Improved apple latent spherical virus-induced gene silencing in multiple soybean genotypes through direct inoculation of agro-infiltrated Nicotiana benthamiana extract.

BACKGROUND: Virus induced gene silencing (VIGS) is a powerful genomics tool for interrogating the function of plant genes. Unfortunately, VIGS vectors often produce disease symptoms that interfere with the silencing phenotypes of target genes, or are frequently ineffective in certain plant genotypes or tissue types. This is especially true in crop plants like soybean [Glycine max (L.) Merr]. To address these shortcomings, we modified the inoculation procedure of a VIGS vector based on Apple latent spherical virus (ALSV). The efficacy of this new procedure was assessed in 19 soybean genotypes using a soybean Phytoene desaturase (GmPDS1) gene as the VIGS target. Silencing of GmPDS1 was easily scored as photo-bleached leaves and/or stems. RESULTS: In this report, the ALSV VIGS vector was modified by mobilizing ALSV cDNAs into a binary vector compatible with Agrobacterium tumefaciens-mediated delivery, so that VIGS-triggering ALSV variants could be propagated in agro-infiltrated Nicotiana benthamiana leaves. Homogenate of these N. benthamiana leaves was then applied directly onto the unifoliate of young soybean seedlings to initiate systemic gene silencing. This rapid inoculation method bypassed the need for a particle bombardment apparatus. Among the 19 soybean genotypes evaluated with this new method, photo-bleaching indicative of GmPDS1 silencing was observed in nine, with two exhibiting photo-bleaching in 100% of the inoculated individuals. ALSV RNA was detected in pods, embryos, stems, leaves, and roots in symptomatic plants of four genotypes. CONCLUSIONS: This modified protocol allowed for inoculation of soybean plants via simple mechanical rubbing with the homogenate of N. benthamiana leaves agro-infiltrated with ALSV VIGS constructs. More importantly, inoculated plants showed no apparent virus disease symptoms which could otherwise interfere with VIGS phenotypes. This streamlined procedure expanded this functional genomics tool to nine soybean genotypes.

Use of Agrobacterium expressing green fluorescent protein to evaluate colonization of sonication-assisted Agrobacterium-mediated transformation-treated soybean cotyledons.

Colonization and infection of soybean cotyledons by Agrobacterium tumefaciens and subsequent elimination of bacteria from cotyledons were monitored using bacteria expressing green fluorescent protein (GFP). GFP provided a quick, non-destructive method to evaluate, in real time, Agrobacterium colonization of cotyledon surfaces as well as infection of internal cells. GFP was first detected 7 h following inoculation of the cotyledon. By 36 h, GFP expression was very intense, and was limited to the adaxial surface of the cotyledon. Expression of GFP also served as a useful indicator of successful elimination of the bacterium from plant tissue following antibiotic treatment.

Transient expression and stable transformation of soybean using the jellyfish green fluorescent protein.

Embryogenic soybean [Glycine max (L.) Merrill.] suspension cultures were bombarded with five different gene constructions encoding the jellyfish (Aequorea victoria) green fluorescent protein (GFP). These constructions had altered codon usage compared to the native GFP gene and mutations that increased the solubility of the protein and/or altered the native chromophore. All of the constructions produced green fluorescence in soybean cultures upon blue light excitation, although a soluble modified red-shifted GFP (smRS-GFP) was the easiest to detect based on the brightness and number of foci produced. Expression of smRS-GFP was visible as early as 1.5 h after bombardment, with peak expression at approximately 6.5 h. Large numbers of smRS-GFP-expressing areas were visible for 48 h postbombardment and declined rapidly thereafter. Stably transformed cultures and plants exhibited variation in the intensity and location of GFP expression. PCR and Southern hybridization analyses confirmed the presence of introduced GFP genes in stably transformed cultures.

Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons: optimization of transient expression.

