scholarly journals Kriopreservasi Tanaman Obat Langka Purwoceng dengan Teknik Enkapsulasi-Vitrifikasi

2016 ◽  
Vol 14 (2) ◽  
pp. 49
Author(s):  
Ika Roostika ◽  
Suci Rahayu ◽  
Novianti Sunarlim
Keyword(s):  
Liquid Nitrogen ◽  
Incubation Period ◽  
Optimization Method ◽  
Endangered Medicinal Plant ◽  
In Vitro Shoots ◽  
Long Term Preservation ◽  
Cryopreservation Protocol ◽  
Cacl2 Solution

<p>Pruatjan (Pimpinella pruatjan Molk.) is an Indonesian endangered medicinal plant, so that it is highly protected. Cryopreservation can be applied to this plant for long-term preservation. The aim of this research was to obtain a method of encapsulation-vitrification by optimizing each step in cryopreservation protocol i.e. preculture, loading, dehydration with and without freezing in liquid nitrogen. The best treatment of each step would be applied in the following step. On preculture experiment, in vitro shoots were planted on the Driver and Kuniyaki (DKW) basal media containing 0.3 M sucrose and incubated for 1, 2, 3, 4, and 5 days. After those incubation period, shoot tips were encapsulated with 2.5% Na-alginate and soaking for 15 minutes in 100 ppm CaCl2 solution before planting. On loading experiment, precultured explants were loaded in DKW basal solution containing 2 M glycerol and 0.4 M sucrose for 0, 30, 60, and 90 minutes. On dehydration experiment, preculturead and loaded explants were dehydrated with PVS2 solution PVS2 (DKW + 30% glycerol + 15% DMSO + 15% ethyleneglicol + 0.4 M sucrose) for 0, 30, 60, 90, and 120 minutes. The parts of them were freezed in liquid nitrogen (-196oC). The result showed that cryopreservation through encapsulation-vitrification technique could be applied on pruatjan. The best preculture treatment was 5 days incubation period. The best loading treatment was 30 minutes. The best dehydration treatment was 90 minutes. The successful level of this research was still low (10%) so that it needs optimization method.</p><p> </p><p><strong>Abstrak</strong></p><p>Purwoceng (Pimpinella pruatjan Molk.) adalah tanaman obat langka asli Indonesia yang hampir punah sehingga harus dilindungi. Kriopreservasi dapat diterapkan pada tanaman ini untuk penyimpanan jangka panjang. Tujuan penelitian adalah untuk memperoleh teknik enkapsulasi-vitrifikasi dengan melakukan optimasi dari tiap-tiap tahapan kriopreservasi yang meliputi perlakuan prakultur, loading, dehidrasi sebelum dan setelah pembekuan dalam nitrogen cair. Perlakuan yang terbaik kemudian diterapkan pada tahapan percobaan berikutnya. Pada perlakuan prakultur, tunas in vitro ditanam pada media Driver dan Kuniyaki (DKW) dengan penambahan sukrosa 0,3 M dengan masa inkubasi 1, 2, 3, 4, dan 5 hari. Setelah itu, pucuk yang berukuran 0,5 cm dienkapsulasi dengan Na-alginat 2,5% (yang mengandung media regenerasi) dalam larutan CaCl2 100 ppm selama 15 menit sebelum penanaman kembali. Pada percobaan loading, terlebih dahulu eksplan diprakultur kemudian direndam dalam larutan DKW + gliserol 2 M + sukrosa 0,4 M dengan durasi rendam selama 0, 30, 60, dan 90 menit. Pada percobaan dehidrasi, eksplan diprakultur dan loading terlebih dahulu, kemudian direndam dalam larutan krioprotektan PVS2 (DKW + gliserol 30% + DMSO 15% + etilen glikol 15% + sukrosa 0,4 M ) selama 0, 30, 60, 90, dan 120 menit. Eksplan tersebut sebagian dibekukan dalam nitrogen cair (-196oC) dan sebagian lainnya tidak dibekukan. Hasil penelitian menunjukkan bahwa kriopreservasi secara enkapsulasi-vitrifikasi berpeluang diterapkan pada tanaman purwoceng. Perlakuan prakultur terbaik adalah 5 hari. Perlakuan loading terbaik adalah 30 menit dan perlakuan dehidrasi terbaik 90 menit. Tingkat keberhasilan ini masih rendah (10%) sehingga diperlukan optimasi metode.</p>

scholarly journals Effect of the Duration of Liquid Nitrogen Storage on the Regrowth of Blackberry Cryopreserved by Droplet Vitrification

