Microprojectile Bombardment Research Articles

Functional complementation of a periila ω3 fatty acid desaturase under the seed-specific SeFAD2 promoter

Functional characterization of the fatty acid desaturase genes and seed-specific promoters is prerequisite for altering the unsaturated fatty acid content of oilseeds by genetic manipulation. The ω-6 fatty acid desaturase (FAD2) and ω-3 fatty acid desaturase (FAD3) catalyze extra-plastidial desaturation of oleic acid to linoleic acid and linoleic acid to linolenic acid, respectively. These are major constituents in seed storage oils. Here, we report the complementation of a perilla linoleic acid desaturase (PrFAD3) cDNA under the seed-specific sesame FAD2 (SeFAD2) promoter in the Arabidopsis fad3 mutant. PrFAD3 is functionally active and the SeFAD2 promoter is applicable for modifying fatty acid composition in developing seeds. Transient expression of the GUS gene under that promoter in the developing seeds and leaves of sesame, soybean, and corn via microprojectile bombardment indicated that the SeFAD2 promoter likely will be useful for altering the seed phenotypes of dicot and monocot crops.

Large-scale gene expression pattern analysis, in situ, in Caenorhabditis elegans

Observation of gene expression in situ provides a direct connection between the genetic information in the genome sequence and the fully determined developmental cell lineage of Caenorhabditis elegans. Green Fluorescent Protein (GFP) reporters have been fused with many C. elegans genes, in large-scale projects, by conventional DNA ligation, PCR stitching, Gateway recombination and recombineering. These reporter gene fusions have then been used in C. elegans transformation either by microinjection or microprojectile bombardment. So far, the developmental distributions of GFP, as driven by the C. elegans DNA to which the reporter gene has been attached, have been determined simply from direct examination of the transgenic strains by epifluorescence microscopy. Automation of GFP expression pattern determination promises improvements in both quality and quantity of this data type, facilitating the handling of such expression pattern data within computer databases. As with the descriptions of the developmental cell lineage and the genome sequence, a complete description of gene expression patterns will provide a vital knowledge framework through which a full understanding of the development of this animal can emerge.

A comparison of strategies for transformation with multiple genes via microprojectile-mediated bombardment

The stable insertion and expression of multiple transgenes in crops is highly desirable, as the manipulation of complex agronomic traits and the introduction of novel biosynthetic pathways are dependent upon it. This study was performed to explore the frequency and efficiency of introducing multiple genes in soybean by using somatic embryogenesis and microprojectile bombardment transformation. The co-transformation frequency of six selectable marker or reporter genes (GusA, bleomycin resistance, glufosinate resistance, hygromycin resistance, green fluorescent protein, and kanamycin resistance) were followed throughout the T0, T1, and T2 generations. Three bombardment strategies were compared to determine the best method to generate transgenic plants that express the introduced transgenes and have a simple insertion pattern that would facilitate any downstream breeding. The plasmid bombardment treatments were (1) a six-gene-containing plasmid, (2) an equimolar treatment of five individual plasmids that collectively contained the six transgenes of interest (genes of glufosinate and hygromycin resistance were on the same plasmid), and (3) a 1:9 ratio mixture of the five plasmids, in which the plasmid containing the selectable marker used in the regeneration process, hygromycin resistance, was used in ninefold excess to all the other plasmids. Of the six bombardments performed per plasmid treatment, the results of seven independent events for the six-gene plasmid, four events for the 1:9 treatment, and a single regenerated event for the equimolar treatment indicate that containing all the transgenes on one plasmid just had an advantage in terms of frequency of a successful transformation events. Based on Southern analysis, the only events that contained all six transgenes was the one obtained by the equimolar treatment. No event was obtained that expressed all six transgenes, and certain transgenes seem to be non-randomly lost, namely gusA, bleomycin resistance, and glufosinate resistance, regardless of treatment. The addition of elements to optimize the expression of each gene cassette when multiple genes are in close proximity needs to be further investigated.

