Estimation of Genetic Parameters for in vitro Culture Traits and Selection Best Progenies for Tenera Oil Palm Tissue Culture

Abstract Clonal propagation of oil palm through in vitro culture is a potential approach to fulfill the demand of oil palm elite planting materials. However, the incidence of floral abnormality known as “Mantled” from oil palm derived from in vitro culture which was around 5%-80%, hampered the commercialization of this clonal oil palm planting materials. EGAD1, a defensin gene detected in oil palm, was reported to be expressed in significantlyhigher in callus cultures initiated from mantledpalms compared with those obtained from normally flowering individuals. As a part of research work to develop a molecular marker for early detection of abnormality in oil palm derived tissue culture, this research was aimed to isolate and analyze the sequences of the 5’ flanking region of EGAD1 gene of the normal and mantled oil palm. The research was initiated by expression analysis of EGAD1 at the flower and fruit of normal and mantled phenotypes, followed by isolation of the 5’ flanking region of the gene by genomic PCR. The sequences of PCR product were then aligned by ClustalW from BioEdit. The results showed that mantled phenotype of flower and fruit accumulated mRNA EGAD1 higher than that of normal phenotype. Differences between the two DNA sequences were detected at the bases of 141, 188 dan 198, which implied on the differences of the restriction map. These differences give a possibility to develop a molecular marker for detection of the abnormality on oil palm derived from tissue culture, based on the RFLP technique.Abstrak Perbanyakan kelapa sawit melalui kultur jaringan merupakan salah satu pendekatan yang sangat potensial untuk memenuhi permintaan bibit unggul kelapa sawit. Namun terjadinya abnormalitas pembungaan yang dikenal sebagai bunga mantled pada tanaman kelapa sawit hasil kultur jaringan, menjadi hambatan komersialisasi bibit tersebut. Gen EGAD1, yaitu gen defensin yang diidentifikasi merupakan salah satu gen pada kelapa sawit yang ekspresinya dilaporkan jauh lebih tinggi pada kalus yang diinduksi dari tanaman kelapa sawit abnormal dibandingkan dengan pada kalus asal kelapa sawit normal. Sebagai bagian dari usaha pengembangan pelacak molekuler untuk deteksi dini abnormalitas kelapa sawit hasil kultur jaringan, penelitian ini bertujuan untuk mengisolasi dan menganalisis perbedaan sekuen DNA daerah 5’ flanking gen EGAD1 dari buah normal dan buah mantled. Penelitian dimulai dengan analisis ekspresi EGAD1 pada jaringan bunga dan buah normal dan mantled dengan RT- PCR, dilanjutkan dengan isolasi daerah 5’flanking EGAD1 dengan PCR genomik. Sekuen produk PCR kemudian disejajarkan melalui ClustalW BioEdit. Hasil penelitian menunjukkan bahwa bunga dan buah mantled mengakumulasikan mRNA EGAD1 lebih tinggi dibanding-kan dengan bunga dan buah normal. Terdapat perbedaan sekuen DNA pada daerah 5’ flanking dari gen tersebut antara buah normal dengan buah mantel, yaitu pada basa ke-141, 188 dan 198, yang berimplikasi pada perbedaan peta restriksi kedua sekuen. Hal ini memberi peluang untuk pengembangan suatu pelacak deteksi abnormalitas pada tanaman kelapa sawit hasil kultur jaringan, yang berbasis pada teknik RFLP.

