OPTIMASI DAN EVALUASI METODE KRIOPRESERVASI PURWOCENG

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

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Biocompatibility Evaluation of Hollow Pollen Grains/Fe3 O4 Nanoparticles Composites as Potential Medical Devices

International Journal of Nanoscience ◽

10.1142/s0219581x21500484 ◽

2021 ◽

Author(s):

Solmaz Zakhireh ◽

Yadollah Omidi ◽

Younes Beygi-Khosrowshahi ◽

Ayoub Aghanejad ◽

Jaleh Barar ◽

...

Keyword(s):

Morphological Analysis ◽

Pollen Grains ◽

Building Blocks ◽

Drug Carriers ◽

Sem Analysis ◽

Narrow Region ◽

In Vitro Biocompatibility ◽

Apoptosis Related Gene

Recently, pollen grains (PGs) have been introduced as drug carriers and scaffolding building blocks. This study aimed to assess the in-vitro biocompatibility of Pistacia vera L. hollow PGs/Fe3O4 nanoparticles (HPGs/Fe3O4NPs) composites using human adipose-derived mesenchymal stem cells (hAD-MSCs). In this regard, iron oxide nanoparticles (Fe3O4NPs) were assembled on the surface of HPGs at different concentrations. The biocompatibility of the prepared composites was assessed through MTT assay, apoptosis-related gene expression and field emission scanning electron microscopy (FE-SEM) analysis. Compared to the bare HPGs, the HPGs/Fe3O4NPs exhibited a biphasic impact on hAD-MSCs. The composite containing 1% Fe3O4NPs demonstrated no cytotoxicity up to 21 days while higher Fe3O4NPs contents and long-term exposure revealed adverse effects on the hAD-MSCs’ growth. The obtained result was verified by the qRT-PCR and morphological analysis carried out through FE-SEM which suggests that a narrow region below 1% Fe3O4NPs may be the optimum choice for medicinal applications of HPGs/Fe3O4NPs microdevices.

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Long-term maintenance of in vitro cultures affects growth and secondary metabolism of St. John's Wort

Ciência Rural ◽

10.1590/s0103-84782010001000010 ◽

2010 ◽

Vol 40 (10) ◽

pp. 2115-2121 ◽

Cited By ~ 5

Author(s):

Adriana de Andrade Figueiró ◽

Cynthia Manira Correa ◽

Leandro Vieira Astarita ◽

Eliane Romanato Santarém

Keyword(s):

Phenolic Compounds ◽

Secondary Metabolism ◽

Adventitious Shoots ◽

In Vitro Shoots ◽

Culture Production ◽

Short And Long Term ◽

Short Term Experiment ◽

Term Maintenance

Hypericum perforatum is a traditional medicinal plant with wound healing and antidepressant properties. Efficiency of micropropagation is often related to the long term maintenance of tissues in culture, which may alter the secondary metabolism of plants. The objective of this study was to evaluate growth and secondary metabolism of in vitro shoots of H. perforatum on short and long term maintenance of cultures (30 and 100 days). The effect of BA and NAA supplementation was evaluated during 30 days of culture. Adventitious shoots were cultivated on MS medium supplemented with 4.4mM BA alone or in combination with 0.05mM NAA for 30 days. A hormone-free medium was used as control. Shoots cultivated for 100 days were maintained in presence of 4.4mM BA. Biomass, multiplication of shoots, contents of phenolic compounds, flavonoids and hypericin were evaluated. No difference between BA and BA+NAA was observed on growth, multiplication of shoots and levels of flavonoids at the end of 30 days of culture. Production of phenolic compounds was promoted by addition of BA+NAA to the medium, whereas hypericin was increased by the presence of BA. The time of culture (30 and 100 days) affected all the parameters analyzed, except the levels of flavonoids in the short term experiment.

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Incorporation of the factor IX Padua mutation into FIX-Triple improves clotting activity in vitro and in vivo

Thrombosis and Haemostasis ◽

10.1160/th13-02-0154 ◽

2013 ◽

Vol 110 (08) ◽

pp. 244-256 ◽

Cited By ~ 20

Author(s):

Chung-Yang Kao ◽

Shu-Jhu Yang ◽

Mi-Hua Tao ◽

Yung-Ming Jeng ◽

I-Shing Yu ◽

...