The N-terminal presequence from F1-ATPase β-subunit of Nicotiana plumbaginifolia efficiently targets green fluorescent fusion protein to the mitochondria in diverse commercial crops

Approximately 10-15% of plant nuclear genes appear to encode mitochondrial proteins that are directed to mitochondria by specific targeting signals. Reports on the heterologous function of these targeting signals are generally limited to one or a few species, with an emphasis on model plants such as tobacco and Arabidopsis. Given their sequence diversity and their insufficient testing in commercially important crops (including monocotyledonous crops), the extent to which these signals can be relied on for biotechnological purposes across species remains to be established. This study provides the experimental verification of a mitochondrial signal that is functional across diverse crop species, including five monocots (sugarcane, wheat, corn, sorghum and onion) and seven dicots (cucumber, cauliflower, tomato, capsicum, pumpkin, coriander and sunflower). In all 12 crops, transient assays following microprojectile bombardment showed that the N-terminal mitochondrial presequence from F1-ATPase β-subunit (ATPase-β) of Nicotiana plumbaginifolia Viv. targeted green fluorescent fusion protein to the mitochondria. The transient assay results in sugarcane were confirmed in stably transformed root cells. The ATPase-β signal should be a useful metabolic engineering tool for directing recombinant proteins to the mitochondrial matrix in diverse plant species of commercial interest.

PLANT TISSUE CULTURE: PAST, PRESENT AND PROSPECTS FOR THE FUTURE

Modern biotechnology owes much to its roots derived from plant tissue culture and micropropagation. Indeed, the landmark publication by Gottlieb Haberlandt (1902), who is arguably referred to as the Father of Tissue Culture, is often cited as the origin and emergence of plant tissue culture and its subsequent applications. Success of biotechnological approaches is dependent on regeneration of intact plants following genetic modification, generally by micropropagation. Contributions of Knudson, Thimann and Went, White, Gautheret, Skoog and others are discussed in a historical perspective. Examples of currently employed uses of plant tissue culture, especially practical applications of micropropagation are presented, which enables science to transition into an evaluation of what lies ahead. Potential uses of plant tissue culture and biotechnology to further our understanding of plant physiology, how plants function and resolution of legal issues are presented. Agrobacterium-mediated genetic transformation along with other modes of transformation such as micro-projectile bombardment have been accomplished. Incorporation of disease and stress resistance and other horticulturally important traits is a logical outcome to be anticipated in the future. Use of molecular technologies for identification of genotypes, clones and their ancestors will enable researchers and producers to verify identity and parentage of propagules, whether produced by conventional or modern propagation methods. No doubt yet to be imagined applications of plant biotechnology will emerge as the XXI st Century continues to unfold.

High efficiency of stable genetic transformation in Dendrobium via 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.

A simple and effective method for protein subcellular localization using Agrobacterium-mediated transformation of onion epidermal cells

A modified Agrobacterium-mediated transformation protocol has been successfully used for transient expression of the intrinsically fluorescent proteins and their fusion proteins in onion epidermis. The mean of the transformed cells rate per peel is about 10.5±0.9%, while that of the particle bombardment method is at the range 2.0±0.4%. To compare with the prevailing method of micro-projectile bombardment, the modified Agrobacterium-mediated transformation may provide with higher efficiency and even more simplified manipulability on a lower budget.

Bcl-xL transformed peanut (Arachis hypogaea L.) exhibits paraquat tolerance

The human Bcl-xL gene was transformed into peanut cultivar Georgia Green via microprojectile bombardment. Following selection on hygromycin-containing medium and regeneration, eighty hygromycin-resistant callus clusters were recovered. Southern blot analysis of ten fertile lines revealed multiple insertions of the Bcl-xL transgene in most lines. Western blot analysis of primary plants and T1 progenies demonstrated detectable levels of Bcl-xL expression in four transgenic lines. We could not detect Bcl-xL protein in other tested lines even though transcripts were identified by RT-PCR and northern blot. Three of the western-positive transgenic lines either were sterile or the progenies lost the expressive copy of Bcl-xL. Only T1 progenies from line BX25-4-2a-19 continued to express an intermediate level of Bcl-xL. This line demonstrated paraquat tolerance at the 5 microM level. Tolerance to salt of T1 and T2 seeds from seven other transgenic lines also was tested, but no tolerance was found in these lines. A high level of Bcl-xL transgene expression may be deleterious to plant growth and development even though the gene may confer tolerance to other abiotic and biotic stresses such as drought and pathogens.