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Analisis sekuen DNA daerah 5’–EGAD1 dari buah kelapa sawit normal dan abnormal hasil kultur jaringan Analysis of DNA sequences of the 5’-flanking EGAD1 from normal and abnormal fruit from tissue culture derived oil palm

E-Journal Menara Perkebunan ◽

10.22302/iribb.jur.mp.v78i2.63 ◽

2016 ◽

Vol 78 (2) ◽

Author(s):

Asmini BUDIANI

Keyword(s):

Tissue Culture ◽

Molecular Marker ◽

In Vitro Culture ◽

Dna Sequences ◽

Oil Palm ◽

Research Work ◽

Callus Cultures ◽

Flanking Region ◽

Planting Materials

Abstract Clonal propagation of oil palm through in vitro culture is a potential approach to fulfill the demand of oil palm elite planting materials. However, the incidence of floral abnormality known as “Mantled” from oil palm derived from in vitro culture which was around 5%-80%, hampered the commercialization of this clonal oil palm planting materials. EGAD1, a defensin gene detected in oil palm, was reported to be expressed in significantlyhigher in callus cultures initiated from mantledpalms compared with those obtained from normally flowering individuals. As a part of research work to develop a molecular marker for early detection of abnormality in oil palm derived tissue culture, this research was aimed to isolate and analyze the sequences of the 5’ flanking region of EGAD1 gene of the normal and mantled oil palm. The research was initiated by expression analysis of EGAD1 at the flower and fruit of normal and mantled phenotypes, followed by isolation of the 5’ flanking region of the gene by genomic PCR. The sequences of PCR product were then aligned by ClustalW from BioEdit. The results showed that mantled phenotype of flower and fruit accumulated mRNA EGAD1 higher than that of normal phenotype. Differences between the two DNA sequences were detected at the bases of 141, 188 dan 198, which implied on the differences of the restriction map. These differences give a possibility to develop a molecular marker for detection of the abnormality on oil palm derived from tissue culture, based on the RFLP technique.Abstrak Perbanyakan kelapa sawit melalui kultur jaringan merupakan salah satu pendekatan yang sangat potensial untuk memenuhi permintaan bibit unggul kelapa sawit. Namun terjadinya abnormalitas pembungaan yang dikenal sebagai bunga mantled pada tanaman kelapa sawit hasil kultur jaringan, menjadi hambatan komersialisasi bibit tersebut. Gen EGAD1, yaitu gen defensin yang diidentifikasi merupakan salah satu gen pada kelapa sawit yang ekspresinya dilaporkan jauh lebih tinggi pada kalus yang diinduksi dari tanaman kelapa sawit abnormal dibandingkan dengan pada kalus asal kelapa sawit normal. Sebagai bagian dari usaha pengembangan pelacak molekuler untuk deteksi dini abnormalitas kelapa sawit hasil kultur jaringan, penelitian ini bertujuan untuk mengisolasi dan menganalisis perbedaan sekuen DNA daerah 5’ flanking gen EGAD1 dari buah normal dan buah mantled. Penelitian dimulai dengan analisis ekspresi EGAD1 pada jaringan bunga dan buah normal dan mantled dengan RT- PCR, dilanjutkan dengan isolasi daerah 5’flanking EGAD1 dengan PCR genomik. Sekuen produk PCR kemudian disejajarkan melalui ClustalW BioEdit. Hasil penelitian menunjukkan bahwa bunga dan buah mantled mengakumulasikan mRNA EGAD1 lebih tinggi dibanding-kan dengan bunga dan buah normal. Terdapat perbedaan sekuen DNA pada daerah 5’ flanking dari gen tersebut antara buah normal dengan buah mantel, yaitu pada basa ke-141, 188 dan 198, yang berimplikasi pada perbedaan peta restriksi kedua sekuen. Hal ini memberi peluang untuk pengembangan suatu pelacak deteksi abnormalitas pada tanaman kelapa sawit hasil kultur jaringan, yang berbasis pada teknik RFLP.

