High frequency in vitro regeneration of adventitious shoots in daylilies (Hemerocallis sp) stem tissue using thidiazuron.

BACKGROUND: Daylilies are a lucrative crop used for its floral beauty, medicinal proprieties, landscaping, fire prevention, nutritional value, and research. Despite the importance, daylilies remain extremely challenging for multiplying in vitro. The response difficulty is exacerbated because a few good protocols for daylilies micropropagation are generally difficult to reproduce across genotypes. An efficient strategy, currently applied at Langston University, is to systematically explore individual tissues or organs for their potential to micropropagation. This article is a partial report of the investigation carried out under room environmental conditions and focuses on developing an efficient daylilies in vitro propagation protocol that uses the stem tissue as the principal explant. RESULTS: In less than three months, using thidiazuron, the use of the stem tissue as the in vitro experimental explant was successful in inducing multiple shoots several folds greater than current daylilies shoot organogenesis protocols. The study showed that tissue culture can be conducted successfully under unrestricted room environmental conditions as well as under the controlled environment of a growth chamber. It also showed that splitting lengthwise stem explants formed multiple shoots several folds greater than cross-sectioned and inverted explants. Shoot conversion rate was mostly independent of the number of shoots formed per explants. The overall response was explant and genotype-dependent. Efficient responses were observed in all thidiazuron treatments. CONCLUSION: An efficient protocol, which can be applied for mass multiple shoots formation using the daylilies stem tissue as the main explant, was successfully developed. This could lead to a broad and rapid propagation of the crop under an array of environmental conditions to meet the market demand and hasten exogenous gene transfer and breeding selection processes.

De novo next-generation sequencing, assembling and annotation of Arachis hypogaea L. Spanish botanical type whole plant transcriptome.

Peanut is a major agronomic crop within the legume family and an important source of plant oil, proteins, vitamins, and minerals for human consumption, as well as animal feed, bioenergy, and health products. Peanut genomic research effort lags that of other legumes of economic importance, mainly due to the shortage of essential genomic infrastructure, tools, resources, and the complexity of the peanut genome. This is a pioneering study that explored the peanut Spanish Group whole plant transcriptome and culminated in developing unigenes database. The study applied modern technologies, such as, normalization and next-generation sequencing. It overall sequenced 8,308,655,800 nucleotides and generated 26,048 unigenes amongst which 12,302 were annotated and 8,817 were characterized. The remainder, 13,746 (52.77 %) unigenes, had unknown functions. These results will be applied as the reference transcriptome sequences for expanded transcriptome sequencing of the remaining three peanut botanical types (Valencia, Runner, and Virginia), which is currently in progress, RNA-seq, exome identification, and genomic markers development. It will also provide important tools and resources for other legumes and plant species genomic research.

A novel mRNA level subtraction method for quick identification of target-orientated uniquely expressed genes between peanut immature pod and leaf.

Subtraction technique has been broadly applied for target gene discovery. However, most current protocols apply relative differential subtraction and result in great amount clone mixtures of unique and differentially expressed genes. This makes it more difficult to identify unique or target-orientated expressed genes. In this study, we developed a novel method for subtraction at mRNA level by integrating magnetic particle technology into driver preparation and tester-driver hybridization to facilitate uniquely expressed gene discovery between peanut immature pod and leaf through a single round subtraction. The resulting target clones were further validated through polymerase chain reaction screening using peanut immature pod and leaf cDNA libraries as templates. This study has resulted in identifying several genes expressed uniquely in immature peanut pod. These target genes can be used for future peanut functional genome and genetic engineering research.

Cloning, sequence and functional analysis of goat ATP-binding cassette transporter G2 (ABCG2).

ATP-binding cassette transporter G2 (ABCG2) gene encodes a protein that has a wide variety of substrates and is responsible for the active secretion of clinically and toxicologically important molecules into milk. Although known in many species, this marks the first time this gene product has been reported in goats. In this study, we cloned and sequenced goat ABCG2 gene complete coding sequence and predicted its putative translated protein structure with implicative functional domains. One six-transmembrane span on C-terminal region and at least one coiled-coil domain on N-terminal were predicted and compared primarily with those of other closely related species. In addition, three conserved cysteines (in positions 595, 606, and 611) were determined toward the C-terminal of goat's ABCG2. Two known functional motifs were identified in goat's protein through comparative studies with other species. The goat ABCG2 relative expression profile revealed that the gene expression was a function of lactation stage and parallel to goat lactation curve.

Construction and characterization of a goat mammary gland cDNA library.