Capsid Protein-Mediated Recruitment of Host DnaJ-Like Proteins Is Required for Potato Virus Y Infection in Tobacco Plants

The capsid protein (CP) of potyviruses is required for various steps during plant infection, such as virion assembly, cell-to-cell movement, and long-distance transport. This suggests a series of compatible interactions with putative host factors which, however, are largely unknown. By using the yeast two-hybrid system the CP from Potato virus Y (PVY) was found to interact with a novel subset of DnaJ-like proteins from tobacco, designated NtCPIPs. Mutational analysis identified the CP core region, previously shown to be essential for virion formation and plasmodesmal trafficking, as the interacting domain. The ability of NtCPIP1 and NtCPIP2a to associate with PVY CP could be confirmed in vitro and was additionally verified in planta by bimolecular fluorescence complementation. The biological significance of the interaction was assayed by PVY infection of agroinfiltrated leaves and transgenic tobacco plants that expressed either full-length or J-domain-deficient variants of NtCPIPs. Transient expression of truncated dominant-interfering NtCPIP2a but not of the functional protein resulted in strongly reduced accumulation of PVY in the inoculated leaf. Consistently, stable overexpression of J-domain-deficient variants of NtCPIP1 and NtCPIP2a dramatically increased the virus resistance of various transgenic lines, indicating a critical role of functional NtCPIPs during PVY infection. The negative effect of impaired NtCPIP function on viral pathogenicity seemed to be the consequence of delayed cell-to-cell movement, as visualized by microprojectile bombardment with green fluorescent protein-tagged PVY. Therefore, we propose that NtCPIPs act as important susceptibility factors during PVY infection, possibly by recruiting heat shock protein 70 chaperones for viral assembly and/or cellular spread.

Geminivirus-induced gene silencing of the tobacco retinoblastoma-related gene results in cell death and altered development

The retinoblastoma-related protein (RBR) is required for cell cycle control and differentiation and is expressed throughout the life of plants and animals. In this study, the tomato golden mosaic virus (TGMV) geminivirus vector was used to silence NbRBR1 in Nicotiana benthamiana by microprojectile bombardment into fully developed leaves. Similar to previous results using agroinoculation of a tobacco rattle virus silencing vector [Park et al. (Plant J 42:153, 2005)], developmental defects caused by disruptions in cell size and number were seen in new growth. Leaf midvein cross-sections showed tissue-specific differences in size, cell number, and cell morphology. While cortical cell numbers decreased, size increased to maintain overall shape. In contrast, xylem parenchyma cells increased approximately three fold but remained small. Normally straight flowers often curved up to 360 degrees without a significant change in size. However, the most striking phenotype was cell death in mature cells after a delay of 3-4 weeks. Trypan blue staining confirmed cell death and demonstrated that cell death was absent in similarly treated leaves of wild type TGMV-inoculated plants. Quantitative RT-PCR confirmed that the mature TGMV:RBR-inoculated leaves still maintained reduced accumulation of RBR transcript at 4 weeks compared to controls. The results suggest that either inappropriate activation of the cell cycle is lethal in plants or that RBR has other functions, unrelated to the cell cycle. The results also demonstrate that continual transcription of RBR is necessary for cell survival.

Soybean dwarf virus-resistant transgenic soybeans with the sense coat protein gene

We transformed a construct containing the sense coat protein (CP) gene of Soybean dwarf virus (SbDV) into soybean somatic embryos via microprojectile bombardment to acquire SbDV-resistant soybean plants. Six independent T(0) plants were obtained. One of these transgenic lines was subjected to further extensive analysis. Three different insertion patterns of Southern blot hybridization analysis in T(1) plants suggested that these insertions introduced in T(0) plants were segregated from each other or co-inherited in T(1) progenies. These insertions were classified into two types, which overexpressed SbDV-CP mRNA and accumulated SbDV-CP-specific short interfering RNA (siRNA), or repressed accumulation of SbDV-CP mRNA and siRNA by RNA analysis prior to SbDV inoculation. After inoculation of SbDV by the aphids, most T(2) plants of this transgenic line remained symptomless, contained little SbDV-specific RNA by RNA dot-blot hybridization analysis and exhibited SbDV-CP-specific siRNA. We discuss here the possible mechanisms of the achieved resistance, including the RNA silencing.

ACTIN BINDING PROTEIN29 fromLiliumPollen Plays an Important Role in Dynamic Actin Remodeling

