Deden Sukmadjaja
Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian

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Development of Transgenic Sugarcane Resistant to Stem Borer by Transforming cry1Ab-cry1Ac Fusion Gene through Agrobacterium tumefaciens Transformation Method Deden Sukmadjaja; Sri Koerniati; Auliya Lukman
Jurnal AgroBiogen Vol 17, No 1 (2021): JUNE
Publisher : Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumber Daya Genetik Pertanian

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.21082/jbio.v17n1.2021.p11-24

Abstract

Bulu Lawang (BL) is a sugarcane variety preferred by farmers in Indonesia due to its high yield, but this cultivar is susceptible to shoot and stem borer insect pests. Genetic engineering using cry1Ab and cry1Ac fusion gene is an effort to generate BL varieties resistant to the insect pests. This study aimed to 1) transform T-DNA containing cry1Ab-cry1Ac fusion gene into sugarcane genome by using Agrobacterium tumefaciens method, 2) obtain selection media composition of callus transformants, and 3) obtain transformation efficiency comparison of A. tumefaciens strains EHA105 and GV3101. The research was conducted at the Laboratory of Cell and Tissue Biology, the Laboratory of Molecular Biology, and the greenhouse of the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, Bogor from March to August 2019. Research activities consisted of four parts namely 1) callus induction and Agrobacterium culture preparation containing pCambia-5300_OaRbcS-prom-cTP-cry1Ab-cry1Ac plasmid, 2) callus incubation in cocultivation and resting media, 3) selection and differentiation of shoots on regeneration media, and 4) molecular analysis using PCR method. Results showed that the composition of media, both for selection and regeneration processes of putative plant transformants, was the key to the success of this experiment. A. tumefaciens strain EHA105 resulted in higher transformation efficiency (11.1%) compared to that of strain GV3101 (9.0%). Molecular analysis showed that cryIAb-cryIAc fusion gene was successfully inserted into the sugarcane genome suggesting that the transgenic plant containing cry1Ab-cry1Ac fusion gene was obtained. The putative transgenic plants need further assay through bioassay tests to verify its resistance phenotype to the insect pests.
Uji Efikasi Teknik Kultur Meristem dan Kemoterapi untuk Eliminasi Sugarcane Streak Mosaic Virus (SCSMV) pada Tebu Ika Roostika; Sedyo Harsono; Darda Efendi; Deden Sukmadjaja; Cece Suhara
Buletin Tanaman Tembakau, Serat & Minyak Industri Vol 8, No 2 (2016): Oktober 2016
Publisher : Balai Penelitian Tanaman Pemanis dan Serat

