scholarly journals Perkembangan kalus embriogenik sagu (Metroxylon sagu Rottb.) pada tiga sistem kultur in vitro Development of embryogenic callus of sago (Metroxylon sagu Rottb.) on three systems of in vitro culture

2016 ◽  
Vol 76 (1) ◽  
Author(s):  
Pauline D KASI ◽  
. SUMARYONO
Keyword(s):  
In Vitro Culture ◽  
Liquid Medium ◽  
Embryogenic Callus ◽  
Somatic Embryos ◽  
Temporary Immersion System ◽  
Embryogenic Cells ◽  
Temporary Immersion ◽  
Friable Embryogenic Callus ◽  
Metroxylon Sagu

Summary Embryogenic callus of sago (Metroxylon sagu Rottb.) has been grown on three systems of in vitro culture i.e. agar-solidified medium, liquid medium, and temporary immersion system (TIS) medium to observe and compare the development of embryogenic callus over one passage of six weeks.  A-half gram of embryogenic callus was cultured on a modified MS medium containing 10 mg/L   2,4-D and 0.1 mg/L kinetin. For histological studies, embryogenic callus was fixed in FAA and embedded in paraplast wax. Serial sections were stained with safranin 1% and observed microscopically. By the end of culture period, the development of embryogenic callus in TIS medium was relatively better than those of the other two media.  Fresh weight of callus in liquid medium and TIS increased by 6.5-fold, while on agar-solidified medium increased by 5.4-fold in six weeks.  About 40% of callus in liquid medium and TIS and 20% of callus on agar solidified medium have changed into somatic embryos at globular stage. Histology structure of embryogenic callus of the three systems of in vitro culture shows different pattern. On agar-solidified medium, secondary callus and friable embryogenic callus that consist of meristematic cells were formed. In contrast, more embryogenic cells were formed in liquid medium and TIS to support maturation process to somatic embryos. Therefore, temporary immersion system and liquid medium are recommended for maturation of embryogenic callus, whereas agar-solidified medium is for proliferation of embryogenic callus of sago.  Ringkasan Kalus embriogenik sagu (Metroxylon sagu Rottb.) telah ditumbuhkan pada tiga sistem kultur in vitro yaitu medium padat, medium cair, dan medium dengan sistem perendaman sesaat (SPS) untuk mempelajari dan mem-bandingkan perkembangan dari kalus embrio-genik selama periode enam minggu. Setengah gram kalus embriogenik dikulturkan pada medium MS modifikasi yang mengandung  2,4-D 10 mg/L dan kinetin 0,1 mg/L.  Untuk studi histologi, kalus embriogenik difiksasi dengan FAA dan embedding menggunakan lilin paraplast. Irisan diwarnai dengan safranin 1% dan diamati menggunakan mikroskop. Pada akhir periode kultur, pertumbuhan kalus pada medium dengan SPS lebih baik dibandingkan dengan medium cair dan padat. Bobot basah kalus pada  medium cair dan SPS meningkat 6,5 kali sedangkan pada medium padat meningkat 5,4 kali dalam waktu enam minggu. Sebanyak 40% kalus pada medium cair dan SPS serta 20% kalus pada medium padat berubah menjadi embrio somatik fase globuler. Struktur histologi kalus embriogenik pada ketiga jenis sistem kultur in vitro menunjukkan pola yang berbeda. Pada medium padat terjadi pembentukan kalus sekunder dan kalus embriogenik remah yang terdiri atas sel-sel meristematik. Sebaliknya pada medium cair dan SPS pembentukan sel embriogenik lebih banyak yang menunjang proses pendewasaan menjadi embrio somatik. Oleh karena itu, medium cair dan SPS direkomendasikan untuk pendewasaan kalus embriogenik, sedangkan medium padat untuk proliferasi kalus embriogenik sagu. 

scholarly journals Perkembangan kalus embriogenik sagu (Metroxylon sagu Rottb.) pada tiga sistem kultur in vitro Development of embryogenic callus of sago (Metroxylon sagu Rottb.) on three systems of in vitro culture