Villin/gelsolin/fragmin superfamily proteins have been shown to function in tip-growing plant cells. However, genes encoding gelsolin/fragmin do not exist in the Arabidopsis thaliana and rice (Oryza sativa) databases, and it is possible that these proteins are encoded by villin mRNA splicing variants. We cloned a 1006-bp full-length cDNA from Lilium longiflorum that encodes a 263-amino acid predicted protein sharing 100% identity with the N terminus of 135-ABP (Lilium villin) except for six C-terminal amino acids. The deduced 29-kD protein, Lilium ACTIN BINDING PROTEIN29 (ABP29), contains only the G1 and G2 domains and is the smallest identified member of the villin/gelsolin/fragmin superfamily. The purified recombinant ABP29 accelerates actin nucleation, blocks barbed ends, and severs actin filaments in a Ca(2+)- and/or phosphatidylinositol 4,5-bisphosphate-regulated manner in vitro. Microinjection of the protein into stamen hair cells disrupted transvacuolar strands whose backbone is mainly actin filament bundles. Transient expression of ABP29 by microprojectile bombardment of lily pollen resulted in actin filament fragmentation and inhibited pollen germination and tube growth. Our results suggest that ABP29 is a splicing variant of Lilium villin and a member of the villin/gelsolin/fragmin superfamily, which plays important roles in rearrangement of the actin cytoskeleton during pollen germination and tube growth.

Resistance of transgenic papaya plants to Papaya ringspot virus

The coat protein gene isolated from Papaya ringspot virus, Thai isolate, was used to generate transgenic papayas. A binary vector containing the coat protein gene under the control of a 35S promoter, was constructed and transformed into somatic embryos of papaya cultivar Khak Dum by microprojectile bombardment. Eight transgenic lines were identified from 1980 bombarded calli of papaya somatic embryos under kanamycin selection. Integration of the transferred genes into kanamycin resistant papaya calli was verified by PCR amplification of the coat protein gene, GUS assays and Southern blot hybridization. Although the coat protein gene was detected in all transgenic lines, only line G2 was found to be highly resistant to virus. This resistant line showed high degree of rearrangement of the inserted coat protein expression cassette while the coat protein gene itself had a deletion of 166 bp on the 3' end of its sequence. Although the transcription of the coat protein gene was detected in all transgenic lines by RT-PCR, only two transgenic papayas expressed the intact coat protein. Moreover, in the resistant line G2 the amount of the truncated coat protein mRNA was significantly decreased. These results point to an RNA mediated mechanism of coat protein mediated resistance in papaya, probably based on post-transcriptional gene silencing.

Downregulation of theNbNACa1Gene Encoding a Movement-Protein-Interacting Protein Reduces Cell-to-Cell Movement ofBrome mosaic virusinNicotiana benthamiana

The 3a movement protein (MP) plays a central role in the movement of the RNA plant virus, Brome mosaic virus (BMV). To identify host factor genes involved in viral movement, a cDNA library of Nicotiana benthamiana, a systemic host for BMV, was screened with far-Western blotting using a recombinant BMV MP as probe. One positive clone encoded a protein with sequence similarity to the alpha chain of nascent-polypeptide-associated complex from various organisms, which is proposed to contribute to the fidelity of translocation of newly synthesized proteins. The orthologous gene from N. benthamiana was designated NbNACa1. The binding of NbNACa1 to BMV MP was confirmed in vivo with an agroinfiltration-immunoprecipitation assay. To investigate the involvement of NbNACa1 in BMV multiplication, NbNACa1-silenced (GSNAC) transgenic N. benthamiana plants were produced. Downregulation of NbNACa1 expression reduced virus accumulation in inoculated leaves but not in protoplasts. A microprojectile bombardment assay to monitor BMV-MP-assisted viral movement demonstrated reduced virus spread in GSNAC plants. The localization to the cell wall of BMV MP fused to green fluorescent protein was delayed in GSNAC plants. From these results, we propose that NbNACa1 is involved in BMV cell-to-cell movement through the regulation of BMV MP localization to the plasmodesmata.

Heterologous signals allow efficient targeting of a nuclear‐encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots (Arabidopsis, broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.

Single chain antibody fragments for ocular use produced at high levels in a commercial wheat variety

We are investigating the use of single chain antibody fragments (scFv) in eye drops for diagnosis and treatment of eye diseases. For ocular use, recombinant proteins must be free of bacterial endotoxin that causes inflammation in the eye. We required a means of generating high yields of scFvs with little endotoxin contamination. Using microprojectile bombardment we produced transgenic lines of the commercial wheat variety, Westonia, that express two scFvs that bind to CD4 or CD28 on the surface of rat thymocytes. A high level of expression of active scFv in the range 50-180 microg/g was measured by quantitative flow cytometry in crude extracts made from mature seeds. The levels of expression were stable over four generations of transgenic plants and mature seeds were stored for one year with little loss of scFv activity. Substantial purification of scFv was achieved by immobilised metal affinity chromatography. Compared to bacterial extracts, crude transgenic seed extracts contained only a small amount of endotoxin (150 EU/ml) that will be easily removed by purification. The transgenic wheat lines express functional scFv at levels comparable to production in bacteria and promise to be superior to bacteria for production of scFv pharmaceuticals for ocular use.

