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Genetic and Phenotypic Analyses of a Papaver somniferum T-DNA Insertional Mutant with Altered Alkaloid Composition  [PDF]
Noriaki Kawano,Fumiyuki Kiuchi,Nobuo Kawahara,Kayo Yoshimatsu
Pharmaceuticals , 2012, DOI: 10.3390/ph5020133
Abstract: The in vitro shoot culture of a T-DNA insertional mutant of Papaver somniferum L. established by the infection of Agrobacterium rhizogenes MAFF03-01724 accumulated thebaine instead of morphine as a major opium alkaloid. To develop a non-narcotic opium poppy and to gain insight into its genetic background, we have transplanted this mutant to soil, and analyzed its alkaloid content along with the manner of inheritance of T-DNA insertion loci among its selfed progenies. In the transplanted T 0 primary mutant, the opium (latex) was found to be rich in thebaine (16.3% of dried opium) by HPLC analysis. The analyses on T-DNA insertion loci by inverse PCR, adaptor-ligation PCR, and quantitative real-time PCR revealed that as many as 18 copies of T-DNAs were integrated into a poppy genome in a highly complicated manner. The number of copies of T-DNAs was decreased to seven in the selected T 3 progenies, in which the average thebaine content was 2.4-fold that of the wild type plant. This may indicate that the high thebaine phenotype was increasingly stabilized as the number of T-DNA copies was decreased. In addition, by reverse transcription PCR analysis on selected morphine biosynthetic genes, the expression of codeine 6- O-demethylase was clearly shown to be diminished in the T 0 in vitro shoot culture, which can be considered as one of the key factors of altered alkaloid composition.
Molecular Analysis and Genomic Organization of Major DNA Satellites in Banana (Musa spp.)  [PDF]
Jana ?í?ková, Eva H?ibová, Lenka Humplíková, Pavla Christelová, Pavla Suchánková, Jaroslav Dole?el
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0054808
Abstract: Satellite DNA sequences consist of tandemly arranged repetitive units up to thousands nucleotides long in head-to-tail orientation. The evolutionary processes by which satellites arise and evolve include unequal crossing over, gene conversion, transposition and extra chromosomal circular DNA formation. Large blocks of satellite DNA are often observed in heterochromatic regions of chromosomes and are a typical component of centromeric and telomeric regions. Satellite-rich loci may show specific banding patterns and facilitate chromosome identification and analysis of structural chromosome changes. Unlike many other genomes, nuclear genomes of banana (Musa spp.) are poor in satellite DNA and the information on this class of DNA remains limited. The banana cultivars are seed sterile clones originating mostly from natural intra-specific crosses within M. acuminata (A genome) and inter-specific crosses between M. acuminata and M. balbisiana (B genome). Previous studies revealed the closely related nature of the A and B genomes, including similarities in repetitive DNA. In this study we focused on two main banana DNA satellites, which were previously identified in silico. Their genomic organization and molecular diversity was analyzed in a set of nineteen Musa accessions, including representatives of A, B and S (M. schizocarpa) genomes and their inter-specific hybrids. The two DNA satellites showed a high level of sequence conservation within, and a high homology between Musa species. FISH with probes for the satellite DNA sequences, rRNA genes and a single-copy BAC clone 2G17 resulted in characteristic chromosome banding patterns in M. acuminata and M. balbisiana which may aid in determining genomic constitution in interspecific hybrids. In addition to improving the knowledge on Musa satellite DNA, our study increases the number of cytogenetic markers and the number of individual chromosomes, which can be identified in Musa.
Optimization of DNA isolation and PCR protocol for RAPD analysis of banana / plantain (Musa spp.)
Das B.K.,Jena R. C.,Samal K.C.
International Journal of Agriculture Sciences , 2009,
Abstract: Genetic analysis of plants relies on high yields of pure DNA samples. Here we present the optimizationof DNA isolation and Polymerase chain reaction (PCR) conditions for Random Amplified Polymorphic DNA (RAPD)analysis of banana/plantain. The leaf of banana contains high level of polysaccharides, poly phenols and secondarymetabolites. The extracted DNA from these cultivars when subjected to PCR is often problematic, especially whenmature tissues are used for DNA extraction. In order to overcome these problems a protocol has been developed,availing on a high salt concentration and on the combination of Polyvinyl pyrrolidone (PVP) and Cetyl trimethylammonium bromide (CTAB) in the extraction buffer, in order to prevent the solubilization of polysaccharides andpolyphenols during the DNA extraction method. It also involves successive long term chloroform: Isoamylalcoholextractions, an long term RNAse treatment with all steps carried out at Room temperature (RT). Using this method,DNA was extracted from different banana species including young leaves, old leaves, frosted old leaves andwithered old leaves. The yield of DNA ranged from 1-2 μg / μl per gram of the leaf sample / tissue and the purityratio was between 1.6-1.7 indicating minimal levels of contaminating metabolites. The technique is ideal for isolationof DNA from different plant species / cultivars and the isolated DNA were used for RAPD analysis. The optimizationof RAPD protocol was based on the use of 50 ng of template DNA, higher concentration of MgCl2 (3 mM) andlower concentration of primer (0.6μM), Taq DNA polymerase (1.5 units) and an annealing temperature of 35oC,which resulted, optimal amplification. In all PCR reactions Reproducible amplifiable products were observed. Thusthe results indicate that the optimized protocol for DNA isolation and PCR was applicable to plant species belongingto different genera and this process is suitable for further work on diversity analysis. Furthermore, here we usedsuitable DNA isolation protocol for RAPD analysis to study the genetic variation in the future in Musaceae speciesgrown in Orissa.