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Influence of Indole acetic acid and Tryptophan on production of Vinblastine and Vincristine of Catharanthus roseous callus cells in the accumulation media of In Vitro tissue culture

Baghdad Science Journal ◽

10.21123/bsj.7.3.1113-1119 ◽

2010 ◽

Vol 7 (3) ◽

pp. 1113-1119

Author(s):

Baghdad Science Journal

Keyword(s):

Tissue Culture ◽

High Performance Liquid Chromatography ◽

Acetic Acid ◽

Liquid Chromatography ◽

In Vitro Culture ◽

High Performance ◽

Indole Acetic Acid ◽

Culture Technique

This study on the plant of Ain –AL Bason Catharanthus roseous showed the ability of callus cells that is produced by In Vitro culture technique and transformed to the accumulated media (MS 40gm/L sucrose ,2gm/L IAA Indole acetic acid , 0.5gm/L Tryptophan) to produce Vinblastine and Vincristine compounds. Extraction, purification and quantitive determination of Vinblastine and Vincristine compounds using High performance liquid chromatography technique (HPLC)were carried out. The results showed that the highest concentration of Vinblastine and Vincristine compounds were ( 4.653,12.5 (ppm /0.5 dry Wight respectively from transformed callus cells from MS 40 gm /L sucrose , 2 gm / L NAA Naphthaline acetic acid .

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ISOLATION AND CHARACTERIZATION OF MDR BACTERIA FROM IN VITRO CULTURE OF BACOPA MONNIERA AND SUPPLEMENTATION OF NATURAL EXTRACTS TO CONTROL BACTERIAL CONTAMINATION

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2016v8i11.13593 ◽

2016 ◽

Vol 8 (11) ◽

pp. 102

Author(s):

Yash Sharma ◽

Manish Bhardwaj ◽

Anshita Nagar ◽

Neeta Bhagat

Keyword(s):

Tissue Culture ◽

In Vitro Culture ◽

Plant Tissue Culture ◽

Plant Tissue ◽

Bacterial Contamination ◽

Resistant Bacteria ◽

Bacopa Monniera ◽

Natural Extracts ◽

Isolation And Characterization

Objective: The purpose of the present study was to isolate and characterize bacterial contamination from in vitro culture of Bacopa monniera callus culture.Methods: The two selected isolates (1 and 2) were identified by morphological, biochemical and molecular (16S rRNA gene sequencing) methods. Beside this antibiotic resistance was also determined.Results: The isolates were identified as closely related to Enterobacter cloacae (KU350623) (Isolate 1) and Myroides odoratimimus (KU382740) (Isolate 2). The isolate 1 and 2 were found to be resistant to streptomycin (25 mcg), dapsone (10 mcg), erythromycin (15 mcg), chloroamphenicol (25 mcg) and ampicillin (10 mcg). Supplementation of the natural extract was tested to control the contamination due to these multi drug resistant bacteria. Aqueous and alcoholic leaf extracts of Azadirachta indica was added to plant tissue culture media i.e. MS media in order to control contamination.Conclusion: The present studies suggest using natural extracts supplementation as a promising strategy to control the in vitro bacterial contamination in plant tissue culture.

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Identification of Stevioside Using Tissue Culture-Derived Stevia (Stevia rebaudiana) Leaves

Biochemistry Insights ◽

10.4137/bci.s30378 ◽

2015 ◽

Vol 8s2 ◽

pp. BCI.S30378 ◽

Cited By ~ 1

Author(s):

Ziaul Karim ◽

Daisuke Uesugi ◽

Noriyuki Nakayama ◽

M. Monzur Hossain ◽

Kohji Ishihara ◽

...

Keyword(s):

Tissue Culture ◽

In Vitro Culture ◽

Large Scale ◽

Stevia Rebaudiana ◽

Potential Method ◽

Modern Technology ◽

Commercial Production ◽

Sugar Levels ◽

Disease Free

Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for identification of stevioside from tissue culture-derived stevia leaf. Stevioside in the sample was identified using HPLC by measuring the retention time. The percentage of stevioside content in the leaf samples was found to be 9.6%. This identification method can be used for commercial production and industrialization of stevia through in vitro culture across the world.

