TRANSFORMASI GEN ALBUMIN BUNGA MATAHARI (Helianthus annuuss L.) DENGAN VEKTOR Agrobacterium tumefaciens SECARA IN PLANTA PADA TANAMAN KEDELAI

Penggunaan A. tumefaciens untuk maksud mengintroduksi DNA albumin bunga matahari ke dalam sel tanaman kedelai didasari atas kemampuan alami dari A.tumefaciens untuk mentransfer suatu fragmen yang spesifik dari DNA plasmid (T-DNA ) ke dalam sel tanaman lalu berintegrasi pada genom sel tanaman inang. Hasil kloning gen albumin yang ditumbuhkan pada bakteri E.coli selanjutnya ditransfer ke A. tumefaciens LBA4404, melalui metode triparental mating. A. tumefaciens LBA4404 (pAL4404, pSW600 ) yang dihasilkan kemudian ditransformasikan pada tanaman kedelai secara in planta. Hasil analisis transformasi pada tanaman kedelai transgenic secara in planta dapat dibuktikan dengan analisis PCR.

Keefektifan Agrobacterium mentransfer gen P5CS ke dalam kalus tebu klon PS 851 Effectiveness of Agrobacterium to transfer P5CS gene into sugarcane callus PS 851 clone

Summary Transformation of a P5CS gene construct into plant cells coupled with regeneration for transgenic plantlets should develop sugarcane tolerant to drought stress. The purpose of the research is to increase the effectiveness and efficiency of Agrobacterium transferring the gene into sugarcane callus. In this method, recombinant plasmid of pBI-P5CS could be transferred into host cells of Agrobacterium LBA4404 through triparental mating with pRK2013 helper. The parameters were tested to increase the effectiveness and efficiency of Agrobacterium transferring the gene into sugarcane callus were the addition of antioxidant and 1.0% glucose, callus age (2, 3, and 4 weeks), medium pH (4.5; 5.0; and 5.6), treated with air dry for 30 minutes, wetting agent of silwet with and without short vacuum treatment, and acetosyringone consentration (100, 500, and 1000 mg/L). Identification of the transgene in sugarcane was conducted by PCR using spesific primers, and the expression was tested by measuring of the proline content. The result showed that addition of acetosyringone 100 ppm or more, P5CS transfer into the sugarcane explants by Agrobacterium was effective. The genetic transformation could be optimized by selecting proper age of calli, which was four weeks after sub-culture. The effectiveness could be maintained and slightly improved by inoculation at pH 4.5, addition 1.0% glucose, wetting agent of silwet with short vacuum treatment, or treated with air drying for 30 minutes. In vitro cultures for transgenic regeneration required addition of antioxidant to prevent browning in the culture media. The amplified DNA fragment demonstrated that the gene was transferred into sugarcane plantlets, and P5CS gene expression showed increasing proline content in transgenic sugarcane plantlets.Ringkasan Transformasi transgen P5CS yang diikuti dengan regenerasi tanaman transgeniknya diper-kirakan mampu menghasilkan tanaman tebu transgenik yang toleran terhadap cekaman kekeringan. Penelitian ini bertujuan untuk me-ningkatkan efektivitas dan efisiensi Agro-bacterium mentransfer gen P5CS ke dalam kalus tebu. Dalam metode ini, plasmid rekombinan pBI-P5CS berhasil dengan baik ditransformasi-kan ke dalam sel. Agrobacterium LBA4404 dengan pendekatan triparental mating meng-gunakan helper pRK2013. Parameter yang diuji untuk meningkatkan kondisi efektif dan efisien dalam transfer gen P5CSke dalam kalus tebu adalah penambahan antioksidan dan glukosa 1,0%, umur kalus (2, 3, dan 4 minggu), pH medium (4,5; 5,0; dan 5,6), pengeringan kalus 30 menit, bahan pembasah silwet tanpa dan dengan pemakuman, dan konsentrasi aseto-siringon (100, 500, dan 1000 mg/L). Pengujian keberadaan transgen P5CS dilakukan dengan PCR menggunakan primer spesifik, sedangkan ekspresinya diuji dengan mengukur kandungan prolin dari tanaman tebu. Hasil percobaan menunjukkan bahwa dengan penambahan asetosiringon 100 ppm atau lebih, penggunaan Agrobacterium terbukti efektif dan efisien dalam transfer konstruk transgen P5CS ke dalam eksplan kalus tebu. Transformasi dapat dioptimalkan dengan memilih eksplan kalus tebu yang baik, yaitu yang umur subkulturnya empat minggu. Efektivitasnya juga dapat dijaga atau sedikit ditingkatkan dengan inokulasi pH 4,5, penambahan glukosa 1,0%, bahan pembasah silwet dengan pemakuman, ataupun pemberian perlakuan pengeringan udara selama 30 menit. Kultur kalus transgenik memerlukan penambahan antioksidan untuk mencegah terjadinya pen-cokelatan. Adanya fragmen DNA hasil amplifikasi dengan primer spesifik P5CS menunjukkan pada tanaman tebu telah terdapat gen P5CS. Demikian pula dengan ekspresi gen P5CS, menunjukkan adanya peningkatan kandungan prolin pada tanaman tebu transgenik.

