COLD HARDENING VS ABA AS A PRETREATMENT FOR MERISTEM CRYOPRESERVATION

Cold hardening is an effective method for conditioning meristems for cryopreservation. ABA plays a role in hardening and produces increased hardiness in suspension cultured cells. This study was designed to determine if growth, in vitro, on ABA (5×10-5 M) for one week, would substitute for one week of cold hardening, and if ABA would provide additional conditioning when added in combination with cold hardening treatments. In vitro plantlets of Rubus spp. were grown for one week with or without cold hardening and with or without ABA. Meristems from these plants were frozen at 0.8C* min-1 to -35 C, then plunged into LN2, thawed, and plated on recovery medium. One month after thawing, cold-hardened plants with and without ABA treatment had recovery rates of up to 83%. Survival of plants grown at room temperature ranged from zero to 8% and zero to 28% for plants grown on ABA at room temperature. At the rates tested, ABA is less effective than cold hardening in conditioning apical meristems of in vitro Rubus plants for cryopreservation and provides no additional protection to cold-hardened meristems.

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Cryopreservation of Limonium serotinum apical meristems from in vitro plantlets using droplet-vitrification

Scientia Horticulturae ◽

10.1016/j.scienta.2011.07.001 ◽

2011 ◽

Author(s):

Giuseppe Barraco ◽

Isabelle Sylvestre ◽

Giovanni Iapichino ◽

Florent Engelmann

Keyword(s):

Apical Meristems ◽

Droplet Vitrification ◽

In Vitro Plantlets

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Factors Affecting the In Vitro Storage of Strawberry

HortScience ◽

10.21273/hortsci.30.4.871b ◽

1995 ◽

Vol 30 (4) ◽

pp. 871B-871 ◽

Cited By ~ 2

Author(s):

Barbara M. Reed

Keyword(s):

Cold Hardening ◽

Factors Affecting ◽

Plant Condition ◽

In Vitro Storage ◽

In Vitro Plantlets ◽

And Storage

Fragaria germplasm, stored at 4C as cold-hardened in vitro plantlets, was rated for condition on a 0–5 scale at 9, 12, and 19 months. Plants with ratings ≥2 were healthy enough to remain in storage. Benzyladenine (BA) at 0, 1, 2.5, and 5 μM and storage in darkness or with a 12-h photoperiod were examined for a group of four genotypes. Means of plant condition ratings over all treatments and genotypes were best (3.4) at 9 and 12 months and declined to 2.2 at 19 months. Increasing concentrations of BA were correlated with higher condition ratings compared to those without BA, and plants stored with a 12-h photoperiod had higher ratings than those stored in the dark. At 9 and 12 months, plants grown with BA and a 12-h photoperiod had the highest ratings. Five different Fragaria genotypes were used to study the effect of photoperiod and cold-hardening on condition ratings of stored plantlets. Cold-hardened plants had higher condition ratings (2.1) than those stored without cold-hardening (1.8). Plants stored with a 12-h photoperiod had higher mean condition ratings (2.2) than those stored in the dark (1.7). Storage with cold-hardening and a 12-h photoperiod resulted in improved plant condition. The extent of improvements was genotype-dependent.

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Expression Pattern of Genes Encoding Farnesyl Diphosphate Synthase and Sesquiterpene Cyclase in Cotton Suspension-Cultured Cells Treated with Fungal Elicitors

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1999.12.12.1095 ◽

1999 ◽

Vol 12 (12) ◽

pp. 1095-1104 ◽

Cited By ~ 39

Author(s):

Chang-Jun Liu ◽

Peter Heinstein ◽

Xiao-Ya Chen

Keyword(s):

