"Tissue Culture Blog": Laboratory made Gorontalo North Sea Grass

Geographical Location North Gorontalo's support for the development of various kOMIDITI superior. Eg seaweed, North Gorontalo newly bloomed two years were selected to serve as a laboratory where the sea grass. Thursday last week, PT. Helmi Karagenan Indonesia (HKI) and the Commission on two Deprov Gorontalo Gorontalo SEA received by the North in discussing the program.
The arrival of two Deprov Commission and PT. HKI is intended to develop cooperation between the North and PT Gorontalo IPR Gorut which will be made as Seaweed Laboratory in Gorontalo. IPR parties disclose the reason they chose this area, because Gorut only area that has the longest coastline in Gorontalo, namely the 320 km long coast. Later the fishermen in two district in which Gorut Kwandang and Orchid will be given seaweed seedlings freely by PT HKI seedlings.
IPR party also plans not only provide the seeds, but will provide guidance how to develop a good seaweed. After the seaweed is ready for the harvest, the HKI will buy a standard national price. Because the main constraints Seaweed Farmers Fishermen have been said of Indra, is a marketing issue, where there is no formal company that would accommodate the fishermen Seaweed.
Alhamdulillah this project and received a positive response from the Regency Gorut, because not only seaweed, but the PT HKI plans to make Gorut the Marine and Fisheries Laboratory Gorontalo Province. Indra hoped that the cooperation, marine potentials in Gorut Investment Plus can be for this region. Indra also said thank you for the Provincial Government Commission two main Gorontalo Provincial Parliament who had to mediate this cooperation. The plan in the near Regency Gorut determine the proper location for the nursery location make. gpinfo Posted by ujilamadi at 22:31 0 comments Tuesday, December 22, 2009 Regeneration mutants

Posted by ujilamadi at 20:58 0 comments Transformation of Sweet Potato (Ipomoea batatas) with Gen-PinII and SPFMV CP
Sweet potato (Ipomoea batatas (L.) Lam.) Is an additional food or rice substitute that has received public attention, and a world food crop products which ranked seventh. Besides the food, sweet potato is used as industrial raw materials, such as for making flour, molasses, animal feed, and alcohol. The main constraint in increasing production of sweet potato is pest boleng (Cylas formicarius F.) and stripe virus disease (Sweet Potato Feathery Mottle Virus, SPFMV). Boleng pests can cause loss of 60-100%. SPFMV attack can reduce the results up to 30%, sometimes even not producing at all.
Plant breeding programs have a lot of sweet potatoes is good for improving quality, improving results and to obtain resistance to pests or diseases. However, until now not been found varieties resistant to pest boleng or boleng disease, because there was no source of sweet potato resistance genes against pests or disease. Conventional Plant Breeding also difficult, because the selection process takes a long time, the number of accessions that much, incompatibility problems, sterility, seed bag viabili-low, and pests are heksaploid. Techniques of biotechnology through genetic engineering is an option that can be taken to support and complement the glorification of the program. Transformation studies to include resistance genes against pests boleng or SPFMV virus is a new vehicle for assembling plants resistant transgenic sweet potato pest or disease virus boleng. Transformation studies to assemble potato plants resistant to pests boleng done using the source gene from potato plants, namely proteinase inhibitor gene (pinII). Resistance to viral diseases can be obtained by introducing the gene coat protein (CP-SPFMV) is derived from the virus. Transformation or transfer of a functional foreign gene to obtain transgenic potato plants can be done by using several methods, such as a bacterial vector Agrobacterium tumefaciens or particle bombardment.
