Biolistic-mediated Transformation Research Articles

CRISPR/Cas9-mediated editing of the phytoene desaturase gene in Vanilla planifolia enabling targeted domestication

ABSTRACT Vanilla is an undomesticated species and the botanical source of vanilla extract. There has been increasing demand to expand vanilla cultivation in the Americas to strengthen the vanilla supply chain. Recent development in vanilla genomics resources has opened opportunities for interrogation of vanilla diversity and discovery of candidate genes responsible for key traits. The ability to precisely change genomic sequences could be a valuable tool for domesticating vanilla while enabling functional genomics. Here we report the successful biolistic-mediated transformation of seed-derived callus and CRISPR/Cas9 gene editing of the phytoene desaturase (PDS) gene in vanilla. Sequencing of leaves from regenerated plantlets displaying the expected albino phenotype showed short deletions from 1 to 163 base pairs in the phytoene desaturase gene at each of three target loci. The ability to apply gene editing to vanilla can now be used in combination with the vanilla genome to create targeted knockouts to support the domestication of vanilla.

Overexpression of Choline Oxidase Gene in Three Filial Generations of Rice Transgenic Lines.

Glycinebetaine (GB) accumulation in many halophytic plants, animals, and microorganisms confers abiotic stress tolerance to salinity, drought, and extreme temperatures. Although there are a few genetic and biochemical pathways to synthesize GB, but isolation of a single gene Choline Oxidase (codA) from Arthrobacter spp. have opened a new hatch to engineer the susceptible plants. The effects of overexpressed codA gene, through multiple copy insertion and GB accumulation on salinity tolerance in rice were studied. Seed-derived embryogenic calli of 'Tarom Molaie' cultivar were targeted with two plasmids pChlCOD and pCytCOD both harboring the codA gene using the biolistic mediated transformation. The regenerated T0 plants were screened by PCR analysis. A line containing three copies of codA gene and harboring pChlCOD and pCytCOD was identified by Southern blot analysis. The expression of codA gene in this transgenic line was then confirmed by RT-PCR. The Mendelian segregation pattern of the inserted sequences was accomplished by the progeny test using PCR. The effects of overexpression of codA on salinity tolerance were evaluated at germination and seedling stage using T2-pChl transgenic line and control seeds in the presence of 0, 100, 200, and 300 mM NaCl. Finally, leaf growth dynamics of T2-pChlCOD transgenic line and control line under hydroponic conditions in the presence of 0, 40, 80, and 120 mM NaCl were assessed. The seed germination experiment results showed that the transformed seeds had a higher germination rate than the controls under all salinity treatments. But also, the leaf growth dynamics showed that the control plants had a more favorable leaf growth dynamic in all of the treatments. Although, the transgenic lines (T0, T1 and T2) exhibited lower performance than the wild type, the transgenic line varied for GB and choline contents and increasing codA gene copy number led to increased GB content. In a salinity sensitive crop such as rice, GB may not significantly contribute to the plant protection against salt stress. Also, insufficiency of choline resources as GB precursor might have affected the overall growth ability of the transgenic line and resulted in decreased leaf growth dynamics.

A competence of embryo-derived tissues of tetraploid cultivated wheat species Triticum dicoccum and Triticum timopheevii for efficient and stable transgenesis mediated by particle inflow gun

