Self‐fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs^†

Abstract

A system for enhanced induction of somatic embryo‐genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self‐fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long‐term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L‐phosphinothricin (L‐PPT) to obtain herbicide‐resistant self‐fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L‐PPT whereas none of the plants showed β‐glucuronidase (GUS) activity. Molecular analysis of PAT‐positive plants confirmed stable integration of both bar and gus genes in R0 and R1 progeny plants. Segregation of the PAT activity and herbicide resistance in R1 progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho‐transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R1 progeny.

An efficient wheat transformation procedure: transformed calli with long-term morphogenic potential for plant regeneration

“…Using this calculation, an overall transformation efficiency of 28% was obtained with our protocol. Regardless, the minimum transformation efficiency (6.3%) reported here is higher than those previously reported (0.1-2.5%) (Weeks et al 1993;Nehra et al 1994;Altpeter et al 1996;Barro et al 1997;Cheng et al 1997;Dobrzanska et al 1997;Takumi and Shimada 1997). The higher transformation rates reported here are likely due to the induction of long-term morphogenic callus and the early application of high selection pressure.…”

“…Different concentrations (from 0.5 to 8.0 mg/l) of 2,4-D in wheat callus induction media have been reported Vasil 1982, 1983;Sears and Deckard 1982;Ou et al 1989;Nabors 1990, 1991;Vasil et al 1992;Vasil et al 1993;Weeks et al 1993;Nehra et al 1994;Fellers et al 1995;Altpeter et al 1996;Maes et al 1996;Dobrzanska et al 1997). For the most part, these studies indicate that 2,4-D concentrations of 1.0-2.0 mg/l are optimum to induce embryogenic callus from scutellum tissue of immature embryos.…”

“…Most bialaphos-resistant calli became apparent only after 2 months of culture under a high selection pressure (5.0 mg/l of bialaphos). Thus, a callus induction period of 2-4 weeks after bombardment may not provide enough time for most transformed cells to outgrow non-transformed ones, resulting in escapes and chimeric plants (Weeks et al 1993;Nehra et al 1994;Altpeter et al 1996). Furthermore, the early application of the high selection pressure facilitated the growth of transformed cells by inhibiting growth of untransformed cells.…”

“…These manipulations generally produce many regenerants but few true transformants. The transformation efficiencies are usually 0.1-2.5% (Weeks et al 1993;Nehra et al 1994;Altpeter et al 1996;Barro et al 1997;Dobrzanska et al 1997;Takumi and Shimada 1997). Using these methods, several hundred to thousands of immature embryos or explants are typically bombarded to obtain about ten transgenic T0 plants.…”

Identification and Efficient Utilization of Antibiotics for the Development of a Stable Transformation System in Porphyra yezoensis (Bangiales, Rhodophyta)

“…The availability of a useful selection protocol, which depends on the choice of a selection agent such as antibiotics and herbicides, is generally a prerequisite for the development of a stable transformation system [16]. Thus, the identification of selection agents such as antibiotics and herbicides was carried out in P. yezoensis [17], and it resulted in the indication of the non-sensitivity of P. yezoensis gametophytes to many kinds of antibiotics at concentrations up to 5 mg ml-1 in culture medium; however, the reproducibility of the test results was not examined.…”