Number Of Bombardments Research Articles

Particle bombardment – mediated gene transfer and GFP transient expression in Setaria viridis

ABSTRACTSetaria viridis is one of the most important model grasses in studying monocot plant biology. Transient gene expression study is a very important tool in plant biotechnology, functional genomics, and CRISPR-Cas9 genome editing technology via particle bombardment. In this study, a particle bombardment-mediated protocol was developed to introduce DNA into Setaria viridis in vitro leaf explants. In addition, physical and biological parameters, such as helium pressure, distance from stopping screen to the target tissues, DNA concentration, and number of bombardments, were tested and optimized. Optimum concentration of transient GFP expression was achieved using 1.5 ug plasmid DNA with 0.6 mm gold particles and 6 cm bombardment distance, using 1,100 psi. Doubling the bombardment instances provides the maximum number of foci of transient GFP expression. This simple protocol will be helpful for genomics studies in the S. viridis monocot model.

Transformasi Genetik Kedelai dengan Gen Proteinase Inhibitor II Menggunakan Teknik Penembakan Partikel

<p class="p1">An experiment was conducted at the Molecular Biology and Genetic Engineering Laboratory of BB-Biogen, Bogor with an objective to obtain transgenic soybean plants containing the <em>proteinase inhibitor </em>II (<em>pin</em>II) gene. The experiment consisted of three steps, i.e., optimalization of the soybean transformation technique using the <em>gus </em>gene; transformation of soybean using the <em>pin</em>II gene, and molecular analysis of the transformed soybean plants. Two type of explants (young embryo and cotyledon) were bombarded with <em>p</em>RQ6 plasmid containing the <em>gus </em>gene with the following treatment: Helium gas pressure (1100 psi and 1300 psi), shoot distance (5 and 7 cm), and number of bombardment (1x and 2x). The result of <em>gus </em>assay indicated that the best bombardment was done on young cotyledon explants with 1100 psi Helium pressure, shoot distance 5 cm, and 1x bombardment. Transformation of the soybean explant using the <em>pin</em>II gene (inside the <em>p</em>TWa plasmid) was conducted using the best bombardment treatment from the first activity. Two plants from c.v. Wilis (WP<span class="s1">1</span>, WP<span class="s1">2</span>) and three plants from c.v. Tidar (TP<span class="s1">1</span>, TP<span class="s1">2</span>, TP<span class="s1">3</span>) were recovered from regeneration and selection of the transformed explants. Molecular analysis of the regenerated plants using the PCR technique showed that only WP<span class="s1">2 </span>contained the <em>pin</em>II gene. This plant was fertile and will be used for further evaluation.</p>

Optimization of particle bombardment parameters for DNA delivery into the male flowers of banana

The possibility of increasing the efficiency of banana transformation was investigated by particle bombardment of the male flowers of banana plants for constitutive expression of gfp gene. The effects of particle bombardment parameters, such as acceleration pressure, bombardment distance, chamber vacuum pressure, gold microcarrier size, gold quantity, DNA quantity, number of bombardments and pre-culture were examined. Single cauliflower-like bodies (CLBs) clusters, induced from meristemic parts of Musa sapientum cv. Nangka (AAB) male flowers, were bombarded by pCambia1304 plasmid carrying gfp gene driven by the CaMV 35S promoter. Optimal transient expression of green-fluorescent protein (GFP) was obtained when the three-day old cultured tissues were bombarded two times at 1100 psi helium pressure. However, the highest GFP expression was observed when 9 cm was applied as bombardment distance with 28 mmHg chamber vacuum pressure. Gold particle with 1 μm diameter at 60 μg/μL concentrations coated with 1.5 μg/μL of DNA have been used as the optimum bombardment parameter since GFP expression was significantly different compared to other conditions. Application of optimized condition proved effective for the generation of stable transgenic banana plants. PCR and southern blot analyses confirmed the presence and integration of gfp gene in genomic DNA of transformed plants. Transformation frequency achieved with the optimized protocol was 7.5% which was significantly higher than the conventional protocol.

