Chief Editor: Muhammad Ahkam Subroto. Associate Editors: Bambang Sunarko, Adi Santoso, Satya Nugroho, Wien Kusharyoto. Editorial Assistant: Siti Elly Faisholyah. Consulting Editors: Asrul Muhammad Fuad, Nina Artanti, Ines Irene Atmosukarto, Totik Sri Mariani, Novik Nurhidayat, Tri Muji Ermayanti, Sri Koerniati (Indonesian Center for Agriculture Biotechnology and Genetic Resources Research and Development (ICABIOGRAD) Department of Agriculture). Cover: Wien Kusharyoto
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Content

Modifications of Plasmids and Cry Genes of Bacillus thuringiensis subsp. kurstaki HD-1 after Treatment with Ethylmethanosulfonate (EMS) and UV Light
Eddy Jusuf

Abstract
Bacillus thuringiensis subsp. kurstaki HD-1 is a potential insecticidal bacterium producing five types of d-endotoxin crystal proteins. This bacterium is widely commercialized due to its wide spectrum toxicity against both Lepidopteran and Dipteran larvae. The objective of this work was to create autolysin deficient mutant causing cell fails to lyse. This mutant yield intact cells within spore and d-endotoxin crystal protein protected inside, from which an undamaged active bio-insecticide would be obtained. Two methods of mutagenesis, 2% of ethylmethanosulfonate and 10, 25, and 50 seconds of UV light exposure, resulted in mot- (loss of motility) mutation. Observation showed that 14 of 20 survived mutants have lost some of its plasmids (varied from one to five), while the other six maintained their plasmids. By employing the polymerase chain reaction (PCR) the change on the cry genes was studied.

Keywords: UV and EMS mutagenesis, autolysin deficient mutant, plasmid, cry genes, Bacillus thuringiensis subsp. kurstaki HD-1.


Cytological Analysis of Root Cultures of Artemisia cina
Tri Muji Ermayanti, Oktavia Yanti and Erwin Al Hafiizh

Abstract
Artemisia cina is a medicinal plant species producing bioactive compounds which are potential as antitumor, antifungal and antibacterial. The aim of this study was to analyze the stability of chromosome numbers in root cultures of A. cina. Transformed root culture was established by infection of leaves of A. cina with Agrobacterium rhizogenes strains 07-20001, ATCC-15834, A4 and A. tumefaciens strain R1000. Roots isolated from glasshouse plants, plantlets grown in solid and liquid MS medium were utilized for investigation of chromosome examination of untransformed roots. Chromosome examination was conducted by squashing method and chromosome numbers were calculated under microscope. The results showed that both untransformed and transformed roots had instability in the chromosome number, but had the modal number of chromosome x=8 with the diploid number of 2n = 4x = 32. Roots isolated from glasshouse plants of A. cina had 53.7% of cells with the diploid numbers of 2n = 32, and 46.3% of cells had chromosome numbers ranged from 2n = 22 to 2n = 64. Untransformed roots isolated from plantlets cultured in solid media had only 36.1% of cells with chromosome number of 2n = 32, and untransformed roots grown in liquid medium had 49.4% of cells with 2n = 32. The chromosome numbers of A. cina transformed roots was affected by strains of Agrobacterium. Roots transformed with the bacterium strain 07-20001 showed the highest in normal chromosome numbers of 2n = 32 (62.4%) followed by roots transformed with strains ATCC-15834 (61.9%), R1000 (43.6%) and A4 (43.0%). The range of the chromosome number of untransformed roots was from 2n=17 to 2n=64, whilst that of transformed roots was from 2n=11 to 2n=66.

Keywords: untransformed roots, transformed roots, chromosome number, genetic stability, Artemisia cina.


Denitrification of Activated Sludge in The Presence of Different Organic Substrates
Dwi Agustiyani and Takao Yamagishi

Abstract
The effect of organic carbon on denitrifying activity was studied in batch reactor. Four reactors were operated in parallel under anoxic condition in four different donor electrons, which were acetic acid (Reactor A), methanol (Reactor M), phenol (Reactor P), and glucose (Reactor G). The reactors were fed with the artificial waste, which contain 721.8 mg/l NaNO3. The concentration of organic carbon added to the reactors were varied from TOD:N ratio of 0.5:1; 1:1, 1.5:1, to 2:1. The denitrification activity was estimated by measuring the reduction rate of nitrogenous oxide and N2O gas production. The denitrification capacity of adapted-sludge was also investigated, and the rates were estimated from the cumulative N2O (without acetylene inhibition) and N2 gas production. Reduction rate of nitrogenous oxide in all reactors increased during the investigation; the increase reduction rate were correlated to the increase of organic carbon concentration. The maximum reduction rate of nitrogenous oxide in reactor A was higher than those of the others. However, reduction rate in reactor M was more constant, so that nitrogenous oxides existed in this reactor was removed faster. The highest potential denitrification rate (N2O production) was observed in sludge of reactor A. However, N2 gas recovery from nitrate and nitrite transformed by sludge of reactor M was the highest. Linear correlation between nitrogenous oxide reduction with gas production was observed in reactor A, M and P, but not in reactor G.

Keywords: Denitrifying microorganisms, denitrification, nitrogenous oxide, activated-sludge.


Development of Somatic Embryo in Lithospermum erythrorhizon Siebb. et Zucc and the Study on the Effect of Methyl Jasmonate on Its Maturation
Totik Sri Mariani, Octavia Ramayanti, Kazufumi Yazaki and Hiroshi Miyake

Abstract
A research on the somatic embryogenesis in Lithospermum erythrorhizon has been conducted. Embryogenic callus was inoculated in EIM9 liquid medium (modification of LS medium), i.e. L2PVP (initiation medium) and the development of somatic embryo was observed. Ten µM and 100 µM methyl jasmonate was added into L3PVP medium (differentiation medium) for somatic embryo maturation. The purposes of this research were to observe the development of somatic embryo and to observe the effect of methyl jasmonate on maturation of the somatic embryo in L. erythrorhizon. The results showed that somatic embryogenesis in L. erythrorhizon derived from single cells differentiated further forming proembryo, globular, heart, torpedo and cotyledon stage. Treatment with 10 µM and 100 µM methyl jasmonate induced maturation of somatic embryos (cotyledon stage) after transferring them to embryo development medium (L4PVP).

Keywords: Somatic embryogenesis, Lithospermum erythrorhizon, methyl jasmonate.


PCR Amplification of Ornithine Decarboxylase (ODC) Gene Fragment from Tobacco (Nicotiana tabacum L.) cv. Temanggung
Ira Djajanegara, Sabar Pambudi, Retno Lestari and Nina Artanti

Abstract
In order to create an antisense construct of the gene encoding Ornithine Decarboxylase (ODC) from tobacco (Nicotiana tabacum L.) cv. Temanggung, the target gene must be isolated. In this paper, we present the PCR amplification of a fragment from putative gene encoding ODC from tobacco cv. Temanggung. Leaf genomic DNA was isolated and used as the template for PCR. PCR optimization was done by adjusting the annealing temperature and the cycle number. Verification of the fragment obtained was also done using the second primer pairs.

Keywords: Ornithine decarboxylase, tobacco, PCR.