The success of transformation marked by the success of inserting the gene sequence is introduced into the plant genome, can be expressed, and still preserved in the whole process the next cell division. Attempts to confirm the integrity of the introduced genes and determine the number of copies in the genome of plants, and determining the gene may function correctly or not. The identification of transformed plant tissue can be done with some existing techniques, among them using PCR techniques. Cell or tissue that has been transformed separated from the network that are not transformed to avoid the occurrence of tissue dichotume.Di addition, cells that are not transformed will grow better than cells that are transformed so that should be done with dibuang.Seleksi subculture that few believed that tissue or living cells or escaped from the selection (selected by media containing the herbicide or antibiotics) are not escaped. Types of agents or materials used for the selection depends on the selection of genes used. This selection gene may be an antibiotic such as neomycin phosphotransferase (NPT II) which causes resistance to antibiotics kanamisin, or the bar gene that causes resistance to herbicides such as basta (PPT) and bialafos. In addition to selectable marker, transformation was also done by including a reporter gene (reporter genes). There are several reporter genes used for transformation, including GUS (β-glucoridase), LUC (luciferase), and antosianin.
In the year 2002 has been done pinII gene transformation and gene SPFMV CP-through technique A. tumefaciens and has obtained several transforman who escaped from the media and managed diaklimatisasi selection. Further identification was carried out the integration of CP gene-pinII or SPFMV into potato plants transgenic putatif using PCR techniques. With this technique, pinII or SPFMV CP sequences can be amplified using DNA from the gene-specific primers flanking the gene, which can detect the presence of the target gene in the genome of potato plants.
Proteinase inhibitor gene (pin) is a gene encoding anti-nutritional compounds that can inhibit proteolytic enzymes work (proteinase) in the insect's stomach. This gene can be used to assemble the pest resistant transgenic plants. If it successfully transferred the gene into the chromosomes of plants and can be expressed very well, then the insects eat the plant will be disrupted digestive system, stunted growth and eventually die if the high-level penghambatannya.
Boleng pest of sweet potatoes Cylas formicarius and viral diseases caused by Sweet Potato Feathery Mottle Virus (SPFMV) is a constraint in potato cultivation, which can cause loss of 30-100%. Hama boleng (Cylas formicarius) is one of the important pest that attacks potato plants and have the very wide spread. Early symptoms of this pest in the form of small holes on the surface of the tubers, which then spread to the inside and cause the tubers to rot and bitter. This pest can cause very large losses (60-100%). Among the 15 kinds of virus disease in sweet potato, Sweet Potato Feathery Mottle Virus (SPFMV) is a major viral disease that attacks the leaves and cause stains klorotik with the purple spots, and the level of damage may reduce the results up to 30%.
To get the sweet potato plants that have resistance to pests boleng C. formicarius can be done by inserting a gene proteinase inhibitors (pinII) which is a single gene, into the genome of potato plants. because the protein with a single gene code more easily introduced into a plant. As for resistance to viral diseases by inserting SPFMV coat protein gene of SPFMV. This resistance gene insertion can be done with gene transformation techniques through particle bombardment or by Agrobacterium tumefaciens. System transformation with A. tumefaciens has been widely used as an efficient, relatively cheap, and stable in introducing a gene. Some things to note in the process of transformation through A. is tumefaciens T-DNA transfer into the core of the target plant, the T-DNA integration into the target plant's genome and the transformed gene expression. Presence / Gus reporter gene expression can be detected through testing Histokimia tissue by soaking in a solution transforman ß-glucuronidase. While the identification of the target gene integration can be confirmed by molecular analysis, for example by PCR technique or Southern blot. With PCR techniques, DNA sequencing of the gene or genes pinII-SPFMV CP flanked by specific oligonucleotide primers of these genes can be amplified a lot of repetition of the cycle in the PCR process, so that its presence can be detected in the plant genome. Positive plants containing the target gene remains to be tested or the expression level of effectiveness against the target pest or disease through bioasai.
Evaluation of sweet potato resistance to pests boleng be judged by the number of holes on the surface of the tuber gerekan, the percentage of tuber surface area of damaged or broken bulbs depth. Having gained some putativ transgenic crops with genes for transformation of both genes pinII-SPFMV CP, then in fiscal year 2000 Dinar et al do initial research for molecular testing, the optimization of DNA extraction techniques and PCR sweet potato and sweet potato resistance testing against pests boleng. In addition, transformation with gene activity or gene pinII-SPFMV CP still continue to get the number of plants more.
