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

Abstract

Summary
Genetic engineering of robusta coffee for
resistance to pathogenic fungi is considered to be
one of the potential approaches to overcome the
problem at robusta coffee plantation caused by
pathogenic fungi. This research was aimed to
introduce chitinase (CHI) gene into embryogenic
calli of robusta coffee and regenerate the
plantlets. Embryogenic calli were co-cultivated
with Agrobacterium tumefaciens LBA4404
harboring pCAMBIA1301 which contains
chitinase gene under 35S promoter. In this
research four concentrations (0, 50, 100 and
150 mg/L) of acetosyringone (AC) were used in
the co-cultivation medium. Selection for
transformed calli was conducted by gradually
increasing the concentration of hygromicin from
5 to 25 mg/L. Somatic embryo (SE) was induced
from callus on the medium containing a
combination of BAP 5 mg/L and IAA (0, 0.25 or
0.50 mg/L). Integration CHI in plant genome was
examined by GUS assay and PCR. The result
revealed that among the four AC concentrations
tested, 100 mg/L gave the highest percentage of
calli growing on the selection medium (42.5%).
BAP concentration of 5 mg/L alone was the most
effective for inducing of SE from transformed
calli with the highest percentage of 43.1% and
average number SE of 8.8 ± 3. The strongest
GUS expression on the calli at 3 days after
transformation and the calli grown on selection
medium containing 150 mg/L AC, which were
56.5% and 40% respectivelly. PCR analysis
showed that 7 out of 12 plantlets tested,
contained CHI gene. From this research 28
transgenic plantlets of robusta coffee were
obtained

Ringkasan
Rekayasa genetika untuk merakit tanaman
kopi robusta tahan jamur pathogen dipandang
merupakan salah satu pendekatan alternatif yang
potensial untuk mengatasi masalah pada
perkebunan kopi robusta akibat serangan jamur
patogen. Penelitian ini bertujuan untuk meng-
introduksikan gen kitinase (CHI) ke dalam kalus
embriogenik kopi robusta dan regenerasinya
menjadi planlet, sebagai upaya untuk merakit
tanaman kopi robusta tahan serangan jamur.
Kalus embriogenik diko-kultivasi dengan
Agrobacterium tumefaciens LBA4404 pembawa
pCAMBIA1301 yang mengandung gen kitinase
di bawah kontrol promotor 35S. Pada percobaan

ini, empat konsentrasi asetosiringon (AS) (0, 50,
100 dan 150 mg/L) digunakan dalam medium ko-
kultivasi. Seleksi kalus hasil transformasi
dilakukan dengan peningkatan konsentrasi higro-
misin secara bertahap dari 5 mg/L sampai
25 mg/L. ES diinduksi dari kalus pada medium
yang mengandung BAP 5 mg/L dan IAA (0; 0,25
dan 0,50 mg/L). Integrasi gen CHI ke dalam
genom tanaman dianalisis melalui uji GUS dan
PCR. Hasil penelitian menunjukkan bahwa dari
keempat konsentrasi AS yang diuji, AS 100 mg/L
ternyata menghasilkan persentase tertinggi kalus
yang tumbuh pada medium seleksi (42,5%).
Konsentrasi BAP 5 mg/L tanpa penambahan IAA
efektif menginduksi ES dari kalus hasil
transformasi dengan persentase tertinggi 43,1%
dan rata-rata jumlah ES 8,8±3. Ekspresi GUS
tertinggi dideteksi pada kalus tiga hari setelah
transformasi dan kalus yang tumbuh di medium
seleksi yang mengandung AS 150 mg/L,
masing-masing 56,5% dan 40,0 %. Analisis PCR
menunjukkan bahwa 7 planlet dari 12 planlet
yang diuji, membawa gen CHI. Dari penelitian
ini dihasilkan 28 planlet kopi robusta transgenik.