THE USE OF GREEN FLUORESCENCE PROTEIN GENE FOR DETECTING THE OCCURENCE OF ENDOPHYTE DIAZOTROPHIC BACTERIA IN SUGARCANE TISSUE

by WIWIK E. WIDAYATI

Pusat Penelitian Perkebunan Gula Indonesia, Pasuruan/Indonesian Sugar Research Institute
alamat korespondensi : P3GI Telp. 0343-421086, Fax. 0343 421178, w_e_widayati@yahoo.com

ABSTRAK

Tujuan dari penelitian ini adalah mendeteksi keberadaan isolat bakteri diazotrof endofit
dalam jaringan tebu dengan menggunakan penanda gen gfp. Sebanyak 4 isolat bakteri
diazotrof endofit yang telah diperoleh dari penelitian sebelumnya yaitu isolat Klebsiellasp
10.2.3, Bacillussp. NB12, Pseudomonassp 10 K1 dan Acinetobactersp 10 K2 digunakan
dalam penelitian ini. Deteksi keberadaan keempat isolat tersebut, dilakukan dengan
menggunakan penanda gen green fluorescence protein (gfp), kemudian dinokulasikan pada
tebu varietas PS 851 yang ditanam di rumah kaca sampai umur 35 hari. Keberadaan bakteri
diazotrof endofit dalam jaringan tebu ditunjukkan dengan visualisasi koloni bakteri yang
berada dalam akar dan daun tebu menggunakan mikroskop fluorescencedan jumlah bakteri
pada bagian akar, batang dan daun tebu. Hasil yang diperoleh menunjukkan bahwa
penandaan gen gfppada bakteri diazotrof endofit telah berhasil dilakukan baik pada bakteri
gram positif maupun bakteri gram negatif. Penyisipan gen gfptersebut tidak merubah
kemampuan endofitik bakteri diazotrof yang diuji serta bersifat tetap walau disimpan secara
in vitrodalam tenggang waktu cukup lama. Visualisasi koloni bakteri diazotrof endofit dalam
akar dan sap daun menggunakan mikroskop fluorescen tampak berbentuk mikrokoloni.
Hasil reisolasi bakteri diazotrof endofit pada tebu berumur 15 hari menunjukkan bahwa
bakteri diazotrof endofit tersebut terdistribusi pada seluruh bagian tanaman tebu yaitu
dijumpai mulai di daerah perakaran tebu, batang dan di daun tebu. Acinetobactersp 10 K2
dan Pseudomonassp 10 K1 memiliki tingkat kesesuaian yang lebih tinggi dengan tanaman
tebu dari pada isolat Klebsiellasp 10.2.3 dan Bacillussp. NB12 karena pada umur tebu 35
hari masih dijumpai pada bagian akar, batang dan daun tebu.

Key words : endofit, bakteri diazotrofic, gen gfpdan tebu

ABSTRACT

The aim of this research was to detect the occurrence of endophyte diazotrophic bacteria
in sugarcane tissue by using green fluorescence protein gene marker. Klebsiella sp 10.2.3,
Bacillus sp. NB12, Pseudomonas sp 10 K1 and Acinetobacter sp 10 K2 as endophyte
diazotrophic bacteria isolated from sugarcane were used in this research. The fourth isolates
of endophyte diazotrophic bacteria were marked with green fluorescence protein (gfp) gene
and inoculated to sugarcane bud chip of PS 851. The bud chip was kept grow until 35 days
old at a glass house. The occurrence of endophyte diazotrophic bacteria in sugarcane tissue
was showed by visualization of endophyte diazotrophic bacteria colony in sugarcane root
tissue, leaf sap and the amount of endophyte diazotrophic bacteria in sugarcane tissue
(leaf, stalks and sugarcane root at 15 and 35 days old). The results showed that gfp gene
has been inserted successfully into the four bacterial chromosomes either gram positive or
gram negative. Endophytic properties of the bacteria did not change by the insertion of gfp
gene and these bacteria were stable during in vitro storage for a long period. Visualization of
the fourth isolates in sugarcane root tissue and leaf sap of sugarcane at 15 days old showed
that the fourth isolates presence as a micro colony. Acinetobacter sp 10 K2 and Pseudomonas
sp 10 K1 were found distributed on leaf stalks and root of sugarcane at 35 days old. It
seems that Acinetobacter sp 10 K2 and Pseudomonas sp 10 K1 have higher compatibility
to sugarcane than Klebsiella sp 10.2.3 and Bacillus sp. NB12 isolates

Key words : endophyte, diazotrophic bacteria, gfp gene,and sugarcane

INDONESIAN SUGAR RESEARCH JOURNAL  Vol. 44, No. 3, September 2008
Terakreditasi B, SK LIPI No. 536/D/2007 tgl. 26 Juni 2007