Keyword(s):

Gene Therapy ◽

Replacement Therapy ◽

Therapeutic Potential ◽

Factor Ix ◽

Adeno Associated Virus ◽

Viral Dose ◽

Better Than

SummaryUsing gain-of-function factor IX (FIX) for replacement therapy for haemophilia B (HB) is an attractive strategy. We previously reported a high-activity FIX, FIX-Triple (FIX-V86A/E277A/R338A) as a good substitute for FIX-WT (wild-type) in protein replacement therapy, gene therapy, and cell therapy. Here we generated a new recombinant FIXTripleL (FIX-V86A/E277A/R338L) by replacing the alanine at residue 338 of FIX-Triple with leucine as in FIX-Padua (FIX-R338L). Purified FIX-TripleL exhibited 22-fold higher specific clotting activity and 15-fold increased binding affinity to activated FVIII compared to FIXWT. FIX-TripleL increased the therapeutic potential of FIX-Triple by nearly 100% as demonstrated with calibrated automated thrombogram and thromboelastography. FIX-TripleL demonstrated a normal clearance rate in HB mice. The clotting activity of FIX-TripleL was consistently 2- to 3-fold higher in these mice than that of FIX-Triple or FIXR338L. Gene delivery of adeno-associated virus (AAV) in HB mice showed that FIX-TripleL had 15-fold higher specific clotting activity than FIX-WT, and this activity was significantly better than FIX-Triple (10-fold) or FIX-R338L (6-fold). At a lower viral dose, FIX-TripleL improved FIX activity from sub-therapeutic to therapeutic levels. Under physiological conditions, no signs of adverse thrombotic events were observed in long-term AAV-FIX-treated C57Bl/6 mice. Hepatocellular adenomas were observed in the high- but not the medium- or the lowdose AAV-treated mice expressing FIX-WT or FIX-Triple, indicating the advantages of using hyperfunctional FIX variants to reduce viral doses while maintaining therapeutic clotting activity. Thus, incorporation of the FIX Padua mutation significantly improves the clotting function of FIX-Triple so as to optimise protein replacement therapy and gene therapy.

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Short-term injection of antiapoptotic cytokine combinations soon after lethal γ-irradiation promotes survival

Blood ◽

10.1182/blood-2002-06-1634 ◽

2003 ◽

Vol 101 (7) ◽

pp. 2609-2616 ◽

Cited By ~ 110

Author(s):

Francis Hérodin ◽

Philippe Bourin ◽

Jean-François Mayol ◽

Jean-Jacques Lataillade ◽

Michel Drouet

Keyword(s):

Lethal Dose ◽

Survival Rates ◽

Protective Effects ◽

Γ Irradiation ◽

Term Survival ◽

Hematopoietic Stem ◽

Better Than

Recovery from radiation-induced (RI) myelosuppression depends on hematopoietic stem and progenitor cell survival and the active proliferation/differentiation process, which requires early cytokine support. Single cytokine or late-acting growth factor therapy has proved to be inefficient in ensuring reconstitution after severe RI damage. This work was aimed at evaluating the in vivo survival effect of combinations of early-acting cytokines whose antiapoptotic activity has been demonstrated in vitro: stem cell factor (SCF [S]), FMS-like tyrosine kinase 3 ligand (FLT-3 ligand [F]), thrombopoietin (TPO [T]), interleukin-3 (IL-3 [3]), and stromal derived factor-1 (SDF-1). B6D2F1 mice underwent total body irradiation at 8 Gy cesium Cs 137 γ radiation (ie, lethal dose 90% at 30 days) and were treated soon after irradiation, at 2 hours and at 24 hours, with recombinant murine cytokines, each given intraperitoneally at 50 μg/kg per injection. All treatments induced 30-day survival rates significantly higher than control (survival rate, 8.3%). 4F (SFT3) and 5F (4F + SDF-1) were the most efficient combinations (81.2% and 87.5%, respectively), which was better than 3F (SFT, 50%), TPO alone (58.3%), and SDF-1 alone (29.2%) and also better than 4F given at 10 μg/kg per injection (4F10, 45.8%) or as a 50 μg/kg single injection at 2 hours (4Fs, 62.5%). Despite delayed death occurring mainly from day 150 on and possible long-term hematopoiesis impairment, half the 30-day protective effects of 4F and 5F were preserved at 300 days. Our results show that short- and long-term survival after irradiation depends on appropriate multiple cytokine combinations and at optimal concentrations. The proposal is made that an emergency cytokine regimen could be applied to nuclear accident victims as part of longer cytokine treatment, cell therapy, or both.

