Judul: Soybean transformation with proteinase inhibitor 11 gene through agrobacterium and particle bombardment
Abstrak: The objectives of these research were (1) to obtain the best protocols of soybean transformation using Agrobacterhm-mediated bransformation and Particle Bombardment, (2) to transfer and regenerate soybean explants transformed with the proteinase inhibitor I1 gene, and to obtain soybean plants that contained the proteinme inhibitor II gene and resistant to pod barer. Research was included three experiments i.e 1) Soybean transformation using gus gen, 2) Soybean trmsfarmation using proteinase inhibitor (pin) II gene, and 3) Evaluation of soybean transformant plants. Two soybean cdtivars (Wilis and Tidar) were used in this experiments. Both cdtivars were chosen because it shown good response on early in vitro plant regeneration study and very popular to the farmers, especially cv. Wilis. On the first activity, soybean explants were inoculated with A. tumefaciens strain EHA 105 which is contained plasmid pCambia 1301 with p s gene in T-DNA region. The treatments were included an optical density (0.5; 1 and 1.5), inoculation time (60 and 90 minutes), co-cultivation time (3 and 5 days) and type of explants (young embryo and cotyledon). Result indicated that the best protocol for inoculation was using young cotyledon as explants with 1 x 10' celYrnl of Agrobacterium for 90 minutes inoculation and 5 days co-cdtivation. While on the particle bombardment method, young embryos and cotyledons were bombarded with pRQ6 plasmid. Treatment were included Helium gas pressure (1 1 00 and 1 300 psi), shooting distance (5 and 7 cm) and number of bombardment (once and twice). The best treatment was using young cotyledon as explant, bombarded once or twice with gold particles at 1 100 psi of the Helium gas pressure and 5 crn of shooting distance. On the second activity, 1539 young cotyledon explants from Wilis and 984 explants from Tidar were inoculated with A. tumefaciens which is contained proteinase inhibitor II gene on plasmids pGApin LI construct. Result from plant regeneration and selection of tsansformant explants on media with 200 mgA Kariamycine (spesific for nptII gene selection) indicated that c.v Wilis were better than Tidar, because its produced more plantlets/plants 8 plants (coded: A W I -AW8), while Tidar produced only one plant (coded: AT& On the Particle Bombardment experiments, 1274 young cotyledon explants of Wilis and 1 763 explants of Tidar were bombarded using gold particle coated with pTWa plasmids (contained pinn and bar genes). Result of plant regeneration and selection of transformant explants on media with 3 mg~l Basta (spesific for bar gene selection) indicated that cv. Tidar produced three plants (TPI-TP3) while cv.Wi1is produced only 2 plants (WPI and WP2). On the third activity, transformant plants were evaluated through molecular and bioassay analysis. Molecular analysis of nine soybean plants obtained from Agrobacterium-mediated transformation (8 Wilis and 1 Tidar) using Polymerase Chain Reaction (PCR) technique indicated that only event ATI (Tidar) was positive to pin I1 gene test, while 8 plants from event AW (Wilis) were negative. PCR test of five soybean plants obtained from bombardments experiments (2 Wilis and 3 Tidar ) indicated that no plants were positive to pin 11 gene, except WP2 (Wilis). Seeds fiom these positive
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Judul: Optimasi Transformasi Eksplan Tebu menggunakan Gen P5CS melalui Agrobacterium tumefaciens
Abstrak: Genetic transformation is one of the attempt in generating sugarcane (Saccharum officinarum L.) that are tolerant to drought stress. P5CS gene has a role in prolin biosynthesis, the amino acid that accumulating in water stress condition. Transformation of a P5CS gene construct into plant cells in conjunction with regeneration for transgenic plantlets should develop sugarcane tolerant to drought stress. The aim of this research is to generate drought-tolerant sugarcane through optimization transformation which includes the strain of Agrobacterium tumefaciens, a good source of sugarcane eksplant and varieties. This study was done through verification of IBRIEC (Indonesian Biotechnology Research Institute for Estate Crops) P5CS gene collection A. Tumefaciens