ACCELERATION OF PREPARING HEALTHY SUGAR CANE SEEDLINGS THROUGH MICRO CUTTING MULTIPLICATION

SRIWINARSIH DAN EKA SUGIYARTA

Pusat Penelitian Perkebunan Gula Indonesia, Jl Pahlawan 25 Pasuruan

ABSTRAK

Penyediaan bibit varietas tebu unggul baru secara cepat mempunyai peranan yang
penting dalam menghambat terjadinya proses degenerasi klonal. Perbanyakan bibit melalui
bagal mikro mempunyai keunggulan antara lain lebih cepat dan efisien dalam pengiriman
bibit. Penelitian yang bertujuan untuk mengetahui tingkat multiplikasi bagal mikro telah
dilaksanakan di hardening kebun percobaan Pusat Penelitian Perkebunan Gula Indonesia,
Pasuruan. Penelitian disusun menurut rancangan acak kelompok dengan 4 perlakuan
kombinasi varietas dan prosedur kultur jaringan. Dua macam varietas yang digunakan adalah
PS 851 dan PS 951 dan terdapat 2 prosedur kultur jaringan yaitu kultur pucuk dan kultur
kalus. Setiap perlakuan diulang 3 kali. Penelitian ini terdiri atas 2 bagan percobaan masingmasing menggunakan polibag kecil (17,5 x 8 cm) dan polibag besar (18 x 23 cm).
Hasil penelitian menunjukkan bahwa jumlah batang per rumpun semakin menurun
seiring dengan semakin bertambahnya umur tanaman. Jumlah mata tunas viable per rumpun
asal kultur pucuk lebih baik pada perlakuan polibag kecil. Sementara itu pada polibag besar
tidak nyata, tergantung varietasnya. Tingkat multiplikasi bagal mikro dari bibit polibag besar
lebih tinggi dibandingkan dengan polibag kecil. Pada umur 4 bulan tingkat multiplikasi varietas
PS 851 pada polibag kecil berkisar antara 28–37 kali dan PS 951 adalah 19 kali. Tingkat
multiplikasi varietas PS 851 pada polibag besar berkisar antara 38–43 kali dan untuk PS
951 berkisar antara 21–27 kali. Pada umur 6 bulan tingkat multiplikasi bagal mikro dari bibit
polibag kecil adalah 26–41 kali dan 16–33 kali masing-masing untuk varietas PS 851 dan
PS 951. Sementara itu tingkat multiplikasi bagal mikro dari bibit polibag besar adalah 39–
42 dan 28–33 kali berturut-turut untuk PS 851 dan PS 951. Tingkat multiplikasi PS 851 lebih
tinggi dibandingkan dengan PS 951.

Kata kunci: tebu, bagal mikro, multiplikasi, kultur jaringan

ABSTRACT
Rapid preparation of new superior sugar cane variety has an important role in inhibiting
the occurrence of clone degeneration process. Seedling multiplication through micro cutting
has several advantages including its delivery that is rapid and more efficient. This research
was aimed to study the micro cutting multiplication which has been conducted in the hardening
of Pasuruan experiment station of Indonesian Sugar Research Institute. The research was
arranged as randomized block design with 4 combination treatments of varieties and tissue
culture procedures. There were 2 varieties used as a treatment i.e. PS 851 and PS 951 as
well as 2 tissue culture methods namely callus culture and shoot tip culture. Every treatment
was replicated 3 times. Experiment was conducted using small polybags (17,5 x 8 cm) and
large polybags (18 x 23 cm).
The results showed that the number of stalks per stool decreased by increasing the
plant age. The number of viable shoots per stool of shoot culture was better when the
plantlets were planted in small polybags compared to large polybags. Meanwhile the plants
planted in large polybags did not show significant result depending on the varieties.
Multiplication level of seedlings in large polybags was higher than that of small polybags. At
the age of 4 months, multiplication level of PS 851 and PS 951 in small polybags was
between 28-37 times and 19 times, respectively. Mean while multiplication level of PS 851
in large polybags was between 38-43 times and for PS 951 was in the range of 21-27 times.
At the age of 6 months, multiplication level of seedlings in small polybags was 26-41 times
and 16-33 times for PS 851 and PS 951, respectively, while in large polybags, it was 39-42
and 28-33 times for PS 851 and PS 951, respectively. The fact showed that multiplication
level of PS 851 was higher compared to PS 951.

Key words: cane, micro cuttings, multiplication, tissue culture

MPG/NDONESIAN SUGAR RESEARCH JOURNAL Vol. 44 No. 3 September 2008: 155-165
Pusat Penelitian Perkebunan Gula Indonesia/Indonesian Sugar Research Institute
Jl. Pahlawan 25, Pasuruan 67126
Telp. 0343-421086, Fax: 0343-421178
E-mail : mpg.p3gi@gmail.com; Website: http://www.p3gi.net