Infection by agnoprotein-negative mutants of polyomavirus JC and SV40 results in the release of virions that are mostly deficient in DNA content
Ilker K Sariyer, Abdullah S Saribas, Martyn K White, Mahmut Safak
Virology Journal , 2011, DOI: 10.1186/1743-422x-8-255
Abstract: Analysis of both viral protein expression and replication using Pt mutant of each virus revealed that both processes were substantially down-regulated in the absence of agnoprotein compared to wild-type (WT) virus. Complementation studies in cells, which are constitutively expressing JCV agnoprotein and transfected with the JCV Pt mutant genome, showed an elevation in the level of viral DNA replication near to that observed for WT. Constitutive expression of large T antigen was found to be not sufficient to compensate the loss of agnoprotein for efficient replication of neither JCV nor SV40 in vivo. Examination of the viral release process for both JCV and SV40 Pt mutants showed that viral particles are efficiently released from the infected cells in the absence of agnoprotein but were found to be mostly deficient in viral DNA content.The results of this study provide evidence that agnoprotein plays an important role in the polyomavirus JC and SV40 life cycle. Infection by agnoprotein-negative mutants of both viruses results in the release of virions that are mostly deficient in DNA content.A large number of studies indicate that the small regulatory proteins of many viruses play important roles in various aspects of viral infection cycle, including replication [1-3], transcription [4-10], translation [11], export of viral transcripts from nucleus to cytoplasm [12], viral assembly [13] and release of viral particles [14,15]. In addition, these proteins may also modulate host-cell functions by deregulating the expression of key cellular genes [16]. Therefore, such regulatory proteins are important for successful completion of the viral life cycle and study of their regulatory roles in viral life cycle is critically important for understanding of the viral replication process and the disease progression that respective viruses induce in their host.The late coding region of human polyomavirus JC (JCV) and simian virus 40 (SV40) encodes a small regulatory phosphoprotein
Kun Li,Shiyu Fu,Huaiyu Zhan,Yao Zhan
BioResources , 2010,
Abstract: An analysis of the chemical composition and anatomical structure of banana pseudo-stem was carried out using Light Microscopy (LM), Scanning Electron Microscopy (SEM), and Confocal Laser Scanning Microscopy (CLSM). The chemical analysis indicated there is a high holocellulose content and low lignin content in banana pseudo-stem compared with some other non-wood fiber resources. These results demonstrate that the banana pseudo-stem has potential value for pulping. In addition, we report for the first time from using LM and CLSM that banana stems possess a structure involving helicoidal fibers separated by barrier films.