Keefektifan Agrobacterium mentransfer gen P5CS ke dalam kalus tebu klon PS 851 Effectiveness of Agrobacterium to transfer P5CS gene into sugarcane callus PS 851 clone

Summary Transformation of a P5CS gene construct into plant cells coupled with regeneration for transgenic plantlets should develop sugarcane tolerant to drought stress. The purpose of the research is to increase the effectiveness and efficiency of Agrobacterium transferring the gene into sugarcane callus. In this method, recombinant plasmid of pBI-P5CS could be transferred into host cells of Agrobacterium LBA4404 through triparental mating with pRK2013 helper. The parameters were tested to increase the effectiveness and efficiency of Agrobacterium transferring the gene into sugarcane callus were the addition of antioxidant and 1.0% glucose, callus age (2, 3, and 4 weeks), medium pH (4.5; 5.0; and 5.6), treated with air dry for 30 minutes, wetting agent of silwet with and without short vacuum treatment, and acetosyringone consentration (100, 500, and 1000 mg/L). Identification of the transgene in sugarcane was conducted by PCR using spesific primers, and the expression was tested by measuring of the proline content. The result showed that addition of acetosyringone 100 ppm or more, P5CS transfer into the sugarcane explants by Agrobacterium was effective. The genetic transformation could be optimized by selecting proper age of calli, which was four weeks after sub-culture. The effectiveness could be maintained and slightly improved by inoculation at pH 4.5, addition 1.0% glucose, wetting agent of silwet with short vacuum treatment, or treated with air drying for 30 minutes. In vitro cultures for transgenic regeneration required addition of antioxidant to prevent browning in the culture media. The amplified DNA fragment demonstrated that the gene was transferred into sugarcane plantlets, and P5CS gene expression showed increasing proline content in transgenic sugarcane plantlets.Ringkasan Transformasi transgen P5CS yang diikuti dengan regenerasi tanaman transgeniknya diper-kirakan mampu menghasilkan tanaman tebu transgenik yang toleran terhadap cekaman kekeringan. Penelitian ini bertujuan untuk me-ningkatkan efektivitas dan efisiensi Agro-bacterium mentransfer gen P5CS ke dalam kalus tebu. Dalam metode ini, plasmid rekombinan pBI-P5CS berhasil dengan baik ditransformasi-kan ke dalam sel. Agrobacterium LBA4404 dengan pendekatan triparental mating meng-gunakan helper pRK2013. Parameter yang diuji untuk meningkatkan kondisi efektif dan efisien dalam transfer gen P5CSke dalam kalus tebu adalah penambahan antioksidan dan glukosa 1,0%, umur kalus (2, 3, dan 4 minggu), pH medium (4,5; 5,0; dan 5,6), pengeringan kalus 30 menit, bahan pembasah silwet tanpa dan dengan pemakuman, dan konsentrasi aseto-siringon (100, 500, dan 1000 mg/L). Pengujian keberadaan transgen P5CS dilakukan dengan PCR menggunakan primer spesifik, sedangkan ekspresinya diuji dengan mengukur kandungan prolin dari tanaman tebu. Hasil percobaan menunjukkan bahwa dengan penambahan asetosiringon 100 ppm atau lebih, penggunaan Agrobacterium terbukti efektif dan efisien dalam transfer konstruk transgen P5CS ke dalam eksplan kalus tebu. Transformasi dapat dioptimalkan dengan memilih eksplan kalus tebu yang baik, yaitu yang umur subkulturnya empat minggu. Efektivitasnya juga dapat dijaga atau sedikit ditingkatkan dengan inokulasi pH 4,5, penambahan glukosa 1,0%, bahan pembasah silwet dengan pemakuman, ataupun pemberian perlakuan pengeringan udara selama 30 menit. Kultur kalus transgenik memerlukan penambahan antioksidan untuk mencegah terjadinya pen-cokelatan. Adanya fragmen DNA hasil amplifikasi dengan primer spesifik P5CS menunjukkan pada tanaman tebu telah terdapat gen P5CS. Demikian pula dengan ekspresi gen P5CS, menunjukkan adanya peningkatan kandungan prolin pada tanaman tebu transgenik.