A lactating goat mammary gland cDNA library was constructed by using a modified commercially available cDNA library construction kit protocol. The resulting clones were sequenced and functionally analyzed through cross-species genomic comparison to assess (1) the capacity and functional quality of the constructed library for subsequent research and (2) the efficiency of the procedural modifications. The study resulted in the construction of a high-quality mammary gland cDNA library, which was characterized by (1) the total recombinants number of 1.4 x 10(7) colony-forming units (cfus) that was at least 10 times greater than the number expected from the application of the standard kit protocol, (2) the recombinants rate of 96%, and (3) the average insert size of 1,082 bp. BLAST analysis of sequenced clones against GenBank databases determined 55.7% of clone redundancy, 22 known function gene clusters, and 29 novel gene clusters. The analysis of the primary gene expression profile showed that 59% of the tested clones were genes that coded for milk proteins while 16% of the clones coded for ribosomal, metabolism, immune response, and translation proteins. The remaining 25% of the tested clones were described as novel genes. Cross-species comparison showed that 77% of characterized gene clusters were successfully identified by using resources from other ruminants and unrelated species. This outcome is in consonance with the common belief that the genomic resources that have been generated across species are potentially powerful tools that could be used for enhancing the molecular understanding of less genomically studied species, such as goat.

Evaluation of peanut genotypes for in vitro plant regeneration using thidiazuron.

A major limitation with the available protocols for in vitro regeneration of peanut (Arachis hypogaea L.) is their narrow application to very few select genotypes. Here, we report a protocol that can be applied across a broad spectrum of peanut market types, explant types and geographic regions using thidiazuron (TDZ). The effect of the timing of TDZ application to the culturing of both zygotic embryos and subsequent plantlet explants on MS medium is also reported. An extended use of TDZ and at a higher concentration (30 m/l) resulted in the greatest explant shoot average (approximately 13). However, a limited application of TDZ (10 d) was sufficient to induce shoot formation in peanut. Hypocotyl was the best explant type that induced the greatest shoot average (15) across market types followed by lamina (7.4). Spanish and Valencia were the most efficient market groups that induced shoots across explant types, consistently.

Normal/disease-paired cDNA library subtraction for molecular marker development.

This study consists of a novel strategy for the identification of potential cancer exclusively expressed genes, which might lead to the development of valuable diagnostic molecular markers. Normal- and cancer-paired tissues from the same patient were collected and subjected to the construction of primary complementary deoxyribonucleic acid (cDNA) libraries. The cancer cDNA library was generated by subtracting normal cDNAs from the primary cancer library. The remaining clones in the subtracted cancer library were sequenced and Basic Local Alignment Search Tool (BLAST)-analyzed against current nonredundant and est_human databases in the GenBank. The clones that had no matches with any known gene sequences except the human genomic sequences were identified as ideal candidates for potential molecular marker development. The candidate marker sequence and associated primer sequences were identified on the target clone sequence of the region with the least, or no, matched homologs. The specificity of the markers was measured by a polymerase chain reaction test experiment with the DNA templates from different human normal tissues, genomic DNA, and additional patients' tissues. Results from bioinformatical and experimental approaches used suggest that the methodology has a high potential to identify cancer exclusive transcripts. Thus, it might result in the development of diagnostic markers.

Mouse brain full-length cDNA library construction by negative selection of intact mRNAs.

Synthesis of full-length cDNA libraries is an essential step for the study of gene function. The method for selecting the intact mRNA directly affects the number of full-length transcripts. We have developed a novel method for intact mRNA selection based on the elimination of uncapped mRNAs. A negative-selection strategy that removes both uncapped mRNA and other non-mRNA molecules that present a phosphate at the 5'-end has been applied in the mRNA purification procedures. Briefly, after performing a standard mRNA purification, a biotinylated oligoribonucleotide is ligated to the 5-end phosphate of uncapped mRNAs. Streptavidin extraction is then performed to remove truncated and non-mRNAs from the intact mRNAs. By comparing random sequencing results of mouse brain full-length and standard cDNA libraries, there was a significant increase of full-length clones with the modified procedure. The results showed that the full-length library contained more than 68% full-length clones with the 5'-end positions ranging between -485 to +100 compared to the standard library with 33% of full-length clones and 5'-end positions ranging between -233 to +100. The data were analyzed using the t-test with the significance level set at p<0.05. The statistical results showed that there were significant differences between two libraries in both 5'-end position and mRNA size (p<0.05).

Enhancing DNA electrotransformation efficiency in escherichia coli DH10B electrocompetent cells

Electrotransformation also known as electroporation is the most reliable and efficient tool for plasmid DNA uptake. Electrotransformation efficiency is function of many factors which include (1) number of cell washes prior to electroporation, (2) electroporation cell number, (3) electroporation DNA amount, and (4) cell growth phase. Those factors have limitedly been concomitantly investigated in E. coli DH10B strain. This study is aimed to explore above key factors to define the optimal conditions for high electrotransformation efficiency. The results showed that electrotransformation efficiency of E. coli DH10B was enhanced to 1.5 x 10(9) cfu/ug by washing cells three times with 15 ml of 10 percent glycerol. This washed off extra salts from cell suspension and enhanced electrotransformation by preventing arcing and enhancing cell resistance while ensuring minimal level of conductivity. Early exponential phase at 0.15 OD600 was the best growth phase for enhancing electrotransformation of E. coli DH10B. The results also showed that higher electrotransformation efficiency was similarly achieved when 0.5 x 10(10) and 0.6 x 10(10) cell numbers were electroporated with DNA amount ranging from 10 to 40 pg. This study confirmed the optimal conditions for electro competent cell preparation and plasmid DNA electrotransformation, which can result highest transformation efficiency.