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Temporary Immersion System Improves Regeneration of in vitro Irradiated Recalcitrant Rice Embryogenic Calli

10.20944/preprints202112.0408.v1 ◽

2021 ◽

Author(s):

Alejandro Hernández-Soto ◽

Jason Pérez-Chávez ◽

Rebeca Fait-Zuñiga ◽

Randall Rojas-Vásquez ◽

Andres Gatica-Arias ◽

...

Keyword(s):

Gamma Ray ◽

Lethal Dose ◽

Radiation Condition ◽

Embryogenic Calli ◽

Temporary Immersion System ◽

Semisolid Medium ◽

Regeneration Rate ◽

In Vitro Establishment

The development of gamma rays mutant rice lines would be a solution for introducing variability in already farmer using varieties. In vitro gamma (60Co) mutagenesis reduces chimeras and allows a faster selection of desired traits but requires laboratory process optimization. The objective of the present work was the in vitro establishment of a recalcitrant rice embryogenic calli, the determination of its sensitivity to gamma radiation (Co-60), sequencing MATK and Rubisco for identification purposes, as well as generation optimization. The radiosensitivity of embryogenic calli resulted in an LD50 of 110Gy, while the 20% lethal dose was 64Gy. All sequenced genes matched perfectly with already reported MATK and Rubisco O. sativa genes with a clear SNP that identifies the local variety related to the southeast Asia Region. Callus induction improves with an MS with 2mg/L 2,4D, and the regeneration was achieved with an MS medium with 3mg/L BAP and 0,5mg/L NAA. The optimized radiation condition was 60Gy with an 83% regeneration in a semisolid medium, allowing a balance between mutation and regeneration. When increased to 80Gy, the regeneration rate falls to 29%. An immersion system (RITA®) of either 60 or 120 seconds every 8hours allowed a systematic and homogeneous total regeneration of the recalcitrant line, in contrast with the semisolid medium that resulted in positive but irregular regeneration. Other well-known recalcitrant cultivars, CR1821, CR1113 also had an improving regeneration in the immersion system, demonstrating its potential use for recalcitrant materials. To our knowledge, this is the first report on using an immersion system to allow regeneration of gamma-ray mutants from recalcitrant rice materials.

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Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparan sulfates: requirement for optimal binding interactions of heparan sulfate with early-acting cytokines and matrix proteins

Blood ◽

10.1182/blood.v95.1.147.001k28_147_155 ◽

2000 ◽

Vol 95 (1) ◽

pp. 147-155

Author(s):

Pankaj Gupta ◽

Theodore R. Oegema ◽

Joseph J. Brazil ◽

Arkaduisz Z. Dudek ◽

Arne Slungaard ◽

...

Keyword(s):