Sonication-assisted Agrobacterium-mediated transformation (SAAT) tremendously improves the efficiency of Agrobacterium infection by introducing large numbers of microwounds into the target plant tissue. Using immature cotyledons of soybean as explants, we evaluated the effects of the following parameters on transient ß-glucuronidase (GUS) activity: cultivars, binary vectors, optical density of Agrobacterium during infection, duration of sonication treatment, co-culture conditions, length of explant preculture and addition of acetosyringone during co-culture. The extent of tissue disruption caused by sonication was also determined. The highest GUS expression was obtained when immature cotyledons were sonicated for 2 s in the presence of Agrobacterium (0.11 OD600nm) followed by co-cultivation with the abaxial side of the explant in contact with the culture medium for 3 days at 27°C. The addition of acetosyringone to the co-culture medium enhanced transient expression. No differences were observed when different cultivars or binary vectors were used. Cotyledons sonicated for 2 s had moderate tissue disruption, while the longer treatments resulted in more extensive damage.

Sonication-assisted Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue.

Successful transformation of plant tissue using Agrobacterium relies on several factors including bacterial infection, host recognition, and transformation competency of the target tissue. Although soybean [Glycine max (L.) Merrill] embryogenic suspension cultures have been transformed via particle bombardment, Agrobacterium-mediated transformation of this tissue has not been demonstrated. We report here transformation of embryogenic suspension cultures of soybean using "Sonication-Assisted Agrobacterium-mediated Transformation" (SAAT). For SAAT of suspension culture tissue, 10-20 embryogenic clumps (2-4 mm in diameter) were inoculated with 1 ml of diluted (OD600nm 0.1-0.5) log phase Agrobacterium and sonicated for 0-300 s. After 2 days of co-culture in a maintenance medium containing 100 µM acetosyringone, the medium was removed and replaced with fresh maintenance medium containing 400 mg/l Timentin®. Two weeks after SAAT, the tissue was placed in maintenance medium containing 20 mg/l hygromycin and 400 mg/l Timentin®, and the medium was replenished every week thereafter. Transgenic clones were observed and isolated 6-8 weeks following SAAT. When SAAT was not used, hygromycin-resistant clones were not obtained. Southern hybridization analyses of transformed embryogenic tissue confirmed T-DNA integration.

Intron-mediated enhancement of gene expression in maize (Zea mays L.) and bluegrass (Poa pratensis L.).

We report a strength comparison of a large variety of monocot and dicot intron-containing fragments inserted in the 5' untranslated leader, between the CaMV 35S promoter and the uidA gene (coding for the ß-glucuronidase: GUS). Relative strengths of the intron-containing fragments were evaluated by comparing transient GUS expression after particle bombardment in embryogenic maize and bluegrass suspension cultures. Our results confirm a dramatic dependence on the presence of an intron for chimeric gene expression in both species. On average, the maize first intron of ubi1 provided the highest enhancement of gene expression in maize and bluegrass (71- and 26-fold enhancement, respectively). Half of the introns tested affected gene expression differently in bluegrass and maize. This suggests that the intron-mediated enhancement of gene expression generally obtained with maize may not be fully applicable to all monocots. We also report enhancement of gene expression (92-fold) in a monocot species by a dicot intron (chsA intron).

Transformation of 12 different plasmids into soybean via particle bombardment.

Particle bombardment offers a simple method for the introduction of DNA into plant cells. Multiple DNA fragments may be introduced on a single plasmid or on separate plasmids (co-transformation). To investigate some of the properties and limits of co-transformation, 12 different plasmids were introduced into embryogenic suspension culture tissue of soybean [Glycine max (L.) Merrill] via particle bombardment. The DNAs used for co-transformation included 10 plasmids containing KFLP markers for maize and 2 plasmids separately encoding hygromycin-resistance and ß-glucuronidase. Two weeks following bombardment with the 12 different plasmids, suspension culture tissue was placed under hygromycin selection. Hygromycin-resistant clones were isolated after an additional 5 to 6 weeks. Southern hybridization analysis of 26 hygromycin-resistant embryogenic clones verified the presence of introduced plasmid DNAs. All of the co-transforming plasmids were present in most of the transgenic soybean clones and there was no preferential uptake and integration of any of the plasmids. The copy number of individual plasmids was approximately equal within clones but highly variable between clones. While some clones contained as few as zero to three copies of each plasmid, others clones contained as many as 10 to 15 copies of each of the 12 different plasmids.

Identification of a homeobox-containing gene with enhanced expression during soybean (Glycine max L.) somatic embryo development.

