Efficient particle bombardment-mediated transformation of Cuban soybean (INCASoy-36) using glyphosate as a selective agent

Soybean is highly affected by weeds in tropical countries, causing significant losses in yields. Transgenic herbicide resistant soybeans have been produced in a limited number of varieties and parental lines. This study was conducted to obtain glyphosate herbicide resistant transgenic soybean plants through particle bombardment of embryonic axes in a Cuban variety. Shoot regeneration in 25 mg/L of glyphosate occurred within a short period and plantlets developed roots in a medium without selection pressure, which favored the in vitro growth of plants at a transformation frequency of 3.1–6.0?%. Expression and integration of the cp4epsps gene was confirmed in the progeny by an immune-detection assay, PCR and Southern blot. All greenhouse evaluated transgenic soybean lines (T₁) displayed tolerance to 1.25 Kg/ha of glyphosate. Growth and seed development of transformed plants was similar to untransformed plants. The regeneration procedure using embryonic axes combined with the efficient selection of shoots in glyphosate enabled the production of transgenic plants of this Cuban genotype, showing high tolerance to the herbicide, good efficiency and reproducibility.     

Multiple shoot induction and plant regeneration from embryonic axes of cotton

Cytokinins are involved in shoot development of plants. Events of multiple bud formation and shoot development in apical embryonic axes of cotton treated for 2 or 20 days with the cytokinin benzyladenine (BA), were compared with the development of untreated control axes. Meristematic regions (supernumerary vegetative buds) were observed in axes treated for 20 days with BA. An average of 3.4 shoots per embryonary axis was obtained when explants were cultured on medium supplemented with 3 mg l-1 BA. Higher and lower concentrations of the growth regulator yielded fewer shoots per explant. Results shown in this report suggest that BA is directly responsible for re-programming the embryonic apical meristem axes of cotton toward the production of multiple buds and subsequent shoot development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biolistic-mediated genetic transformation of cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis>) and stable Mendelian inheritance of transgenes

We describe a novel system of exploiting the biolistic process to generate stable transgenic cowpea (Vigna unguiculata) plants. The system is based on combining the use of the herbicide imazapyr to select transformed meristematic cells after physical introduction of the mutated ahas gene (coding for a mutated acetohydroxyacid synthase, under control of the ahas 5' regulatory sequence) and a simple tissue culture protocol. The gus gene (under control of the act2 promoter) was used as a reporter gene. The transformation frequency (defined as the total number of putative transgenic plants divided by the total number of embryonic axes bombarded) was 0.90%. Southern analyses showed the presence of both ahas and gus expression cassettes in all primary transgenic plants, and demonstrated one to three integrated copies of the transgenes into the genome. The progenies (first and second generations) of all self-fertilized transgenic lines revealed the presence of the transgenes (gus and ahas) co-segregated in a Mendelian fashion. Western blot analysis revealed that the GUS protein expressed in the transgenic plants had the same mass and isoelectric point as the bacterial native protein. This is the first report of biolistic-mediated cowpea transformation in which fertile transgenic plants transferred the foreign genes to next generations following Mendelian laws.     

Direct DNA transfer using electric discharge particle acceleration (ACCELL™ technology)

Direct DNA transfer methods based on particle bombardment have revolutionized plant genetic engineering. Major agronomic crops previously considered recalcitrant to gene transfer have been engineered using variations of this technology. In many cases variety-independent and efficient transformation methods have been developed enabling application of molecular biology techniques to crop improvement. The focus of this article is the development and performance of electric discharge particle bombardment (ACCELL™) technology. Unique advantages of this methodology compared to alternative propulsion technologies are discussed in terms of the range of species and genotypes that have been engineered, and the high transformation frequencies for major agronomic crops that enabled the technology to move from the R&D phase to commercialization. Creation of transgenic soybeans, cotton, and rice will be used as examples to illustrate the development of variety-independent and efficient gene transfer methods for most of the major agronomic crops. To our knowledge, no other gene transfer method based on particle bombardment has resulted in variety-independent and practical generation of large numbers of independently-derived crop plants. ACCELL™ technology is currently being utilized for the routine transfer of valuable genes into elite germplasm of soybean, cotton, bean, rice, corn, peanut and woody species.     

Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.     