Eksplan used to study the transformation of leaf pieces and petiol taken from the culture eksplan sources (mother stocks) Jewel sweet potato varieties, while the leaves and tubers of transgenic plant sweet potatoes putativ respectively used as materials for molecular analysis and bioasai. Bombardment transformation methods using gene gene gun (Biolistic PDS 1000/He) Biorad. Conditions used for the shooting of pressure 1100 psi, twice the number of shots with a range of 7 and 9 cm. The shooting using co-transformation system pTwa (pinII, Bars) with pRQ6 (Gus, HPT) on the Molarity 2: 1. Transformation for resistance to pests using Agrobacterium strain LBA4404 containing pGA643pin with kanamisin antibiotic resistance gene. While transformation for resistance to viral diseases SPFMV using pMON10574-1 plasmid containing the coat protein gene SPFMV, nptII, and uidA.
Map pRQ6 plasmid carrying the genes gus and concessions.
Map pTWa plasmid that carries genes and gene pinII bar.
Potato DNA extraction using CTAB method of modification. Extraction buffer consisting of 100 mM Tris HCl, 50 mM EDTA, 500 mM NaCl, 0.3% ß-mercaptoethanol, 2% CTAB, with the addition of PVP 50 mg. Purification of DNA by adding a mixture of chloroform and octanol (24: 1). Test quantities of DNA concentration using a spectrophotometer. Obtained DNA used for PCR analysis with PCR machine PROGRAMMABLE Thermal Controller. Hama Bioasai to Boleng C. formicarius do with infestations with 5 pairs of imago per tuber. Observations tuber damage done 1 week after infestation by counting the number of holes gerekan.
The process of transformation through particle bombardment is random, so there is the possibility of tissue / cell does not participate transformed so that no resistance to the selection media. In addition, damage to cells / tissue due to physical factors during the shooting process will be greater than the A. tumefaciens.
Agrobacterium transformation by providing the possibility of a transformed network larger, in addition to integration is a simple (low copy number), are relatively cheap, and reprodusibel. In ransformasi with SPFMV CP gene for resistance to SPFMV virus diseases, with over terekspresinya Gus Gus gene assay and gene nptII (kanamisin) gives hope that the target gene-SPFMV CP has joined integrated, since all three genes are present in T-DNA is transferred by Agrobacterium into the plant.
Extraction of DNA modification combines sweet potatoes specific methods for plants containing high polysaccharide and polyphenol. Polysaccharides will complicate the extraction technique for forming a gelatinous matrix that DNA is difficult and can not be separated teramplifikasi on PCR by inhibiting the activity of Taq polymerase. Some of the modifications made to such treatment during the dark leaves of the plant 12-48 hours before the extracted DNA, the addition of ß-mercaptoetanol and PVP (polivinilpirolidon) in extraction buffer. PVP is a solid polymer with high molecular weight polyphenol that will bind through hydrogen bonding that can be separated from DNA. Can also be added in the DEAE-cellulose purification to eliminate proteins, polysaccharides, and polyphenols. The quantity of DNA concentration was measured using a spectrophotometer at OD A260/A280 ratio. DNA samples of sweet potatoes can be cut by several restriction enzymes such as BGL II, Hae III, and Xho.
From this initial study obtained results that the transformation through A. tumefaciens to produce 18 transgenic plants putativ sweet potatoes transformed with the gene pinII and 6 transgenic plants transformed putativ with SPFMV CP gene. Preliminary study for the extraction of DNA molecular produce the quantity of DNA (A260/A280) of 0,7-1,8 mcg / μl. Bioasai on 32 potato tubers against pests boleng in the laboratory showed an average score of tuber damage 4,4-4,5; Cylas percentage mortality at higher putativ plants compared with control plants, while the number of live larvae and pupa found in plants putativ less than the control plants.
