scholarly journals Isolation and culture of leaf protoplasts from Tunisian grapes

OENO One ◽  
2003 ◽  
Vol 37 (3) ◽  
pp. 145 ◽  
Author(s):  
Ahmed Mliki ◽  
Rahma Jardak ◽  
Götz M. Reustle ◽  
Abdelwahed Ghorbel
Keyword(s):  
Cell Division ◽  
Plant Regeneration ◽  
Aspergillus Niger ◽  
Shoot Culture ◽  
Protoplast Isolation ◽  
Penicillium Funiculosum ◽  
Experimental Conditions ◽  
Grape Varieties ◽  
Leaf Protoplasts

<p style="text-align: justify;">Experimental conditions for leaf protoplast isolation and culture were optimised for <em>in vitro</em> plants deriving from shoot culture of two Tunisian grape varieties, Sakasly and Muscat d’Alexandrie (<em>Vitis vinifera</em> L.). The best yields were obtained from leaves of 4 to 5 weeks old <em>in vitro</em> plants, digested for 13 hours under 25 rpm agitation with an enzymatic mixture containing 0.25 % cellulase of <em>Aspergillus niger</em>, 0.25 % cellulase of <em>Penicillium funiculosum</em>, 0.5 % cellulysin of <em>Trichoderma viridae</em>, and 0.2 % macerozyme R-10 of <em>Rhizopus</em> sp. More than 50 % of the purified protoplasts had a diameter of 30-40 μm and were rich in chloroplasts. Best aptitude for cell division was found in protoplasts immobilised in sodium alginate layers at a density of 0.5x10<sup>6</sup> cell/ml, cultivated in CPW-13 medium containing 4 mg/l of NOA and 0.88 mg/l of TDZ. The variety Muscat d’Alexandrie gave better yield whereas Sakasly showed better cell division rates. Formation of micro and macrocallus have been obtained, but the oxidation of the medium has to be solved in order to promote plant regeneration.</p>

Plant regeneration from in vitro -cultured seedling leaf protoplasts of Actinidia eriantha Benth

1998 ◽  
Vol 17 (10) ◽  
pp. 819-821 ◽  
Author(s):  
Y.-J. Zhang ◽  
Y.-Q. Qian ◽  
X.-J. Mu ◽  
Q.-G. Cai ◽  
Y.-L. Zhou ◽  
...  
Keyword(s):  
Plant Regeneration ◽  
Seedling Leaf ◽  
Leaf Protoplasts

scholarly journals Plant regeneration from protoplast of Brazilian citrus cultivars

2000 ◽  
Vol 35 (4) ◽  
pp. 727-732 ◽  
Author(s):  
FERNANDA JANUZZI MENDES DA GLORIA ◽  
FRANCISCO DE ASSIS ALVES MOURÃO FILHO ◽  
BEATRIZ MADALENA JANUZZI MENDES
Keyword(s):  
Plant Regeneration ◽  
Adventitious Shoots ◽  
Cell Suspension Cultures ◽  
Protoplast Isolation ◽  
Direct Organogenesis ◽  
Malt Extract ◽  
Embryogenic Cell Suspension ◽  
Embryogenic Cell ◽  
Leaf Mesophyll

A procedure is described to regenerate plants from protoplasts of Brazilian citrus cultivars, after isolation, fusion and culture. Protoplasts were isolated from embryogenic cell suspension cultures and from leaf mesophyll of seedlings germinated in vitro. The enzyme solution for protoplast isolation was composed of mannitol (0.7 M), CaCl2 (24.5 mM), NaH2PO4 (0.92 mM), MES (6.15 mM), cellulase (Onozuka RS - Yakult, 1%), macerase (Onozuka R10 - Yakult, 1%) and pectolyase Y-23 (Seishin, 0.2%). Protoplast culture in liquid medium after chemical fusion lead to the formation of callus colonies further adapted to solid medium. Somatic embryo formation occurred spontaneously after two subcultures, on modified MT medium supplemented with 500 mg/L of malt extract. Well defined embryos were germinated in modified MT medium with addition of GA3 (2.0 muM) and malt extract (500 mg/L). Plant regeneration was also achieved by adventitious shoots obtained through direct organogenesis of not well defined embryos in modified MT medium with addition of malt extract (500 mg/L), BAP (1.32 muM), NAA (1.07 muM) and coconut water (10 mL/L). Plantlets were transferred to root medium. Rooted plants were transferred to a greenhouse for further adaptation and development.

scholarly journals Assembly Dynamics of FtsZ Rings in Bacillus subtilis and Escherichia coli and Effects of FtsZ-Regulating Proteins

