Split-gene system for hybrid wheat seed production.

Hybrid wheat plants are superior in yield and growth characteristics compared with their homozygous parents. The commercial production of wheat hybrids is difficult because of the inbreeding nature of wheat and the lack of a practical fertility control that enforces outcrossing. We describe a hybrid wheat system that relies on the expression of a phytotoxic barnase and provides for male sterility.

The production of male-sterile wheat plants through split barnase expression is promoted by the insertion of introns and flexible peptide linkers.

The successful use of transgenic plants depends on the strong and stable expression of the heterologous genes. In this study, three introns (PSK7-i1 and PSK7-i3 from Petunia and UBQ10-i1 from Arabidopsis) were tested for their ability to enhance the tapetum-specific expression of a split barnase transgene. We also analyzed the effects of introducing multiple copies of flexible peptide linkers that bridged the fusion domains of the assembled protein.

phiC31 integrase-mediated site-specific recombination in barley.

The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase.

Split-transgene expression in wheat.

The establishment of traits that result from the concerted expression of complementing transgene fragments is a feasible tool for trait control or gene flow control in plants. This chapter describes the methodology for producing herbicide-resistant and pollen-sterile wheat plants by the intein-mediated assembly of inactive precursor protein fragments (protein trans-splicing). We suggest the design of intein-containing vectors for split-transgene expression.

Transgene excision from wheat chromosomes by phage phiC31 integrase.

The Streptomyces phage phiC31 integrase was tested for its ability to excise transgenic DNA from the wheat genome by site-specific recombination. Plants that stably express phiC31 integrase were crossed to plants carrying a target construct bearing the phiC31 recognition sites, attP and attB. In the progeny, phiC31 recombinase mediates recombination between the att sites of the target locus, which results in excision of the intervening DNA.

RNAi suppression of the morphine biosynthetic gene salAT and evidence of association of pathway enzymes.

Papaver somniferum L. was transformed with an RNAi construct designed to reduce transcript levels of the gene encoding the morphine biosynthetic enzyme, salutaridinol 7-O-acetyltransferase (SalAT). RNA interference of salAT led to accumulation of the intermediate compounds, salutaridine and salutaridinol, in a ratio ranging from 2:1 to 56:1.

Intein-mediated protein assembly in transgenic wheat: production of active barnase and acetolactate synthase from split genes.

Engineering traits by the assembly of non-functional gene products is a promising tool for modern plant biotechnology. In this article, we describe the establishment of male sterility and herbicide resistance in wheat (Triticum aestivum) by complementing inactive precursor protein fragments through a split intein system. N- and C-terminal fragments of a barnase gene from Bacillus amyloliquifaciens were fused to intein sequences from the Synechocystis sp.

Expression of active Streptomyces phage phiC31 integrase in transgenic wheat plants.

Site-specific recombination systems are becoming an important tool for the genetic modification of crop plants. Here we report the functional expression of the Streptomyces phage-derived phiC31 recombinase (integrase) in wheat. T-DNA constructs containing a phiC31 integrase transgene were stably transformed into wheat plants via particle gun bombardment.