Transformasi kopi robusta (Coffea canephora) dengan gen kitinase melalui Agrobagterium tumefaciens LBA4404 Transformation of robusta coffee (Coffea canephora) with chitinase gene mediated by Agrobacterium tumefaciens LBA4404
Authors: SISWANTO . , OKTAVIA Fetrina , BUDIANI Asmini , , SUDARSONO . , PRIYONO . , MAWARDI Surip 
Published: September 2018

SummaryGenetic engineering of robusta coffee forresistance to pathogenic fungi is considered to beone of the potential approaches to overcome theproblem at robusta coffee plantation caused bypathogenic fungi. This research was aimed tointroduce chitinase (CHI) gene into embryogeniccalli of robusta coffee and regenerate theplantlets. Embryogenic calli were co-cultivatedwith Agrobacterium tumefaciens LBA4404harboring pCAMBIA1301 which containschitinase gene under 35S promoter. In thisresearch four concentrations (0, 50, 100 and150 mg/L) of acetosyringone (AC) were used inthe co-cultivation medium. Selection fortransformed calli was conducted by graduallyincreasing the concentration of hygromicin from5 to 25 mg/L. Somatic embryo (SE) was inducedfrom callus on the medium containing acombination of BAP 5 mg/L and IAA (0, 0.25 or0.50 mg/L). Integration CHI in plant genome wasexamined by GUS assay and PCR. The resultrevealed that among the four AC concentrationstested, 100 mg/L gave the highest percentage ofcalli growing on the selection medium (42.5%).BAP concentration of 5 mg/L alone was the mosteffective for inducing of SE from transformedcalli with the highest percentage of 43.1% andaverage number SE of 8.8 ± 3. The strongestGUS expression on the calli at 3 days aftertransformation and the calli grown on selectionmedium containing 150 mg/L AC, which were56.5% and 40% respectivelly. PCR analysisshowed that 7 out of 12 plantlets tested,contained CHI gene. From this research 28transgenic plantlets of robusta coffee wereobtainedRingkasanRekayasa genetika untuk merakit tanamankopi robusta tahan jamur pathogen dipandangmerupakan salah satu pendekatan alternatif yangpotensial untuk mengatasi masalah padaperkebunan kopi robusta akibat serangan jamurpatogen. Penelitian ini bertujuan untuk meng-introduksikan gen kitinase (CHI) ke dalam kalusembriogenik kopi robusta dan regenerasinyamenjadi planlet, sebagai upaya untuk merakittanaman kopi robusta tahan serangan jamur.Kalus embriogenik diko-kultivasi denganAgrobacterium tumefaciens LBA4404 pembawapCAMBIA1301 yang mengandung gen kitinasedi bawah kontrol promotor 35S. Pada percobaanini, empat konsentrasi asetosiringon (AS) (0, 50,100 dan 150 mg/L) digunakan dalam medium ko-kultivasi. Seleksi kalus hasil transformasidilakukan dengan peningkatan konsentrasi higro-misin secara bertahap dari 5 mg/L sampai25 mg/L. ES diinduksi dari kalus pada mediumyang mengandung BAP 5 mg/L dan IAA (0; 0,25dan 0,50 mg/L). Integrasi gen CHI ke dalamgenom tanaman dianalisis melalui uji GUS danPCR. Hasil penelitian menunjukkan bahwa darikeempat konsentrasi AS yang diuji, AS 100 mg/Lternyata menghasilkan persentase tertinggi kalusyang tumbuh pada medium seleksi (42,5%).Konsentrasi BAP 5 mg/L tanpa penambahan IAAefektif menginduksi ES dari kalus hasiltransformasi dengan persentase tertinggi 43,1%dan rata-rata jumlah ES 8,8±3. Ekspresi GUStertinggi dideteksi pada kalus tiga hari setelahtransformasi dan kalus yang tumbuh di mediumseleksi yang mengandung AS 150 mg/L,masing-masing 56,5% dan 40,0 %. Analisis PCRmenunjukkan bahwa 7 planlet dari 12 planletyang diuji, membawa gen CHI. Dari penelitianini dihasilkan 28 planlet kopi robusta transgenik.