Figure 1 - uploaded by Yuri Jorge Peña-Ramírez
Content may be subject to copyright.
PCR analysis of Cedrela odorata rrn16-rrn23 region and localization of primers. A. Agarose gel electrophoresis . Lane 1 was loaded with the amplification product (~5700 bp) amplified using the 16S-F/IRB23S primers and corresponding to the C. odorata chloroplast region used as flankers for transgene insertion. B. Lane 2 was loaded with the PCR product of the amplification of region rrn16-trnI using the CLB-1/CLB-2 primers. Lane 3 was loaded with the PCR product of the amplification of region trnA-rrn23 using the CLB-3/CLB-4 primers . Lane 4 was loaded with the PCR product of the amplification of region 3' end rrn16 to 5' end rrn23 using the CLB-6/CLB-7 primers . Lane molecular weight markers in A) and B) corresponds to a 1 kbp ladder. C. Graphic representation of the rrn16-rrn23 region showing the name and position of the primers used in this study. The position of the 16S-F primer was upstream of the sequence at a non-determined position (ND). The horizontal bar indicates the GC% content according to the reference bar shown at the bottom. Braces below sequences indicate the accession numbers for regions rrn16-trnI and trnA-rrn23 .
Source publication
The forest tree Spanish cedar (Cedrela odorata L.) is well-known for its high-value timber; however, this species is attacked by the shoot borer (Hypsipyla grandella) during its early years of development, resulting in branched stems and making the plants useless for high-quality wood production. The generation of resistant varieties expressing ent...
Similar publications
Mahogany is the collective international trade name for the high-value tropical and subtropical timber tree species of the family Meliaceae. Mahogany species are noted for their deep red-brown heartwood and are widely used in the construction, boat building, interior decoration (particularly paneling and floor tiles), and in the manufacture of furn...
Desde el año 2002 la Universidad Nacional Agraria La Molina (UNALM) asume el compromiso de ser Autoridad Científica de la Convención sobre el comercio internacional de especies amenazadas de flora y fauna silvestre (CITES) para flora maderable, donde diseña una estrategia de trabajo para conocer las poblaciones de las especies de Swietenia macrophy...
Determination of favorable light habitat condition per species and life stage is transcendental for both ex situ and in situ conservation strategies of endangered forest tree species, and for their utilization as plantation trees. This becomes especially important when planting material is scarce. Here, we studied the responses in biomass allocatio...
Concentration dynamics and nutrient accumulation in above ground biomass components of Cedrela odorata L. in Costa Rica. Present study was conducted aiming to study nutrient concentration and accumulation dyna mics on Cedrela odorata L. (Meliaceae) trees to improve knowledge and management on tropical forestry plantations. False time series (chrono...
Citations
... Conversely, as a consequence of gene transfer events, the original functional gene sequence of ndhF was relocated to the exon region of nad5. The rrn16-rrn23 regions were used to construct transformation vectors, indicating that the rrn16-rrn23 chloroplast region might affect transformation efficiency (Lopez-Ochoa et al., 2015). The lost tRNAs in mitogenomes can be replaced by tRNAs inserted into other organelles (Sloan et al., 2010). ...
Introduction
Prunus pedunculata (Prunoideae: Rosaceae), a relic shrub with strong resistance and multiple application values, is endangered in China. Extensive research had been devoted to gene expression, molecular markers, plastid genome analysis, and genetic background investigations of P. pedunculata. However, the mitochondrial genome of this species has not been systematically described, owing to the complexity of the plant mitogenome.
Methods
In the present research, the complete mitochondrial genome of P. pedunculata was assembled, annotated, and characterized. The genomic features, gene content and repetitive sequences were analyzed. The genomic variation and phylogenetic analysis have been extensively enumerated.
Results and discussion
The P. pedunculata mitogenome is a circular molecule with a total length of 405,855 bp and a GC content of 45.63%, which are the smallest size and highest GC content among the known Prunus mitochondrial genomes. The mitogenome of P. pedunculata encodes 62 genes, including 34 unique protein-coding genes (PCGs, excluding three possible pseudogenes), three ribosomal RNA genes, and 19 transfer RNA genes. The mitogenome is rich in repetitive sequences, counting 112 simple sequence repeats, 15 tandem repeats, and 50 interspersed repetitive sequences, with a total repeat length of 11,793 bp, accounting for 2.91% of the complete genome. Leucine (Leu) was a predominant amino acid in PCGs, with a frequency of 10.67%, whereas cysteine (Cys) and tryptophan (Trp) were the least adopted. The most frequently used codon was UUU (Phe), with a relative synonymous codon usage (RSCU) value of 1.12. Selective pressure was calculated based on 20 shared PCGs in the mitogenomes of the 32 species, most of which were subjected to purifying selection (Ka/Ks < 1), whereas ccmC and ccmFn underwent positive selection. A total of 262 potential RNA editing sites in 26 PCGs were identified. Furthermore, 56 chloroplast-derived fragments were ascertained in the mitogenome, ranging from 30 to 858 bp, and were mainly located across IGS (intergenic spacer) regions or rRNA genes. These findings verify the occurrence of intracellular gene transfer events from the chloroplast to the mitochondria. Furthermore, the phylogenetic relationship of P. pedunculata was supported by the mitogenome data of 30 other taxa of the Rosaceae family. Understanding the mitochondrial genome characteristics of P. pedunculata is of great importance to promote comprehension of its genetic background and this study provides a basis for the genetic breeding of Prunus.