transformation (Wang 2006), sugarcane explants transformation (Sain 1994) and sugarcane transformant regeneration. Verification of P5CS gene collection was PCR analysed by specific primers P5CS full lenght. PCR and gel electrophoresis showed a single band agarose and the same size of 2.4 kb with positive control. It is convinced that the P5CS gene has been isolated and transformed into A. tumefaciens. IBRIEC collection P5CS genes in plasmid pBI121 were arranged in a recombinant construct of PBI-P5CS transformed into A. tumefaciens strain GV3101, LBA4404 and AGL1. Recombinant constructs PBI- P5CS from Vigna aconitifolia (V. aconitifolia) successfully transformed into cell A. tumefaciens were grown with appropriate antibiotics. Strain GV3101 and LBA4404 used 50 ppm kanamycin and rifampicin respectively, while antibiotics were added in AGL1 strain is 50 ppm kanamycin, rifampicin and ampicillin respectively. The existence of P5CS gene in A. tumefaciens was tested by observing the growth of transformed colonies in media selection and colony PCR analysis using specific primers P5CS full lenght. The results showed that A. tumefaciens transformants were grown in selection media. PCR and gel electrophoresis analysis showed a single band agarose and same size of 2.4 kb with positive control. This showed the success of the transformation A. tumefaciens and used to explants sugar cane transformation. Sugarcane explants used in this research was included calli grown in solid medium, embryogenic calli and somatic embryos with MS medium derived from Temporary Immersion System (TIS) culture with three varieties Kidang Kencana, PS 881 and PS 891. Co-cultivation medium used solid MS medium with acetosyringon in room temperature for two days. Transgenes selection was done in solid MS with antibiotics 50 ppm kanamycin and 500 ppm cefotaxime. Explants were not transformed cultured in MS media as positive control negative control while planted in the media selection. Observation of sugarcane transforman in selection media was calculated the percentage growth in 16th weeks and 32th weeks. The existence of P5CS gene in transgenic shoots was tested with GUS histochemical assay, PCR using conserved regions of specific primers P5CS and NPTII. GUS histochemical test for the presence of P5CS gene constructs showed positive results in the presence of blue staining. DNA amplification showed that expected bent size at 1.2 kb and 700 bp for primers NPTII. The results of this examination proved that both transgenes were inserted in the sugarcane genomes. The test results are stable sugarcane transformants through histochemical GUS test and PCR analysis showed stable transformants sugarcane. Growth
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Judul: Karakteristik Anatomi Skelet Kepala Buaya senyulong (Tomistoma schlegelii).
Abstrak: Buaya sepit atau senyulong (Tomistoma schlegelii) merupakan jenis buaya asli Indonesia yang berhabitat di sungai-sungai Sumatera, Kalimantan, dan juga pedalaman Sulawesi. Salah satu ciri khas buaya senyulong adalah bentuk moncong yang runcing dan sempit, namun hingga saat ini informasi mengenai struktur skelet kepala buaya senyulong sangat terbatas. Penelitian ini bertujuan untuk mempelajari karakteristik anatomi skelet kepala buaya senyulong yang dikatikan dengan fungsi dan perilakunya. Penelitian dilakukan secara eksploratif dengan melakukan pengamatan, pencatatan, dan pengambilan gambar dari skelet kepala buaya senyulong. Skelet kepala berukuran panjang 70 cm, lebar 29 cm, dan tingggi 17 cm dengan permukaan kasar pada os nasale, os frontale, os maxilla, os mandibula, os lacrimale, os jugale. Jumlah gigi pada buaya ini sebanyak 78 buah, terdiri dari 4 buah gigi premaxila, 16 buah gigi maxila, dan 19 buah gigi mandibula. Pada regio orbitalis ditemukan dua pasang fenestrae temporales yang berkembang baik. Fenestra supratemporale merupakan lubang oval yang membentang di sepanjang os parietale. Fenestra infratemporale berbentuk segitiga dan dibatasi oleh os squamosum, os postorbitale, os jugulare, dan os quadratum. Os mandibula hewan ini mempunyai symphysis mandibularis yang panjang yaitu garis yang melintang di corpus mandibulae. Secara fungsional ukuran symphysis mandibularis berkaitan dengan adaptasi hewan terhadap diet makan.
Keyword: kepala, moncong, Tomistoma schlegelii