Heparan Sulfate ◽

Biologically Active ◽

Matrix Proteins ◽

Hematopoietic Progenitor Cell ◽

Heparin Binding ◽

Heparan Sulfates ◽

Kinetics Of ◽

Better Than

We have shown that stromal O-sulfated heparan sulfate glycosaminoglycans (O-S-GAGs) regulate primitive human hematopoietic progenitor cell (HPC) growth and differentiation by colocalizing heparin-binding cytokines and matrix proteins with HPC in stem cell “niches” in the marrow microenvironment. We now show that long-term culture-initiating cells (LTC-IC) are maintained for 5 weeks in the absence of stroma when O-S-GAGs are added to IL-3 and either MIP-1 or PF4 (LTC-IC maintenance without GAGs, 32 ± 2%; with GAGs, 95 ± 7%; P < .001). When cultured with 5 additional cytokines, O-S-GAGs, IL-3, and MIP-1, LTC-IC expanded 2- to 4-fold at 2 weeks, and 92 ± 8% LTC-IC were maintained at 5 weeks. Similar results were seen when PF4 replaced MIP-1. Although O-S-GAG omission did not affect 2-week expansion, only 20% LTC-IC were maintained for 5 weeks. When O-S-heparin was replaced by completely desulfated-, N-sulfated (O-desulfated), or unmodified heparins, LTC-IC maintenance at week 5 was not better than with cytokines alone. Unmodified- and O-S-heparin, but not desulfated- or N-sulfated heparin, bound to MIP-1, IL-3, PF4, VEGF, thrombospondin, and fibronectin. However, the affinity of heparin for thrombospondin and PF4, and the association and dissociation rates of heparin for PF4, were higher than those of O-S-heparin. We conclude that (i) although cytokines may suffice to induce early expansion, adult human LTC-IC maintenance for longer than 1 month requires O-S-GAGs, and (ii) HPC support may depend not only on the ability of GAGs to bind proteins, but also on optimal affinity and kinetics of interactions that affect presentation of proteins in a biologically active manner to progenitors. (Blood. 2000;95:147-155)

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Kriopreservasi Tanaman Obat Langka Purwoceng dengan Teknik Enkapsulasi-Vitrifikasi

Buletin Plasma Nutfah ◽

10.21082/blpn.v14n2.2008.p49-56 ◽

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

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. AbstrakPurwoceng (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.

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Synthesis and Antibacterial Evaluation of Novel 1,3,4-Oxadiazole Derivatives Containing Sulfonate/Carboxylate Moiety

Molecules ◽

10.3390/molecules25071488 ◽

2020 ◽

Vol 25 (7) ◽

pp. 1488

Author(s):

Lei Wang ◽

Xia Zhou ◽

Hui Lu ◽

Xianfu Mu ◽

Linhong Jin

Keyword(s):

Antibacterial Activity ◽

Inhibitory Effect ◽

Sem Analysis ◽

Leaf Blight ◽

Xanthomonas Oryzae ◽

Bacterial Leaf Blight ◽

Xanthomonas Axonopodis ◽

Better Than

In order to discover new lead compounds with high antibacterial activity, a series of new derivatives were designed and synthesized by introducing a sulfonate or carboxylate moiety into the 1,3,4-oxadiazole structure. Antibacterial activity against two phytopathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas axonopodis pv. citri (Xac), was assayed in vitro. The preliminary results indicated that ten compounds including 4a-1-4a-4 and 4a-11-4a-16 had good antibacterial activity against Xoo, with EC50 values ranging from 50.1-112.5 µM, which was better than those of Bismerthiazol (253.5 µM) and Thiodiazole copper (467.4 µM). Meanwhile, 4a-1, 4a-2, 4a-3 and 4a-4 demonstrated good inhibitory effect against Xanthomonas axonopodis pv. citri with EC50 values around 95.8-155.2 µM which were better than those of bismerthiazol (274.3 µM) and thiodiazole copper (406.3 µM). In addition, in vivo protection activity of compound 4a-2 and 4a-3 against rice bacterial leaf blight was 68.6% and 62.3%, respectively, which were better than bismerthiazol (49.6%) and thiodiazole copper (42.2%). Curative activity of compound 4a-2 and 4a-3 against rice bacterial leaf blight was 62.3% and 56.0%, which were better than bismerthiazol (42.9%) and thiodiazole copper (36.1%). Through scanning electron microscopy (SEM) analysis, it was observed that compound 4a-2 caused the cell membrane of Xanthomonas oryzae pv. oryzae ruptured or deformed. The present results indicated novel derivatives of 5-phenyl sulfonate methyl 1,3,4-oxadiazole might be potential antibacterial agents.

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Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparan sulfates: requirement for optimal binding interactions of heparan sulfate with early-acting cytokines and matrix proteins

Blood ◽

10.1182/blood.v95.1.147 ◽

2000 ◽

Vol 95 (1) ◽

pp. 147-155 ◽

Cited By ~ 69

Author(s):

Pankaj Gupta ◽

Theodore R. Oegema ◽

Joseph J. Brazil ◽

Arkaduisz Z. Dudek ◽

Arne Slungaard ◽

...

