Plastid transformation in soybean.

The biotechnological potential of plastid genetic engineering has been illustrated in a limited number of higher plant species. We have developed a reproducible method to generate plastid transformants in soybean (Glycine max), a crop of major agronomic importance. The transformation vectors are delivered to embryogenic cultures by the particle gun method and selection performed using the aadA antibiotic resistance gene. Homoplasmy is established rapidly in the selected events without the need for further selection or regeneration cycles, and genes of interest can be expressed at a high level in green tissues. This is a significant step toward the commercial application of this technology.

Translocation of aprotinin, a therapeutic protease inhibitor, into the thylakoid lumen of genetically engineered tobacco chloroplasts.

Aprotinin, a bovine protease inhibitor of important therapeutic value, was expressed in tobacco plastid transformants. This disulphide bond-containing protein was targeted to the lumen of thylakoids using signal peptides derived from nuclear genes which encode lumenal proteins. Translocation was attempted via either the general secretion (Sec) or the twin-arginine translocation (Tat) pathway. In both cases, this strategy allowed the production of genuine aprotinin with its N-terminal arginine residue. The recombinant protease inhibitor was efficiently secreted within the lumen of thylakoids, accumulated in older leaves and was bound to trypsin, suggesting that the three disulphide bonds of aprotinin are correctly folded and paired in this chloroplast compartment. Mass spectrometric analysis indicated that translocation via the Sec pathway, unlike the Tat pathway, led predominantly to an oxidized protein. Translocation via the Tat pathway was linked to a slightly decreased growth rate, a pale-green leaf phenotype and supplementary expression products associated with the thylakoids.

Generation and characterization of soybean and marker-free tobacco plastid transformants over-expressing a bacterial 4-hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance.

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is part of the biosynthetic pathway leading to plastoquinone and vitamin E. This enzyme is also the molecular target of various new bleaching herbicides for which genetically engineered tolerant crops are being developed. We have expressed a sensitive bacterial hppd gene from Pseudomonas fluorescens in plastid transformants of tobacco and soybean and characterized in detail the recombinant lines. HPPD accumulates to approximately 5% of total soluble protein in transgenic chloroplasts of both species. As a result, the soybean and tobacco plastid transformants acquire a strong herbicide tolerance, performing better than nuclear transformants. In contrast, the over-expression of HPPD has no significant impact on the vitamin E content of leaves or seeds, quantitatively or qualitatively. A new strategy is presented and exemplified in tobacco which allows the rapid generation of antibiotic marker-free plastid transformants containing the herbicide tolerance gene only. This work reports, for the first time, the plastome engineering for herbicide tolerance in a major agronomic crop, and a technology leading to marker-free lines for this trait.

Stability of soybean recombinant plastome over six generations.

The stability of a plastid transgene has been evaluated in soybean transformants over six generations. These transformants had integrated the aadA selection cassette in the intergenic region between the rps12/7 and trnV genes. Three independent homoplasmic T0 transformation events were selected and ten plants from each event propagated to generation T5 in the absence of selection pressure. No transgene rearrangement nor wild-type plastome were detected in generation T5 by Southern blot analysis. All tested progenies were uniformly resistant to spectinomycin. Therefore, soybean transformants of generations T0 and T5 appear to be genetically and phenotypically identical.

Generation of fertile transplastomic soybean.

We describe here the development of a plastid transformation method for soybean, a leguminous plant of major agronomic interest. Chloroplasts from embryogenic tissue of Glycine max have been successfully transformed by bombardment. The transforming DNA carries a spectinomycin resistance gene (aadA) under the control of tobacco plastid regulatory expression elements, flanked by two adjacent soybean plastome sequences allowing its targeted insertion between the trnV gene and the rps12/7 operon. All generated spectinomycin resistant plants were transplastomic and no remaining wild type plastome copies were detected. No spontaneous mutants were obtained. The transformation efficiency is similar to that of tobacco plastids. All transplastomic T0 plants were fertile and T1 progeny was uniformly spectinomycin resistant, showing the stability of the plastid transgene. This is the first report on the generation of fertile transplastomic soybean.