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PROTOPLAST FUSION BETWEEN WHITE AND BROWN OYSTER MUSHROOMS Djajanegara, Ira; Masduki, Agus
Indonesian Journal of Agricultural Science Vol 11, No 1 (2010): April 2010
Publisher : Indonesian Agency for Agricultural Research and Development - MOA

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Abstract

Genetic crossing of white oyster mushroom (Pleurotus floridae)to introduce longer storage life trait can only be done withinindividuals in this particular species. However, longer storagelife trait is possessed by brown oyster mushroom (Pleurotuscystidiosus). Therefore, a protoplast fusion experiment betweenwhite and brown oyster mushrooms was conducted to obtain anoyster mushroom strain showing high productivity and longstorage life. The experiment was done at the biology laboratoryof the University of Al Azhar Indonesia from May 2008 toAugust 2009. Protoplast fusion was done by isolating protoplastfrom 5-day old monokaryotic mycelia grown in potato dextrosebroth (PDB). Around 3.15 x 105 protoplasts ml-1 were harvestedusing mixture of cellulase Onozuka R-10 (1%) and macerozymeR-10 (1%) from brown oyster mushroom with 80.61% viability.Similarly, 3.71 x 105 protoplasts ml-1 were harvested using lysingenzyme (2%) from white oyster mushroom with 83.68% viability.Protoplast fusions were conducted using 40% PEG6000 for10 minutes. The candidate fusants were then screened usingminimum regeneration media (MRM). There were 22 coloniesgrew on MRM media and four colonies (FS1, FS2, FS3, and FS4)showed clamp connection as well as primordia formation to bechosen as candidate fusants. However, isozyme studies usingmalate dehydrogenase and acid phosphatase as marker enzymesconfirmed that only FS1 and FS2 were the hybridized products.The two colonies showed different mycelia growth patterns andhyphae sizes, fruit body morphology and productivity comparedto their parents. These two fusants, however, did not indicatethe presence of longer storage life trait as expected despite ahigher productivity achieved by FS1. In this study, the protoplastfusion only yielded higher productivity strain of mushroomwith different colors without any changes in storage life.
Alternative Oxidase (AtAox) c78s Mutant Expression at Escherichia coli (SASX41DB) Djajanegara, Ira
Indonesian Journal of Biotechnology Vol 12, No 1 (2007)
Publisher : Universitas Gadjah Mada

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Abstract

Alternative oxidase (AOX) is the terminal oxidase operating in the mitochondrial electron transport chain. Theenzyme is activated by organic acid such as pyruvate and by reduction process. Based on sequences alignment ofalternative oxidase gene (Aox) found in several organisms, there are 2 conserved cysteine residues. In order toinvestigate the importance of those cysteine residues on the activity of AOX, mutation at cysteine residue number 78of Aox gene isolated from Arabidopsis thaliana (AtAox) was conducted. Cysteine at position number 78 was changedinto serine and the c78s mutant was expressed in Escherichia coli strain SASX41DB. This particular E. coli strain isunable to grow aerobically unless transformed with Arabidopsis Aox gene (AtAox). Expression studies on c78smutant showed that this mutant cannot be oxididized and can not be activated by pyruvic acid. This mutant isacivated by succinate instead of pyruvate. Mutation at cysteine closer to the N residue is affecting both organic acidand redox activation. Therefore, it is concluded that cysteine residue closer to the N residue is the site for bothactivation by pyruvate as well as activation by reduction process.Keywords : Alternative oxidase, site-directed mutation, SASx41DB, cysteine residues
PRIMER-PRIMER BARU UNTUK MENGAMPLIFIKASI GEN PENGKODE PROTEIN AMPLOP VIRUS DENGUE STRAIN CH53489 Djajanegara, Ira
BERITA BIOLOGI Vol 10, No 2 (2010)
Publisher : Research Center for Biology-Indonesian Institute of Sciences

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (694.396 KB) | DOI: 10.14203/beritabiologi.v10i2.1969

Abstract

Restriction site of BamHI and Sail must be added in order to express the gene encoding envelope protein of dengue virus strain CH53489 (gene E) into expression vector pMAL-p2x. This approach required the PCR technique for amplification as well as restriction sites addition. However, PCR amplification is prone to error due to the process of misincorporation eventhough using Platinum taq polymerase. Therefore, it is important to be concern that there will be no alteration of the gene especially for biopharmaceutical purposes such as recombinant vaccine. This experiment was aimed to design several primers of DenV-M F, D3-1715s, D3-2117s, D3-1911c and DenV-M R for full length sequencing of the amplified products. Primers were designed in silico using Oligo Explorer and tried in vitro to check the ability of the primers to produce fragments. The sequencing results showed that the amplified product suffered from misincorporation during amplification (98.9% homology). However, the 3-D protein structure prediction did not show any major changes in the protein structure. Further analysis of the expressed protein is required to be used for biopharmaceutical purposes.
Pcr Amplification of Ornithine Decarboxylase (ODC) Gene Fragment from Tobacco (Nicotiana tabacum L.) cv. Temanggung Djajanegara, Ira; Pambudi, Sabar; Lestari, Retno; Artanti, Nina
ANNALES BOGORIENSES Vol 9, No 2 (2004): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.1234/11

Abstract

In order Lo 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 i olated 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 .  
PEMANFAATAN LIMBAH BUAH PISANG DAN AIR KELAPA SEBAGAI BAHAN MEDIA KULTUR JARINGAN ANGGREK BULAN (Phalaenopsis amabilis) TIPE 229 Djajanegara, Ira
Jurnal Teknologi Lingkungan Vol. 11 No. 3 (2010)
Publisher : Center for Environmental Technology - Agency for Assessment and Application of Technology

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (393.15 KB) | DOI: 10.29122/jtl.v11i3.1182

Abstract

Coconut water and rotten banana fruits are commonly found in traditional markets as organic wastes. One way to overcome the problems caused by these organic wastes is to convert these unuseful matter into an important and economically useful matter by using them as components of tissue culture media. One important commodity that is usually propagated by tissue culture is Phalaenopsis orchid type 229 (Phalaenopsis amabilis). Therefore, it would be more benefit to substitute the expensive chemicals with organic wastes such as coconut water and banana puree.In this experiment, addition of coconut water and banana pure to the minimum media containing commercial fertilizer red Polyhyponex, sucrose and commercial agar did not show any inhibition of Phalaenopsis orchid plantlet growth. This probably caused by sufficient macro and micro nutrients provided by those organic matter and Polyhyponexfertilizer. Moreover, addition of 100 mL/L of coconut water and 100 mg/L banana puree gave the optimum leaf and adventitious shoot formation. On the other hand, addition of 150 mL/L coconut water gave the optimum height and root formation. In this case,growing Phalaenopsis orchid plantlet should be done in 2 subculture period. The first subculture is to obtain maximum amount of leaf and shhot formation while the second subculture is to obtain optimum height and root formation.Key words : Waste, coconut water, banana puree, tissue culture, Phalaenopsis orchi