Cultured Cells ◽

De Novo ◽

Constitutive Expression ◽

Farnesyl Diphosphate ◽

Farnesyl Diphosphate Synthase ◽

Phytopathogenic Fungus ◽

Suspension Cultured Cells ◽

Genes Encoding ◽

Sesquiterpene Cyclase

Cotton plants accumulate sesquiterpene aldehydes in pigment glands. The two enzymes farnesyl diphosphate synthase (FPS) and (+)-δ-cadinene synthase (CAD), a sesquiterpene cyclase, are involved in the biosynthesis of these secondary metabolites. A full-length cDNA (garfps) encoding FPS was isolated from Gossypium arboreum and identified by in vitro enzymatic assay of the garfps protein heterologously expressed in Escherichia coli. Treatment of G. arboreum suspension-cultured cells with an elicitor preparation obtained from the phytopathogenic fungus Verticillium dahliae dramatically induced transcription of both FPS and CAD, paralleling the accumulation of the sesquiterpene aldehydes in these cells. For G. australe, a wild species from Australia, the V. dahliae elicitor preparation also caused an induction of FPS but only a low rate of induction of CAD, apparently because of a constitutive expression of the sesquiterpene cyclase gene in suspension-cultured cells. Two transcripts and proteins of FPS were detected in the elicited G. australe cells; the smaller FPS seemed to be de novo synthesized after elicitation. Furthermore, G. australe-cultured cells accumulated the cadinene, instead of sesquiterpene aldehydes, indicating that the biosynthetic pathway leading to sesquiterpene aldehydes was absent or blocked after FPP cyclization.

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Survival of in Vitro-grown Apical Meristems of Pyrus Following Cryopreservation

HortScience ◽

10.21273/hortsci.25.1.111 ◽

1990 ◽

Vol 25 (1) ◽

pp. 111-113 ◽

Cited By ~ 40

Author(s):

Barbara M. Reed

Keyword(s):

Polyethylene Glycol ◽

Liquid Nitrogen ◽

Survival Rates ◽

Geographic Origin ◽

Pyrus Communis ◽

Cold Hardening ◽

Cooling Rates ◽

Apical Meristems ◽

Pyrus Communis L

Apical meristems of four pears (Pyrus communis L. cv. Beurre Hardy, P. koehnei Schneider, P. cossonii Coss. and Dur., and a hybrid, P. dimorphophylla Makino × P. fauriei Schneider) were tested for their ability to survive immersion in liquid nitrogen. Plantlets were grown in vitro at 25C or cold-hardened for 1 week at – 1C before cooling at rates of 0.1, 0.3, 0.5, and 0.8 C/rein to –40C, followed by plunging the vials into liquid nitrogen. Vials were thawed for 1 min at 40C. A cryoprotectant mixture of polyethylene glycol, glucose, and dimethylsulfoxide (DMSO) was used. Regrowth of meristems ranged from 0% to 61% for plants grown at 25C and from 5% to 95% for cold-hardened plants. Cold-hardening significantly improved the recovery rates of all species tested. Survival rates increased as cooling rates decreased. Survival rates were not linked to the geographic origin of the species tested.

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In vitro induction of the formation of tracheary elements from suspension-cultured cells of the conifer Cryptomeria japonica

Trees ◽

10.1007/s00468-014-1139-2 ◽

2015 ◽

Vol 29 (4) ◽

pp. 1283-1289 ◽

Cited By ~ 5

Author(s):

Yusuke Yamagishi ◽

Hiromu Uchiyama ◽

Takenao Sato ◽

Kei Kitamura ◽

Joto Yoshimoto ◽

...

Keyword(s):

Cryptomeria Japonica ◽

Cultured Cells ◽

Tracheary Elements ◽

Suspension Cultured Cells ◽

In Vitro Induction

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Differential patterns of arabinosylation by membranes of suspension-cultured cells of Phaseolus vulgaris (French bean) after subculture or elicitation

Biochemical Journal ◽

10.1042/bj2220427 ◽

1984 ◽

Vol 222 (2) ◽

pp. 427-435 ◽

Cited By ~ 20

Author(s):

G P Bolwell

Keyword(s):