2016 ◽  
Vol 76 (1) ◽  
Author(s):  
Pauline D KASI ◽  
. SUMARYONO
Keyword(s):  
In Vitro Culture ◽  
Liquid Medium ◽  
Embryogenic Callus ◽  
Somatic Embryos ◽  
Temporary Immersion System ◽  
Embryogenic Cells ◽  
Temporary Immersion ◽  
Friable Embryogenic Callus ◽  
Metroxylon Sagu

Summary Embryogenic callus of sago (Metroxylon sagu Rottb.) has been grown on three systems of in vitro culture i.e. agar-solidified medium, liquid medium, and temporary immersion system (TIS) medium to observe and compare the development of embryogenic callus over one passage of six weeks.  A-half gram of embryogenic callus was cultured on a modified MS medium containing 10 mg/L   2,4-D and 0.1 mg/L kinetin. For histological studies, embryogenic callus was fixed in FAA and embedded in paraplast wax. Serial sections were stained with safranin 1% and observed microscopically. By the end of culture period, the development of embryogenic callus in TIS medium was relatively better than those of the other two media.  Fresh weight of callus in liquid medium and TIS increased by 6.5-fold, while on agar-solidified medium increased by 5.4-fold in six weeks.  About 40% of callus in liquid medium and TIS and 20% of callus on agar solidified medium have changed into somatic embryos at globular stage. Histology structure of embryogenic callus of the three systems of in vitro culture shows different pattern. On agar-solidified medium, secondary callus and friable embryogenic callus that consist of meristematic cells were formed. In contrast, more embryogenic cells were formed in liquid medium and TIS to support maturation process to somatic embryos. Therefore, temporary immersion system and liquid medium are recommended for maturation of embryogenic callus, whereas agar-solidified medium is for proliferation of embryogenic callus of sago.  Ringkasan Kalus embriogenik sagu (Metroxylon sagu Rottb.) telah ditumbuhkan pada tiga sistem kultur in vitro yaitu medium padat, medium cair, dan medium dengan sistem perendaman sesaat (SPS) untuk mempelajari dan mem-bandingkan perkembangan dari kalus embrio-genik selama periode enam minggu. Setengah gram kalus embriogenik dikulturkan pada medium MS modifikasi yang mengandung  2,4-D 10 mg/L dan kinetin 0,1 mg/L.  Untuk studi histologi, kalus embriogenik difiksasi dengan FAA dan embedding menggunakan lilin paraplast. Irisan diwarnai dengan safranin 1% dan diamati menggunakan mikroskop. Pada akhir periode kultur, pertumbuhan kalus pada medium dengan SPS lebih baik dibandingkan dengan medium cair dan padat. Bobot basah kalus pada  medium cair dan SPS meningkat 6,5 kali sedangkan pada medium padat meningkat 5,4 kali dalam waktu enam minggu. Sebanyak 40% kalus pada medium cair dan SPS serta 20% kalus pada medium padat berubah menjadi embrio somatik fase globuler. Struktur histologi kalus embriogenik pada ketiga jenis sistem kultur in vitro menunjukkan pola yang berbeda. Pada medium padat terjadi pembentukan kalus sekunder dan kalus embriogenik remah yang terdiri atas sel-sel meristematik. Sebaliknya pada medium cair dan SPS pembentukan sel embriogenik lebih banyak yang menunjang proses pendewasaan menjadi embrio somatik. Oleh karena itu, medium cair dan SPS direkomendasikan untuk pendewasaan kalus embriogenik, sedangkan medium padat untuk proliferasi kalus embriogenik sagu. 

scholarly journals Pengaruh TDZ terhadap induksi embrio somatik sagu (Metroxylon sagu Rottb.) pada tiga metode kultur berbeda (Effect of TDZ on the somatic embryo induction of sago palm (Metroxylon sagu Rottb.) in three different culture methods)

2018 ◽  
Vol 86 (1) ◽  
Author(s):  
Imron Riyadi ◽  
Darda EFENDI ◽  
Bambang S PURWOKO ◽  
Djoko SANTOSO
Keyword(s):  
Somatic Embryo ◽  
Shoot Apical Meristem ◽  
Embryogenic Callus ◽  
Somatic Embryos ◽  
Apical Meristem ◽  
Culture Methods ◽  
Sago Palm ◽  
Callus Differentiation ◽  
Metroxylon Sagu