Homeotic gene are key 'switches' that control developmental processes. Homeotic genes containing the consensus 'homeobox' domain have been identified from a number of organisms including Drosophila melanogaster, Caenorhabditis elegans, Homo sapiens, and Zea mays. Although homeotic genes have been demonstrated to be important in embryo development of some insects, amphibians, and mammals, there are no reports of their involvement in plant embryogenesis. Here, we report the isolation and characterization of a cDNA clone for a homeobox-containing gene expressed in somatic embryos of soybean. The cDNA (Sbh1 for soybean homeobox-containing gene) was isolated using maize Knotted-1 (Kn1) cDNA as a heterologous probe. The Sbh1 cDNA clone is 1515 bp long which is the approximate size of its transcript. Within the homeodomain, the amino acid sequence of a helix-turn-helix structure, and invariant and conserved residues were identified. The deduced SBH1 protein shares a high amino acid identity with KN1 protein (47.0% overall and 87.5% for the homeodomain). Southern hybridization analysis indicated that Sbh1 is a member of a small gene family. The expression of Sbh1 is development- and tissue-specific. The transcript of Sbh1 was present in early-stage somatic embryos, increased prior to cotyledon formation and decreased thereafter. Sbh1 was weakly expressed in soybean stems and hypocotyl but was not detected in other plant tissues and nonembryogenic materials. The enhanced expression during embryogenesis, the homology with maize Kn1 gene, and the regulatory nature of homeodomain proteins suggest that the SBH1 protein plays an important role in plant embryo development.

Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize.

The effects of osmotic conditioning on both transient expression and stable transformation were evaluated by introducing plasmid DNAs via particle bombardment into embryogenic suspension culture cells of Zea mays (A188 × B73). Placement of cells on an osmoticum-containing medium (0.2 M sorbitol and 0.2 M mannitol) 4 h prior to and 16 h after bombardment resulted in a statistically significant 2.7-fold increase in transient ß-glucuronidase expression. Under these conditions, an average of approximately 9,000 blue foci were obtained from 100 µl packed cell volume of bombarded embryogenic tissue. Osmotic conditioning of the target cells resulted in a 6.8-fold increase in recovery of stably transformed maize clones. Transformed fertile plants and progeny were obtained from several transformed cell lines. We believe the basis of osmotic enhancement of transient expression and stable transformation resulted from plasmolysis of the cells which may have reduced cell damage by preventing extrusion of the protoplasm from bombarded cells.

Induction of Glutamine Synthetase Activity in Nonnodulated Roots of Glycine max, Phaseolus vulgaris, and Pisum sativum.

Nitrate or ammonium fertilization significantly increased glutamine synthetase (GS) activity in nonnodulated roots of French bean (Phaseolus vulgaris), soybean (Glycine max), and pea (Pisum sativum). Western analysis revealed substantial GS antibody-positive protein in root extracts that had minimal GS activity, indicating that an inactive form of GS may be present in nonfertilized plants.

Development of the particle inflow gun for DNA delivery to plant cells.

A simple and inexpensive particle bombardment device was constructed for delivery of DNA to plant cells. The Particle Inflow Gun (PIG) is based on acceleration of DNA-coated tungsten particles using pressurized helium in combination with a partial vacuum. The particles are accelerated directly in a helium stream rather than being supported by a macrocarrier. Bombardment parameters were partially optimized using transient expression assays of a ß-glucuronidase gene in maize embryogenic suspension culture and cowpea leaf tissues. High levels of transient expression of the ß-glucuronidase gene were obtained following bombardment of embryogenic suspension cultures of corn and soybean, and leaf tissue of cowpea. Stable transformation of embryogenic tissue of soybean has also been obtained using this bombardment apparatus.

Growth and disinfestation of 6 different bacteria in embryogenic suspension cultures of cotton.