2017 ◽  
Vol 66 (1-2) ◽  
pp. 44-50
Author(s):  
Tatjana Vujović ◽  
Đurđina Ružić ◽  
Radosav Cerović
Keyword(s):  
Liquid Nitrogen ◽  
Room Temperature ◽  
Shoot Tips ◽  
Lower Survival Rate ◽  
Droplet Vitrification ◽  
Vitrification Solution ◽  
Long Term Preservation ◽  
Direct Immersion

SummaryIn vitro shoot tips of the blackberry cultivar ‘Čačanska Bestrna’ were cryopreserved using the droplet vitrification technique. Upon loading (30 min) in a solution of 1.9 M glycerol and 0.5 M sucrose, the explants were dehydrated for 40 min on ice with the PVS A3 vitrification solution (glycerol 37.5%, dimethyl sulfoxide 15%, ethylene glycol 15% and sucrose 22.5%) and for 40 min at room temperature with the PVS3 solution (glycerol 50% and sucrose 50%). They were subsequently frozen in individual microdroplets of vitrification solution, by direct immersion in liquid nitrogen (LN), and kept therein for 2, 4, 8 and 24 h. The explant rewarming was performed in an unloading solution (0.8 M sucrose) for 30 min at room temperature. The duration of LN exposure did not exert significant effects on the survival and regrowth of explants in both types of vitrification solutions. The survival and regrowth of cryopreserved shoot tips dehydrated with PVS3 solution ranged between 90–95% and 80–90%, respectively. However, dehydration with PVS A3 resulted in a lower survival rate (80–90%) and a considerably lower regrowth rate (55–65%) of explants. Monitoring the shoots regenerated in the in vitro culture revealed their normal capacity for multiplication and rooting in comparison with the controls, which fully confirms the purpose of cryopreservation in the long-term preservation of plant material.

Effect of long-term preservation of basidiomycetes on perlite in liquid nitrogen on their growth, morphological, enzymatic and genetic characteristics

2010 ◽  
Vol 114 (11-12) ◽  
pp. 929-935 ◽  
Author(s):  
Ladislav Homolka ◽  
Ludmila Lisá ◽  
Ivana Eichlerová ◽  
Vendula Valášková ◽  
Petr Baldrian
Keyword(s):  
Liquid Nitrogen ◽  
Genetic Characteristics ◽  
Long Term Preservation

scholarly journals OPTIMASI DAN EVALUASI METODE KRIOPRESERVASI PURWOCENG

2020 ◽  
Vol 19 (3) ◽  
pp. 147
Author(s):  
IKA ROOSTIKA ◽  
IRENG DARWATI ◽  
RITA MEGIA
Keyword(s):  
Visual Observation ◽  
Sem Analysis ◽  
Combined Treatments ◽  
Embryogenic Calli ◽  
Regeneration Rate ◽  
Adenine Sulphate ◽  
In Vitro Shoots ◽  
Better Than

