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Endophytic can be defined as a bacterium that colonize internal plant tissue without showing any negative symptoms towards their host (Schulz et al., 2006). These bacteria usually gain entry through the roots, flowers, stems and cotyledons (Kobayashi et al., 2000). Some of the endophytic bacteria gives a positive effect towards their host plant, such as promoting growth (Sturz et al., 1997) and improve the host's ecological adaptation towards environment (Schulz et al., 1995). In addition to these positive finding as mentioned , some of the endophytic bacteria also revealed that they can provide protection for their host from pathogens and animal (Clay et al.,1985).
Based on previous research, endophytic bacteria have been isolated from variety of healthy plants species such as herbaceous crop plants (Lodewyckx et al., 2002a, 2001b; Malinowski et al., 2004 ; Mastretta et al., 2009) and different grass species (Zinniel et al., 2002; Dalton et al., 2004). The most common genera cultivable endophytic bacteria that have been found are Pseudomonaceae, Burkholderiaceae and Enterobacteriaceace (Mastretta et al., 2006). The most recent similar study was conducted and completed by a group of researchers which have isolated endophytic from celery (D'Amico et al., 2008). They have detected twenty-three isolated endophytic fungi and among the isolated species are Acremonium, Alternaria, Fusarium and Plectosporium. However, for this research the focus is on endophytic bacteria and use sample plant in Tees Valley. This research also hope to discover new species of endophyte bacteria in celery because endophytic bacteria may not be identical even from the same species or host (Lu et al., 2004; Rodrigues et al., 2004). I sincerely hope that this study could contribute significantly in term of research materials and findings to the University's Biotechnology department and Teesside University students and present and future as currently because there are not much studies and research materials on endophytic bacteria from different types of plants. This effort could be my personnel contribution to the University and the local community and help others in same and related field to have a better understanding of the subject matter.
The sample that will be used for this research is Apium graveolens (celery) and the parts that will be used are stems, leaves and roots which are to be collected by using a sterilized cutter. This sample has to be collected directly from the soil within 24 hours to avoid the sample being contaminated by the surrounding environment, for example humidity and temperature conditions.
Isolation of endophytic bacteria
All parts of the celery shall be separated individually into roots, stems, and leaves and each part is subjected to a surface sterilization. This is a very critical and crucial process because the endophytic bacteria have to be carefully protected and then subsequently to remove unwanted organisms (Hallmann et al., 2006). This procedure shall start with submersing all parts of the sample into 0.5 % of sodium hypochlorite and subsequently with 70% alcohol (Arnold et al., 2001). Each of this steps shall take approximately about 2 minutes and after the steps completed the sample will be rinsed with sterilize distilled water. Parts of the celery were then air-dried and cut into small segment which is about 2cm per segment and placed onto Nutrient Agar in triplicates. The plates shall be then be incubated for 3 days at room temperature to allow the bacteria to grow.
Identification and Characteristic
These steps are described by Mudili, 2007. Prior to this step, a smear preparation need to be prepared. This process need to be prepared properly and with care. Subsequently, the next step is observation procedure by using microscopic and identifies the characteristic of the bacteria. Firstly, a small amount of isolated endophytic bacteria will be transfer onto slide by using wire loop. Then the bacteria will be fixed heated to allow the specimen to adhere to the slide and let air-dried for one minute.
The Gram-staining procedure will be start using crystal violet, which will be covered on the smear for one minute and washed off with distilled water. Subsequently, the smear will be covered with iodine solution for one minute and wash off again with distilled water. Iodine or mordant will fixed the crystal violet to the bacterial cell wall. As the next step, ethanol will be added onto the smear for twenty seconds and rinse under running tap water. Finally, the specimen will be covered with Safranin solution for twenty seconds and wash it again under running tap water. The specimen will blot dry by using filter paper and later observed under microscope.
Endophytic bacteria DNA extraction
This method was described by Kalia et al., 1998. Bacterial cells will be transferred into Eppendorf tubes containing 1.5 ml TNE buffer and later will be wash thoroughly by extensive vortexing. Glass beads also will be included for clump-forming bacteria. Later cell will be harvested by using centrifugation at 15,000 rpm for 5 minutes. The pellet will be placed in 2ml ice-cold 70 % ethanol, and mixed, subsequently incubated on ice for 20 minutes. Subsequently, the cell will be collected by centrifugation and place in buffer and incubate with ice for about 1 hour by moderately shaking it. This tube will be keep at -20oC for 20 minutes. Next, the tubes will immediately be transferred to water bath (68oC) and incubate for 10 minutes. Approximately, 53Î¼l of 10% of sodium lauryl sulfate will be added and incubate for about 15 minutes. The tubes will be incubating further for 15 minutes at 68oC and later will be keeping at -20oC for 30 minutes. Next, 1 volume of chloroform: isoamyl alcohol (24:1, v/v) will be added and the tubes will be placed on a flat rocking platform for 5 minute. Phases will be separated by centrifuge at 10,000 rpm for 10 minutes. The DNA will be purified firstly with phenol:chloroform extraction secondly followed by another extraction with chloroform:isoamyl alcohol. The DNA will be precipitate at -20oC for 1 hour after ice-cold absolute ethanol will be added. The genomic DNA will be forming clumps as the ethanol will be added drop by drop. Finally, DNA will be recovered by centrifuge at 15,000 rpm for 15 minutes. The DNA pellet will be washed in ice-cold ethanol, dried in air and suspended in 50-250 Î¼l of TE buffer. The concentration and purify of DNA isolated can be determine by using spectrophotometrically (Hitachi, Tokyo, Japan) which by taking the ratio of absorbance at 260 nm (A260) and 280 nm (A280)
PCR amplification of 16S rRNA gene
This method will be performed using DGGE primers 357FGC-518R (Muyzer et al., 1993). Amplifications will be carried out by using primers, DNA template, 1x reaction buffer, MgCl2, Taq DNA polymerase, dNTP in a 50Î¼l PCR mixture with grade water. PCR condition will be at 95 °C for 5 min, follow by 10 cycles of 94 °C/30 s; 55 °C/30 s; 72 °C/60 s, 25cycles of 92 °C/30 s; 52 °C/30 s; 72 °C/60 s and 10 min at 72 °C ( Dillon et al., 2007). During preliminary steps, PCR will be carried out as described by Webster et al., 2006. In this reaction, the universal bacteria primers will be used. The PCR samples will be amplify using PTC-200 thermal cycles (MJ Research Co., USA) and for the final PCR product will be check on a 1.2 % agarose gel.
