RESUMO
Although Agrobacterium-mediated transformation protocols for many economically important plant species have been well established, protocol for a number of flowering plants including Anthurium andraeanum remains challenging. In this study, we report success in generating transgenic Anthurium andraeanum cv Arizona using Agrobacterium GV3101 strain harboring a binary vector carrying gfp as a reporter gene. The possibility of facilitating the screening process for transgenic plants expressing functional proteins using gfp marker was explored. In order to realize high transformation efficiency, different explant sources including undifferentiated callus pieces and petioles were compared for their regeneration efficiency and susceptibility to Agrobacterium-mediated transformation. We also optimized the concentration of AS added to co-cultivation media. Genomic PCR revealed that 11 of the 22 resistant plantlets regenerated on selective medium were successfully transformed. Green fluorescence was observed using a fluorescence microscope in 7 of the 11 PCR-positive plants, indicating GFP was expressed stably in the transformed Anthurium andraeanum. The highest transformation efficiency obtained in this study was 1.71 percent (percentage of explants with transgenic shoots in total explants) when callus explants were used as starting material and 125 umol l-1 AS was added during the co-cultivation process.
Assuntos
Araceae/genética , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Rhizobium/genética , DNA de Plantas/isolamento & purificação , Técnicas de Cocultura , Genes Reporter , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Plantas Geneticamente Modificadas/genética , Regeneração , Transformação GenéticaRESUMO
The exact localization of an insertion in the genome of transgenic plants obtained by Agrobacterium-mediated transformation is an integral part of most experiments aimed at studying these types of mutants. There are several methods for isolating unknown nucleotide sequences of genomic DNA which flank the borders of T-DNA integrated in the genome of plants. However, all the methods based on PCR have limitations which in some cases do not permit the desired objective to be achieved. We have developed a new technique for isolating flanking sequence tags (FSTs) via modified inverse PCR. This method is highly efficient and simple, but also retains the advantages of previously well-documented approaches.
Assuntos
Arabidopsis/genética , Primers do DNA , DNA Bacteriano , DNA de Plantas/isolamento & purificação , Etiquetas de Sequências Expressas/metabolismo , Mutagênese Insercional , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase/métodosRESUMO
In order to investigate the mutation characteristics and to further examine the genetic variation of mutant sunflower (Helianthus annuus) obtained in plants grown from seeds exposed to space conditions, only the mature tissues such as leaf and flower could be used for DNA extraction after mutation characteristics were confirmed. The rich contents of polysaccharides, tannins, secondary metabolites, and polyphenolics made it difficult to isolate high-quality DNA from mature leaves of sunflower according to previous reports. Based on the comparison of the differences in previously reported protocols, a modified method for the extraction of high-quality DNA was developed. Using this protocol, the DNA isolated from sunflower was high in quality and suitable for restriction digestion (EcoRI, HindII, BamHI), random amplified polymorphic DNA study and further molecular research. Therefore, the modified protocol was suitable for investigating the genetic variation of sunflower using mature leaf DNA.
Assuntos
DNA de Plantas/genética , Genoma de Planta , Helianthus/genética , DNA de Plantas/isolamento & purificação , Folhas de Planta/genética , Variação Genética , Helianthus/crescimento & desenvolvimento , Mutação , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
Five published DNA extraction protocols were compared for their ability to produce good quality DNA from fresh and herbarium leaves of several species of the genus Dalbergia. The leaves of these species contain high amounts of secondary metabolites, which make it difficult to perform a clean DNA extraction and thereby interfering with subsequent PCR amplification. The protocol that produced the best DNA quality in most of the Dalbergia species analyzed, utilizes polyvinylpyrrolidone to bind the phenolic compounds, a high molar concentration of NaCl to inhibit co-precipitation of polysaccharides and DNA, and LiCl for removing RNA by selective precipitation. The DNA quality of herbarium specimens was worse than that for fresh leaves, due to collecting conditions and preservation of samples. We analyzed 54 herbarium specimens, but the recovered DNA allowed successful PCR amplification in only eight. For the genus Dalbergia, the herbarium is an important source of material for phylogenetic and evolutionary studies; due to the occurrence of the different species in various geographical regions in Brazil, it is difficult to obtain fresh material in nature. Our results demonstrated that for Dalbergia species the methods used for the collection and preservation of herbarium specimens have a mayor influence on DNA quality and in the success of phylogenetic studies of the species
Assuntos
Bancos de Espécimes Biológicos , DNA de Plantas/isolamento & purificação , Dalbergia/química , Folhas de Planta/química , Dalbergia/genética , Filogenia , Folhas de Planta/genética , Reação em Cadeia da Polimerase , Manejo de Espécimes/métodosRESUMO
For studying genetic diversity in natural populations of Terminalia, a medicinal plant, our attempts to isolate high quality DNA using several previously reported protocols and even modifications were unsuccessful. We therefore combined CTAB based isolation, and column based purification step, to isolate DNA from Terminalia arjuna. The DNA isolated using this standardized protocol was high in quality and suitable for restriction digestion and generation of random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP).
Assuntos
DNA de Plantas/isolamento & purificação , Variação Genética , Polimorfismo de Fragmento de Restrição , Técnica de Amplificação ao Acaso de DNA Polimórfico , Terminalia/genética , DNA de Plantas/genética , Compostos de Cetrimônio , Marcadores Genéticos , Genética Populacional , Plantas Medicinais/genética , Reprodutibilidade dos TestesRESUMO
OBJECTIVE@#The feasibility of Papaver somniferum L. cultivars identification was explored by TD-RAPD technique.@*METHOD@#Genomic DNA was extracted by improved CTAB method. One sample of species from Papaver somniferum L in xishuangbanna area. was studied by using TD-RAPD method.@*RESULT@#We established an optimal method of extracting genomic DNA. Six primers were picked out from 10 primers.@*CONCLUSION@#TD-RAPD could be applied to researches of molecular marker of Papaver somniferum L. TD-RAPD technique provide a method to constitute DNA database of Papaver somniferum L. and conclude the source of opium poppy.
Assuntos
Humanos , Primers do DNA , DNA de Plantas/isolamento & purificação , Estudos de Viabilidade , Papaver/genética , Folhas de Planta/genética , Reação em Cadeia da Polimerase , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Especificidade da EspécieRESUMO
A CDNA library was constructed using mRNA isolated from mature corm tissue of taro (Colocasia esculenta). By differential screening, four cDNA clones, pCE1, pCE2, pCE3 and pCE4, complementary to moderately abundant corm mRNAs, were selected. These were used as probes to study the expression of the corresponding genes in different taro tissue. Northern analysis of transcripts indicated that their expression is highly enhanced in the corm and that they encode mRNAs with 0.70 kb, 0.80 kb, 0.75 kb and 1.20 kb, respectively. Dot blot hybridizations revealed that clones pCE1 to 4 bear inserts homologous to mRNAs that accumulate to 1.5 percent, 1.0 percent, 0.40 percent and 0.20 percent respectively, of the total poly (A)+ mRNA present in mature corms. The four genes are differentially regulated in taro tissue. Their transcripts were detected at lower levels in the steady state mRNA of petiole, lamina and roots, except in the case of pCE3 whose mRNA could not be detected neither in petiole nor in lamina.