Muh. Alias L. Rajamuddin, Muh. Alias L.
Postgraduated student of Bogor Agricultural University, Bogor. Polytechnic Agricultural of Pangkep, South Sulawesi.

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Evaluation of different promoters driving the GFP reporter gene in seaweed Kappaphycus alvarezii Rajamuddin, Muh. Alias L.; Alimuddin, A.; Widyastuti, Utut; Faizal, Irvan
Indonesian Journal of Biotechnology Vol 19, No 2 (2014)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (366.674 KB) | DOI: 10.22146/ijbiotech.9304

Abstract

Promoter regulates expression level of foreign gene in transgenic organism. This study was performed to select asuitable promoter as the fi rst step towards production of valuable trait-enhanced seaweed by transgenic technology. Greenfl uorescent protein (GFP) gene was used as a reporter to determine the activity of promoter in seaweed Kappaphycusalvarezii. GFP gene constructs driven by cytomegalovirus (pCMV-GFP), caulifl ower mosaic virus (pCaMV-GFP),medaka β-actin (pmBA-GFP) and Japanese fl ounder keratin (pJfKer-GFP) promoters were introduced by electroporationmethod. Electroporation was performed using a gene pulser (BIORAD) with voltage of 300 V, pulse length of 0.5 ms,pulse numbers of 4, and pulse interval of 0.1 s. Promoter activity was determined by analyzing GFP gene expressionlevel using a fl uorescent microscope. The results showed that CMV regulated highest number of fi lament callus(34.10%±1.49) expressing GFP at medium to strong fl uorescence levels. CaMV promoter had relatively similar activitywith CMV, but lower number of fi lament callus expressing GFP (10.48%±0.25). mBA promoter drove GFP expressionat medium level and similar number of fi lament callus (8.85%±2.31) expressing GFP with CaMV, while JfKer promoterhad lowest activity by means in number of fi lament callus expressing GFP (4.79%±0.26) and GFP expression level. PCRanalysis for transgenic confi rmation showed a DNA band of PCR product from pCMV-GFP and pCaMV-GFP expressingfi lament callus in the same size (about 0.6 kb) with positive control of plasmid. Thus, CMV and CaMV promoters wasan appropriate promoter and foreign gene could be transferred to fi lament callus by electroporation method. Combiningthis achievement with developing a culture method of fi lament callus to be thallus, stable transgenic breeding in K.alvarezii can be feasible.
KAJIAN INDUKSI KALUS RUMPUT LAUT Kappaphycus alvarezii UNTUK PRODUKSI EMBRIO SOMATIK Rajamuddin, Muh. Alias L.; Jaya, Andi Asdar; Ridwan, Ridwan; Suryati, Emma
Jurnal Riset Akuakultur Vol 5, No 2 (2010): (Agustus 2010)
Publisher : Pusat Riset Perikanan, Badan Riset dan Sumber Daya Manusia Kelautan dan Perikanan

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (217.066 KB) | DOI: 10.15578/jra.5.2.2010.211-219

Abstract

Untuk mendukung program transgenesis pada rumput laut, embrio somatik dapat digunakan sebagai material untuk transfer gen baik secara individu sel ataupun kluster sel embriogenik, sehingga mempercepat keberhasilan dengan peluang transformasi yang lebih tinggi. Penelitian ini bertujuan untuk mengkaji induksi kalus rumput laut K. alvarezii untuk produksi sel embrio somatik (e.s.) dengan beberapa rasio zat pengatur tumbuh (ZPT) dan konsentrasi agar media induksi, sampai sel menjadi filamen. Penelitian terdiri atas dua tahap: Tahap (1) induksi kalus, dengan rasio ZPT asam indol asetat (IAA):kinetin = 0,5:0,0 mg/L; 1,0:1,0 mg/L; dan 2,0:0,2 mg/L dengan konsentrasi agar media induksi = 0,6%; 0,8%; 1,0%; dan 1,5%. Tahap (2) regenerasi massa sel e.s., dengan rasio IAA:kinetin = 0,1:1,0 mg/L; 0,0:0,1 mg/L dan tanpa ZPT dengan konsentrasi agar media = 0,4%; 0,6%; dan 0,8%. Untuk perkembangan sel-sel e.s. lebih lanjut dipelihara pada kultur cair. Hasil penelitian menunjukkan pada tahap induksi kalus, rasio IAA: kinetin = 1:1 mg/L dengan konsentrasi agar media 0,8% dan 1,0% menghasilkan persentase induksi kalus tertinggi (90%). Pada tahap regenerasi massa sel e.s., ZPT tidak berpengaruh terhadap perkembangan massa sel e.s., di mana tanpa ZPT dengan konsentrasi agar 0,6% memperlihatkan perkembangan tertinggi (rata-rata diameter massa sel 5 mm). Pada media cair, perkembangan sel e.s. dari single cell ukuran 3-4 mm menjadi filamen-filamen ukuran rata-rata 0,5 mm dapat dicapai dalam satu bulan kultur. Keberhasilan produksi sel e.s. K. alvarezii, selain sebagai material untuk transfer gen juga dapat dijadikan acuan dalam produksi benih rumput laut kultur jaringan.To support the program of seaweed transgenesis, somatic embryo can be used as a materials for gene transfer purpose either by individual or cluster of cells in accelerating the higher rate of transformation. This research aims to study the callus induction of seaweed K. alvarezii for production of somatic embrio (s.e) cell by different ratio of growth regulators (GR) and agar media concentrations. The study consists of two stages: stage (1) callus induction to the cells filament using GR of indol acetic acid (IAA):kinetin in ratio of  0.5:0.0 mg/L; 1.0:1.0 mg/L; and 2.0:0.2 mg/L on the media agar concentration of 0.6%; 0.8%; 1.0%; and 1.5%, and stage (2) regeneration of the cell mass of s.e, using GR of IAA:kinetin in ratio of 0.1:1.0 mg/L; 0.0:0.1 mg/L on the media agar concentration of 0.4%; 0.6%; and 0.8%. For further maintenance, the s.e cells were cultured in liquid media. The results of callus induction showed that the ratio of IAA:kinetin (1:1) on the media agar concentration of 0.8% and 1.0% produced the highest callus induction (90%). The study of mass cell regeneration of s.e showed did not different of GR IAA:kinetin ratio of the cell mass development, but the media agar concentration of 0.6% and 0.4% showed the higher growth of cell mass (in diameter size of 4-5 mm). The development of cells culture of s.e from the size of 3-4 mm to filament of 0.5 mm could be reached in one month of culture period. The success of K. alvarezii s.e production will be helpful not only as a material for gene transfer but also as a reference on tissue culture for seed production of seaweed.