Utut Widyastuti

Efficient genetic transformation of Jatropha curcas L. by means of vacuum infiltration combined with filter-paper wicks.

Jatropha curcas L. (Jatropha) is a promising plant for the production of biodiesel in arid regions. To improve its productivity, it is important to understand the molecular functions of key genes in Jatropha, and to seek ways to modify important agronomic traits of Jatropha via molecular breeding. However, Jatropha is notorious for its recalcitrance to transformation. Thus, an improved protocol for efficient and reproducible Agrobacterium tumefaciens-mediated transformation is essential for molecular biology research and breeding in this species. Because resistance of Jatropha to A. tumefaciens infection is one of the key factors limiting transformation efficiency, we attempted to improve infection efficiency by comparing various co-culture conditions with the use of a β-glucuronidase (GUS) assay. The LBA4404 strain of A. tumefaciens was more efficient than the EHA105 strain for transformation of Jatropha. Vacuum infiltration of an A. tumefaciens suspension with Jatropha explants, combined with co-cultivation on filter-paper wicks moistened with co-culture medium instead of on solid medium, significantly improved transformation efficiency. In these conditions, GUS-positive sectors were observed in 93.0 ± 23.6% of infected explants. Moreover, a variant of the yellow fluorescent protein gene, Venus, was used as a visible marker, which proved effective for discrimination of candidate transgenic shoots from escape shoots. Transgene integration was confirmed by means of polymerase chain reaction and Southern hybridization analyses. Transgenic shoots were regenerated from 23% of explants from the vacuum-filter-paper treatment, which is sufficient for practical use.

Genetic Diversity of Boeseman's Rainbowfish (Melanotaenia Boesemani) Reared in Indonesian Farms Compared to Endangered Natural Populations

Endemic to two lakes (Ayamaru and Uter) of West Papua (Indonesia), the Boesemans Rainbowfish Melanotaenia boesemani Allen & Cross, 1980 is a very popular ornamental freshwater fish. As a result, this rainbowfish species faces great threats and is on the red list of endangered species. Therefore, rearing of this species in aquaculture systems appears to be a promising solution to limit capture of wild specimens and prevent its extinction. Although its reproduction cycle has been controlled for more than 30 years, very few farms still raise M. boesmani, probably due to the problems reported by the farmers, such as decline of production, higher proportion of females per spawning, loss of coloration, lower growth rate and fecundity. Using 12 microsatellites previously developed for this species, comparison of genotypes within six farms around Jakarta indicated that all reared strains originated from Ayamaru Lake. No deficit in heterozygotes was evidenced, suggesting that there was no major inbreeding in these reared populations. Genotype analysis also suggested that M. boesemani species is a metapopulation composed of genetically differentiated populations. Altogether, these results indicate that the problems experienced by the farmers are due not to inbreeding depression but to other factors such as inadequate management and/or poor water quality. Yet, increasing aquaculture production is probably the most effective way to alleviate the pressure that M. boesemani faces in its natural environment.

Development of twelve novel polymorphic microsatellite DNA markers for the Boeseman’s rainbowfish (Melanotaenia boesemani) and tests for their cross-utility in 21 rainbowfish species from West Papua (Indonesia)

We developed 12 microsatellite markers for the endangered Boeseman’s rainbowfish (Melanotaenia boesemani). Twenty-eight individuals from the type locality at Ayamaru Lake were examined, and all loci were polymorphic with a number of alleles per locus varying from 3 to 18. Average observed and expected heterozygosities were 0.681 and 0.678, respectively. Cross-species amplification was successfully obtained for 21 Melanotaenia species, with a number of alleles per locus ranging between 1 and 20. Average observed and expected heterozygosities varied between 0.105 and 0.708 and 0.118–0.755, respectively. Only 21 inbreeding coefficient (Fis) values presented a significant homozygote excess among the 264 locus-by-locus calculated values. Tests for genotyping errors revealed that four of these 21 significant Fis values could be explained by the presence of null alleles. These new microsatellite markers appear highly reliable for further conservation purposes or population genetic studies of the many rainbowfish endangered species.