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INDUKSI DAN REGENERASI KALUS KELADI TIKUS (Typonium flagelliforme. Lodd. ) SECARA IN VITRO

Jurnal Penelitian Tanaman Industri ◽

10.21082/jlittri.v13n4.2007.142-146 ◽

2020 ◽

Vol 13 (4) ◽

pp. 142 ◽

Cited By ~ 2

Author(s):

SITTI FATIMAH SYAHID ◽

NATALINI NOVA KRISTINA

Keyword(s):

Tissue Culture ◽

Genetic Variation ◽

Carbon Source ◽

Genetic Variability ◽

In Vitro Culture ◽

Basic Medium ◽

Randomized Design ◽

Completely Randomized Design ◽

Group B

ABSTRAK Keladi tikus umumnya diperbanyak secara vegetatif sehingga ragam genetiknya sempit. Penelitian peningkatan keragaman genetik pada keladi tikus melalui kultur in vitro telah dilakukan di Laboratorium Kultur Jaringan, Balai Penelitian Tanaman Obat dan Aromatik (Balittro) Bogor pada bulan April sampai Desember 2005. Bahan tanaman yang digunakan adalah daun steril keladi tikus in vitro. Media dasar yang digunakan adalah Murashige and Skoog (MS) yang diperkaya vitamin dari group B. Sebagai sumber energi digunakan sukrosa sebanyak 30 g/l. Penelitian terdiri dari dua tahap yaitu induksi dan regenerasi kalus. Perlakuan yang diuji pada tahap I adalah beberapa taraf konsentrasi auksin (2,4-D) secara tunggal maupun kombinasi dengan sitokinin (kinetin) terhadap induksi kalus yaitu : 2,4-D 0,1 mg/l; 2,4-D 0,5 mg/l; 2,4-D 1,0 mg/l; 2,4-D 0,1 + kinetin 0,1 mg/l; 2,4-D 0,5 mg/l + kinetin 0,1 mg/l; 2,4-D 1.0 mg/l + kinetin 0,1 mg/l; 2,4-D 0,1 mg/l + kinetin 0,3 mg/l; 2,4-D 0,5 mg/l +kinetin 0,3 mg/l dan 2,4-D 1,0 mg/l + kinetin 0,3 mg/l. Tahap II adalah beberapa taraf konsentrasi benzyl adenin untuk regenerasi kalus. Penelitian disusun menggunakan rancangan acak lengkap dengan pola faktorial dan lima ulangan, dan setiap ulangan terdiri dari satu eksplan. Faktor pertama adalah asal kalus dan faktor kedua adalah beberapa taraf konsentrasi BA yaitu : BA 0,1 mg/l ; BA 0,3 mg/l dan BA 0,5 mg/l. Parameter yang diamati adalah waktu inisiasi kalus, struktur dan warna kalus, jumlah tunas serta penampilan kultur secara visual. Hasil penelitian menunjukkan bahwa kalus asal eksplan daun dapat diinduksi pada perlakuan 2,4-D 1,0 mg/l + kinetin 0,1 mg/l dan 2,4-D 1,0 mg/l + kinetin 0,3 mg/l dengan waktu inisiasi 8 sampai 10 minggu setelah perlakuan. Regenerasi kalus terbaik diperoleh pada medium 2,4-D 1,0 mg/l + kinetin 0,3 mg/l mengandung BA 0,3 mg/l dengan rata-rata tunas dan daun yang dihasilkan sebanyak 13,2 tunas dan 4,4 daun. Kata kunci : Keladi tikus, Typonium flagelliforme Lodd., induksi, regenerasi kalus, in vitro ABSTRACT Induction and regeneration of Rodent tuber calli through in vitro culture Rodent tuber plant (Typonium flagelliforme Lodd) is commonly propagated vegetatively, the repro its genetic variation is narrow. A research to increase the genetic variability of the plant was conducted in Tissue Culture Laboratory of the Indonesian Medicinal and Aromatic Research Institute, Bogor from April to December 2005. The leaf of Rodent tuber in vitro used as an explants. Murashige and Skoog (MS) medium used as basic medium, supplemented with vitamin from B group, sucrose 30 g/l was added into the medium as carbon source. The research consist of two steps : 1) calli induction and 2) calli regeneration. The treatment tested in first step : 2.4-D 0.1 mg/l; 2.4-D 0.5 mg/l; 2.4-D 1,0 mg/l; 2.4-D 0.1 + kinetin 0.1 mg/l; 2.4-D 0.5 mg/l + kinetin 0.1 mg/l; 2.4- D 1.0 mg/l + kinetin 0,1 mg/l; 2.4-D 0.1 mg/l + kinetin 0.3 mg/l; 2.4-D 0.5 mg/l + kinetin 0.3 mg/l and 2.4-D 1.0 mg/l + kinetin 0.3 mg/l. In the second steps, several concentration of BA were tested i.e: BA 0,1 mg/l ; BA 0,3 mg/l and BA 0,5 mg/l. The experiment was arranged in completely randomized design with factorial pattern. Each treatment consist of five replications. The parameters observed were time of calli initiation, texture, colour of calli and number of shoot and leaves in regeneration. The result showed that calli can be induced on 2.4-D 1.0 mg/l + kinetin 0.1 mg/l and 2.4-D 1.0 mg/l + kinetin 0.3 mg/l, eight to ten weeks after culture. The best medium for shoots regeneration contains 2.4- D 1.0 mg/l + kinetin 0.3 mg/l with 0.3 mg/l BA, with mean result of 13.2 shoots and 4.4 leaves. Key words : Rodent tuber, Typonium flagelliforme Lodd. bl , induction, regeneration, calli, in vitro