Transformation of HVA1 Gene Into Malus hupenensis var. `Pingyitiancha'

Malus hupenensis var. `Pingyitiancha' is an important apple stock with many good characteristics, including waterloggig resistance, cold resistance, salt resistance and so on. The three group gene-HVA1 come from barley was transformed into `Pingyitiancha' mediated by Agrobacterium tumefaciens and transformed regeneration plants were obtained in this research. The HAV1 gene cloned from plasmid containing it (offered by Dr. Guo Weidong) by PCR with high fidelity pfu Taq DNA polymerase. It was ligated between BamH 1 and Sac 1 site in PUC118 vector, and identified by electrophoresis after digested with BamH 1 and Sac 1. Through nuclear sequence detecting, it is confirmed that the HAV1 gene cloned in this research is 703bp.This fragment was ligated with 11kb fragment from pB121 plasmid and constructed pBHA vetor. The pBHA vector was introduced in A.tum LBA4404 by triparental mating and the binary vector was obtained. It is cinfirmed that HVA1 gene had been insert in T-DNA by in situ hybirdization. Using `Pingyitiancha' shoot apex, mediated by A. tum. System, the HAV 1 gene was transformed into the plant. Kam resistance regeneration plants were obtained, 6 of them were confirmed as transformation plants by PCR and dot blot.

Transformation of methylotrophic bacteria by electroporation

An efficient system for electroporation of the methylotrophic bacteria Hyphomicrobium facilis, Hyphomicrobium denitrificans, Methylobacillus glycogenes, Methylobacterium extorquens, and Methylophilus methylotrophus is described. It could be demonstrated that vectors based on the broad-host-range plasmid pBBR1 could be transferred into these strains. Plasmid pBBR1KAN (3.9 kb), a kanamycin-resistant derivative of pBBR1, was suitable for transformation experiments in these methylotrophic bacteria. Transformation efficiencies up to 104transformants/μg plasmid pBBR1KAN were obtained. The broad-host-range plasmid pLA2917 was transferred into Hyphomicrobium species by a triparental mating. However, this plasmid was integrated into the genome of Hyphomicrobium spp. Plasmids pLA2917, pKT231, pSUP2021, pRZ705, and phage DNA could not be transferred in Hyphomicrobium spp. by electroporation under the conditions applied.Key words: Hyphomicrobium, transformation, methylotrophic bacteria, plasmid pBBR1, broad-host-range vector.