene in response to exogenous ABA, MeJA and SA. The phylogenetic tree analysis revealed that SmERF1 is most similar to AP2/ERF VII subgroup members. The transient expression of the SmERF1::GFP fusion protein indicated that the SmERF1 was exclusively localized to the nucleus. The transcript of SmERF1 highly accumulated when the plants were treated with MeJA, while accumulated slightly in response to exogenous ABA, salicylic acid. These results suggest hormone such as ABA, MeJA and SA signaling pathways can be involved in the activation and inhibition of the SmERF1.

Transfer of a β-glucuronidase marker gene to triticale (xTriticosecale Wittmack) via particle bombardment (biolistic) method

Abstract This study was carried out to investigte the biotechnology laboratory of Department of Field Crops, Ankara University with the aim of investigating the optimum parameters of gene transfer to mature embryo and leaf segment explants of Triticale (x Triticosecale Wittmack) via particle bombardment (Biolistic) method. Mature embryo and leaf segment explants obtained from Triticale (x Triticosecale Wittmack) were bombardment with accelerated gold or tungsten particles coated with the plasmid pBI221.23 containing the b -glucuronidase (GUS: u idA ) marker gene. Different bombardment distances from the stopping plate (6, 9, 12, cm) and different rupture disk pressures (900, 1100, 1550 psi ) were used as physical parameters. Blue cells expressing the b -glucuronidase gene were detected by using a histochemical assay. The variation in the number of blue spots in bombarded explants was used to determine the gene transfer efficiency. The mature embryos of triticale were found to be more amenable for the direct delivery of foreign DNA by the particle bombardment technique than the leaf segments. Also the gold particles were more suitable than tungsten particles in the gene transfer efficiency. The highest gene transfer results were obtained in mature embryos when 6 cm bombardment distance and 1100 psi rupture disk pressure were used.