Keyword(s):

Heparan Sulfate ◽

Biologically Active ◽

Matrix Proteins ◽

Hematopoietic Progenitor Cell ◽

Heparin Binding ◽

Heparan Sulfates ◽

Kinetics Of ◽

Better Than

Abstract We have shown that stromal O-sulfated heparan sulfate glycosaminoglycans (O-S-GAGs) regulate primitive human hematopoietic progenitor cell (HPC) growth and differentiation by colocalizing heparin-binding cytokines and matrix proteins with HPC in stem cell “niches” in the marrow microenvironment. We now show that long-term culture-initiating cells (LTC-IC) are maintained for 5 weeks in the absence of stroma when O-S-GAGs are added to IL-3 and either MIP-1 or PF4 (LTC-IC maintenance without GAGs, 32 ± 2%; with GAGs, 95 ± 7%; P < .001). When cultured with 5 additional cytokines, O-S-GAGs, IL-3, and MIP-1, LTC-IC expanded 2- to 4-fold at 2 weeks, and 92 ± 8% LTC-IC were maintained at 5 weeks. Similar results were seen when PF4 replaced MIP-1. Although O-S-GAG omission did not affect 2-week expansion, only 20% LTC-IC were maintained for 5 weeks. When O-S-heparin was replaced by completely desulfated-, N-sulfated (O-desulfated), or unmodified heparins, LTC-IC maintenance at week 5 was not better than with cytokines alone. Unmodified- and O-S-heparin, but not desulfated- or N-sulfated heparin, bound to MIP-1, IL-3, PF4, VEGF, thrombospondin, and fibronectin. However, the affinity of heparin for thrombospondin and PF4, and the association and dissociation rates of heparin for PF4, were higher than those of O-S-heparin. We conclude that (i) although cytokines may suffice to induce early expansion, adult human LTC-IC maintenance for longer than 1 month requires O-S-GAGs, and (ii) HPC support may depend not only on the ability of GAGs to bind proteins, but also on optimal affinity and kinetics of interactions that affect presentation of proteins in a biologically active manner to progenitors. (Blood. 2000;95:147-155)

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Photoperiod Improves Long-term Survival of In Vitro-stored Strawberry Plantlets

HortScience ◽

10.21273/hortsci.37.5.811 ◽

2002 ◽

Vol 37 (5) ◽

pp. 811-814 ◽

Cited By ~ 5

Author(s):

Barbara M. Reed

Keyword(s):

Cold Storage ◽

Shoot Cultures ◽

Storage Medium ◽

Term Survival ◽

Cold Acclimatization ◽

Long Term Survival ◽

Germplasm Collections ◽

Better Than

Cold storage is important for managing in vitro germplasm collections. Strawberry shoot cultures can typically be held at 4 °C for 9 to 24 months before they require repropagation. Concentration of BA in the storage medium, pre-storage cold acclimatization (CA), and exposure to a photoperiod during storage were studied to determine conditions for improved strawberry culture storage. Fragaria shoot cultures stored at 4 °C were rated for plantlet condition on a 0-5 scale at 9, 12, and 19 months. Four species were CA and stored on medium with 0, 1, 2.5, or 5 μm BA either in darkness or under a 12-hour photoperiod. Mean ratings over all treatments and genotypes were best at 9 and 12 months (3.4) and declined at 19 months (2.2). BA in the storage medium significantly improved ratings for two species at 9 and 12 months, but ratings were not significantly different at 19 months. At 19 months of storage, shoot cultures stored with a photoperiod were rated significantly better (P ≤ 0.05) than those grown in darkness. Five Fragaria genotypes stored on medium without BA were used to study the effect of photoperiod and CA on ratings of stored plantlets. CA-shoot cultures stored for 9 or 12 months were rated significantly better than non-CA cultures. After 12 and 19 months storage, three of the five genotypes stored under a 12-hour photoperiod had significantly higher ratings than those stored in the dark (P ≤ 0.01), but by 19 months CA was nonsignificant. Overall, the addition of a photoperiod improved the condition of Fragaria shoot cultures stored at 4 °C. Chemical name used: N6-benzyladenine (BA).

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