Transformation of peanut with a soybean vspB promoter‐uidA chimeric gene. I. Optimization of a transformation system and analysis of GUS expression in primary transgenic tissues and plants

Abstract

Direct DNA delivery via microprojectile bombardment has become an established approach for gene transfer into peanut (Arachis hypogaea L.). To optimize our transformation protocol and to simultaneously explore the function of a heterologous promoter whose activity is developmentally regulated, embryogenic cultures from three peanut cultivars were bombarded with two plasmid constructs containing a uidA gene controlled by either a soybean vegetative storage protein gene promoter or a cauliflower mosaic virus 35S promoter. We found that GUS transient expression was useful to predict stable transformation and confirmed that image analysis could provide a quick and efficient method for semi‐quantitation of transient expression. One hundred and sixty hygromycin‐resistant cell lines were recovered from and maintained on selective medium, and those tested by Southern blot analysis showed integration of the foreign gene. Over 200 transgenic plants were regenerated from 38 cell lines. More than 100 plants from 32 cell lines flowered and 79 plants from 19 cell lines produced pods. Over 1000 R1 seeds were harvested. Analysis of expression in primary transgenic plants showed that GUS expression driven by the vspB promoter was modulated by chemical and positional information.

Antifungal Activity in Transgenic Peanut (Arachis hypogaeaL.) Conferred by a Nonheme Chloroperoxidase Gene

“…Similar results were previously shown by Yang et al (1998), who reported that 92% of cell lines PCR-positive for the hygromycin resistance gene also showed PCR amplification of the nucleocapsid protein gene of tomato spotted wilt virus, and Wang et al (1998) who reported that 92% of hygromycin-resistant and PCR-positive cell lines showed PCR amplification of the vspB promoter-driven reporter gene, b-glucuronidase. It is likely that the non-selected gene linked with hph was disrupted in some instances during plasmid DNA integration.…”

“…Tissues that were actively growing on liquid selection medium were transferred to agar medium containing 20 mg/mL hygromycin, and individual pieces were assigned a unique cell line number. Thirteen bombardment experiments with 107 plates yielded 147 hygromycin-resistant cell lines for an average of 1.37 cell lines per plate, which is within the reported peanut transformation efficiency range of 0.8 to 4.6 hygromycin resistant callus lines per bombardment (Higgins et al 2004;Ozias-Akins et al 1993;Wang et al 1998;Yang et al 1998). It is difficult to get accurate transformation efficiency data based on somatic embryo numbers bombarded since clusters of somatic embryos contain variable numbers, and some cell lines may have originated from the same transformation event but were separated due to agitation during liquid selection.…”

Transgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system

“…Plasmid DNA was isolated from recombinant E. Coli by the alkaline lysis method and was purified with 13% PEG solution [8]. Particles coated with plasmid [1] were bombarded into cotyledons using a PDS 1000/helium-driven apparatus (Bio-Rad, CA, USA). Each bombardment delivered approximately 1 μg DNA and 300 μg gold particle 1.0 mm in diameter.…”

“…Genetic engineering provides a prospective way to reduce certain problems by transferring individual genes for pest resistance or other traits into elite germplasm of a cultivated species. Transgenic peanut plants have been produced by particle bombardment [1] and via Agrobacterium-mediated transformation [2]. Microprojectile bombardment uses high velocity particles to penetrate cell walls and to deliver DNA into intact plant cells.…”

Integration and expression of Bluetongue VP2 gene in somatic embryos of peanut through particle bombardment method

“…Transgenic peanut plants have been produced by particle bombardment [4] and via Agrobacterium-mediated transformation [5]. Microprojectile bombardment uses high velocity particles to penetrate cell walls and to deliver DNA into intact plant cells.…”