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Deteksi Kestabilan Genetik Ramet Kelapa Sawit Hasil Kultur In Vitro Menggunakan SSR

Jurnal Agronomi Indonesia (Indonesian Journal of Agronomy) ◽

10.24831/jai.v44i1.12507 ◽

2016 ◽

Vol 44 (1) ◽

pp. 83

Author(s):

Yuni Fitri Cahyaningsih ◽

Ni Made Armini Wiendi ◽

Dan Nurita Toruan-Mathius

Keyword(s):

Tissue Culture ◽

Stability Analysis ◽

Oil Palm ◽

Genetic Stability ◽

Genetic Similarity ◽

Qualitative Data ◽

Elaeis Guineensis ◽

Group Method ◽

Pair Group

ABSTRACT Commercial production of oil palm ramet requires the guarantee of high genetic stability. The objectives of this research were to determine 1) genetic diversity of ortet as source of explant, and 2) genetic stability of ramet derived from ortet propagated through tissue culture. Genetic stability analysis was done using ramet from five Tenera (D×P) oil palm ortets.As many as 20 ramets were randomly chosen from each ortet. A total of 100 ramets were used for genetic stability analysis. Genetic similarity analysis was analyzed using NTSyspc version 2.1 software with method Similarity for Qualitative Data and Unweighted Pair Group Method Aritmatic (UPGMA). The results indicated 20 SSR primer pairs were polymorphic and could form 44 alleles. As many as 80% of ramets from IS 3 ortet showed genetic similarity ranged from 97-100% to the ortet. All ramets derived from IS 10, IS 20 and IS 40 ortet had 90-100% of genetic similarity to its respective ortet. Futhermore, 95% of ramets from IS 39 ortet had 97-100% of genetic similarity to the ortet. Keywords: Elaeis guineensis Jacq., genetic similarity, tissue culture

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A REVISED METHOD FOR SUCKER STERILIZATION TO SUPPORT THE IN VITRO PROPAGATION OF SAGO PALM (Metroxylon sagu Rottb.)