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.21082/btsm.v8n2.2016.55-64

Abstract

Penggunaan benih bebas virus merupakan salah satu cara pengendalian penyakit virus. Jaringan tanaman dapat dibebaskan dari virus melalui aplikasi teknik eliminasi virus, seperti termoterapi, kemoterapi, kultur meristem, dan krioterapi. Tujuan penelitian ini adalah untuk menguji respon varietas tebu terhadap perlakuan teknik kultur meristem dan kemoterapi dengan bahan antiviral, serta untuk mengetahui efektivitasnya dalam mengeliminasi virus sugarcane streak mosaic virus (SCSMV) pada tebu. Penelitian ini dilakukan pada April−November 2015 di Laboratorium Kultur Jaringan, Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian dan Laboratorium Virologi, Fakultas Pertanian, Universitas Gadjah Mada. Penelitian terdiri atas tiga tahap, yaitu 1) Deteksi virus dari tanaman induk, 2) Aplikasi teknik kultur meristem dan kemoterapi, serta 3) Indeksing virus. Bahan tanaman yang digunakan adalah sebelas varietas tebu (GMP3, PS865, dan Kentung asal Bogor, PS862 dan Cening asal Cirebon, PS881 asal Jember, PSJK922 asal Malang, serta PS864, PS881, PSJK922, PSJT941 asal Pati). Deteksi virus dilakukan secara RT-PCR dengan primer universal MJ dan primer spesifik SCSMV. Bahan antiviral yang digunakan untuk kemoterapi adalah Ribavirin (0 dan 25 µg/l). Hasil uji RT-PCR menggunakan primer universal MJ menunjukkan bahwa empat varietas (GMP3 asal Bogor, PS864 dan PSJT941 asal Pati, serta Cening asal Cirebon) terinfeksi oleh Potyvirus. Empat varietas lainnya (PS862 asal Cirebon, PS881 asal Jember, PSJK922 asal Malang, dan Kentung asal Bogor) terbukti terserang virus SCSMV berdasarkan uji RT-PCR dengan primer spesifik. Seluruh meristem mampu beregenerasi membentuk tunas. Penggunaan Ribavirin 25 µg/l tidak menyebabkan penurunan daya tumbuh meristem (50−100%), bahkan seluruh varietas mampu bermultiplikasi tunasnya dibandingkan dengan kontrol yang hanya memiliki daya tumbuh 0−100%, dan tidak semua varietas mampu bermultiplikasi tunasnya. Secara tunggal, aplikasi teknik kultur meristem tidak mampu mengeliminasi virus SCSMV, namun jika dikombinasikan dengan perlakuan kemoterapi maka virus SCSMV dapat tereliminasi dengan efikasi sebesar 44,4%. The use of virus-free seedling is an option for controlling viral disease that can be obtained through the application of viral elimination method. Plant tissues can be eliminated from virus infection by applying virus thermotherapy, chemotherapy, meristem culture, and cryotherapy. The research objectives were to examine the response of sugarcane varieties to meristem culture treatments and antiviral agent and also to determine the efficacy rate of both techniques in eliminating SCSMV disease. The study was conducted atTissuseCulture Laboratory, Indonesian Center for Agricultural Biotechnology and Genetic Resources Research andDevelpoment, and also at Virology Laboratory, Faculty of Agriculture, Gadjah Mada University.   This study consisted of three activities: 1) Virus detection of the mother plants, 2) Application of meristem culture and chemotherapy, and 3) Virus indexing. The plant material used was eleven varieties of sugarcane (GMP3, PS865, and Kentung from Bogor, PS862 and Cening from Cirebon, PS881 from Jember, Malang PSJK922 origin, as well as the PS864, PS881, PSJK922, PSJT941 from Pati). Virus detection was performed by RT-PCR assay with universal primer of MJ and specific primers of SCSMV. Antiviral used for chemotherapy was Ribavirin (0 and 25 µg/l). The result of RT-PCR using universal primers MJ showed that four varieties (GMP3 from Bogor, PS864 and PSJT941 from Pati, and Cening from Cirebon) were infected by Potyvirus. Based on RT-PCR assay with specific primer, four other varieties (PS862 from Cirebon, PS881 from Jember, PSJK922 from Malang, and Kentung from Bogor) were infected by SCSMV. All of meristems were able to regenerate to form shoots. The use of Ribavirin (25µg/l) did not decrease the growth rate of meristems and the shoots of all of the varieties could be multipied compared to control where the shoots could not be multiplied in all varietis. The application of meristem culture technique was not able to eliminate the SCSMV, but when it was combined with chemotherapy treatment, the SCSMV virus could be eliminated with the efficacy rate of 44.4%. 
Pengaruh PVP dan DIECA terhadap Regenerasi Meristem Tebu Ika Roostika; Rara Puspita Dewi Lima Wati; Deden Sukmadjaja
Buletin Tanaman Tembakau, Serat & Minyak Industri Vol 7, No 1 (2015): April 2015
Publisher : Balai Penelitian Tanaman Pemanis dan Serat

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.21082/bultas.v7n1.2015.9-14

Abstract

Tebu (Saccharum officinarum L.) merupakan tanaman yang diperbanyak secara vegetatif sehingga berisiko besar akan terjadinya akumulasi virus di dalam jaringan tanaman. Kultur meristem merupakan salah satu teknik eliminasi virus yang umum digunakan, namun seringkali meristem memiliki daya hidup dan daya re-generasi yang rendah. Salah satu penyebabnya adalah karena akumulasi senyawa fenol. Akumulasi senyawa tersebut dapat direduksi melalui penggunaan senyawa adsorben dan antioksidan. Penelitian ini bertujuan un-tuk mengetahui pengaruh polyvinylpyrrolidone (PVP) dan diethyldithiocarbamate sodium (DIECA) terhadap regenerasi meristem tebu. Bahan tanaman yang digunakan adalah tebu PS864. Eksplan yang digunakan adalah meristem dengan 1–2 primordia daun yang diisolasi di bawah mikroskop. Perlakuan meliputi PVP (100 dan 300 mg/l) dan DIECA (0 dan 20 mg/l) serta kombinasi antara kedua zat tersebut, dengan 3 ulang-an (botol) dan setiap botol terdiri atas 3 meristem. Hasil penelitian menunjukkan bahwa peningkatan kon-sentrasi PVP atau kombinasi perlakuan PVP dan DIECA dapat meningkatkan persentase eksplan hidup, daya regenerasi, dan jumlah tunas. Kombinasi perlakuan PVP 300 mg/l dan DIECA 20 mg/l merupakan perlakuan terbaik karena persentase hidup dan daya regenerasi eksplan yang paling tinggi (100%) dengan jumlah tu-nas 3,8 tunas/eksplan. Being vegetatively propagated, sugar cane faces a high risk of virus accumulation. Meristem culture is one method that usually applied for virus elimination. However, it often has low survival and regeneration rate due to an accumulation of phenolic compounds. Accumulation of those compounds can be reduced by apply adsorbent antioxidant. This research aimed at evaluating the effect of PVP and DIECA on the regeneration capacity of meristem. The plant material was sugar cane PS864. Meristems with 1─2 primoridial leaves were used as the explants and isolated under microscope. The PVP (100−300 mg/l) and DIECA (0− 20 mg/l), or combined treatment of both antioxidants were used as treatments. Each treatment was replicated 3 times (bottles), and each bottle contained 3 meristems. The result showed that the higher concentration of PVP or combined treatment of PVP and DIECA could increase the percentage of survival, regeneration rate, and number of shoot. The combined treatment of 300 mg/l PVP, and 20 mg/l DIECA produced the highest level of survival rate (100%) which yielded 3.8 shoots/explants.
Uji Efikasi Teknik Kultur Meristem dan Kemoterapi untuk Eliminasi Sugarcane Streak Mosaic Virus (SCSMV) pada Tebu Ika Roostika; Sedyo Harsono; Darda Efendi; Deden Sukmadjaja; Cece Suhara
Buletin Tanaman Tembakau, Serat & Minyak Industri Vol 8, No 2 (2016): Oktober 2016
Publisher : Balai Penelitian Tanaman Pemanis dan Serat