... In the current experiment, we successfully screened the transplastomic lines of S. dulcis using an efficient selection process on spectinomycin antibiotic. Stable integration of chloroplast transformation was achieved in tobacco, lettuce, Cedrelaodorata, Capsicum and Marchantia polymorpha L. using the insertion sites of trnA and trnI fragments from IR region (Bock and Maliga 1995;Chiyoda et al. 2007;K Mühlbauer et al. 2002;Lelivelt et al. 2005;López-Ochoa et al. 2015;Staub and Maliga 1992). In our lab, the same trnA/trnI of IR region was targeted to recover transplastomic lines of Momordica charantia by a Cucumis sativus based heterologous vector (CuIA) (Narra et al. 2018b) and obtained 2 transplastomic lines from 15 bombarded plates using petiole explants. ...
The high expression level of industrial and metabolically important proteins in plants can be achieved by plastid transformation. The CaIA vector, a Capsicum-specific vector harboring aadA (spectinomycin resistance), is a selectable marker controlled by the PsbA promoter, and the terminator is flanked by the trnA and trnI regions of the inverted repeat (IR) region of the plastid. The CaIA vector can introduce foreign genes into the IR region of the plastid genome. The biolistic method was used for chloroplast transformation in Scoparia dulcis with leaf explants followed by antibiotic selection on regeneration medium. Transplastomes were successfully screened, and the transformation efficiency of 3 transgenic lines from 25 bombarded leaf explants was determined. Transplastomic lines were evaluated by PCR and Southern blotting for the confirmation of aadA insertion and its integration into the chloroplast genome. Seeds collected from transplastomes were analyzed on spectinomycin medium with wild types to determine genetic stability. The increased chloroplast transformation efficiency (3 transplastomic lines from 25 bombarded explants) would be useful for expressing therapeutically and industrially important genes in Scoparia dulcis L.
Keywords : Scoparia dulcis L., Heterologous vectors, CaIA plastid vector, Inverted repeat region, Transplastomic lines
... The first ever high-frequency plastid transformation was successfully established in Nicotiana tabacum (Svab et al. 1990) and many other reports revealed its suitability as model experimental plant system for chloroplast transformation (Gottschamel et al. 2013). Apart from tobacco, chloroplast transformation was also reported in other plants such as potato (Sidorov et al. 1999), rapeseed (Cheng et al. 2010), tomato (Ruf et al. 2001), cabbage (Liu et al. 2007), lettuce (Lelivelt et al. 2005), rice (Lee et al. 2006), Cedrela odorata (López-Ochoa et al. 2015), scoparia , and bitter melon (Narra et al. 2018). However, the development of chloroplast transformation has been extended to many other plant systems, until today, this technology remains challenging and limited to achieve due to Srinivas Kota and Raghuvardhan Lakkam contributed equally. ...
... The trnfM and trnG sites of LSC region were used to design the chloroplast transformation vector in tobacco (Bock and Maliga 1995;Madanala et al. 2012) and tomato (Ruf et al. 2001). In addition, trnA and trnI fragments from IR region were used to develop new chloroplast transformation vector in tobacco (Bock andMaliga 1995, K Mühlbauer et al. 2002;Staub and Maliga 1992), and other species such as lettuce (Lelivelt et al. 2005), Marchantia polymorpha L. (Chiyoda et al. 2007), and Cedrela odorata (López-Ochoa et al. 2015). Construction of speciesspecific plastid vectors is not always necessary, as the plastid genome is very conserved among different plant species, but they will significantly increase transformation efficiency (Maliga 2004;Svab et al. 1990). ...