BackgroundThe ability to engineer cereal crops by gene transfer technology is a powerful and informative tool for discovering and studying functions of genes controlling environmental adaptability and nutritional value. Tetraploid wheat species such as emmer wheat and Timopheevi wheat are the oldest cereal crops cultivated in various world areas long before the Christian era. Nowadays, these hulled wheat species are gaining new interest as donors for gene pools responsible for the improved grain yield and quality, tolerance for abiotic and biotic stress, resistance to pests and disease. The establishing of efficient gene transfer techniques for emmer and Timopheevi wheat may help in creation of modern polyploid wheat varieties.ResultsIn the present study, we describe a robust protocol for the production of fertile transgenic plants of cultivated emmer wheat (Russian cv. ‘Runo’) using a biolistic delivery of a plasmid encoding the gene of green fluorescent protein (GFP) and an herbicide resistance gene (BAR). Both the origin of target tissues (mature or immature embryos) and the type of morphogenic calli (white or translucent) influenced the efficiency of stable transgenic plant production in emmer wheat. The bombardment of nodular white compact calluses is a major factor allowed to achieve the highest transformation efficiency of emmer wheat (on average, 12.9%) confirmed by fluorescence, PCR, and Southern blot. In the absence of donor plants for isolation of immature embryos, mature embryo-derived calluses could be used as alternative tissues for recovering transgenic emmer plants with a frequency of 2.1%. The biolistic procedure based on the bombardment of immature embryo-derived calluses was also successful for the generation of transgenic Triticum timopheevii wheat plants (transformation efficiency of 0.5%). Most of the primary events transmitted the transgene expression to the sexual progeny.ConclusionThe procedures described here can be further used to study the functional biology and contribute to the agronomic improvement of wheat. We also recommend involving in such research the Russian emmer wheat cv. ‘Runo’, which demonstrates a high capacity for biolistic-mediated transformation, exceeding the previously reported values for different genotypes of polyploid wheat.

Heterologous Expression of the Constitutive Disease Resistance 2 and 8 Genes from Poncirus trifoliata Restored the Hypersensitive Response and Resistance of Arabidopsis cdr1 Mutant to Bacterial Pathogen Pseudomonas syringae.

Huanglongbing (HLB), also known as citrus greening, is the most destructive disease of citrus worldwide. In the United States, this disease is associated with a phloem-restricted bacterium, Candidatus Liberibacter asiaticus. Commercial citrus cultivars are susceptible to HLB, but Poncirus trifoliata, a close relative of Citrus, is highly tolerant of HLB. Isolating P. trifoliata gene(s) controlling its HLB tolerance followed by expressing the gene(s) in citrus is considered a potential cisgenic approach to engineering citrus for tolerance to HLB. Previous gene expression studies indicated that the constitutive disease resistance (CDR) genes in P. trifoliata (PtCDRs) may play a vital role in its HLB tolerance. This study was designed to use Arabidopsis mutants as a model system to confirm the function of PtCDRs in plant disease resistance. PtCDR2 and PtCDR8 were amplified from P. trifoliata cDNA and transferred into the Arabidopsis cdr1 mutant, whose resident CDR1 gene was disrupted by T-DNA insertion. The PtCDR2 and PtCDR8 transgenic Arabidopsis cdr1 mutant restored its hypersensitive response to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) expressing avrRpt2. The defense marker gene PATHOGENESIS RELATED 1 (PR1) expressed at much higher levels in the PtCDR2 or PtCDR8 transgenic cdr1 mutant than in the non-transgenic cdr1 mutant with or without pathogen infection. Multiplication of Pst DC3000 bacteria in Arabidopsis was inhibited by the expression of PtCDR2 and PtCDR8. Our results showed that PtCDR2 and PtCDR8 were functional in Arabidopsis and played a positive role in disease resistance and demonstrated that Arabidopsis mutants can be a useful alternate system for screening Poncirus genes before making the time-consuming effort to transfer them into citrus, a perennial woody plant that is highly recalcitrant for Agrobacterium or biolistic-mediated transformation.

Induction of competent cells for Agrobacterium tumefaciens-mediated stable transformation of common bean (Phaseolus vulgaris L.).