Expression analysis of Jatropha curcas L. almt genes under low pH and aluminum stress

In the previous study, the six populations of Jatropha curcas L. were grown on an acid Al-toxic subsoil (pH 4.3) media in the greenhouse and drained with nutrient solutions containing 3.2 mM AlCl3 to characterize their differences in response to Al toxicity. The Al treatment significantly affected all growth parameters. Statistical analysis on Al tolerance growth parameters showed a grouping pattern among the six Jatropha populations. They can be grouped into two clusters based on similar sensitivity to Al toxicity; those two groups represent the slightly tolerant and sensitive population to 3.2 mM AlCl3 following treatment for a month. Although grouped as Al-sensitive population, IP-2P has superior traits in productivity and those become the reason to use it as plant material to develop transgenic Jatropha plant. For the preliminary analysis, in this study IP-2P population was selected to subjected to gene expression analysis by qRT PCR. Quantitative real-time RT PCR analysis verified almt gene is prefer...

Introduction of the Serine Green Fluorescent Protein (sGFP) Gene into Pyricularia grisea Race dc4 Isolated from Digitaria ciliaris using Agrobacterium tumefaciens-mediated Genetic Transformation

Gene serin Green Fluorescent Protein (sGFP) has been used to monitor gene expression specific tagged proteins that has implication for fungal cell study. This research aimed to introduce sGFP gene into genome of P. grisea dc4 from D. ciliaris using A. tumefaciens. Plasmid sGFP was introduced into A. tumefaciens by triparental mating method (TPM). Genetic transformation was performed by co-cultivating spore P. grisea dc4 with A. tumefaciens LBA4404–pCAMB-sGFP. Pyricularia grisea dc4 transformant was selected by using selection medium that contains 300 µg/ml hygromycin. The integration of sGFP gene into genome was confirmed by PCR using sGFPs spesific primer pair, sGFP-Nos terminator primer pair and β-Tubulin primer pair as internal control. Expression of sGFP from P. grisea dc4 transformant were detected with blue light fluorescent microscope.

Growth Characteristicsof Physic Nut (Jatropha Curcas L.) Added by Endophyte Fungal on Post Tin Mining Land

Tin mining activity caused canging in physical and chemical characteristic of the soil that were not suitable for growth of plants.The objective of this experiment was to study accessions of Jatropha curcas planted on post tin mining land which were given endophyte. This research was conducted in a Sinar Baru village TS 133, district of Bangka, Bangka Belitung province for field research conducted in May 2007 to April 2008. The experimentas a factorial experiment in the design of the randomized complete block design with three replications. The main plot is 7 accessions consisting of: accession Madiun, Ponorogo, Jember, Dompu, Lampung, Bengkulu, andSukabumi, while the subplot of the land without giving endophyte fungal (control) and the provision of endophyte fungal in baglog 250 g. Each experimental unit contained four plants per plot.The result showed that vegetative growth the highest for the former tin mining land given endophyte fungal vary in some accessions. Accession to the highest Sukabumi: plant height, branch number, plant dry weight, dry weight of the shoot, and root dry weight, the largest diameter have Jember accession, accession Dompu had the highest canopy diameter, and the accession of Lampung has the lowest ratio of shoot roots.

Isolation, Cloning and Characterization of Actin-Encoding cDNAs from Jatropha curcas L. IP-2P

Actin is a major component of the plant cytoskeleton, so all cells contain this protein. Actin is expressed constitutively and is involved in basic housekeeping functions required for cell maintenance. Because of this, it has been frequently used as an internal control to normalize changes in gene expressions analysis. Actually, the information of nucleotide sequence of actin gene of Jatropha curcas L. population IP-2P from Indonesia is not available yet. The objective of this research was to isolate, clone and characterize cDNA of actin genes of J. curcas IP-2P. Three partial actin gene sequences had been successfully isolated by PCR using total cDNA as template, and actin primer designed from conserved region of Arabidopsis thaliana. Nucleotide sequence analysis showed that the length of JcACT fragment is 610, 534, and 701 bp encoding 203, 177, and 234 amino acids respectively. Local alignment analysis based on mRNA sequences shows that JcACT fragment shares 98% similarity with actin mRNA of Hevea brasiliensis and 99% with actin mRNA of Ricinus communis. Based on deduced amino acid sequence, JcACT is 100% identical to actins from Prunus salicina, Gossypium hirsutum, and Betula luminifera. Even though these clones of cDNA are not completed yet, they can be used as reference in J. curcas L. gene expression analysis.