In early studies of selected low transforman transformation caused by particle bombardment techniques are random, so not all transformed cells or tissue and may be used basta concentration is too high, so it would kill the cells or tissues are transformed. Therefore, the concentration basta need to be reduced again to less than 0.5 mg / l and thus more effective for selecting the cell or tissue transforman. The selection and concentration of the appropriate selection is very important in the process of transformation because it will provide a stringent selection for cells and tissues transforman. In addition, selectors agents can also inhibit the growth of cells or tissue transforman, so that the growth or regeneration requires a longer time compared with controls.
Dinar et al conduct further research in 2002 by using previous research results transforman. The difference in this study is the addition of additional treatment asetosiringone solution of 200 mM at A3 (pre-treatment) and the co-cultivation medium. The aim is to condition eksplan so ready to be transformed so that it will increase the efficiency of antibiotic transformasi.Seleksi transforman use kanamisin 50 mg / l with two subculture periods. Results showed that the selected transforman diminishing eksplan even selected not all can regenerate. Because the selectors agents in addition to inhibiting the cells or tissue that is not transformed can also slow the growth of cells or tissue transforman. According to research results Prakash and Varadarajan (1992) that the petiole is the sweet potato plant is suitable or appropriate for transformation through A. tumefaciens.
Results of transformation after tested with gus gene shows that there is no real difference between treatments with or without the addition of asetosiringone and 2 construction plasmid used for transformation. When compared with the previous year of research results achieved by the transformation efficiency is higher, and it ranged between 20-56%. With terekspresinya gus gene is expected to target genes expressed, too, because there are CPSPFMV genes in T-DNA in a plasmid construction with gus and nptII genes.
As a result of the transformation of sweet potato research in 2002 through 2001 atupun particle bombardment putatif not produce transgenic plants, whereas the A. tumefaciens has produced some putatif transgenic plants transformed with the gene or genes pinII-SPFMV CP. To further A. tumefaciens with research focused on the transformation of the modifying some parameters of transformation, in order to obtain the number of plants that transforman more so the possibility to obtain transgenic plants will be greater.
Studies conducted in 2003, but attempted transformation of sweet potato varieties in Jewel varieties are also made to BIS 182-81. In previous studies Jewel varieties have been successfully regenerated and transformed, while the Bus 182-81 regeneration system not yet optimal. Transformation for virus resistance to SPFMV using pMON10574 or pMON10575-1-2 which contains the gene nptII, uidA and CP (Coat Protein)-SPFMV. Eksplan dikulturkan on MSO medium (Skoog without substance Murashiga growth regulators) as a source eksplan.
Eksplan who respond positively to good test indicated by the presence of blue color in the cell or tissue tested, which showed positive results of transformation. Based on the number of blue spots per eksplan, the addition of 100 μM can asetosiringon improve regional / area of the transformed network, although the number of positive eksplan less than asetosiringon adding 200 μM.
The composition of regeneration medium and selection agent is the determining factor for putatif transgenic plant regeneration of sweet potato callus transformed with the gene pinII done through organogenesis and embryogenesis using eksplan leaves and sweet potato varieties petiol Jewel. Just as the transformation with the CP gene SPFMV, regeneration by organogenesis using two kinds of media, namely R1 (MS + 0.2 mg / l kinetin) and R2 (MS + 0.2 mg / l 2ip).