Efficient in vitro plant regeneration from shoot apices and gene transfer by particle bombardment in Jatropha curcas

An efficient and reproducible in vitro plant regeneration system from shoot apices was developed in Jatropha curcas. Benzylaminopurine (BAP; 2.5 μM) was most effective in inducing an average of 6.2 shoots per shoot apex. Incorporation of gibberellic acid (GA3; 0.5 μM) to basal medium was found essential for elongation of shoots. The BAP-habituated mother explants continuously produced shoots during successive subculture without any loss of morphogenic potential. The shoots rooted efficiently on half-strength MS medium. The rooted plantlets were acclimatized with more than 98 % success and the plants transferred to soil:compost in nursery showed no sign of variation compared to the seed-grown plants. The whole process of culture initiation to plant establishment was accomplished within 5-6 weeks. A genetic transformation system in J. curcas was established for the first time, using bombardment of particles coated with plasmid pBI426 with a GUS-NPT II fusion protein under the control of a double 35S cauliflower mosaic virus (CaMV) promoter. The β-glucuronidase (GUS) activity in J. curcas shoot apices was significantly affected by the gold particle size, bombardment pressure, target distance, macrocarrier travel distance, number of bombardments, and type and duration of osmotic pre-treatment. The proliferating bombarded shoot apices were screened on medium supplemented with 25 mg dm-3 kanamycin and surviving shoots were rooted on medium devoid of kanamycin. The integration of the transgene into genomic DNA of transgenic plants was confirmed by PCR and Southern blot hybridization. The transgenic plants showed insertion of single to multiple copies of the transgene.

Optimization and Transformation of Garden Balsam, Impatiens balsamina, Mediated by Microprojectile Bombardment

In this study, a transformation system was developed by initially optimizing the microprojectile bombardment parameters for cotyledonous explants. The parameters optimized were combination of distance from stopping screen to target tissue and helium pressure, number of bombardment, preculture duration prior to bombardment, DNA amount, osmoticum treatment and post-bombardment incubation time. Determination of minimal inhibitory concentration for efficient selection of transformants was also carried out for hygromycin. Different concentrations of hygromycin (25. 50, 75 and 100 mg L-1) were tested against different ages of explants (0, 1, 3 and 5 weeks) for efficient selection of transformants. Using the optimized parameters, transformation of cotyledons was carried out followed by selection on 75 mg L-1 hygromycin at different weeks of post-bombardment. Transgenic plants were successfully regenerated from bombarded explants and confirmed via histochemical GUS assay and PCR analysis.

Stable genetic transformation of castor (Ricinus communis L.) via particle gun-mediated gene transfer using embryo axes from mature seeds

The first successful attempt to produce stably transformed castor plants through direct gene transfer using particle gun (BioRad) is described. Decotyledonated embryos from mature seeds were germinated and the embryonic axis was induced to proliferate on Murashige and Skoog (MS) medium supplemented with 0.5 mg l(-1) thidiazuron (TDZ) and subjected to bombardment after 5-7 days of pre-incubation. The physical parameters for transient transformation were optimized using the UidA gene encoding beta-glucuronidase (GUS) as the reporter gene and with hygromycin-phosphotransferase (hptII) gene as selectable marker. Statistical analysis revealed that helium pressure, target distance, osmoticum, microcarrier type and size, DNA quantity, explant type and number of bombardments had significant influence on transformation efficiency, while the effect of genotype was non-significant. Of the different variables evaluated, embryonic axes from mature seeds, a target distance of 6.0 cm, helium pressure of 1,100 psi, 0.6 microm gold microcarriers, single time bombardment and with both pre- and post-osmoticum were found ideal. Selection of putative transformants was done on MS medium supplemented with 0.5 mg l(-1) BA and hygromycin (20, 40 and 60 mg l(-1)) for 3 cycles. The stable integration of the incorporated gene into castor genome was confirmed with PCR and Southern analysis of T0 and T1 plants. Transformation frequency in terms of plants grown to maturity and showing the presence of the introduced genes was 1.4%. The present results demonstrate the possibility of transformation of embryonic meristematic tissues of castor through particle delivery system.