Stable transformation of common bean (Phaseolus vulgaris L.) has been successful, to date, only using biolistic-mediated transformation and shoot regeneration from meristem-containing embryo axes. In this study, using precultured embryo axes, and optimal co-cultivation conditions resulted in a successful transformation of the common bean cultivar Olathe using Agrobacterium tumefaciens strain EHA105. Plant regeneration through somatic embryogenesis was attained through the preculture of embryo axes for 12 weeks using induced competent cells for A. tumefaciens-mediated gene delivery. Using A. tumefaciens at a low optical density (OD) of 0.1 at a wavelength of 600 nm for infection and 4-day co-cultivation, compared to OD600 of 0.5, increased the survival rate of the inoculated explants from 23% to 45%. Selection using 0.5 mg L-1 glufosinate (GS) was effective to identify transformed cells when the bialaphos resistance (bar) gene under the constitutive 35S promoter was used as a selectable marker. After an 18-week selection period, 1.5% -2.5% inoculated explants, in three experiments with a total of 600 explants, produced GS-resistant plants through somatic embryogenesis. The expression of bar was confirmed in first- and second-generation seedlings of the two lines through reverse polymerase chain reaction. Presence of the bar gene was verified through genome sequencing of two selected transgenic lines. The induction of regenerable, competent cells is key for the successful transformation, and the protocols described may be useful for future transformation of additional Phaseolus germplasm.

Agrobacterium- and Biolistic-Mediated Transformation of Maize B104 Inbred.

Genetic transformation of maize inbred genotypes remains non-routine for many laboratories due to variations in cell competency to induce embryogenic callus, as well as the cell's ability to receive and incorporate transgenes into the genome. This chapter describes two transformation protocols using Agrobacterium- and biolistic-mediated methods for gene delivery. Immature zygotic embryos of maize inbred B104, excised from ears harvested 10-14days post pollination, are used as starting explant material. Disarmed Agrobacterium strains harboring standard binary vectors and the biolistic gun system Bio-Rad PDS-1000/He are used as gene delivery systems. The herbicide resistant bar gene and selection agent bialaphos are used for identifying putative transgenic type I callus events. Using the step-by-step protocols described here, average transformation frequencies (number of bialaphos resistant T0 callus events per 100 explants infected or bombarded) of 4% and 8% can be achieved using the Agrobacterium- and biolistic-mediated methods, respectively. An estimated duration of 16-21weeks is needed using either protocol from the start of transformation experiments to obtaining putative transgenic plantlets with established roots. In addition to laboratory in vitro procedures, detailed greenhouse protocols for producing immature ears as transformation starting material and caring for transgenic plants for seed production are also described.

Biolistic transformation of oil palm using the phosphomannose isomerase (pmi) gene as a positive selectable marker

The selectable marker system based on the Escherichia coli phosphomannose isomerase (pmi) gene was evaluated for the genetic transformation of oil palm. Four transformation vectors were constructed for transforming oil palm. The transformation vectors both carried the pmi gene alone or in combination with the β-glucuronidase (gusA) gene, and were driven by either the maize ubiquitin promoter (pMI11 and pMI11G) or the CaMV35S promoter (pMI3 and pMI3G). The four transformation vectors were transformed into oil palm embryogenic calli via biolistic-mediated transformation. For stable transformation experiments, bombarded oil palm embryogenic calli were selected on a medium supplemented with mannose as the only carbon source (without sucrose) one month after bombardment. Transformed embryogenic calli that survived selection on mannose were later isolated, proliferated and regenerated into whole plantlets on a regeneration medium containing mannose. The status of the regenerated transgenic plantlets was confirmed by polymerase chain reaction (PCR) and Southern hybridization. Transgene expression was detected by reverse transcription (RT)-PCR analysis. The results of this study indicate that a mannose-based selection system can be successfully used in oil palm transformation.

Optimization of Key Bombardment Parameters in Biolistic Mediated Transformation in Wheat

Biolistic mediated method is an important approach in wheat genetic transformation. The objective of this study was to optimize the bombardment parameters and establish an efficient and stable biolistic transformation system using common wheat (Triticum aestivum L.) variety Kenong 199 with 12 treatments of parameter combinations composed of gold particle size, target distance, and acceleration pressure. The results indicated that different combinations of parameters had different effects on the transient expression of GUS gene encoding β-glucuronidase, regeneration of calli, and final transformation efficiency. The parameter combination of 0.6 μm gold, 5.5 cm target distance, and 650 psi acceleration pressure exhibited good regeneration and the highest level GUS transient expression. Furthermore, stable expression of GUS gene in transgenic lines was confirmed by histochemical staining assay. Of the 28 transgenic T0 plants obtained, 12 were from the treatment with the above optimized parameter combination, with an average transformation efficiency of 2.45%.

Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer

Expression of genes in plant chloroplasts provides an opportunity for enhanced production of target proteins. We report the introduction and expression of a fusion DPT protein containing immunoprotective exotoxin epitopes of Corynebacterium diphtheriae, Bordetella pertussis, and Clostridium tetani in tobacco chloroplasts. Using biolistic-mediated transformation, a plant-optimized synthetic DPT gene was successfully transferred to tobacco plastomes. Putative transplastomic T0 plants were identified by PCR, and Southern blot analysis confirmed homoplasmy in T1 progeny. ELISA assays demonstrated that the DPT protein retained antigenicity of the three components of the fusion protein. The highest level of expression in these transplastomic plants reached 0.8% of total soluble protein. To assess whether the functional recombinant protein expressed in tobacco plants would induce specific antibodies in test animals, a mice feeding experiment was conducted. For mice orally immunized with freeze-dried transplastomic leaves, production of IgG and IgA antibodies specific to each toxin were detected in serum and mucosal tissues.

Expression of Cholera Toxin B Subunit in Transgenic Rice Endosperm

The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1% of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced only in the rice endosperm demonstrated strong affinity for G(M1)-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine.

Arabitol dehydrogenase as a selectable marker for rice

Arabitol dehydrogenase has been adapted for use as a plant selectable marker. Arabitol is a five-carbon sugar alcohol that can be used by E. coli strain C, but not by the laboratory K12 strains. The enzyme converts the non-plant-metabolizable sugar arabitol into xylulose, which is metabolized by plant cells. Rice was transformed with a plant-expression-optimized synthetic gene using Biolistic-mediated transformation. Selection on 2.75% arabitol and 0.25% sucrose yielded a transformation efficiency (9.3%) equal to that obtained with hygromycin (9.2%). Molecular analyses showed that the atlD gene was integrated into the rice genome of selected plants and was inherited in a Mendelian manner. This study indicates that arabitol could serve as an effective means of plant selection.

The development of a nuclear male sterility system in wheat. Expression of the barnase gene under the control of tapetum specific promoters

Nuclear male sterility within Triticum aestivum is considered as the ideal basis for the development of a hybridization system for wheat. We engineered nuclear male sterility in wheat by introducing the barnase gene under the control of tapetum-specific promoters derived from corn and rice. A biolistic-mediated transformation method, based on the use of the poly(ADP-ribose)polymerase inhibitor niacinamide, was set up which enriched for low-copy integrations (1–3 copies). Most of these copies were not linked and segregated in the next generation.

Expression of a Late Embryogenesis Abundant Protein Gene, HVA1, from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice.

A late embryogenesis abundant (LEA) protein gene, HVA1, from barley (Hordeum vulgare L.) was introduced into rice suspension cells using the Biolistic-mediated transformation method, and a large number of independent transgenic rice (Oryza sativa L.) plants were generated. Expression of the barley HVA1 gene regulated by the rice actin 1 gene promoter led to high-level, constitutive accumulation of the HVA1 protein in both leaves and roots of transgenic rice plants. Second-generation transgenic rice plants showed significantly increased tolerance to water deficit and salinity. Transgenic rice plants maintained higher growth rates than nontransformed control plants under stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by stress and by improved recovery upon the removal of stress conditions. We also found that the extent of increased stress tolerance correlated with the level of the HVA1 protein accumulated in the transgenic rice plants. Using a transgenic approach, this study provides direct evidence supporting the hypothesis that LEA proteins play an important role in the protection of plants under water-or salt-stress conditions. Thus, LEA genes hold considerable potential for use as molecular tools for genetic crop improvement toward stress tolerance.