Expression of a Bacillus thuringiensis cry1Ab gene in transgenic white spruce and its efficacy against the spruce budworm (Choristoneura fumiferana)

A synthetic version of the cry1Ab gene from Bacillus thuringiensis (Bt) was introduced into white spruce (Picea glauca) by microprojectile bombardment. A plasmid carrying the cry1Ab gene, driven by a ubiquitin (maize) promoter, was co-transferred with a plasmid containing the gus–nptII fusion gene as a screenable selection marker. Molecular analysis of the transgenic lines showed a high level (more than 90%) of co-integration of the cry1Ab gene with the screenable marker. A wide range of expression levels of the cry1Ab gene and corresponding endotoxin was obtained. Accumulation of the Cry1Ab protein was evaluated in embryogenic tissue, the needles of somatic seedlings and in the needles of 5-year-old field-grown trees of individual lines. Laboratory and field insect feeding trials suggest that several spruce transgenic lines were lethal to spruce budworm larvae.

Soybean Mosaic Virus Resistance in Transgenic Soybean Caused by Post-transcriptional Gene Silencing

Somatic embryos of Glycine max (L.) Merrill cultivar ‘Jack’ were co-transformed with the coat protein (CP) gene of attenuated isolates of Soybean mosaic virus (SMV) and hygromycin phosphotransferase gene by microprojectile bombardment. CP gene was detected in eleven transgenic plants, and three independent lines highly resistant to SMV were obtained in a previous study. One of these lines, line No. 55, which was assumed to have acquired RNA-mediated resistance, was selected for further gene expression analysis of T4 and T5 plants in relation to their viral resistance. The resistant plants contained a lower level of transgene-derived RNA than the susceptible ones. On the other hand, based on RNA analysis after mechanical inoculation, SMV-specific RNAs were detected faintly in the resistant plants, while large amounts of RNAs were found in the susceptible ones. During the development of the resistant lines, SMV CP sequence-specific small interfering RNAs (siRNAs) appeared initially in the leaves of the first leaf stage but not in those of the primary stage, and were detected thereafter in the leaves of later stages constantly. The presence of the siRNAs before SMV inoculation was strongly correlated with the resistant phenotype of the lines tested.

Infection, transfection, and co-transfection of baculoviruses by microprojectile bombardment of larvae

The use of baculoviruses as expression vectors for heterologous proteins has been practically limited to the use of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). In this work, infection, transfection and co-transfection events with the baculoviruses AcMNPV and Trichoplusia ni granulovirus (TnGV) were accomplished by bombardment of T. ni first-instar larvae with microprojectiles coated with virions, viral DNA, and viral DNA and a transfer vector, respectively. A series of shooting conditions were tested until positive results were obtained. The use of 1.6 μm gold particles at 900 psi shooting pressure, 400 Torr vacuum, 7 cm distance to target, on sets of 20 first-instar larvae held in a 16 mm diameter container, proved to be the best shooting conditions. Typical infection symptoms were shown by larvae when shot with viruses or viral DNA from AcMNPV or TnGV. Co-transfected recombinant AcMNPV and TnGV were identified by the formation of occlusion bodies and GFP, respectively, in bombarded larvae. This technique opens a wide range of possibilities, not only to use an extensive number of baculoviruses as expression vectors for heterologous proteins, but also be used to infect, transfect or co-transfect a wide variety of viruses into animal cells.

Ornamental Plant Transformation

Ornamental plant transformation has advanced considerably in the last decade. Now over 40 genera have been reported to be transformed. The primary methods of creating transgenic ornamental species have been Agrobacterium tumefaciens-medmtedtransformation and microprojectile bombardment. The vast majority of reports indicate the use of Agrobacteriummedmtedtransformation employing binary vectors and virhelper plasmids or supervirulence genes. Many reports are of transformation with the uidA reporter gene driven by the 35S cauliflower mosaic virus promoter, but recent efforts are now focusing on trait genes including disease resistance, flower color, flower longevity, floral scent and plant habit. Greater use of tissue specific and inducible promoters promises to enhance the functionality and usefulness of introduced trait genes. While technical challenges for production of transgenic ornamental plants still exist, the greatest challenges to realizing the potential benefits of transgenic ornamental plants are questionable public acceptance of transgenic plants and the prohibitive costs of generating environmental impact data needed to gain regulatory clearance.