Growth of 6 different common laboratory bacteria (Escherichia coli, Flavobacterium balustrum, Xanthomonas maltophilia, Enterobacter cloacae, Pseudomonas fluorescens, and Agrobacterium tumefaciens) in a bacterial medium, fresh plant medium, and "spent" plant media was initially measured. In all cases, bacteria grew best in the bacterial medium followed by the fresh plant medium. The spent plant medium did not support growth of the bacteria and apparently was actively toxic to bacterial cells. Proliferating, embryogenic suspension cultures of cotton (Gossypium hirsutum) were then inoculated with these 6 different bacteria. Two to three d following bacterial inoculation, embryogenic tissues were placed in various concentrations of bleach for various amounts of time, rinsed with sterile water, and placed on a bacterial culture medium. Clumps of embryogenic tissue which showed no visible bacterial growth after 3 d of culture were then transferred to an agar-solidified plant tissue culture medium to determine viability of bleachdisinfested tissues. Viable, single pieces of the disinfested embryogenic tissue were then used to reinitiate embryogenic suspension cultures. Treatment of contaminated tissue with a 1% bleach solution for 1-5 min resulted in the highest recovery of viable, disinfested tissues using 5 of the 6 bacteria. It was not possible to remove F. balustrum from clumps of embryogenic tissue without also killing the plant tissue.

Transformation of cotton (Gossypium hirsutum L.) via particle bombardment.

Embryogenic suspension cultures of cotton (Gossypium hirsutum L.) were subjected to particle bombardment, where high density particles carrying plasmid DNA were accelerated towards the embryogenic plant cells. The plasmid DNA coating the particles encoded hygromycin resistance. One to two weeks following bombardment, embryogenic cotton cells were placed in proliferation medium containing 100 µg/ml hygromycin. Clumps of tissue which grew in the presence of hygromycin were subcultured at low density into fresh hygromycin-containing proliferation medium. Following sequential transfer of embryogenic tissue to development and then germination media, plants were recovered from transgenic embryogenic tissue. Southern hybridization confirmed the presence of the hygromycin resistance gene in embryogenic suspension culture tissue and regenerated plants.

Initiation of embryogenic callus and suspension cultures of eastern white pine (Pinus strobus L.).

Embryogenic callus and suspension cultures of eastern white pine (Pinus strobus) have been obtained. The whole female gametophyte was plated on a medium containing 50 mg/l glutamine, 500 mg/l casein hydrolysate, 3% sucrose, 2 mg/1 2,4-D, 1 mg/1 BA and 0.2% Gelrite as a solidifying agent. Embryogenic calli could be seen as early as 5 days following culture. Histological studies indicate proliferation of pre-existing embryogenic tissue in the corrosion cavity followed by extrusion of embryogenic callus through the micropylar end of the gametophyte. Embryogenic suspension cultures were obtained by placing embryogenic callus into liquid medium. Embryogenic suspension cultures were subcultured weekly and proliferated as early-stage embryos with attached suspensors. Embryo development was obtained following transfer of the embryogenic tissue to an auxin-free medium containing 50 mM glutamine, 38 µM abscisic acid, and 6% sucrose. Although embryo development could be consistently obtained, whole plants have not yet been recovered from these somatic embryos.

Plant regeneration from somatic embryogenic suspension cultures of cotton (Gossypium hirsutum L.).

Maintainable, highly embryogenic suspension cultures of cotton (Gossypium hirsutum L. cv. 'Coker 310') have been obtained. Callus cultures were initiated from cotyledonary tissues from aseptically-germinated seedlings. To establish the suspension cultures, callus tissue was placed in a liquid medium containing either 0.5 mg/l picloram or 0.1 mg/l 2,4-dichlorophenoxyacetic acid. For proliferation of the embryogenic suspension, 5 mg/l of 2,4-dichlorophenoxyacetic acid was used. Embryo development took place when the embryogenic tissue was transferred to an auxin-free liquid medium containing 15 mM glutamine. Early embryo development was fairly synchronous and large numbers of somatic embryos were produced. Regenerated plants were fertile and smaller than seed-derived plants.

Apical proliferation of embryogenic tissue of soybean [Glycine max (L.) Merrill].

Somatic embryos and embryogenic tissues were initiated from immature zygotic embryos of soybean [Glycine max (L.) Merrill cv. 'Fayette']. Zygotic embryos were placed on a medium containing 40 mg/l of 2,4-dichlorophenoxyacetic acid and 6% sucrose. Somatic embryos were first seen 4 weeks after cultures were initiated. Following transfer, secondary somatic embryos proliferated directly from the apical or terminal portions of the older primary somatic embryos. Single somatic embryos or clusters of embryos were seen growing directly from the top of older somatic embryos. Light microscopy revealed that these embryos were of surface or subsurface origin. The apical soybean somatic embryo tissue may represent cotyledonary tissue (which has been shown to be most responsive) at a very young and manipulatable state.