Joyce Cartagena

Sequence analysis of the genome of an oil-bearing tree, Jatropha curcas L.

The whole genome of Jatropha curcas was sequenced, using a combination of the conventional Sanger method and new-generation multiplex sequencing methods. Total length of the non-redundant sequences thus obtained was 285 858 490 bp consisting of 120 586 contigs and 29 831 singlets. They accounted for ∼95% of the gene-containing regions with the average G + C content was 34.3%. A total of 40 929 complete and partial structures of protein encoding genes have been deduced. Comparison with genes of other plant species indicated that 1529 (4%) of the putative protein-encoding genes are specific to the Euphorbiaceae family. A high degree of microsynteny was observed with the genome of castor bean and, to a lesser extent, with those of soybean and Arabidopsis thaliana. In parallel with genome sequencing, cDNAs derived from leaf and callus tissues were subjected to pyrosequencing, and a total of 21 225 unigene data have been generated. Polymorphism analysis using microsatellite markers developed from the genomic sequence data obtained was performed with 12 J. curcas lines collected from various parts of the world to estimate their genetic diversity. The genomic sequence and accompanying information presented here are expected to serve as valuable resources for the acceleration of fundamental and applied research with J. curcas, especially in the fields of environment-related research such as biofuel production. Further information on the genomic sequences and DNA markers is available at http://www.kazusa.or.jp/jatropha/.

The Arabidopsis SDG4 contributes to the regulation of pollen tube growth by methylation of histone H3 lysines 4 and 36 in mature pollen

Plant SET domain proteins are known to be involved in the epigenetic control of gene expression during plant development. Here, we report that the Arabidopsis SET domain protein, SDG4, contributes to the epigenetic regulation of pollen tube growth, thus affecting fertilization. Using an SDG4-GFP fusion construct, the chromosomal localization of SDG4 was established in tobacco BY-2 cells. In Arabidopsis, sdg4 knockout showed reproductive defects. Tissue-specific expression analyses indicated that SDG4 is the major ASH1-related gene expressed in the pollen. Immunological analyses demonstrated that SDG4 was involved in the methylation of histone H3 in the inflorescence and pollen grains. The significant reduction in the amount of methylated histone H3 K4 and K36 in sdg4 pollen vegetative nuclei resulted in suppression of pollen tube growth. Our results indicate that SDG4 is capable of modulating the expression of genes that function in the growth of pollen tube by methylation of specific lysine residues of the histone H3 in the vegetative nuclei.

Adventitious shoot regeneration from juvenile cotyledons of a biodiesel producing Plant, Jatropha curcas L

An efficient and rapid adventitious shoot regeneration system from young cotyledons of Jatropha curcas L. was developed. After testing several combinations of plant growth regulators, the highest regeneration frequency was obtained for the medium supplemented with 3 mg/l BA and 0.1 mg/l IBA. This system will be useful for promoting genetic improvement efforts in J. curcas.

Characterization of telomere-subtelomere junctions in Silene latifolia

Telomere-associated regions represent boundaries between the relatively homogeneous telomeres and the subtelomeres, which show much greater heterogeneity in chromatin structure and DNA composition. Although a major fraction of subtelomeres is usually formed by a limited number of highly repeated DNA sequence families, their mutual arrangement, attachment to telomeres and the presence of interspersed unique or low-copy-number sequences make these terminal domains chromosome specific. In this study, we describe the structures of junctions between telomeres and a major subtelomeric repeat of the plant Silene latifolia, X43.1. Our results show that on individual chromosome arms, X43.1 is attached to the telomere either directly at sites corresponding to nucleosome boundaries previously mapped in this sequence, or via other spacer sequences, both previously characterized and newly described ones. Sites of telomere junctions are non-random in all the telomere-associated sequences analysed. These data obtained at the molecular level have been verified using in situ hybridization to metaphase chromosomes and extended DNA fibres.

Physical wounding-assisted Agrobacterium-mediated transformation of juvenile cotyledons of a biodiesel-producing plant, Jatropha curcas L.

The non-edible plant Jatropha curcas L. is one of the most promising feedstock for sustainable biodiesel production as it is not a source of edible vegetable oils, produces high amounts of oil (approx. 30–60% in dry seeds) and does not require high-cost maintenance. However, as with other undomesticated crops, the cultivation of J. curcas presents several drawbacks, such as low productivity and susceptibility to pests. Hence, varietal improvement by genetic engineering is essential if J. curcas is to become a viable alternative source of biodiesel. There is to date no well-established and efficient transformation system for J. curcas. In this study, we tested various physical wounding treatments, such as sonication and sand-vortexing, with the aim of developing an efficient Agrobacterium-mediated transformation for J. curcas. The highest stable transformation rate (53%) was achieved when explants were subjected to 1 min of sonication followed by 9 min of shaking in Agrobacterium suspension. The transformation frequency achieved using this protocol is the highest yet reported for J. curcas.

Bioactive Bead-Mediated Transformation of Plants with Large DNA Fragments

The efficient transformation of plants with large DNA molecules containing a set of useful genes would provide vast possibilities for the genetic improvement of agricultural as well as nonagricultural plants. The development of the bioactive beads (BABs) transformation method has proven useful for introduction of large DNA molecules into plant cells. In this chapter, the BABs transformation method used for the transformation of a 100-kb BAC DNA construct containing wheat genes into rice will be presented. Furthermore, the improved production method for BABs will be described. With the conventional method for producing BABs, the bead size varies, and the larger beads tend to carry fewer DNA molecules than the smaller beads. Thus, in order to facilitate the preparation of BABs with more uniform sizes, a simple set-up -composed of a sine wave sound generator and microsyringe pump was fabricated. Using this bead-maker set-up, uniform and smaller beads could be produced which enhance the transformation efficiency.

Bioactive Beads-Mediated Transformation of Rice with Large DNA Fragments Containing Aegilops tauschii Genes, with Special Reference to Bead-Production Methodology

Plant transformation is a technique that allows us to transfer genes from one species to another in order to introduce new characteristics into the recipient. The plant transformation technique has become widely adopted as a method both to understand plant physiology and to improve plant characteristics. There are now many established gene-transfer methods, both direct and indirect, for the stable introduction of novel genes into plant species. Examples include Agrobacterium-mediated transformation, particle bombardment (biolistic), and protoplast electroporation (Klein and Fitzpatrick-McElligott, 1993; Tzfira and Citovsky, 2006). Recently, a direct gene-transfer method called bioactive beads (BABs)mediated transformation has been developed (Sone et al., 2002; Wada et al., 2011a, 2011b). This method involves immobilization of DNA molecules on alginate beads and their transfer to plant cells with the assistance of a polyethylene glycol (PEG) solution. Alginate, a hydrophilic polysaccharide that solidifies in the presence of Ca2+ ions, is utilized as a barrier membrane to produce calcium-alginate beads for immobilizing high-density DNA molecules. The original procedure for bead production was described in Sone et al. (2002) (Fig. 1). Firstly, a plasmid DNA solution was mixed with a CaCl2 solution. Isoamyl alcohol was then added to a 1.5 ml micro-tube containing an aqueous phase 1% sodium alginate solution to form a water/oil mixture that was emulsified by sonication using an ultrasonic disrupter (UR-20P; Tomy Seiko, Tokyo, Japan) for 10 s. Bioactive beads (BABs) encapsulating DNA molecules were generated after immediately adding a CaCl2 solution containing plasmid DNA into the emulsified solution. The DNA-immobilizing BABs were utilized in combination with PEG solution for protoplast transformation.