Characterization and isolation of a T-DNA tagged banana promoter active during in vitro culture and low temperature stress
Efrén Santos, Serge Remy, Els Thiry, Saskia Windelinckx, Rony Swennen, László Sági
BMC Plant Biology , 2009, DOI: 10.1186/1471-2229-9-77
Abstract: Around 16,000 transgenic cell colonies were screened for baseline luciferase activity at room temperature 2 months after transformation. After discarding positive colonies, cultures were re-screened in real-time at 26°C followed by a gradual decrease to 8°C. The baseline activation frequency was 0.98%, while the frequency of low temperature-responsive luciferase activity was 0.61% in the same population of cell cultures. Transgenic colonies with luciferase activity responsive to low temperature were regenerated to plantlets and luciferase expression patterns monitored during different regeneration stages. Twenty four banana DNA sequences flanking the right T-DNA borders in seven independent lines were cloned via PCR walking. RT-PCR analysis in one line containing five inserts allowed the identification of the sequence that had activated luciferase expression under low temperature stress in a developmentally regulated manner. This activating sequence was fused to the uidA reporter gene and back-transformed into a commercial dessert banana cultivar, in which its original expression pattern was confirmed.This promoter tagging and real-time screening platform proved valuable for the identification of novel promoters and genes in banana and for monitoring expression patterns throughout in vitro development and low temperature treatment. Combination of PCR walking techniques was efficient for the isolation of candidate promoters even in a multicopy T-DNA line. Qualitative and quantitative GUS expression analyses of one tagged promoter in a commercial cultivar demonstrated a reproducible promoter activity pattern during in vitro culture. Thus, this promoter could be used during in vitro selection and generation of commercial transgenic plants.The new generations of transgenic plants require more precisely regulated expression of transferred genes, which calls for the identification and characterization of novel promoters in higher plants. Species-specific promoters can be
Karyotypic and 2C Nuclear DNA Size Instability in vitro Induced Off-Types of East African Highland Banana (Musa AAA East Africa)  [PDF]
Theodosy Msogoya,Brian Grout,Andy Roberts
Biotechnology , 2008,
Abstract: This study was conducted to determine chromosome number and 2C nuclear DNA content in tissue culture induced off-type banana (Musa AAA East Africa) landrace Uganda with tolerance black sigatoka disease, susceptibility to water stress, sparsely black-blotched pseudostems, taller pseudostems, late fruit maturation, altered inflorescence and higher fruit dry matter content. The off-type banana appeared to have higher (p<0.05) frequency of 31 and 32 chromosomes at 15.1 and 13.6%, respectively. Conversely, the frequency of 31 and 32 chromosomes was 12.0 and 9.6% for the micropropagation (MP) derived phenotypically normal plants and 11.8 and 9.5% for the Conventionally Propagation (CP) derived plants with no tissue culture history. Moreover, the off-type banana had lower (p<0.05) leaf 2C nuclear DNA amount of 1.72 pg, whilst the MP and CP derived plants had 1.81 and 1.82 pg, respectively.
Nuclear DNA content of thirty species of Neotropical fishes
Carvalho, Margarida Lima;Oliveira, Claudio;Foresti, Fausto;
Genetics and Molecular Biology , 1998, DOI: 10.1590/S1415-47571998000100009
Abstract: the present paper reports nuclear dna content in 30 neotropical freshwater fish species and summarizes the data on other neotropical species presented in the literature. among neotropical fishes, the nuclear dna content ranges from 1.04 ± 0.09 pg/nucleus in corydoras cf. simulatus (2n = 62) to 248.0 pg/nucleus in lepidosiren paradoxa (2n = 38). a general analysis of the data obtained in the present study for each species showed that dna measurements were practically constant at the individual level, while significant differences were observed among individuals of the same population. this observation was valid for all species analyzed and was more evident in those species that presented other karyotypic particularities such as sex chromosomes or supernumerary chromosomes. the importance of changes in nuclear dna content in the evolutionary process of neotropical fishes is discussed.
Nuclear DNA content of thirty species of Neotropical fishes  [cached]
Carvalho Margarida Lima,Oliveira Claudio,Foresti Fausto
Genetics and Molecular Biology , 1998,
Abstract: The present paper reports nuclear DNA content in 30 Neotropical freshwater fish species and summarizes the data on other Neotropical species presented in the literature. Among Neotropical fishes, the nuclear DNA content ranges from 1.04 ± 0.09 pg/nucleus in Corydoras cf. simulatus (2n = 62) to 248.0 pg/nucleus in Lepidosiren paradoxa (2n = 38). A general analysis of the data obtained in the present study for each species showed that DNA measurements were practically constant at the individual level, while significant differences were observed among individuals of the same population. This observation was valid for all species analyzed and was more evident in those species that presented other karyotypic particularities such as sex chromosomes or supernumerary chromosomes. The importance of changes in nuclear DNA content in the evolutionary process of Neotropical fishes is discussed.
Application of ASI systematic approach on Panyu banana orchard soils

LI Guo-Liang,YAO Li-Xian,FU Chang-Ying,HE Zhao-Huan,TU Shi-Hua,

中国生态农业学报 , 2008,
Abstract: The limiting factors of soil nutrient in three different banana orchard farms, Dongchong, Lingshan and Wanqingsha, in Panyu District, Guangzhou City, were identified by systematic application of indicator plant (banana) and diagnostic soil analysis (soil adsorption trial and pot experiment). Results show lack of soil N in all three banana orchards in the study area. Compared with OPT (Optimal Treatment), yield of N treatment markedly decreases by 59.7%, 46.7% and 55.1% in Dongchong, Lingshan and Wanqingsha respectively. Banana K and Mg content is low, while B is high in the Dongchong and Lingshan banana orchards. P and K are lacking in soils in Wanqingsha banana orchard. However, soil Mn content in the three banana orchard farms is sufficient.
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