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Self‐fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs †
Author(s) -
Nehra Narender S.,
Chibbar Ravindra N.,
Leung Nick,
Caswell Karen,
Mallard Cliff,
Steinhauer Lee,
Baga Monica,
Kartha Kutty K.
Publication year - 1994
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1994.05020285.x
Subject(s) - biology , protoplast , transgene , callus , genetically modified crops , somatic embryogenesis , gene , glufosinate , southern blot , gus reporter system , embryo , fusion gene , somatic cell , botany , microbiology and biotechnology , genetics , embryogenesis , glyphosate , agronomy
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 R 0 and R 1 progeny plants. Segregation of the PAT activity and herbicide resistance in R 1 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 R 1 progeny.