Fertile transgenic wheat from microprojectile bombardment of scutellar tissue

Abstract

A reproducible transformation system for hexaploid wheat was developed based on particle bombardment of scutellar tissue of immature embryos. Particle bombardment was carried out using a PDS 1000/He gun. Plant material was bombarded with the plasmid pDB1 containing the beta-glucuronidase gene (uidA) under the control of the actin-1 promoter of rice, and the selectable marker gene bar (phosphinothricin acetyltransferase) under the control of the CaMV 35S promoter. Selection was carried out using the herbicide Basta (Glufosinate-ammonium). From a total number of 1050 bombarded immature embryos, in seven independent transformation experiments, 59 plants could be regenerated. Putative transformants were screened for enzyme activity by the histochemical GUS assay using cut leaf material and by spraying the whole plants with an aqueous solution of the herbicide Basta. Twelve regenerants survived Basta spraying and showed GUS-activity. Southern-blot analysis indicated the presence of introduced foreign genes in the genomic DNA of the transformants and both marker genes were present in all plants analysed. To date, four plants have been grown to maturity and set seed. Histochemically stained pollen grains showed a 1:1 segregation of the uidA gene in all plants tested. A 3:1 segregation of the introduced genes was demonstrated by enzyme activity tests and Southern blot analysis of R1 plants.

Complex nested promoters control tissue-specific expression of acetyl-CoA carboxylase genes in wheat

“…Transient expression of the GUS reporter gene in wheat protoplasts and in wheat embryos was measured by delivering plasmid DNA by electroporation (11) and particle bombardment (12), respectively. Transgenic wheat lines were generated by the particle-bombardment method (12). GUS activity was determined fluorometrically (transient expression; ref.…”

Genetic Transformation of Wheat: Advances in the Transformation Method and Applications for Obtaining Lines with Improved Bread-Making Quality and Low Toxicity in Relation to Celiac Disease

“…The transformation efficiency obtained with the 4h-post treatment (6.8%) is higher than the reported by other authors that ranged between 0.15-0.5% (Altpeter et al, 1996, Blechl & Anderson, 1996 and between 0.5-1.5% (Barro et al, 1997, Becker et al, 1994, Vasil et al, 1993. However, the transformation capacity is largely genotype-dependent and controlled by a wide number of factors as indicate the range of variation obtained in the different works.…”

“…However, cereal transformation is still difficult due to the number of parameters involved in the technique, and many research works have been focused on the bombardment conditions s u c h a s a m o u n t o f D N A , a m o u n t a n d s i z e o f g o l d p a r t i c l e s , a c c e l e r a t i o n p r e s s u r e , bombardment distance or the osmotic condition of tissues (Altpeter et al, 1996, Becker et al, 1994, Li et al, 2003, Rasco Gaunt & Barcelo, 1998). An osmotic treatment of target tissues for stable transformation results in plasmolysis of cells and restricts damages by preventing extrusion of the protoplasm from bombarded cells (Vain et al, 1993).…”

Somatic Embryogenesis on Plumule and Radicle Explants Obtained from Warm Water Hydroprimed Wheat (Triticum aestivum L.) cv. Kunduru and cv. Cakmak Seeds

“…A review of previous literature suggests variable success in tissue culture of different wheat species using callus of immature embryos (Becker et al 1994;Elena & Ginzo 1988;Maddock et al 1983;Weeks et al 1993) and mature embryos (Bi et al 2007;Patnaik et al 2006). Both plumule and radicle tissue initiated callus induction are affected by types of explant and plant species used in the study at all concentrations of 2,4-D. Callus weight and gain was cultivar and explant specific and largely inconsistent.…”

“…Development of transformation techniques has become one of the focal issues in wheat tissue culture and is becoming an important vehicle in wheat breeding programs throughout the world. It is making use of development of haploids, organogenesis, somatic embryogenesis, selection of somaclonal variants and production of transgenic plants for wheat improvement (Becker et al 1994;Bi et al 2007;Chu et al 1990;Elena & Ginzo 1988;Fellers et al 1995;Maddock et al 1983;Patnaik et al 2006;Rajyalakshmi et al 1991;Sarker & Biswas 2002;Weeks et al 1993). Previous reports suggested the effects of culture medium and its supplements (Mathias & Simpson 1986) tissue (Vasil 1994) and genotype (Hess & Carman 1998) on tissue culture of wheat.…”