AbstractA right combination of cytokinin is able to support the process of callus differentiation to somatic embryo formation in plant somatic embryogenesis. Liquid culture application could increase the efficiency of in vitro culture process on plants. This research aimed to determine the best concentration of TDZ combined with kinetin for callus differentiation to  somatic embryo of sago palm on three culture methods. Plant material used was embryogenic callus derived from tips meristem culture from sucker of Alitir sago palm. Callus was cultured on modified MS media added with: 0.0, 0.1, 0.5 and 1.0 mg/L TDZ combined with 0.5 mg/L kinetin for 12 weeks with subcultures every 6 weeks. Three culture methods used were suspension, temporary immersion system (TIS), and solid media. There were 12 treatments with 4 replicates. The results showed that the highest number of somatic embryos was achieved on TIS culture with 1.0 mg/L TDZ and 0.5 mg/L kinetin in 6 weeks (167.3 embryos/flask) and 12 weeks (389.2 embryos/flask) with its fresh weight of 18.4 g and 29.1 g, respectively. The highset survival rate in final culture (12 weeks) was achieved on TIS culture with 1.0 mg/L TDZ and 0.5 mg/L kinetin (100%). The shortest time for somatic embryos expression was achieved on TIS culture with 1.0 mg/L TDZ and 0.5 mg/L kinetin in two weeks after culture. Histological analysis of early-stage somatic embryos showed the presence of dense and compact cellular arrangements which formed growth spot axis for shoot or SAM (shoot apical meristem) and root or RAM (root apical meristem) that connected each other. [Key words: culture method, embryogenic callus, Metroxylon sagu Rottb., kinetin, sago palm, TDZ]   AbstrakAplikasi kombinasi sitokinin yang tepat dapat mendorong proses diferensiasi kalus membentuk embrio somatik pada proses embriogenesis somatik tanaman. Penggunaan metode kultur cair dapat meningkatkan efisiensi proses kultur in vitro tanaman. Penelitian ini bertujuan untuk menentukan konsentrasi TDZ terbaik dikombinasikan dengan kinetin dalam proses diferensiasi kalus membentuk embrio somatik tanaman sagu pada tiga metode kultur. Bahan tanam penelitian  berupa kalus embriogenik tanaman sagu asal kultur meristem pucuk dari anakan sagu jenis Alitir. Kalus dikulturkan pada media modifikasi dengan penambahan  TDZ dengan konsentrasi 0,1; 0,5; dan 1,0 mg/L dikombinasikan dengan kinetin 0,5 mg/L selama 12 minggu yang disubkultur pada umur 6 minggu. Metode kultur yang digunakan terdiri atas tiga macam yaitu: kultur suspensi, sistem perendaman sesaat (SPS) dan media padat. Perlakuan terdiri atas 12 kombinasi perlakuan dengan empat ulangan. Hasil penelitian menunjukkan bahwa rerata jumlah embrio somatik tertinggi dicapai pada perlakuan metode kultur SPS dengan TDZ 1,0 mg/L baik pada umur kultur 6 minggu (167,3 buah) maupun umur 12 minggu (389,2 buah). Rerata bobot segar tertinggi juga diperoleh pada perlakuan metode kultur SPS dengan TDZ 1,0 mg/L  pada umur kultur 6 minggu (18,4 g) dan  12 minggu (29,1 g). Rerata daya hidup kultur akhir (12 minggu) tertinggi  sebesar 100% diperoleh pada perlakuan SPS. Induksi embrio somatik  tercepat yakni setelah  dua minggu diperoleh pada  metode kultur SPS dengan TDZ 1,0 mg/L dikombinasikan dengan kinetin 0,5 mg/L. Analisis histologi embrio somatik stadium awal  menunjukkan adanya susunan sel yang rapat dan kompak yang menyusun semacam poros atau berkas titik tumbuh tunas atau SAM (shoot apical meristem) maupun akar atau RAM (root apical mersitem) yang saling terhubung.[Kata kunci: kalus embriogenik, metode kultur, kinetin, TDZ, sagu, Metroxylon sagu]