Factors affecting delivery and transient expression of β-glucuronidase gene in Dendrobium Sonia protocorm-like-body

The effect of the biolistic device parameters and other factors affecting delivery and expression of uidA gene in Dendrobium Sonia was investigated. Three week old protocorm like body (PLB) were bombarded with gold microparticles coated with pAHC 25 plasmid harbouring the uidA gene which encodes β -glucuronidase. The factors investigated were the helium pressure, target distance, macrocarrier flight distance to stopping screen, distance from stopping screen to target tissues, vacuum pressure, gold microparticles size, spermidine and calcium on DNA precipitation, and the number of bombardments. Two days after bombardment, the PLB were subjected to histochemical GUS assay, and transient GUS activity was recorded as blue spots using a Leica stereomicroscope. All the factors tested showed significant effects (p<0.05) on the delivery of DNA and expression of the uidA gene in Dendrobium PLB except for calcium. Surviving PLBs were able to grow and regenerate normally into plantlets. Keywords: biolistic transformation, orchid, Dendrobium , protocorm like body, -glucuronidase assay

Optimization of Particle Bombardment Conditions by Monitoring of Transient sGFP(S65T) Expression in Transformed Soybean

In an attempt to increase the efficiency of soybean transformation by particle bombardment, we examined the effects of bombardment parameters such as gold particle size, target distance, acceleration pressure, amount of DNA per bombardment, and number of bombardments. Transgene delivery to embryogenic tissue grown in suspension culture was evaluated by monitoring the transient expression of a gene for a modified form of jellyfish green fluorescent protein [sGFP(S65T)]. Optimal transient expression of sGFP(S65T) was obtained when the tissue was bombarded twice at an acceleration pressure of 7.6 MPa (1,100 psi) and a distance of 6 cm with gold particles that were 0.6 μm in diameter and coated with 0.8 μg of DNA. Application of these optimized conditions proved effective for the generation of stable transgenic soybean plants. Stable transgene integration in the transformants was confirmed by Southern blot analysis. The average transformation efficiency achieved with the optimized protocol was siginificantly higher than that with the conventional protocol.

Transformation of soybean [Glycine max (L.) Merrill] using proliferative embryogenic tissue maintained on semi-solid medium

Transgenic soybean can be efficiently produced by particle bombardment of embryogenic suspension culture material. Unfortunately, the time required to obtain a transformation-competent soybean suspension culture line is often lengthy and can result in reduced fertility of regenerated plants. In addition, establishment and maintenance of embryogenic suspension cultures can be very difficult. The objective of this work was to minimize the time required to obtain transformation-competent embryogenic tissue and optimize DNA delivery into that tissue. Somatic embryos were induced from immature cotyledons of soybean [Glycine max (L.) Merrill cv ‘Jack’] by placement of cotyledons, adaxial side up, on a MS-based induction medium containing 40 mg (181 µM) 2,4-dichlorophenoxyacetic acid (2,4-D) per 1 and 6% sucrose. Embryogenic tissues, which formed from the surface of the cotyledons within 2–4 wk, were transferred to an embryo proliferation medium containing 20 mg (90 µM) 2,4-D per 1 and 3% sucrose. After 4 wk, proliferative embryogenic tissue could be used for transformation via particle bombardment. Desiccation of target tissue, period of subculture prior to bombardment, and the number of bombardments per target tissue were evaluated for enhancement of transient β-glucuronidase (GUS) expression. The highest number of blue foci was observed when the target tissue was desiccated for 10 min in an uncovered Petri plate containing proliferation medium, subcultured on the same day of bombardment, and bombarded three times on a single day. For stable transformation, selection was started 20 d after bombardment using 9 mg hygromycin per 1 for 4 wk, and 18 mg per 1 thereafter. Stably transformed clones were obtained from tissue bombarded once and twice on a single day. GUS assays and Southern hybridization analysis of DNA from putative clones confirmed stable integration of the introduced genes. Fertile transgenic plants were obtained in 11–12 mo following culture initiation.

Optimization of parameters for particle bombardment of embryogenic suspension cultures of cassava (Manihot esculenta Crantz) using computer image analysis.

