A mutant (strain B119) of Agrobacterium tumefaciens with a chromosomal mutation was isolated by transposon (TnS) mutagenesis. The mutant exhibited growth rates on L agar and minimal medium (AB) plates similar to those of the parent strain (strain A208 harboring a nopaline-type Ti plasmid). The (3) containing neomycin. The neomycin-resistant colonies were then transferred with toothpicks onto plates of L agar with and without gentamicin to examine the presence of the pJB4J1 plasmid. About 75% of the colonies were gentamicin sensitive, indicating the absence of the pJB4J1 plasmid. Five thousand neomycinresistant and gentamicin-sensitive transconjugants were assayed for virulence on carrot root disks as previously described (14). Of these transconjugants, 15 were avirulent or very attenuated. Total DNA containing both the Ti plasmid and chromosomal DNA was isolated from those avirulent mutants by the method of Kado and Liu (13). The DNA samples were subjected to agarose gel electrophoresis. The gel was first stained with ethidium bromide to detect the Ti plasmid and chromosomal DNA. Subsequently, the gel was Southern blotted onto a Hybond N+ nylon membrane (Amersham) (21) and hybridized with probe 1, which was prepared by labelling the IS50 segment in TnS by using a [32P]dCTP megaprime DNA labelling system (Amersham). For some mutants, hybridization was detected only with the Ti plasmid, while for others it was detected only with the chromosomal DNA. Nine avirulent mutants had a TnS insertion in the chromosome, while six mutants had it in the Ti plasmid. We selected one strain (B119) among the mutants with the TnS insertion in the chromosome which showed the most distinct avirulent phenotype (Fig. 1). Virulence of strain B119 was also examined on hypocotyls of Cucumis sativus and the stem of Kalanchoe diagremontiana. Virulence assays on hypocotyls of Cucumis sativus were done aseptically according to the method of Matsumoto et al. (17). Virulence was monitored by the size of galls 3 weeks after inoculation. Virulence assays of Kalanchoe diagremontiana were done by making puncture wounds on the stem with a sterile toothpick previously smeared with A. 3208
A chromosomal virulence gene, acvB, of Agrobacterium tumefaciens [J. Bacteriol., 175, 3208-3212 (1993)] was over-expressed in Escherichia coli. A 47-kDa protein was produced and localized in the periplasmic space of E. coli. Amino acid sequence analysis of its N-terminal demonstrated that a signal peptide of 24 amino acids was cleaved from the pre AcvB protein to produce the mature 47-kDa protein. Western-blot analysis using the antiserum against the AcvB protein detected a 47-kDa protein in the periplasmic space only with strain A208 (acvB+). The amount of AcvB protein synthesized was not increased in strain A208 by induction with acetosyringone (100 microM). There was observed no significant difference in induction by acetosyringone of virB::lacZ, virD::lacZ, and virE::lacZ fusion genes regardless of the presence or absence of the acvB gene. The T-strand (lower strand of T-DNA) was detected in strains A208 as well as B119 (acvB-) which were cultured in induction medium containing acetosyringone. AcvB protein bound to single-stranded DNAs with no apparent sequence specificity. The results suggest that AcvB protein binds to the T-strand in periplasm and mediates the transfer of the T-strand from A. tumefaciens to the host plant cell.
The multiple values delivered by protected areas around the world are threatened and in decline. We propose a structured decision science prioritization approach for justifying and guiding increased investments in protected area management to improve outcomes for a suite of important values. Using Bali's only national park, Taman Nasional Bali Barat (TNBB) as a case study, we draw from existing park documentation and 80 participating experts in TNBB's ecology, society and management to define goals that describe a successful outcome for nine core values of the park: threatened species, ecosystem function, ecosystem habitats, scientific research, food and health, spiritual values, traditional fishing, community prosperity, and ecotourism.Participants estimated that without increased investment, the extent of goal achievement is likely to be below 30% for all values at the end of the 15-year planning time frame. However, implementing nine strategies, at an increased annual investment of 5.5 billion Indonesian rupiah (US$385,666) per year, would achieve the goals for all values. The most cost-effective strategies were predicted to be collaboration and planning, monitoring and managing invasive species, followed by establishing and using a research and management fund, adapting to climate change, managing illegal resource use, waste and human impacts, as well as improving the captive breeding program for the iconic and critically endangered bird, curik Bali. Our approach may be useful for systematically comparing costed sets of management investments in other conservation areas worldwide. K E Y W O R D Sbiodiversity, conservation planning, cost-effectiveness analysis, expert elicitation, livelihoods, Priority Threat Management, structured decision making
Bulung sangu (Gracilaria sp.) is commonly consumed as vegetable in Bali. Bulung sangu as other red macroalgae (Rhodophyta) is a source of beneficial nutrient for health. In this study, water and various concentrations (50%, 75% and 100%) of methanol, ethanol, and acetone in water were used as solvent in extraction of bulung sangu. The antioxidant activity, total phenolic content, and total flavonoid content of crude extract of bulung sangu were investigated using various in vitro assay. The extract obtained by 75% of aqueous methanol produced higher extraction yield (27.390 ± 0.414 %). Highest total phenolic content was obtained by the using 100% of acetone (36.738 ± 1.062 mg galic acid equivalent/g ). The extract obtained by 100% of ethanol showed highest total flavonoid content (45.933 ± 0.563 mg quercetin equivalent/g). The same extract also exhibited the strongest antioxidant activity indicated by lowest half maximal inhibitory concentration (IC50) (13.603 ± 0.413 µg/ml) evaluated by using DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity. This IC50 was lower than IC50 of ascorbic acid (18.593 ± 0.135 µg/ml). These results produce the suitable solvent in obtaining optimum phenolic and flavonoid content of bulung sangu. Likewise, the antioxidant activity results indicate that bulung sangu is useful in dietary application with a potential to reduce oxidative stress.
Citrus (Citrus nobilis L.) Propagation Through Culture In Vitro Using Seed From Infected Plants Of Citrus Vein Phloem Degeneration (CVPD) Disease. The sample was taken in Belancan Village, Kintamani District, Bangli Regency, and invitro culture was conducted in UPT. Genetics Resource and Molecular Biology Laboratory in February to June 2018. This research purposes were to get the new citrus plant from citrus seed infected of CVPD disease explants with culture in vitro and found the presence of the bacterium Liberobacter asiaticus in results of citrus plants culture in vitro. The sampling method is using plants appearance. Obtained sample 1 is a plant that shows severe symptoms of CVPD disease and sample 2 plants that show no symptoms of CVPD disease. Seed Sample is cultured with MS0 medium for 8 WAP then proceed to acclimatization. The results of this research showed that the seed explants from both samples were able to grow well and did not have the characteristic of CVPD disease, but explant seed sample 2 in vitro culture result showed that the bacterial DNA bacterium Liberobacter asiaticus pathogen CVPD disease does exist in length 1160bp, so it cannot be expected to produce CVPD-free crops.
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