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (346.938 KB) | DOI: 10.21082/btsm.v8n2.2016.55-64

Abstract

Penggunaan benih bebas virus merupakan salah satu cara pengendalian penyakit virus. Jaringan tanaman dapat dibebaskan dari virus melalui aplikasi teknik eliminasi virus, seperti termoterapi, kemoterapi, kultur meristem, dan krioterapi. Tujuan penelitian ini adalah untuk menguji respon varietas tebu terhadap perlakuan teknik kultur meristem dan kemoterapi dengan bahan antiviral, serta untuk mengetahui efektivitasnya dalam mengeliminasi virus sugarcane streak mosaic virus (SCSMV) pada tebu. Penelitian ini dilakukan pada April−November 2015 di Laboratorium Kultur Jaringan, Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian dan Laboratorium Virologi, Fakultas Pertanian, Universitas Gadjah Mada. Penelitian terdiri atas tiga tahap, yaitu 1) Deteksi virus dari tanaman induk, 2) Aplikasi teknik kultur meristem dan kemoterapi, serta 3) Indeksing virus. Bahan tanaman yang digunakan adalah sebelas varietas tebu (GMP3, PS865, dan Kentung asal Bogor, PS862 dan Cening asal Cirebon, PS881 asal Jember, PSJK922 asal Malang, serta PS864, PS881, PSJK922, PSJT941 asal Pati). Deteksi virus dilakukan secara RT-PCR dengan primer universal MJ dan primer spesifik SCSMV. Bahan antiviral yang digunakan untuk kemoterapi adalah Ribavirin (0 dan 25 µg/l). Hasil uji RT-PCR menggunakan primer universal MJ menunjukkan bahwa empat varietas (GMP3 asal Bogor, PS864 dan PSJT941 asal Pati, serta Cening asal Cirebon) terinfeksi oleh Potyvirus. Empat varietas lainnya (PS862 asal Cirebon, PS881 asal Jember, PSJK922 asal Malang, dan Kentung asal Bogor) terbukti terserang virus SCSMV berdasarkan uji RT-PCR dengan primer spesifik. Seluruh meristem mampu beregenerasi membentuk tunas. Penggunaan Ribavirin 25 µg/l tidak menyebabkan penurunan daya tumbuh meristem (50−100%), bahkan seluruh varietas mampu bermultiplikasi tunasnya dibandingkan dengan kontrol yang hanya memiliki daya tumbuh 0−100%, dan tidak semua varietas mampu bermultiplikasi tunasnya. Secara tunggal, aplikasi teknik kultur meristem tidak mampu mengeliminasi virus SCSMV, namun jika dikombinasikan dengan perlakuan kemoterapi maka virus SCSMV dapat tereliminasi dengan efikasi sebesar 44,4%. The use of virus-free seedling is an option for controlling viral disease that can be obtained through the application of viral elimination method. Plant tissues can be eliminated from virus infection by applying virus thermotherapy, chemotherapy, meristem culture, and cryotherapy. The research objectives were to examine the response of sugarcane varieties to meristem culture treatments and antiviral agent and also to determine the efficacy rate of both techniques in eliminating SCSMV disease. The study was conducted atTissuseCulture Laboratory, Indonesian Center for Agricultural Biotechnology and Genetic Resources Research andDevelpoment, and also at Virology Laboratory, Faculty of Agriculture, Gadjah Mada University.   This study consisted of three activities: 1) Virus detection of the mother plants, 2) Application of meristem culture and chemotherapy, and 3) Virus indexing. The plant material used was eleven varieties of sugarcane (GMP3, PS865, and Kentung from Bogor, PS862 and Cening from Cirebon, PS881 from Jember, Malang PSJK922 origin, as well as the PS864, PS881, PSJK922, PSJT941 from Pati). Virus detection was performed by RT-PCR assay with universal primer of MJ and specific primers of SCSMV. Antiviral used for chemotherapy was Ribavirin (0 and 25 µg/l). The result of RT-PCR using universal primers MJ showed that four varieties (GMP3 from Bogor, PS864 and PSJT941 from Pati, and Cening from Cirebon) were infected by Potyvirus. Based on RT-PCR assay with specific primer, four other varieties (PS862 from Cirebon, PS881 from Jember, PSJK922 from Malang, and Kentung from Bogor) were infected by SCSMV. All of meristems were able to regenerate to form shoots. The use of Ribavirin (25µg/l) did not decrease the growth rate of meristems and the shoots of all of the varieties could be multipied compared to control where the shoots could not be multiplied in all varietis. The application of meristem culture technique was not able to eliminate the SCSMV, but when it was combined with chemotherapy treatment, the SCSMV virus could be eliminated with the efficacy rate of 44.4%. 
Pengaruh PVP dan DIECA terhadap Regenerasi Meristem Tebu Ika Roostika; Rara Puspita Dewi Lima Wati; Deden Sukmadjaja
Buletin Tanaman Tembakau, Serat & Minyak Industri Vol 7, No 1 (2015): April 2015
Publisher : Balai Penelitian Tanaman Pemanis dan Serat