Phaseolus Vulgaris ◽

Cultured Cells ◽

Initial Period ◽

French Bean ◽

Colletotrichum Lindemuthianum ◽

Fungal Elicitor ◽

Arabinogalactan Protein ◽

Suspension Cultured Cells

Suspension-cultured cells of Phaseolus vulgaris (French bean) incorporated [1-3H] arabinose in vivo into high-Mr polymers that could be separated into glycoprotein and polysaccharide. Microsomal membranes from suspension-cultured cells of beans incorporated arabinose from UDP-beta-L-arabinose in vitro into both polysaccharide and glycoprotein. The enzyme involved in arabinan synthesis, arabinan synthase, appeared to be immunologically distinct from the protein:arabinosyltransferase system. Both these activities are inducible, but behave differently with either plant-growth-regulator or fungal-elicitor treatments. After subculture of cells entering the stationary growth phase the arabinan synthase activity reaches much higher values than does that of the protein transferase system during the initial period of cell division and growth, whereas after elicitation at the same growth stage, all the increased incorporation of arabinose occurs into glycoprotein of Mr higher than 200 000 and to a greater extent into a specific glycoprotein of Mr 42 500. Preliminary characterization of these glycoproteins prepared under non-reducing conditions and after acid and alkaline hydrolysis suggests that the high-Mr glycoprotein material is similar to arabinogalactan protein, whereas the lower-Mr material may be a hydroxyproline-rich protein existing as a dimer and that specifically increases during the hypersensitive response of the cells to the fungal elicitor from Colletotrichum lindemuthianum.

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Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100053711 ◽

1982 ◽

Vol 40 ◽

pp. 210-211

Author(s):

M.J. Murphy ◽

R.R. Price ◽

J.C. Sloman

Keyword(s):

Cultured Cells ◽

Human Tumor ◽

Cell Type ◽

Tumor Mass ◽

Initial Cell ◽

Cloning Assay ◽

Human Tumor Cloning ◽

The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

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In Situ Localization of PPAR Gamma and Uncoupling Protein in Mouse Embryo Sections Using Digoxigenin-Labeled Riboprobes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162624 ◽

1996 ◽

Vol 54 ◽

pp. 32-33

Author(s):

C. Jennermann ◽

S. A. Kliewer ◽

D. C. Morris

Keyword(s):

Alkaline Phosphatase ◽

Room Temperature ◽

Uncoupling Protein ◽

Ppar Gamma ◽

Nuclear Hormone Receptor ◽

Full Length ◽

Northern Analysis ◽

Peroxisome Proliferator

Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor superfamily and has been shown in vitro to regulate genes involved in lipid metabolism and adipocyte differentiation. By Northern analysis, we and other researchers have shown that expression of this receptor predominates in adipose tissue in adult mice, and appears first in whole-embryo mRNA at 13.5 days postconception. In situ hybridization was used to find out in which developing tissues PPARg is specifically expressed.Digoxigenin-labeled riboprobes were generated using the Genius™ 4 RNA Labeling Kit from Boehringer Mannheim. Full length PPAR gamma, obtained by PCR from mouse liver cDNA, was inserted into pBluescript SK and used as template for the transcription reaction. Probes of average size 200 base pairs were made by partial alkaline hydrolysis of the full length transcripts. The in situ hybridization assays were performed as described previously with some modifications. Frozen sections (10 μm thick) of day 18 mouse embryos were cut, fixed with 4% paraformaldehyde and acetylated with 0.25% acetic anhydride in 1.0M triethanolamine buffer. The sections were incubated for 2 hours at room temperature in pre-hybridization buffer, and were then hybridized with a probe concentration of 200μg per ml at 70° C, overnight in a humidified chamber. Following stringent washes in SSC buffers, the immunological detection steps were performed at room temperature. The alkaline phosphatase labeled, anti-digoxigenin antibody and detection buffers were purchased from Boehringer Mannheim. The sections were treated with a blocking buffer for one hour and incubated with antibody solution at a 1:5000 dilution for 2 hours, both at room temperature. Colored precipitate was formed by exposure to the alkaline phosphatase substrate nitrobluetetrazoliumchloride/ bromo-chloroindlylphosphate.

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Preparation of cultured astrocytes for x-ray microanalysis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156924 ◽

1989 ◽

Vol 47 ◽

pp. 988-989

Author(s):

N.K.R. Smith ◽

K.E. Hunter ◽

P. Mobley ◽

L.P. Felpel

Keyword(s):

Cultured Cells ◽

Elemental Content ◽

Elemental Distribution ◽

Sprague Dawley ◽

X Ray ◽

Cultured Astrocytes ◽

Freeze Dried ◽

Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

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