An ultrastructural study on the early development of Zea mays somatic embryos

1991 ◽  
Vol 69 (4) ◽  
pp. 858-865 ◽  
Author(s):  
P. F. Fransz ◽  
J. H. N. Schel
Keyword(s):  
Zea Mays ◽  
Early Development ◽  
Embryogenic Callus ◽  
Somatic Embryos ◽  
Zea Mays L ◽  
Light And Electron Microscopy ◽  
Apical Region ◽  
Basal Region ◽  
Embryogenic Cells

Friable embryogenic callus, obtained from immature embryos of Zea mays L., was cultured on N6 medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid, 6 mM proline, and 2% sucrose. Cultured tissue fragments containing several globular embryoids were excised and examined by light and electron microscopy to follow the early development of maize embryoids. The somatic embryos consist of an apical region and a suspensor region. Cells of the apical region are small, cytoplasm rich, and mitotically active. They contain much starch and numerous bundles of microtubules. Suspensor cells are larger and more vacuolated. A high metabolic activity in both cell types is indicated by the presence of many organelles, coated vesicles, and multivesicular bodies. Transition units appear to form intermediate stages between the embryogenic callus cells and the somatic embryo. A transition unit consists of a group of embryogenic cells and shows an apical and a basal region. The unit has many intercellular spaces, and within the cells areas with organelle-free cytosol are frequently observed. Key words: somatic embryogenesis, in vitro culture, ultrastructure, Zea mays L.

scholarly journals Identifikasi dan pencegahan kontaminasi pada kultur cair sistem perendaman sesaat Identification and prevention of contamination in liquid culture of temporary immersion system

2016 ◽  
Vol 82 (2) ◽  
Author(s):  
Masna Maya SINTA ◽  
Imron RIYADI ◽  
. SUMARYONO
Keyword(s):  
In Vitro Culture ◽  
Bacillus Megaterium ◽  
Liquid Culture ◽  
Bacillus Sphaericus ◽  
Small Ring ◽  
Bacillus Firmus ◽  
Temporary Immersion System ◽  
Temporary Immersion ◽  
Bacillus Macerans

AbstractLiquid culture is commonly used to scale up in vitro culture production as well as to optimize the developmental phase of plant in vitro culture. One of the liquid cultures that has been used widely is temporary immersion system (TIS). The main problem of liquid culture is contamination. The use of antibiotics sometimes controls the contaminants less effectively and hinders the growth of plant culture. The purpose of this research was to determine sources of contaminant on whole sequence of TIS to identify and to prevent the emergence of the contaminants. Sampling method was applied to each section and stage of TIS culture and the contaminants found were identified. The results revealed that compartment of TIS was the main source of contaminant (100%). Furthermore, from all components of TIS compartment, washer (a small ring seal connecting screen disc and basket) was the main source of TIS contaminant (41.2%). Four contaminants found were identified as Bacillus macerans, Bacillus megaterium, Bacillus sphaericus and Bacillus firmus. Two times sterilization of washer in an autoclave at temperature of 121 oC and air pressure of 1 kg/cm2 for 20 minutes before and after being installed reduced the contamination level on TIS culture significantly.AbstrakKultur cair umumnya digunakan untuk meningkatkan skala produksi dan mengoptimalkan fase perkembangan kultur in vitro tanaman. Salah satu jenis kultur cair yang banyak digunakan adalah sistem perendaman sesaat (SPS). Masalah utama dalam kultur cair adalah kontaminasi. Penggunaan antibiotika terkadang kurang efektif dalam me-ngendalikan kontaminan dan menghambat pertumbuhan kultur tanaman. Tujuan dari penelitian ini adalah untuk mengetahui sumber kontaminan pada seluruh rangkaian kultur SPS serta mengidentifikasi dan mencegah munculnya kontaminan tersebut. Metode yang digunakan adalah  pengambilan contoh pada tiap bagian dan fase kultur SPS, serta kontaminan yang ditemukan kemudian diidentifikasi. Hasil penelitian memperlihatkan bahwa kompartemen SPS merupakan sumber utama kontaminan (100%). Selanjutnya, dari seluruh komponen kompartemen SPS, washer (cincin penutup yang menghubungkan penyaring dan keranjang) di dalam rangkaian SPS merupakan sumber utama kontaminan (41,2%).  Empat  kontaminan yang ditemukan diidentifikasi sebagai Bacillus macerans, Bacillus megaterium, Bacillus sphaericus dan Bacillus firmus. Sterilisasi cincin penutup sebanyak  dua  kali  dalam  autoklaf pada suhu 121 oC dan tekanan udara 1 kg/cm2selama 20 menit sebelum dan sesudah dirangkai secara nyata menurunkan tingkat konta-minasi pada kultur SPS. 