A minimal DNA cassette as a vector for genetic transformation of soybean (Glycine max)

Currently, the market demands products committed to protecting human health and the environment, known as clean products. We developed a protocol using DNA fragments containing only the gene sequence of interest, to replace the circular vectors containing genes for antibiotic resistance and other undesirable sequences, for obtaining transgenic soybeans for microparticle bombardment. Vector pAC321 was digested with the restriction enzyme PvuII to produce the 6159 bp ahas fragment, which contains the mutated ahas gene from Arabidopsis thaliana (Brassicaceae), under the control of its own promoter and terminator. This gene confers resistance against imazapyr, a herbicidal molecule of the imidazolinone class, capable of systemically translocating and concentrating in the apical meristematic region of the plant, the same region used for the introduction of the transgenes. This fragment was used to generate 10 putative transgenic soybean lines.     

Plasmolysis of precultured immature embryos improves Agrobacterium mediated gene transfer to rice (Oryza sativa L.)

Efficiency of Agrobacterium mediated gene transfer was improved in rice by microprojectile pretreatment. Fertile transgenic plants were easily recovered using this protocol. Since an osmotic treatment is part of the bombardment protocol, we studied the effect of plasmolysis alone (i.e. without any microprojectiles) on transient expression of the gus gene transferred by Agrobacterium to precultured rice embryos. We report here for the first time that plasmolysis alone as a single pretreatment, yielded an even higher number of cells expressing the marker gene than the combination of microprojectile bombardment and osmotic treatment. This indicates that the effect of the osmoticum on gene transfer is independent from bombardment.     

Regeneration of different cultivars of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) via indirect organogenesis

A protocol for in vitro regeneration via indirect organogenesis for Phaseolus vulgaris cv. Negro Jamapa was established. The explants used were apical meristems and cotyledonary nodes dissected from the embryonic axes of germinating seeds. Several auxin/cytokinin combinations were tested for callus induction. The best callus production was obtained with medium containing 1.5 μM 2,4-dichlorophenoxyacetic acid. After 2 weeks of growth calli were transferred to shooting medium containing 22.2 μM 6-benzylaminopurine. Shoots regenerated with a frequency of approximately 0.5 shoots per callus, and upon transfer to rooting medium these shoots produced roots with 100% efficiency. Histological analyses of the regeneration process confirmed the indirect organogenesis pattern. Greenhouse grown regenerated plants showed normal development and were fertile. The protocol was reproducible for other nine P. vulgaris cultivars tested, suggesting a genotype independent procedure.     

Competence of oat ( Avena sativa L.) shoot apical meristems for integrative transformation,inherited expression,and osmotic tolerance of transgenic lines containing hva1

Three oat ( Avena sativa L.) cultivars have been successfully transformed using an efficient and reproducible in vitro culture system for differentiation of multiple shoots from shoot apical meristems. The transformation was performed using microprojectile bombardment with two plasmids (pBY520 and pAct1-D) containing linked ( hva1-bar) and non-linked ( gus) genes. The hva1 and bar genes cointegrated with a frequency of 100% as expected, and 61.6% of the transgenic plants carried all three genes. Molecular and biochemical analyses in R0, R1 and R2 progenies confirmed stable integration and expression of all transgenes. Localization of the GUS protein in R0 and R1 plants revealed that high-expression of gus occurred in vascular tissues and in the pollen grains of mature flowers. The constitutive expression of HVA1 protein was observed at all developmental stages of transgenic plants, and was particularly stronger during the early seedling stages. R2 progeny of five independent transgenic lines was tested in vitro for tolerance to osmotic (salt and mannitol) stresses. As compared to non-transgenic control plants, transgenic plants maintained a higher growth and showed significantly ( P < 0.05) increased tolerance to stress conditions. Less than 10% of transgenic plants showed symptoms of wilting or death of leaves and, when these symptoms present were delayed in transgenic plants as compared to 80% of non-transgenic plants, either wilted or died. These symptoms confirmed the increased in vitro tolerance in hva1-expressing transgenic plants to non-transgenic plants, providing strong evidence that the HVA1 protein may play an important role in the protection of oats against salinity and possible water-deficiency stress conditions.     

