Stable expression of 1Dx5 and 1Dy10 high-molecular-weight glutenin subunit genes in transgenic rye drastically increases the polymeric glutelin fraction in rye flour.

We generated and characterized transgenic rye synthesizing substantial amounts of high-molecular-weight glutenin subunits (HMW-GS) from wheat. The unique bread-making characteristic of wheat flour is closely related to the elasticity and extensibility of the gluten proteins stored in the starchy endosperm, particularly the HMW-GS. Rye flour has poor bread-making quality, despite the extensive sequence and structure similarities of wheat and rye HMW-GS. The HMW-GS 1Dx5 and 1Dy10 genes from wheat, known to be associated with good bread-making quality were introduced into a homozygous rye inbred line by the biolistic gene transfer. The transgenic plants, regenerated from immature embryo derived callus cultures were normal, fertile, and transmitted the transgenes stably to the sexual progeny, as shown by Southern blot and SDS-PAGE analysis. Flour proteins were extracted by means of a modified Osborne fractionation from wildtype (L22) as well as transgenic rye expressing 1Dy10 (L26) or 1Dx5 and 1Dy10 (L8) and were quantified by RP-HPLC and GP-HPLC. The amount of transgenic HMW-GS in homozygous rye seeds represented 5.1% (L26) or 16.3% (L8) of the total extracted protein and 17% (L26) or 29% (L8) of the extracted glutelin fraction. The amount of polymerized glutelins was significantly increased in transgenic rye (L26) and more than tripled in transgenic rye (L8) compared to wildtype (L22). Gel permeation HPLC of the un-polymerized fractions revealed that the transgenic rye flours contained a significantly lower proportion of alcohol-soluble oligomeric proteins compared with the non-transgenic flour. The quantitative data indicate that the expression of wheat HMW-GS in rye leads to a high degree of polymerization of transgenic and native storage proteins, probably by formation of intermolecular disulfide bonds. Even gamma-40k secalins, which occur in non-transgenic rye as monomers, are incorporated into these polymeric structures. The combination 1Dx5 + 1Dy10 showed stronger effects than 1Dy10 alone. Our results are the first example of genetic engineering to significantly alter the polymerization and composition of storage proteins in rye. This may be an important step towards improving bread-making properties of rye whilst conserving its superior stress resistance.

Generation of rye (Secale cereale L.) plants with low transgene copy number after biolistic gene transfer and production of instantly marker-free transgenic rye.

Rye is extremely recalcitrant to tissue culture and genetic transformation. We describe the efficient and reproducible production of stably expressing transgenic rye plants after biolistic gene transfer to callus tissue derived from immature embryos. Key factors in the production of transgenic rye plants include the identification of biolistic gene transfer parameters and a selection protocol, which does not affect its regeneration ability. The bar gene was used as a selectable marker and selection was performed by spraying the regenerated shoots with 0.05% Basta solution without any previous selection of tissue cultures. Based on Southern blot analysis, a total of 21 transgenic rye plants with independent transgene integration patterns were produced. A low transgene copy number was observed in most transgenic plants and 40% of the plants had a single transgene copy insert. The high frequency of single transgene copy inserts might be a consequence of the selection system, which is based on the identification of stably expressing transgenic plantlets rather than stably expressing tissue cultures. All transgenic rye lines with single transgene inserts showed stable transgene expression in sexual progenies, but indications of transcriptional and post-transcriptional gene silencing were observed in few transgenic lines with multiple transgene inserts. Tissue culture-based selection was not necessary for the generation of transgenic rye. The identification of 17 transgenic rye plants without using any selectable marker gene by PCR amplification of transgene sequences is also demonstrated. Instant generation of selectable marker-free transgenic rye avoids a negative impact of selective agents on the transgenic tissue cultures, responds to public concerns on the safety of selectable markers and will support multiple transformation cycles for transgene pyramiding.

Evaluation of rye (Secale cereale L.) inbred lines and their crosses for tissue culture response and stable genetic transformation of homozygous rye inbred line L22 by biolistic gene transfer.

The efficient and reproducible production of stably expressing transgenic rye plants is described. Analysis of the genotype-specific callus culture-response of 21 rye inbred lines, single crosses and a population variety resulted in the identification of the highly responsive inbred line L22. Biolistic transformation experiments were performed using line L22 and the impact of different selection agents on the regeneration capacity was analyzed. Using the selectable marker gene nptII and corresponding paromomycin selection resulted in transformation efficiencies of up to 4.0% of the bombarded explants. A total of 17 independent transgenic rye plants were produced, and their stability and level of transgene expression was analyzed. The majority of these lines showed stable transgene expression. In contrast a few transgenic lines with multiple transgene inserts, provided evidence of transcriptional and post-transcriptional gene silencing.