In the present study, we focused on designing a species-specific chloroplast vector for Capsicum annuum L. and finding out its transformation efficiency compared to a heterologous vector. The plastid transformation vector (CaIA) was designed to target homologous regions trnA and trnI of IR region. A selectable marker gene aadA, whose expression is controlled by psbA promoter and terminator, was cloned between two flanking regions. A heterologous vector pRB95, which targets trnfM and trnG of LSC region along with aadA driven by rrn promoter and psbA terminator, was also used for developing plastid transformation in Capsicum. Cotyledonary explants were bombarded with stabilized biolistic parameters: 900 psi pressure and 9 cm flight distance, and optimized regeneration protocol (0.7 mg/L TDZ + 0.2 mg/L IAA) was used to obtain transplastomic lines on selection medium (300 mg/L spectinomycin). The aadA integration and homoplasmy were confirmed by obtaining 1.2 and 3.7 kb amplicons in CaIA transformants and subsequently verified by Southern blotting, whereas in pRB95 transformants, integration was confirmed by PCR with 1.45 kb and 255 bp amplicons corresponding to aadA integration and flanks, respectively. The transformation efficiencies attained with two plastid vectors were found to be 20%, i.e., 10 transplastomic lines in 50 bombarded plates, with CaIA and 2%, i.e., 1 transplastomic line in 50 bombarded plates, with heterologous pRB95, respectively.
... Recently, Lopez-Ochoa et al. (2015) characterized the chloroplast rrn16-rrn23S region for C. odorata. With this sequence, they developed a transplastomic expression vector suitable for C. odorata and other members in the Meliaceae family. ...
Cedrela odorata L. (Meliaceae) is a timber tree of high economic value. Currently, this species faces serious problems due to selective cutting and environmental degradation, which together have contributed to the decline of C. odorata populations. Establishment of C. odorata commercial plantations on a large scale could alleviate the pressure on natural populations of this species. However, C. odorata plantations are limited due to the susceptibility to the mahogany shoot borer (Hypsipyla grandella Zeller). H. grandella destroys the apical meristems and shoots causing malformations in the stem and subsequently the death of the plant. Genetic improvement of C. odorata by conventional methods is a slow process due to several factors associated with its biology: a long life cycle, recalcitrant seeds, large tree size, low genetic diversity and sexual incompatibility. Therefore, in vitro propagation is the best option for clonal multiplication and assistance to breeding programs of this species. In this review, we present the advances in in vitro tissue culture and genetic transformation of C. odorata. The review includes our own experiences and data obtained in our laboratory. The low reproducibility and regeneration efficiency suggests the recalcitrance of C. odorata. We have established a protocol of somatic embryogenesis (SE) from shoots cultured on WPM medium with 2 mg/L of zeatin. Shoots were placed on different media with zeatin to generate embryogenic callus (80%). Histological analysis revealed the formation of undifferentiated cells, cell clusters with a high rate of mitosis, and the gradual formation of pro-embryonic masses and globular embryos. Transient genetic transformation through Agrobacterium tumefaciens achieved efficiencies of 6.25 to 8.5%. In addition, 250 mg/L of kanamycin was found to be the inhibitory dose to select T0. Despite this progress, it is necessary to optimize the process of somatic embryogenesis for C. odorata. An optimized protocol will allow the development of conservation strategies and synthetic seed technology, the commercial mass propagation, genetic improvement and production of secondary metabolites for C. odorata.
The plastid transformation is used for high level expression of certain metabolically and industrially important recombinant proteins in plants. The vector, pFaadAII, a tobacco based vector system, harbouring a chimeric gene consisting of aadA coding region from Escherichia coli with 5′ 16S rDNA promoter and 3′ untranslated transcript region (UTR) of chlamydomonas rbcL gene, located in between the intergenic regions of rp132 and trnL genes. This vector used for transformation of plastids targets the foreign sequences to the small single-copy region of the plastome. Biolistic mode of approach for chloroplast transformation in Scoparia dulcis L., was achieved by bombarding the leaf explants and spectinomycin based selection system was used for regeneration of transformed plants. Transplastomic lines have been successfully established with overall efficiency of two transgenic lines for twenty-five bombarded explants. Integration of aadA in selection based regenerants was characterized by PCR and protein accumulation analysis along with seedlings experiment obtained from selfing. The chloroplast transformation developed in this plant system will provide scope for research in plastid based metabolic engineering pathways.
Chloroplast transformation vectors require an expression cassette flanked by homologous plastid sequences to drive plastome recombination. The rrn16-rrn23 plastome region was selected and using this region, a new species-specific plastid transformation vector CuIA was developed with pKS+II as a backbone by inserting the rrn16-trnI and trnA-rrn23 sequences from Cucumis sativus L. An independent expression cassette with aadA gene encoding aminoglycoside 3′-adenylyltransferase with psbA controlling elements is added into the trnI-trnA intergenic region that confers resistance to spectinomycin. An efficient plastid transformation in bitter melon (Momordica charantia L.) was achieved by bombardment of petiole segments. The frequency of transplastomic plants yielded using standardized biolistic parameters with CuIA vector was two per 15 bombarded plates, each containing 20 petiole explants. Integration of aadA gene was verified by PCR analysis in transplastomes. Transplastomic technology developed may be a novel approach for high level expression of pharmaceutical traits.
