RESUMEN
In the present work, Agrobacterium tumefaciens-mediated genetic transformation of the model legume Medicago truncatula Gaertn. (barrel medic) was carried out using the pSIM843 vector that contains a Medicago-derived transfer DNA, delineated by a 25-bp sequence homologous to bacterial T-DNA borders. The transfer DNA contains an expression cassette for the nptII (neomycin phosphotransferase) gene and is flanked by an expression cassette for the backbone integration marker gene ipt (isopentenyl transferase). Our results demonstrate that the Medicago-derived RB-like elements efficiently support DNA mobilization from A. tumefaciens to M. truncatula. Kanamycin-resistant shoots with normal phenotype and ipt-shooty lines were recovered at a frequency of 11.7 and 7.8%, respectively. Polymerase chain reaction (PCR) analyses demonstrated that 44.4% of the independent transgenic lines were backbone-free and evidenced the occurrence of backbone-transfer events.
Asunto(s)
ADN Bacteriano/genética , Técnicas de Transferencia de Gen , Medicago truncatula/genética , Transformación Genética , Agrobacterium tumefaciens/genética , ADN de Plantas/genética , Vectores Genéticos , Plantas Modificadas Genéticamente/genética , PlásmidosRESUMEN
SUMMARY: Acrylamide is produced in starchy foods that are baked, roasted or fried at high temperatures. Concerns about the potential health issues associated with the dietary intake of this reactive compound led us to reduce the accumulation of asparagine, one of its main precursors, in the tubers of potato (Solanum tuberosum). This metabolic change was accomplished by silencing two asparagine synthetase genes through 'all-native DNA' transformation. Glasshouse-grown tubers of the transformed intragenic plants contained up to 20-fold reduced levels of free asparagine. This metabolic change coincided with a small increase in the formation of glutamine and did not affect tuber shape or yield. Heat-processed products derived from the low-asparagine tubers were also indistinguishable from their untransformed counterparts in terms of sensory characteristics. However, both French fries and potato chips accumulated as little as 5% of the acrylamide present in wild-type controls. Given the important role of processed potato products in the modern Western diet, a replacement of current varieties with intragenic potatoes could reduce the average daily intake of acrylamide by almost one-third.
Asunto(s)
Acrilamida/análisis , Asparagina/biosíntesis , Silenciador del Gen , Solanum tuberosum/química , Solanum tuberosum/genética , Aspartatoamoníaco Ligasa/genética , Contaminación de Alimentos , Genes de Plantas , Genotipo , Proteínas de Plantas/genética , Tubérculos de la Planta/genética , Tubérculos de la Planta/metabolismo , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/genética , Plásmidos , ARN de Planta/genética , Análisis de Secuencia de Proteína , Transformación GenéticaRESUMEN
Flavonols and caffeoylquinates represent important groups of phenolic antioxidants with health-promoting activities. The genetic potential of potato (Solanum tuberosum) to produce high levels of these dietary compounds has not been realized in currently available commodity varieties. In this article, it is demonstrated that tuber-specific expression of the native and slightly modified MYB transcription factor gene StMtf1(M) activates the phenylpropanoid biosynthetic pathway. Compared with untransformed controls, transgenic tubers contained fourfold increased levels of caffeoylquinates, including chlorogenic acid (CGA) (1.80 mg/g dry weight), whilst also accumulating various flavonols and anthocyanins. Subsequent impairment of anthocyanin biosynthesis through silencing of the flavonoid-3',5'-hydroxylase (F3'5'h) gene resulted in the accumulation of kaempferol-rut (KAR) to levels that were approximately 100-fold higher than in controls (0.12 mg/g dry weight). The biochemical changes were associated with increased expression of both the CGA biosynthetic hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (Hqt) gene and the upstream chorismate mutase (Cm) and prephenate dehydratase (Pdh) genes. Field trials indicated that transgenic lines produced similar tuber yields to the original potato variety Bintje. Processed products of these lines retained most of their phenylpropanoids and were indistinguishable from untransformed controls in texture and taste.