<p>ABSTRAK<br />Optimasi dan evaluasi metode kriopreservasi perlu dilakukan dalam<br />menentukan protokol standar untuk penyimpanan jangka panjang biakan<br />purwoceng. Penelitian ini bertujuan untuk mengetahui pengaruh kombinasi<br />perlakuan pratumbuh, prakultur, dan formulasi media pemulih terhadap<br />daya tumbuh dan daya regenerasi tunas in vitro dan kalus embriogenik<br />serta untuk mengevaluasi metode kriopreservasi melalui observasi<br />morfologi, anatomi, dan sitologi. Penelitian dilakukan di Laboratorium<br />Kultur Jaringan Kelompok Peneliti Biologi Sel dan Jaringan BB Litbang<br />Biogen pada tahun 2008-2009. Teknik kriopreservasi yang digunakan<br />adalah vitrifikasi (untuk apeks) dan enkapsulasi-vitrifikasi (untuk kalus<br />embriogenik). Pada teknik vitrifikasi, tunas pucuk diberi perlakuan<br />pratumbuh dengan sukrosa (3, 4, 5, dan 6%) selama 1 dan 2 minggu,<br />perlakuan prakultur dilakukan pada media yang mengandung sukrosa 0,3<br />M selama 1 dan 3 hari, perlakuan dehidrasi dengan PVS2 diberikan selama<br />15 dan 30 menit, dan media pemulih yang diujikan adalah media dasar MS<br />atau DKW dengan dan tanpa penambahan adenin sulfat 20 ppm. Pada<br />teknik enkapsulasi-vitrifikasi, kalus embriogenik dienkapsulasi terlebih<br />dahulu dengan Na-alginat 3%, perlakuan dehidrasi dengan PVS2 diberikan<br />selama 0, 30, dan 60 menit. Evaluasi metode teknik kriopreservasi<br />dilakukan melalui pengamatan morfologi secara visual, anatomi meristem<br />dengan scanning electron microscope (SEM), pengujian viabilitas dengan<br />fluorescein diacetate (FDA), dan analisis ploidi secara flowcytometry.<br />Hasil penelitian menunjukkan bahwa teknik enkapsulasi-vitrifikasi lebih<br />baik daripada teknik vitrifikasi untuk kriopreservasi purwoceng. Walaupun<br />persentase keberhasilan kriopreservasi rendah (10%), kalus embriogenik<br />purwoceng mampu berproliferasi dan beregenerasi menjadi ribuan embrio<br />somatik dewasa. Evaluasi metode kriopreservasi dengan SEM dan FDA<br />dapat diterapkan untuk memperkirakan keberhasilan teknik kriopreservasi<br />secara dini sedangkan analisis flowcytometry dapat diterapkan untuk<br />menguji stabilitas genetik bahan tanaman pasca-kriopreservasi.<br />Kata kunci: Pimpinella pruatjan Molk., kriopreservasi, SEM, FDA,<br />flowcytometry</p><p>ABSTRACT<br />Optimization and evaluation of cryopreservation methods should be<br />conducted to obtain standard protocol for long term conservation of<br />pruatjan. The objective of this study was to evaluate the effect of<br />combined treatments of pregrowth, preculture, and recovery media to the<br />survival and regeneration rate of in vitro shoots and embryogenic calli and<br />to evaluate the cryopreservation methods by observing the morphological,<br />anatomical, and cytological characters. The techniques of vitrification (for<br />apex) and encapsulation-vitrification (for embryogenic calli) were applied<br />in this study. On vitrification technique, the apical shoots were pregrown<br />on media containing of 3, 4, 5, and 6% sucrose for 1 and 2 weeks,<br />precultured on media containing of 0,3 M sucrose for 1 and 3 days,<br />dehydrated by PVS2 solution for 15 and 30 minutes, and planted on<br />recovery media (MS or DKW basal media supplemented with 20 ppm<br />adenine sulphate). On encapsulation-vitrification technique, embryogenic<br />calli were encapsulated by 3% Na-alginate, dehydrated by PVS2 solution<br />for 0, 30, and 60 minutes. The evaluation of cryopreservation methods was<br />done through visual observation, SEM analysis, viability test, and<br />flowcytometry determination. The result showed that encapsulation-<br />vitrification was better than vitrification technique for cryopreservation of<br />pruatjan. The successful rate of this method was low (10%) but the<br />embryogenic calli could proliferate and regenerate into thousands mature<br />somatic embryos. The evaluation by SEM and FDA can be applied as<br />early detection to estimate the successful of cryopreservation, whereas<br />flowcytometry  analysis  may  determine  the  genetic  stability  of<br />cryopreserved materials.<br />Key words: Pimpinella pruatjan Molk., cryopreservation, SEM, FDA,<br />flowcytometry</p>

96 THE EFFECT OF CHEMICAL DELIPIDATION ON CRYOPRESERVABILITY OF CAT EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 153 ◽  
Author(s):  
T. Tharasanit ◽  
M. Techakumphu
Keyword(s):  
Embryo Cryopreservation ◽  
Blastocyst Formation ◽  
Freezing And Thawing ◽  
Intracellular Lipid ◽  
Embryo Survival ◽  
Senior Research ◽  
In Utero Development ◽  
Long Term Preservation