In planta transformation of pigeon pea: a method to overcome recalcitrancy of the crop to regeneration in vitro

Development of transgenics in pigeon pea remains dogged by poor plant regeneration in vitro from transformed tissues and low frequency transformation protocols. This article presents a non-tissue culture-based method of generating transgenic pigeon pea (Cajanus cajan (L.) Millisp.) plants using Agrobacterium-Ti plasmid-mediated transformation system. The protocol involves raising of whole plant transformants (T₀ plants) directly from Agrobacterium-infected young seedlings. The plumular and intercotyledonary meristems of the seedling axes are targeted for transformation. The transformation conditions optimized were, pricking of the apical and intercotyledonary region of the seedling axes of two-day old germinating seedlings with a sewing needle, infection with Agrobacterium (LBA4404/pKIWI105 carrying uid A and npt II genes) in Winans’ AB medium that was added with wounded tobacco leaf extract, co-cultivation in the same medium for 1h and transfer of seedlings to soilrite for further growth and hardening and subsequent transfer of seedlings to soil in pots in the greenhouse. Out of the 22–25 primary transformants that survived infection-hardening treatments from each of the three experiments, 15 plants on the average established on the soil under greenhouse conditions, showed slow growth initially, nevertheless grew as normal plants, and flowered and set seed eventually. Of the several seeds harvested from all the T₀ plants, six hundred were sown to obtain progeny (T₁) plants and 350 of these were randomly analysed to determine their transgenic nature. PCR was performed for both gus (uid A) and npt II genes. Forty eight of the 350 T₁ plants amplified both transgenes. Southern blot analysis substantiated the integration and transmission of these genes. The protocol ensured generation of pigeon pea transgenic plants with considerable ease in a short time and is applicable across different genotypes/cultivars of the crop and offers immense potential as a supplemental or an alternative protocol for generating transgenic plants of difficult-to-regenerate pigeon pea. Further, the protocol offers the option of doing away with a selection step in the procedure and so facilitates transformation, which is free of marker genes.Key words: Cajanus cajan, Transformation, Tissue culture-independent plant regeneration     

Efficient genotype-independent cotton genetic transformation and genome editing

Cotton(Gossypium spp.) is one of the most important fiber crops worldwide. In the last two decades, transgenesis and genome editing have played important roles in cotton improvement. However,genotype dependence is one of the key bottlenecks in generating transgenic and gene-edited cotton plants through either particle bombardment or Agrobacterium-mediated transformation. Here, we developed a shoot apical meristem(SAM) cell-mediated transformation system(SAMT) that allowed the transformation of r...     

Stable transformation of Phaseolus vulgaris via electric-discharge mediated particle acceleration

Summary Transgenic Phaseolus vulgaris or common bean has been produced using electric-discharge particle acceleration. The method uses particle acceleration to introduce DNA into bean seed meristems. Multiple shoots are then generated and screened to recover transgenic plants at a rate of 0.03% germline transformed plants/shoot. We have been able to recover transgenic plants using both GUS and herbicide screening to introduce the gus, bar, and bean golden mosaic virus coat protein genes into the navy bean cultivar, Seafarer. The transgenic plants have been characterized over 5 generations of self-fertilization with no loss of introduced genes or expression. In addition, several families have been crossed with non-transgenic parents and these plants also show expected inheritance patterns. The introduced bar gene has been shown to confer strong resistance in transgenic beans to basta herbicide application in the greenhouse.Abbreviations BGMV bean golden mosaic virus - PAT phosphinothricin acetyltransferase     

Transformation of soybean via particle bombardment of embryogenic suspension culture tissue

Summary Embryogenic suspension culture tissue of soybean (Glycine max Merrill.) was bombarded with particles coated with plasmid DNAs encoding hygromycin resistance andβ-glucuronidase (GUS). One to two weeks after bombardment, embryogenic tissue was placed in a liquid proliferation medium containing hygromycin. Four to six weeks after bombardment, lobes of yellow-green, hygromycin-resistant tissue, which began as outgrowths on brown clumps of hygromycin-sensitive tissue, were isolated and cultured to give rise to clones of transgenic embryogenic material. In vivo GUS assays of hygromycin-resistant clones showed that the early outgrowths could be negative, sectored, or positive for GUS activity. Transgenic, fertile plants could be routinely produced from the proliferating transgenic embryogenic clones. Southern hybridization analyses confirmed stable transformation and indicated that both copy number and integration pattern of the introduced DNA varied among independently transformed clones. Hybridization analysis of DNA from progeny plants showed genetic linkage of multiple copies of introduced DNA. An average of three transgenic clones were obtained per bombardment making this procedure very suitable for transformation of soybean.     