Tissue derived from embryogenic suspension cultures of cassava was bombarded with microparticles coated with a plasmid containing theuidA gene, which codes forβ-glucuronidase (GUS). After 3 days, the effect of different bombardment parameters was evaluated by comparing the numbers of blue spots that resulted from histological GUS assays. Counting of blue spots was performed using a system comprised of a black and white video camera, a stereoscope and a personal computer. A reproducible counting method was established by optimizing GUS assay conditions, preparation of tissue samples and acquisition of video images in view of attaining the highest possible contrast between the blue spots and the surrounding tissue. The effects of bombardment pressure, microparticle size, number of bombardments, and osmotic pretreatment on GUS expression were investigated. Optimal transient expression of theuidA gene was observed after bombardment at 1100 psi, with a particle size of 1 µm, an osmotic pretreatment and two bombardments per sample. The highest number of blue spots observed was 2400 per square centimeter of bombarded tissue.

Physical parameters affecting transient GUS gene expression in oil palm ( Elaeis guineensis Jacq.) using the biolistic device

Physical parameters for DNA delivery into oil palm embryogenic calli using the biolistic device have been successfully established. Helium pressures, distance from rupture disc to macrocarrier, distance from macrocarrier to stopping plate, distance from stopping plate to target tissue, vacuum pressures, number of bombardments, particle types and sizes and the effect of calcium chloride and spermidine on microcarrier-DNA binding have been optimizied. Embryogénic calli were bombarded with gold or tungsten particles coated with pEmuGN plasmid containing β-glucuronidase gene ( uidA) driven by an Emu promoter. A completely randomized design was used to bombard a total of 200 plates (40 parameters × 5 replications) which were then incubated in a growth chamber at 28 °C. Two days after bombardment, the tissues were stained with GUS assay buffer for 16–20 h at 37 °C and the blue spots were counted under a binocular microscope. Analysis of variance (ANOVA) and Duncan's new multiple range test were used to analyze data from each experiment. All the variables used in this experiment were significantly different except for vacuum pressures and bombardment numbers.

Optimization of Gene Delivery Conditions in Roots of Arabidopsis thaliana by Bombardment-mediated Transformation.

Expression of the β-glucuronidase (GUS) gene in roots of Arabidopsis thaliana collection number C24 was obtained by particle bombardment. Effects of accelerating pressure of projectiles(115 to 200kg/cm2), amount of gold particles (0.2 to 0.6mg per projectile), type of gold particles and number of bombardments (1 to 3 times) on the efficiency of gene expression were studied. Three bombardments of 200kg/cm2 with projectiles having 0.4mg of gold particles (“Tokuriki-3”particles) gave 4856±204 blue spots per ca. 150mg root sections.

Transient Expression of the .BETA.-Glucuronidase Gene in Shoot Primordia of Haplopappus gracilis by Use of a Pneumatic Particle Gun.

Expression of the β-glucuronidase (GUS) gene in shoot primordia of Haplopappus gracilis was obtained by particle bombardment. The efficiency of gene delivery was tested by accelerating the pressure from 115 to 200kg/cm2, the number of bombardments from one to three, the amount of gold particles from 0.1 to 0.4mg per projectile, and the amount of plasmid DNA from 0.2 to 0.8μg/mg gold particles. Double bombardments of 200kg/cm2 with projectiles having 0.2mg of gold particles coated with 8μg DNA/mg of gold gave 884±103 blue spots per petri dish (5.2cm diameter).

GENETIC TRANSFORMATION OF ROSE (ROSA SPP) USING THE BIOLISTIC PROCESS.

Genetic transformation of cut roses may greatly facilitate cultivar improvement programs by shortening the time required to introduce new genes into elite germplasm. The biolistic process offers a very promising method for the genetic transformation of roses. The biolistic process uses high velocity mircoprojectiles (gold or tungsten) to carry foreign DNA into cells. This process has been shown to be useful for genetic transformation of many organisms. The first step in taking advantage of this process is to optimize the factors which affect transformation efficiency. Several factors that have a significant affect on transformation efficiency were examined in an effort to optimize the biolistic process for gene transfer in roses. The factors examined were type of tissue (leaf segments, petioles, callus, etc), bombardment distance, the number of bombardments, DNA construct and microcarrier velocity. The reporter gene, GUS, was used for determining transformation efficiency in this study. GUS was carried on several plasmid constructs which also contained antibiotic resistance (kanamycin or streptomycin. Efficiency of gene transfer was determined by calculating the number of transiently expressing GUS cells for each combination of factors. Results of this study will be discussed and summarized.