RESUMO
Sucrose and its constituent components, glucose and fructose, are major determinants of sweetness in fruits and cooking quality in plant storage organs, such as potato tubers. Accurate monitoring of these sugars provides a predictive assessment of plant product quality. High Performance Liquid Chromatography (HPLC) coupled to a variety of detectors has been the method of choice for selective detection and quantitation of sugars in plant tissues. However, sugar extraction can be time-consuming to obtain a clean and concentrated sample suitable for accurate quantitation. Herein, we took advantage of a modern HPLC system coupled to a highly sensitive high-resolution high-accuracy mass spectrometer to develop a rapid method for measuring glucose, fructose, and sucrose in plant tissues. The method was validated in mature potato tubers and strawberry fruits and it proved to be highly accurate and robust with minimal sample care requirements.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fragaria/química , Espectrometria de Massas/métodos , Solanum tuberosum/química , Açúcares/análise , Frutose/análise , Frutas/química , Glucose/análise , Tubérculos/química , Sacarose/análiseRESUMO
Marker-free methods of plant transformation sacrifice the advantages of a selectable marker during regeneration or add work after regeneration to remove the marker. On the positive side, there is no stably integrated marker gene in the plant genome to present regulatory hurdles or potential biosafety hazards once the plant is released to the environment. A marker-free method that is simple and adaptable to multiple crop species-even asexually propagated species-is presented herein. This method employs an engineered vector that utilizes the isopentenyltransferase (ipt) to drive the regeneration of intragenic cells containing the gene(s) of interest. The ipt gene also acts as a marker to screen against events where the vector backbone is stably integrated.
Assuntos
Agricultura/métodos , Citocininas/genética , Vetores Genéticos , Plantas Geneticamente Modificadas , Transformação Genética , Agrobacterium/genética , Alquil e Aril Transferases/genética , Regulação da Expressão Gênica de Plantas , Técnicas de Transferência de Genes , Marcadores Genéticos , Solanum lycopersicum/genética , Regiões Promotoras Genéticas , Solanum tuberosum/genéticaRESUMO
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.
Assuntos
Quempferóis/biossíntese , Ácido Quínico/análogos & derivados , Solanum tuberosum/genética , Aciltransferases/genética , Aciltransferases/metabolismo , Aminoácidos Aromáticos/metabolismo , Antocianinas/metabolismo , Primers do DNA , Ativação Enzimática , Flavonóis/metabolismo , Perfilação da Expressão Gênica , Engenharia Genética/métodos , Tubérculos/enzimologia , Tubérculos/genética , Tubérculos/metabolismo , Prefenato Desidratase/genética , Prefenato Desidratase/metabolismo , Ácido Quínico/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Solanum tuberosum/enzimologia , Solanum tuberosum/metabolismoRESUMO
Conventional Agrobacterium-mediated transformation methods rely on complex and genotype-specific tissue culture media for selection, proliferation, and regeneration of genetically modified cells. Resulting transgenic plants may not only contain selectable marker genes but also carry fragments of the vector backbone. Here, we describe a new method for the production of transgenic plants that lack such foreign DNA. This method employs vectors containing the bacterial isopentenyltransferase (ipt) gene as backbone integration marker. Agrobacterium strains carrying the resulting ipt gene-containing "cytokinin" vectors were used to infect explants of various Solanaceous plant species as well as canola (Brassica napus). Upon transfer to hormone-free media, 1.8% to 9.9% of the infected explants produced shoots that contained a marker-free T-DNA while lacking the backbone integration marker. These frequencies often equal or exceed those for backbone-free conventional transformation.