scholarly journals Identifikasi dan pencegahan kontaminasi pada kultur cair sistem perendaman sesaat Identification and prevention of contamination in liquid culture of temporary immersion system

2016 ◽  
Vol 82 (2) ◽  
Author(s):  
Masna Maya SINTA ◽  
Imron RIYADI ◽  
. SUMARYONO
Keyword(s):  
In Vitro Culture ◽  
Bacillus Megaterium ◽  
Liquid Culture ◽  
Bacillus Sphaericus ◽  
Small Ring ◽  
Bacillus Firmus ◽  
Temporary Immersion System ◽  
Temporary Immersion ◽  
Bacillus Macerans

AbstractLiquid culture is commonly used to scale up in vitro culture production as well as to optimize the developmental phase of plant in vitro culture. One of the liquid cultures that has been used widely is temporary immersion system (TIS). The main problem of liquid culture is contamination. The use of antibiotics sometimes controls the contaminants less effectively and hinders the growth of plant culture. The purpose of this research was to determine sources of contaminant on whole sequence of TIS to identify and to prevent the emergence of the contaminants. Sampling method was applied to each section and stage of TIS culture and the contaminants found were identified. The results revealed that compartment of TIS was the main source of contaminant (100%). Furthermore, from all components of TIS compartment, washer (a small ring seal connecting screen disc and basket) was the main source of TIS contaminant (41.2%). Four contaminants found were identified as Bacillus macerans, Bacillus megaterium, Bacillus sphaericus and Bacillus firmus. Two times sterilization of washer in an autoclave at temperature of 121 oC and air pressure of 1 kg/cm2 for 20 minutes before and after being installed reduced the contamination level on TIS culture significantly.AbstrakKultur cair umumnya digunakan untuk meningkatkan skala produksi dan mengoptimalkan fase perkembangan kultur in vitro tanaman. Salah satu jenis kultur cair yang banyak digunakan adalah sistem perendaman sesaat (SPS). Masalah utama dalam kultur cair adalah kontaminasi. Penggunaan antibiotika terkadang kurang efektif dalam me-ngendalikan kontaminan dan menghambat pertumbuhan kultur tanaman. Tujuan dari penelitian ini adalah untuk mengetahui sumber kontaminan pada seluruh rangkaian kultur SPS serta mengidentifikasi dan mencegah munculnya kontaminan tersebut. Metode yang digunakan adalah  pengambilan contoh pada tiap bagian dan fase kultur SPS, serta kontaminan yang ditemukan kemudian diidentifikasi. Hasil penelitian memperlihatkan bahwa kompartemen SPS merupakan sumber utama kontaminan (100%). Selanjutnya, dari seluruh komponen kompartemen SPS, washer (cincin penutup yang menghubungkan penyaring dan keranjang) di dalam rangkaian SPS merupakan sumber utama kontaminan (41,2%).  Empat  kontaminan yang ditemukan diidentifikasi sebagai Bacillus macerans, Bacillus megaterium, Bacillus sphaericus dan Bacillus firmus. Sterilisasi cincin penutup sebanyak  dua  kali  dalam  autoklaf pada suhu 121 oC dan tekanan udara 1 kg/cm2selama 20 menit sebelum dan sesudah dirangkai secara nyata menurunkan tingkat konta-minasi pada kultur SPS. 

scholarly journals In Vitro Somatic Embryos Multiplication of Eurycoma longifolia Jack using Temporary Immersion System RITA®

2017 ◽  
Vol 46 (6) ◽  
pp. 897-902 ◽  
Author(s):  
Noor Madihah Mohd ◽  
Hafsah Ja'afar ◽  
Dhiya Dalila Zawawi ◽  
Nadiawati Alias
Keyword(s):  
Somatic Embryos ◽  
Temporary Immersion System ◽  
Temporary Immersion ◽  
Eurycoma Longifolia ◽  
Eurycoma Longifolia Jack

