RESUMO
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.
Assuntos
4-Hidroxifenilpiruvato Dioxigenase/genética , Glycine max/genética , Herbicidas/toxicidade , Nicotiana/genética , Plastídeos/genética , Pseudomonas fluorescens/genética , 4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Tolerância a Medicamentos/genética , Pseudomonas fluorescens/enzimologia , Proteínas Recombinantes/metabolismo , Nicotiana/efeitos dos fármacosRESUMO
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.
Assuntos
Técnicas Genéticas , Glycine max/genética , Plantas Geneticamente Modificadas , Plastídeos/genética , Proteínas Recombinantes/química , Ágar/química , DNA/metabolismo , DNA Intergênico/genética , Resistência a Medicamentos , Vetores Genéticos , Genótipo , Modelos Genéticos , Fenótipo , Plastídeos/metabolismo , Espectinomicina/farmacologia , Fatores de TempoRESUMO
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.