Jurnal Bioteknologi & Biosains Indonesia (JBBI) ◽

10.29122/jbbi.v1i1.548 ◽

2014 ◽

Vol 1 (1) ◽

pp. 21

Author(s):

Teuku Tajuddin ◽

Karyanti . ◽

Tati Sukarnih ◽

Nadirman Haska

Keyword(s):

Tissue Culture ◽

In Vitro Culture ◽

In Vitro Propagation ◽

Large Scale ◽

Culture Method ◽

Large Area ◽

Sago Palm ◽

Superior Genotypes ◽

Metroxylon Sagu

Hutan sagu (Metroxylon sagu Rottb.) dapat ditemukan dalam area yang cukup luas di wilayah Maluku dan Papua. Besarnya keanekaragaman hayati dari pohon sagu dapat dilihat di areal ini. Pohon sagu tumbuh secara alami terutama di daerah dataran atau rawa dengan sumber yang air melimpah. Tanaman sagu dapat diperbanyak dengan metode generatif melalui biji, dan vegetatif melalui tunas anakan. Dalam rangka mendukung perbanyakan pohon induk yang unggul secara in vitro dalam skala besar, perbaikan metode sterilisasi tunas anakan mutlak diperlukan. Tunas anakan muda (15-20 cm) yang diperoleh dari Propinsi Papua digunakan sebagai eksplan. Tujuan percobaan sterilisasi ini dilakukan untuk mendukung perbanyakan pohon sagu secara in vitro. Pada percobaan ini antibiotik digunakan untuk membersihkan jaringan internal eksplan dari jamur dan bakteri. Hasil percobaan ini menunjukkan bahwa campuran alkohol dan antibiotik dapat menekan pertumbuhan kontaminan.Kata kunci: Antibiotik, kontaminan jamur dan bakteri, kultur in vitro, metode sterilisasi, sagu ABSTRACTNatural sago (Metroxylon sagu Rottb.) forest can be found in large area in Maluku and Papua regions. There are wide genetic diversities of sago palm found in these areas. This palm grows along riverbanks and in swampy areas which are not suitable for other crops. Sago palm is propagated generatively by seed and vegetatively by suckers. With the purpose of establishing the in vitro culture method for a large-scale of mass clonally propagation of superior genotypes of sago palm, generating sterilized explants are very important. Young suckers (15-20 cm) obtained from areas of Papua Province were used as explants. The sterilization experiments were carrying out to support the tissue culture of sago palm. Sterilization was conducted using antibiotics in order to get rid of fungi and bacteria from inner part of explants tissues. The results showed that from all sterilization methods tested, the best result was treatment using alcohol and antibiotic as disinfectant agents.Keywords: Antibiotics, fungi and bacteria contaminants, in vitro culture, sterilization method, sago palm

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In vitro culture of sempre-vivas species (Comanthera): a review

Rodriguésia ◽

10.1590/2175-7860202172016 ◽

2021 ◽

Vol 72 ◽

Author(s):

Andressa Priscila Piancó Santos Lima ◽

Alone Lima Brito ◽

José Raniere Ferreira de Santana

Keyword(s):

Tissue Culture ◽

In Vitro Culture ◽

In Vitro Propagation ◽

Economic Importance ◽

In Vitro Conservation ◽

Future Research ◽

In Vitro Cultivation ◽

Natural Form

Abstract The term “sempre-viva” denotes plants whose structures retain their natural form and color after being cut and dried. For these reasons, they are commercially valuable for ornamental purposes. However, due to extractive overexploitation of their inflorescences, some of these species are considered endangered. The genus Comanthera includes the sempre-vivas species with greatest economic importance in Brazil. Previous studies have shown that tissue culture is a workable strategy for in vitro propagation and conservation of species of this genus. However, these studies are still incipient. Therefore, the objective of this review is to summarize the findings on the in vitro cultivation of species of the Comanthera genus, to serve as the basis for future research. The text is structured in two main topics: micropropagation and in vitro conservation.

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