Factors influencing transient gene expression in bean (Phaseolus vulgaris L.) using an electrical particle acceleration device

Summary The parameters influencing transient expression of the betaglucuronidase gene in bean embryonic axes, cotyledons, apical meristems and leaves were evaluated after gene delivery with an electrical particle acceleration device. A calciumspermidine procedure for coating gold particles with DNA resulted in higher levels of GUS expression with lower concentrations of gold particles compared with a calcium phosphate procedure. The DNA concentration, distance between the discharge chamber and the retaining screen and the vacuum in the apparatus also influenced gene delivery. Sections prepared for light and electron microscopy showed the localisation, within target cells, of gold particles used to deliver the DNA. Immunolocalization of foreign gene expression within cells confirmed an even distribution of gene product throughout the cell cytoplasm.Abbreviations BAP 6,benzylaminopurine - BSA bovine serum albumin - MS Murashige and Skoog (1962)     

Isolation of soybean plants with stable transgene expression by visual selection based on green fluorescent protein

Particle bombardment is a common platform for soybean transformation but tends to cause transgene silencing due to the integration of rearranged or multiple copies of transgenes. We now describe the isolation of a total of 44 independent transgenic soybean plants after transformation by particle bombardment with one of two gene constructs, pHV and pHVS. Both constructs contain the hygromycin phosphotransferase gene (hpt) as a selectable marker and a modified glycinin gene (V3-1) for evaluation of homology-dependent silencing of endogenous glycinin genes; pHVS also contains sGFP(S65T), which encodes a modified form of green fluorescent protein (GFP), as a reporter gene in the flanking region of V3-1. Fluorescence microscopy revealed that the leaves of 8 of the 25 independent transgenic plants obtained with pHVS expressed GFP; most of these GFP-positive plants also contained V3-1 mRNA and an increased glycinin content in their seeds, and they exhibited simple banding patterns on Southern blots that were indicative of a low copy number of each of the three transgenes. In contrast, most of the transgenic plants obtained with pHVS that did not express GFP, as well as most of those obtained with pHV, lacked endogenous glycinin in their seeds and exhibited more complex patterns of transgene integration. The use of a reporter gene such as sGFP(S65T) in addition to an antibiotic resistance gene may thus help to reduce the problem of gene silencing associated with direct DNA transformation systems and facilitate the recovery of transgenic plants that stably express the gene of interest.     

Transgenic cotton resistant to herbicide bialaphos

Resistance to bialaphos, a non-selective herbicide, was intro duced into cotton through genetic engineering. A gene encoding phosphinothric in acetyltransferase (bar) from Streptomyces hygroscopicus was inserted into elite varieties of cotton through particle bombardment. Based on the marker gene, -glucuronidase (gus) expression, a total of 18 Pima (Gossypium barbadense), 45 DP50 (G. hirsutum L.), 20 Coker 312 (G. hirsutum) and 2 El Dorado (G. hirsutum) transgenic plants were recovered. Integration of the bar gene into cotton genomic DNA was confirmed by Southern blot analysis and gene expression was confirmed by northern blot and enzyme assays. Herbicide (Basta®) tolerance up to 15 000 ppm was demonstrated in greenhouse trials. The newly introduced herbicide tolerance trait is inherited in a Mendelian fashion in the progenies of germline transformants. This study demonstrates the potential for particle bombardment to introduce commerically important genes directly into elite varieties of cotton. This mode of gene transfer can expedite the introduction of transgenic cotton products into world markets     

Selection<Emphasis Type="Italic">of nptll</Emphasis> transgenic sweetpotato plants using G<Subscript>418</Subscript> and paromomycin

We have used two aminoglycosides, G₄₁₈ and paromomycin, to develop a reliable selection system fornptll transgenic sweet-potato (Ipomoea batatas (L.) Lam.). Embryogenic calli derived from shoot apical meristems were bombarded with gold particles coated with pCAMBIA2301, which contained thenptll andgusA genes. When compared on a kill curve that was based on calli proliferation and cell viability, G₄₁₃-selection proved to be more efficient and had fewer escapes than kanamycin. These bombarded expiants were then selected on G₄₁₈-containing media. The total time required from bombardment to plant establishment in soil was seven to nine months. Multiple copies of the transgene were integrated into the sweetpotato genome. Northern analysis confirmed transgene expression in the regenerated plants, and a paromomycin assay demonstrated that thenptll gene was functionally expressed in transformed sweetpotato. These molecular analyses and assays all showed that selection with G₄₁₈ and paromomycin is reliable. So far, we have produced 69 transgenic events with this system, at a transformation frequency of approx. 1.1%. That efficiency is based on the number of transgenic plants obtained and the amount of calli bombarded. Thus, this selection method that combines G₄₁₈ with paromomycin is now available for selectingnptll transgenic sweetpotato.