Leucojum aestivum L. in vitro bulbs induction and acclimatization

2014 ◽  
Vol 9 (11) ◽  
pp. 1011-1021 ◽  
Author(s):  
Agata Ptak
Keyword(s):  
Liquid Medium ◽  
Physical State ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Microscopy Study ◽  
Temporary Immersion System ◽  
Temporary Immersion ◽  
Leucojum Aestivum ◽  
The One

AbstractThe effects of growth retardants (paclobutrazol and ancymidol), sucrose, GA3 (gibberellic acid) and physical state of the medium (solid and liquid — Rita® temporary immersion system) on in vitro induction of Leucojum aestivum bulbs and their acclimatization were studied. Paclobutrazol, regardless of the physical state of the medium, stimulated the formation of bulbs (99.3%). Under the influence 90 g L−1 of sucrose or paclobutrazol the bulbs with the highest fresh weight (FW) were formed (250 mg and 208.8 mg, respectively). However, the addition of ancymidol to the liquid medium led to obtaining the bulbs showing the highest number of leaves and roots (63.2% and 91.7%, respectively). The scanning microscopy study proved that plants obtained in the medium containing GA3 produced the stomata which most closely resembled to the one observed in the mother plant. Cytometric analysis of all regenerants revealed absence of changes in the nuclear DNA content. The maximum survival rate (100%) was observed for plants derived from liquid medium containing 90 g L−1 of sucrose. Somewhat fewer plants were acclimatized after their cultivations in liquid medium enriched with paclobutrazol or ancymidol. The temporary immersion system led to perform successful ex vitro adaptation of Leucojum aestivum plants.

scholarly journals In Vitro Culture of Rosmarinus officinalis L. in a Temporary Immersion System: Influence of Two Phytohormones on Plant Growth and Carnosol Production

2021 ◽  
Vol 14 (8) ◽  
pp. 747
Author(s):  
Eder Villegas Sánchez ◽  
Mariana Macías-Alonso ◽  
Soraya Osegueda Robles ◽  
Lisset Herrera-Isidrón ◽  
Hector Nuñez-Palenius ◽  
...  
Keyword(s):  
High Performance ◽  
Emerging Infectious Diseases ◽  
Rosmarinus Officinalis ◽  
Wild Plant ◽  
Array Detector ◽  
Phase System ◽  
Naphthaleneacetic Acid ◽  
Temporary Immersion System ◽  
Temporary Immersion

Emerging infectious diseases have become a major global problem with public health and economic consequences. It is an urgent need to develop new anti-infective therapies. The natural diterpene carnosol exhibit a wide variety of interesting antibacterial and antiviral properties, and it is considered a theoretical inhibitor of COVID-19 Mpro. However, this compound is present in the family Lamiaceae in low quantities. To obtain carnosol in concentrations high enough to develop pharmacological studies, we evaluated the efficiency of a micropropagation protocol of Rosmarinus officinalis using a solid medium and a temporary immersion system (TIS), as well as the effect of 6-benzylaminopurine (6-BAP) and α-naphthaleneacetic acid (NAA) on the growth of shoots. Moreover, we developed and validated an analytical method to quantify carnosol using the H-point standard additions method in the high-performance liquid chromatography diode array detector (HPLC-DAD). After 30 days of culture, TIS produced the maximum number of shoots per explant (24.33 ± 1.15) on a liquid medium supplemented with 6-BAP at 5.0 mg L−1. Next, we also evaluated the effect of immersion time and frequency for TIS. After 72 days of culture, the best results were obtained with an immersion cycle of 1 min every 12 h, yielding 170.33 ± 29.40 shoots. The quantification of carnosol on the samples was performed at a flow rate of 1.2 mL min−1 using binary isocratic mobile phase system 60:40 (v/v) 10 mM formic acid (pH 3.0) (A) and acetonitrile (B) on a reverse-phase column. The content of carnosol in the in vitro cultures was around 8-fold higher than in the wild plant. The present study represents an efficient alternative method to obtain carnosol for its pre-clinical and clinical development.

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