Asunto(s)
Quempferoles/biosíntesis , Ácido Quínico/análogos & derivados , Solanum tuberosum/genética , Aciltransferasas/genética , Aciltransferasas/metabolismo , Aminoácidos Aromáticos/metabolismo , Antocianinas/metabolismo , Cartilla de ADN , Activación Enzimática , Flavonoles/metabolismo , Perfilación de la Expresión Génica , Ingeniería Genética/métodos , Tubérculos de la Planta/enzimología , Tubérculos de la Planta/genética , Tubérculos de la Planta/metabolismo , Prefenato Deshidratasa/genética , Prefenato Deshidratasa/metabolismo , Ácido Quínico/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Solanum tuberosum/enzimología , Solanum tuberosum/metabolismoRESUMEN
The novel intragenic approach to genetic engineering improves existing varieties by eliminating undesirable features and activating dormant traits. It transforms plants with native expression cassettes to fine-tune the activity and/or tissue specificity of target genes. Any intragenic modification of traits could, at least in theory, also be accomplished by traditional breeding and transgenic modification. However, the new approach is unique in avoiding the transfer of unknown or foreign DNA. By consequently eliminating various potential risk factors, this method represents a relatively safe approach to crop improvement. Therefore, we argue that intragenic crops should be cleared through the regulatory process in a timely and cost-effective manner.
Asunto(s)
Cruzamiento , Plantas/genética , Agricultura/economía , Productos Agrícolas/genética , Ingeniería Genética , Variación Genética , Plantas Modificadas GenéticamenteRESUMEN
The dominant potato (Solanum tuberosum) variety for French fry production in the United States is the 131-year-old Russet Burbank. Market penetration of the higher yielding and more uniform Ranger Russet variety is limited to about one-fifth of that of the Russet Burbank because of two storage deficits: black spot bruise sensitivity and high levels of cold-induced sweetening. Here, these trait weaknesses are turned into strengths by simultaneously lowering the expression of Ranger Russet's tuber-expressed polyphenol oxidase (Ppo), starch-associated R1, and phosphorylase-L (PhL) genes. This genetic modification was accomplished without inserting any foreign DNA into the plant genome. French fries from the intragenic potatoes also contained reduced amounts of the antinutritional compound acrylamide while, unexpectedly, displaying enhanced sensory characteristics.
Asunto(s)
Manipulación de Alimentos/métodos , Conservación de Alimentos/métodos , Ingeniería Genética/métodos , Plantas Modificadas Genéticamente/genética , Solanum tuberosum/genética , ADN de Plantas/genética , Silenciador del Gen , Tubérculos de la Planta/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Solanum tuberosum/químicaRESUMEN
The transfer of DNA from Agrobacterium to plant cell nuclei is initiated by a cleavage reaction within the 25-bp right border of Ti plasmids. In an effort to develop all-native DNA transformation vectors, 50 putative right border alternatives were identified in both plant expressed sequence tags and genomic DNA. Efficacy tests in a tobacco (Nicotiana tabacum) model system demonstrated that 14 of these elements displayed at least 50% of the activity of conventional Agrobacterium transfer DNA borders. Four of the most effective plant-derived right border alternatives were found to be associated with intron-exon junctions. Additional elements were embedded within introns, exons, untranslated trailers, and intergenic DNA. Based on the identification of a single right border alternative in Arabidopsis and three in rice (Oryza sativa), the occurrence of this motif was estimated at a frequency of at least 0.8x10(-8). Modification of plasmid DNA sequences flanking the alternative borders demonstrated that both upstream and downstream sequences play an important role in initiating DNA transfer. Optimal DNA transfer required the elements to be preceded by pyrimidine residues interspaced by AC-rich trinucleotides. Alteration of this organization lowered transformation frequencies by 46% to 93%. Despite their weaker resemblance with left borders, right border alternatives also functioned effectively in terminating DNA transfer, if both associated with an upstream A[C/T]T[C/G]A[A/T]T[G/T][C/T][G/T][C/G]A[C/T][C/T][A/T] domain and tightly linked cytosine clusters at their junctions with downstream DNA. New insights in border region requirements were used to construct an all-native alfalfa (Medicago sativa) transfer DNA vector that can be used for the production of intragenic plants.