No abstract

Scutellaria ocmulgee (Ocmulgee skullcap) is a rare plant species with medicinal and ornamental value and requires immediate conservation. We report here the first protocol for plant regeneration and Agrobacterium-mediated transformation of S. ocmulgee using leaf and shootderived transverse thin cell layer (tTCL) explants. The effect of MS and B5 salts in combination with varying levels of growth regulators and two carbon sources on shoot proliferation and plant regeneration were studied. Among the various media treatment combinations, the best shoot induction response was observed from leaf-derived tTCL explants on B5 medium supplemented with 6-benzyl aminopurine (BAP), 1-naphthalene acetic acid (NAA), and sucrose whereas shoot-derived tTCL explants produced the maximum number of shoots also on B5 medium supplemented with either BAP or thidiazuron, NAA and maltose. Shoots obtained from both types of explants were rooted on MS medium containing 5.0 lM indole butyric acid. Agrobacterium-mediated genetic transformation protocol was optimized using leaf tTCL explants. A binary plasmid pq35SGR harboring the b glucuronidase and green fluorescent protein/neomycin phosphotransferase II fusion reporter gene was used to optimize transformation parameters. Explants were co-cultivated with Agrobacterium tumefaciens EHA105 and then transferred to shoot induction and regeneration medium to generate transgenic plants. Stable gene expression was observed in transgenic cultures and plants. The presence and integration of transgenes was confirmed by polymerase chain reaction, reverse transcriptase-polymerase chain reaction and Southern blot hybridization.

Paulownia elongata is a short-rotation fast growing tree and is known for high biomass accumulation and carbon sequestration potential. Optimization of protocols for nucleic acid extraction, PCR, RT-PCR, and other molecular biology techniques are required for better understanding of cellulose synthesis and to assess the potential of Paulownia as a biofuel tree. The main objective of this work was to study a putative cellulose synthase amplicon expression under various environmental conditions and evaluate the potentials of Paulownia as a biofuel tree. Using cross-species PCR an amplicon representative of a putative cellulose synthase gene from Paulownia was identified. This 177-bp long DNA sequence was 46% similar with cellulose synthase genes from Arabidopsis as expected. Gene specific primers for this particular Paulownia cellulose synthase gene were designed and reverse transcription PCR was performed to confirm its transcription. We report an inexpensive cDNA dot-blot method to study expression of this gene under various environmental conditions. We observed that cold and, to a lesser extent, heat stress downregulated its expression. This information will help to understand cellulose deposition in plant cell wall under stressful conditions. To the best of our knowledge this is the first characterization of a cDNA sequence from Paulownia elongata.

Paulownia (Paulownia elongata) is a fast growing, multipurpose deciduous hardwood species that grows in a wide range of temperatures from minus 30 degrees celsius to 45 degrees celsius. Seasonal cues influence the secondary growth of tree stems, including cambial activity, wood chemistry, and transition to latewood formation. In this study, a de novo transcriptome approach was conducted to identify the transcripts expressed in vascular cambial tissue from senescent winter and actively growing spring seasons. Illumina paired-end sequenced cambial transcriptome generated 297,049,842 clean reads which finally yielded 61,639 annotated unigenes. Based on non-redundant protein database analyses, Paulownia cambial unigenes shared highest homology (64.8%) with Erythranthe guttata. A total of 35,471 unigenes resulted from KEGG annotation that were mapped to 128 pathways with metabolic pathways dominated among all. Additionally, DEG analysis showed that 2,688 and 7,411 genes were significantly upregulated and downregulated, respectively in spring compared to winter. Interestingly, quite a number of transcripts belonging to heat shock proteins were upregulated in spring season. qPCR expression results of fifteen wood-forming candidate genes involved in hemicellulose, cellulose, lignin, auxin and cytokinin pathways showed that the hemicellulose genes (CSLC4, FUT1, AXY4, GATL1, and IRX19) were significantly upregulated in spring season tissues when compared to winter tissues. In contrast, lignin pathway genes CCR1 and CAD1 were upregulated in winter cambium. Finally, a transcriptome-wide marker analysis identified 11,338 Simple Sequence Repeat (SSRs). The AG/CT dinucleotide repeat predominately represented all SSRs. Altogether, the cambial transcriptomic analysis reported here highlights the molecular events of wood formation during winter and spring. The identification of candidate genes involved in the cambial growth provides a roadmap of wood formation in Paulownia and other trees for the seasonal growth variation.

Paulownia (Paulownia elongata) is a fast-growing, multipurpose deciduous hardwood species that grows in a wide range of temperatures from –30 °C to 45 °C. Seasonal cues influence the secondary growth of tree stems, including cambial activity, wood chemistry, and transition to latewood formation. In this study, a de novo transcriptome approach was conducted to identify the transcripts expressed in vascular cambial tissue from senescent winter and actively growing spring seasons. An Illumina paired-end sequenced cambial transcriptome generated 297,049,842 clean reads, which finally yielded 61,639 annotated unigenes. Based on non-redundant protein database analyses, Paulownia cambial unigenes shared the highest homology (64.8%) with Erythranthe guttata. KEGG annotation of 35,471 unigenes identified pathways enriched in metabolic activities. Transcriptome-wide DEG analysis showed that 2688 and 7411 genes were upregulated and downregulated, respectively, in spring tissues compared to winter. Interestingly, several transcripts encoding heat shock proteins were upregulated in the spring season. RT-qPCR expression results of fifteen wood-forming candidate genes involved in hemicellulose, cellulose, lignin, auxin, and cytokinin pathways showed that the hemicellulose genes (CSLC4, FUT1, AXY4, GATL1, and IRX19) were significantly upregulated in spring season tissues when compared to winter tissues. In contrast, lignin pathway genes CCR1 and CAD1 were upregulated in winter cambium. Finally, a transcriptome-wide marker analysis identified 11,338 Simple Sequence Repeat (SSRs). The AG/CT dinucleotide repeat predominately represented all SSRs. Altogether, the cambial transcriptomic analysis reported here highlights the molecular events of wood formation during winter and spring. The identification of candidate genes involved in the cambial growth provides a roadmap of wood formation in Paulownia and other trees for the seasonal growth variation.