Embryo cryopreservation is a desired technique for long-term preservation of embryos. However, the success rate of cryopreserved in vitro produced cat embryos is currently poor. Until recently, the mechanism underlying the cause of cryoinjury that occurs during cooling and cryopreservation is not well understood. This study aimed to examine the effect of chemical delipidation (forskolin) before cryopreservation of 4- to 8-cell cat embryos on post-thaw embryo survival and in vitro developmental capability. A total of 333 cumulus oocyte complexes (COCs) were matured and fertilized in vitro. At 24 h post-IVF, the presumptive embryos were randomly assigned into one of the following groups: 1) non-frozen control (n = 63); 2) forskolin treatment without freezing (n = 52); 3) freezing without forskolin (n = 77); and 4) freezing after forskolin treatment (n = 89). The embryos were cryopreserved using a programmable controlled-rate freezer. After freezing and thawing, the embryos were subsequently cultured in vitro for a further 6 days. The development competence was assessed by morula and blastocyst rates on Days 5 and 8 of their development, respectively. Percentages of cleaved embryos on Day 2 (IVF = Day 0) did not significantly differ among groups, indicating that there was no adverse effect of forskolin on cleavage rates. Furthermore, blastocyst formation rates of cat embryos treated with forskolin (53.5 ± 3.1) did not significantly differ when compared with non-treated controls (54 ± 9.3). Forskolin-treated embryos survived after cryopreservation at a higher rate than non-forskolin treatment, in terms of survival (93.1 ± 2.6 v. 88.2 ± 1.4), morula (56.9 ± 7.6 v. 40.8 ± 5.7), and blastocyst formation (47.6 ± 6.4 v. 35.6 ± 3.6) rates. It is concluded that partial delipidation of cat embryos before cryopreservation improves the cryopreservability of cat embryos. This study demonstrates that intracellular lipid has an impact on cryopreservability of cat embryos. Further study is required to examine in utero development of these delipidated embryos after embryo transfer. This study was financially supported by the Zoo organisation of Thailand and CHE-TRF Senior Research Scholars RTA-5080010.

Extended Perfusion for Long-Term Preservation of Whole Hearts

1977 ◽  
Vol 35 ◽  
pp. 574-575
Author(s):  
D. E. Philpott ◽  
R. L. Moretti ◽  
G. Harrison ◽  
G. Klein ◽  
S. Abraham ◽  
...  
Keyword(s):  
Present Method ◽  
The Body ◽  
Donor Heart ◽  
Heart Transplants ◽  
Retrograde Perfusion ◽  
Rat Hearts ◽  
Long Term Preservation ◽  
Recipient Selection

One of the problems associated with heart transplants is the maintenance of the donor heart while the recipient is being prepared. The present method, which entails keeping the body of the donor alive as a sort of incubation chamber, is expensive and difficult. Maintenance of the heart in vitro would not only cut costs, but also allow greater flexibility in recipient selection, location and timing of the operation.To determine the effects of preparing hearts for perfusion maintenance, rat hearts were excised and prepared for retrograde perfusion via the aorta. Once excised, the hearts were cooled in iced Tyrode's solution saturated with 95% O2 and 5% CO2 during the preparation for perfusion. Warm Tyrode's (37°) containing 2% plasma was then flushed through the heart at 100mm Hg pressure for 3 min., 15 min., 30 min. and 1 hour.

Development of Probiotic Lactobacillus sporogenes Loaded Biological Macromolecules using Novel Assembly Design for Long-term Preservation as Bio-therapeutic Agent

2021 ◽  
Vol 14 (3) ◽  
pp. 5481-5491
Author(s):  
Himanshu Solanki ◽  
Madhavi P. Ghumare ◽  
Vipul D Prajapati ◽  
Sanjeev R Acharya
Keyword(s):  
Encapsulation Efficiency ◽  
Alginate Beads ◽  
Biological Macromolecules ◽  
Automated Production ◽  
Assembly Design ◽  
Intestinal Delivery ◽  
Structure Surface ◽  
Long Term Preservation

The goal present investigation was to formulate and characterized of biological macromolecules of alginate (ALG) and carboxymethylcellulose sodium (CMC) containing probiotic bacteria of Lactobacillus sporogenes (LS) co-encapsulated with a prebiotics, Bioecolians (α-Gluco-oligosaccharides). The prepared beads were characterized in terms of yield, size, encapsulation efficiency, viabilities in simulated gastric (pH 1.2, 2 hours) and bile (1% w/v, 3 hours) conditions. The beads were also characterized by FTIR, DSC, SEM and XRD to investigate molecular structure, surface properties and morphology of beads. The results showed that spherical beads with size distribution ranging from 1.18 ± 0.11 to 1.45 ± 0.15 mm for ALG and from 1.3 ± 0.12 to 1.5 ± 0.16 mm for ALG-CMC with encapsulation efficiency higher than 90% were achieved. The results indicated that incorporation of carboxymethylcellulose sodium into alginate beads improved viability of the bacteria in simulated gastric conditions as well as bile conditions. According to our in vitro studies, Probiotic beads using combination of ALG-CMC are suitable encapsulating polymer for gastro-intestinal delivery as designed by novel assembly using peristaltic pump for automated production.

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