Denaturing gradient gel electrophoresis (DGGE)
DGGE analysis will be performing accordingly to Webster et al., 2002. DGGE marker will be preparing from a selection of bacterial 16S rRNa gene product to allow gel to gel comparison. The product will be separated using a mixture of polyacrylamide and denaturant gradient between 6 % acrylamide/30 % denaturant and 12 % acrylamide/60 % denaturant. The 100 % denaturing condition will be equivalent to 7M urea and 40% (v/v) formamide. Later, the gel will be poured with the aid of 50 ml gradient mixer (Fisher Scientific, Loughborough, UK) and electrophoresis will be run at 200V for 5 hours at 60oC. The polyacrylamide will be staining with SYBRGold nucleic acid gel stain ( Molecule Probes, Leiden, The Netherlands) for 30 minutes and will be observed under UV light (Dillon et al., 2007). The image should be capture using Gene Genius Bio Imaging System (Syngene, Cambridge, UK)
Tester bacterial strains
Tester strains that will be used for this project are Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Burkholderia cepacia and Pseudomonas aeroginosa. These tester strains will be obtained from the Biotechnology Department at Teesside University.
Screening isolated sample for antibacterial activity
For this screening test, the method that will be used is agar diffusion. The center of Nutrient Agar plate will be made a well by using sterile cork borer. The diameter of the well is 5mm (Lewinstein et al., 2005). All the sample and tester strain will be prepared in broth solution and left for few days at room temperature (Vaidya et al., 2005). Subsequently, the isolated sample will be centrifuged for 10 minutes at 3000 rpm. This process is to obtain the supernatant of the sample before it is poured inside the well. Before the antimicrobial test begins, each of the Nutrient Agar plate need to be swabbed with the tester strains by using sterilizes cotton bud. Each of the isolated broth samples will be poured on the center of the agar and incubate at 37 °C for 24 hours (Lewinstein et al., 2005). The test will be done in triplicates and after 24 hours, the diameter of inhibition zone will be observed and measure if any(s).
For antimicrobial analysis, the data that will be obtained is the number of diameter of inhibition zone between bacteria strains against isolated endophytic bacteria. All the data will be calculated by inserting the data into Minitab.
While for DGGE method, the result will be the numbers of DGGE bands pattern (Xiaoqi et al.,2007). For this analysis, the Labwork software will be used ( Labwork TM software version 4.0,; UVP, UK). Other than that, all statistical data will be use the Mini-tab software which is use to calculate mathematical data for example, averages and correlation.
There will be some limitations during the running of this research. The limitation that could occurs as follows:
Isolation of endophytic bacteria
Bacteria may not growth on the Nutrient Agar if the surface of the disinfected process is not properly prepared. The timing for each steps must be precise and accurate because if the steps exceeded the time allocated (Arnold et al., 2001) the bacteria compound may vanished from the selected sample. The sample should not be submersed inside the hypochlorite solution for too long.
There are a few limitations in doing DGGE method. One of it is to obtain a good result from this method which is needed by using ideal type of marker with a sufficient number of bands because there is some variation between a gradient (Fromin et al., 2002). Other limitation is in reading the number of the bands because it is difficult to interpreted exact amount of population in a community. According to some report, single isolated sample can produce multiple bands by DGGE (Nübel et al., 1996; Satokari et al., 2001) and sometimes single band can show multiple population (Yang et al., 2000).
This limitation can be mitigated by combine DGGE method with other method such as sequencing bands or hybridization with probes which this additional method can reduce the uncertainty of band identification ( Stephen et al., 1998).
Based on the timetable below, this research will be takes about three months to be completed, June until August 2010. The literature review reading and collection will start from the first week until 12 weeks. For the second week of the June, it will be start with search and order of materials and chemicals for this project and followed by health and safety. Subsequently, the project will be fully run after all the material and chemical have been arrived and readily prepared. The experiment will be running for 10 weeks. Data analysis will start to be analyzing one week after the project had been started. The report writing process will start on the first week until the final submission is made.
For this research, samples of celery plant from the farm in Tees Valley will be required. The sample should be directly taken from the soil within for 24 hours. For convenience purpose, possible the farm is not far away from Teesside University, as it will be easy to gather and prepare the sample.