Molecular Analysis of Gene Integration PinII on Sweet Potatoes
PCR program consisted of initial denaturation at 94C for 5 minutes, followed by 35 cycles consisting of denaturation phase of 94 C for 1 minute, 55 primary attachment C for 2 minutes and elongation of DNA at 72C for 1 minute and the cycle ends with a final elongation at 72C for 5 minutes. Primer for the primary pinII gene 1 with sequences 5'-release GGAAGTTAATTTC GTTGCTTACC-3 'and primer 2 with basanya sequence 5'-GCCTTGGGCTCA TCACTCTCTCTCCTTCAC-3'. Primers for genes SPFMV CP-1 is the primary sequence basanya with CCTCAGTTATTGTGGGTGGTGGAG-5'-3 'and primer 2 with the sequence of bases 5'GGTGATGAGCAAGTGTGACATATCCA-3'. PCR amplification results are separated by electrophoresis using 0.9% (w / v) agarose gel and staining with ethidium bromide.
The success of transformation marked by the success of inserting the gene sequence is introduced into the plant genome, can be expressed and maintained in the next generation. Putatif transgenic plants that have been produced by selection on media with antibiotics kanamisin unknown genes containing CP-pinII or SPFMV. Initial confirmation of transgenic plants putatif be very useful, because the plants that do not contain the introduced gene can be directly known, so the only plant that has the desired gene are used for further research. In potato transformation activities in 2002 has 26 plants putatif produced trans-genik who had escaped the selection and suspected to contain genes pinII and 9 plants with SPFMV CP-gene.
PinII gene integration testing with the PCR technique should be using pure DNA to ensure the accuracy of testing. This is because the potato plants contain polysaccharides and polyphenols are high that when not using DNA with a pure state then the fear will interfere in the test mainly to hamper the work of the Taq polymerase enzyme. If this happens then the optimal results of PCR testing may not be obtained. In the CTAB method (which used this), before extracted, dried leaves of sweet potatoes in the oven for 50 C overnight. The treatment was intended to reduce the amount of resin (polyphenols) in the potato leaf tissue, in addition, intended to create a network of dried sweet potato became so easy when done to obtain results penggerusan soft gerusan. Additions made in two CTAB concentration, high and low. High concentration (10% CTAB) was intended to dissolve or remove the DNA from the cell nucleus, whereas the addition of CTAB concentration is low (1% CTAB) was intended to be selective precipitation of DNA.
REFERENCES
Ambarwati, Alberta Dinar., D.W. Utami, D. Damayanti, A. Sisharmini, T.J. Santoso, M. Herman, and Minantyorini.2001. "Transformation, molecular studies, and Sweet Potato Plants Bioasai resistance to pests or disease". Proceeding Seminar on Research and Biotechnology stubs
Ambarwati, A. Dinar., A. Sisharmini, Tri J. Santoso, M. Herman, and Minantyorini.2002. "Transformation of Sweet Potato with pinII Genes and Gene-SPFMV CP". Proceeding Seminar on Research and Biotechnology stubs
Ambarwati, A. Dinar., A. Sisharmini, Tri J. Santoso, M. Herman, and Minantyorini.2003. "Transformation of Sweet Potato with pinII Genes and Gene CPSPFMV via Agrobacterium tumefaciens". Proceeding Seminar on Research and Biotechnology stubs
J. Pardal, Saptowo., G.A. Wattimena, H. Aswidinnoor, M. Herman, E. Listanto, and Slamet.2004. "Transfer proteinase inhibitor II gene in soybean via Agrobacterium tumefaciens vector for resilience against pests borers pods (Etiella zinckenella Tr.)". Journal of Agricultural Biotechnology, Vol. 9, no. 1, pp. 20-28
Sisharmini, Atmitri., A. Dinar Ambarwati, Tri J. Santoso, and M. Herman.2003. "Integration of Molecular Analysis of Genes PinII Sweet Potatoes". Proceeding Seminar on Research and Biotechnology stubs
Sutrisno, Saptowo J. Pardal, Diani Damayanti, M. Herman, Riri Sundasari, and Endang Ibrahim.2003. "Bioasai Transgenic Soybean Crop Pests pinII against borers pods (Etiella zinckenella, Treitschke)". Proceeding Seminar on Research and Biotechnology stubs