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (426.84 KB) | DOI: 10.21082/bultas.v7n1.2015.9-14

Abstract

Tebu (Saccharum officinarum L.) merupakan tanaman yang diperbanyak secara vegetatif sehingga berisiko besar akan terjadinya akumulasi virus di dalam jaringan tanaman. Kultur meristem merupakan salah satu teknik eliminasi virus yang umum digunakan, namun seringkali meristem memiliki daya hidup dan daya re-generasi yang rendah. Salah satu penyebabnya adalah karena akumulasi senyawa fenol. Akumulasi senyawa tersebut dapat direduksi melalui penggunaan senyawa adsorben dan antioksidan. Penelitian ini bertujuan un-tuk mengetahui pengaruh polyvinylpyrrolidone (PVP) dan diethyldithiocarbamate sodium (DIECA) terhadap regenerasi meristem tebu. Bahan tanaman yang digunakan adalah tebu PS864. Eksplan yang digunakan adalah meristem dengan 1–2 primordia daun yang diisolasi di bawah mikroskop. Perlakuan meliputi PVP (100 dan 300 mg/l) dan DIECA (0 dan 20 mg/l) serta kombinasi antara kedua zat tersebut, dengan 3 ulang-an (botol) dan setiap botol terdiri atas 3 meristem. Hasil penelitian menunjukkan bahwa peningkatan kon-sentrasi PVP atau kombinasi perlakuan PVP dan DIECA dapat meningkatkan persentase eksplan hidup, daya regenerasi, dan jumlah tunas. Kombinasi perlakuan PVP 300 mg/l dan DIECA 20 mg/l merupakan perlakuan terbaik karena persentase hidup dan daya regenerasi eksplan yang paling tinggi (100%) dengan jumlah tu-nas 3,8 tunas/eksplan. Being vegetatively propagated, sugar cane faces a high risk of virus accumulation. Meristem culture is one method that usually applied for virus elimination. However, it often has low survival and regeneration rate due to an accumulation of phenolic compounds. Accumulation of those compounds can be reduced by apply adsorbent antioxidant. This research aimed at evaluating the effect of PVP and DIECA on the regeneration capacity of meristem. The plant material was sugar cane PS864. Meristems with 1─2 primoridial leaves were used as the explants and isolated under microscope. The PVP (100−300 mg/l) and DIECA (0− 20 mg/l), or combined treatment of both antioxidants were used as treatments. Each treatment was replicated 3 times (bottles), and each bottle contained 3 meristems. The result showed that the higher concentration of PVP or combined treatment of PVP and DIECA could increase the percentage of survival, regeneration rate, and number of shoot. The combined treatment of 300 mg/l PVP, and 20 mg/l DIECA produced the highest level of survival rate (100%) which yielded 3.8 shoots/explants.