2004 ◽  
Vol 186 (17) ◽  
pp. 5775-5781 ◽  
Author(s):  
David E. Anderson ◽  
Frederico J. Gueiros-Filho ◽  
Harold P. Erickson
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Fluorescence Recovery ◽  
Half Time ◽  
Experimental Conditions ◽  
Z Ring

ABSTRACT FtsZ is the major cytoskeletal component of the bacterial cell division machinery. It forms a ring-shaped structure (the Z ring) that constricts as the bacterium divides. Previous in vivo experiments with green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching have shown that the Escherichia coli Z ring is extremely dynamic, continually remodeling itself with a half time of 30 s, similar to microtubules in the mitotic spindle. In the present work, under different experimental conditions, we have found that the half time for fluorescence recovery of E. coli Z rings is even shorter (∼9 s). As before, the turnover appears to be coupled to GTP hydrolysis, since the mutant FtsZ84 protein, with reduced GTPase in vitro, showed an ∼3-fold longer half time. We have also extended the studies to Bacillus subtilis and found that this species exhibits equally rapid dynamics of the Z ring (half time, ∼8 s). Interestingly, null mutations of the FtsZ-regulating proteins ZapA, EzrA, and MinCD had only modest effects on the assembly dynamics. This suggests that these proteins do not directly regulate FtsZ subunit exchange in and out of polymers. In B. subtilis, only 30 to 35% of the FtsZ protein was in the Z ring, from which we conclude that a Z ring only 2 or 3 protofilaments thick can function for cell division.

Cotyledon-derived diploid and haploid protoplast culture and diploid plant regeneration in Brassica napus cv. ' Topas '

1998 ◽  
Vol 76 (3) ◽  
pp. 530-541
Author(s):  
M Sun ◽  
H Kieft ◽  
AAM van Lammeren
Keyword(s):  
Cell Division ◽  
Brassica Napus ◽  
Plant Regeneration ◽  
Protoplast Culture ◽  
Shoot Formation ◽  
Protoplast Isolation ◽  
Brassica Napus L ◽  
Successful Isolation ◽  
Young Leaves ◽  
Haploid Embryos

The present paper describes a simple and reliable protocol for the successful isolation, purification, culture, and regeneration of diploid cotyledon-derived protoplasts of Brassica napus L. cv. 'Topas'. Various protoplast isolation media, nutrient media, subculture procedures, and protoplast sources were tested under two culture temperatures. Protoplast viability, cell wall regeneration, and cell division were monitored. Single cotyledon-derived protoplasts formed calli in liquid protoplast medium, and when these were subcultured on solid proliferation medium and solid regeneration medium of appropriate composition, plants regenerated either by shoot formation or embryogenesis. Continuous culture at 32°C instead of 25°C favoured the initiation of cell division and cell proliferation but prevented regeneration, although calli maintained regeneration capacity. Viable haploid protoplasts were isolated from cotyledons of heat-shock-induced, microspore-derived haploid embryos and from young leaves of secondary embryos that were formed on microspore-derived embryos. Cell divisions were triggered in the two types of haploid protoplast cultures, and microcalli were formed at high frequencies. Differences between haploid and diploid protoplast cultures are discussed.Key words: cotyledon protoplast culture, haploid culture, plant regeneration.

scholarly journals SOME FACTORS AFFECTING ISOLATION OF MESOPHYLL PROTOPLASTS FROM APPLE (MALUS DOMESTICA BORKH CV. FUJI)

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 693g-694
Author(s):  
Chang-Hoo Lee ◽  
N. I. Hyung ◽  
S. E. Kim
Keyword(s):  
Cell Division ◽  
Culture Media ◽  
Shoot Culture ◽  
Mesophyll Protoplast ◽  
Protoplast Isolation ◽  
Enzyme Treatment ◽  
Planting Density ◽  
Factors Affecting ◽  
Cell Structures ◽  
Viable Protoplast

Experiments were conducted to investigate the factors influencing mesophyll protoplast isolation in `Fuji' apple. Half an hour pretreatment in 0.6M mannitol gave the highest protoplast yield.The enzyme solution containing 2% Cellulase Onozuka R-10 and 0.5% macrozyme R-10 with CPW 0.6M mannitol at pH 5.5 was most effective for protoplast isolation from leaf. Effective incubation time for the enzyme treatment was found to be 15-20 hrs at 25°C in the dark. Use of 1.0-2.0% PVP and 0.5mM MES was essential for higher yield and viability of protoplast. Supplementation of BA and IBA to the shoot culture media gave the higher yield of viable protoplast. From these protoplast, new cell walls were regenerated and 4 cell structures developed from one protoplast by cell division in K8P medium supplemented with 3A and NAA. Planting density higher than 10 protoplasts/ml was required for cell division from protoplast in liquid or 0.5% agarose culture.

scholarly journals Effective callus induction and plant regeneration in callus and protoplast cultures of Nigella damascena L.

2020 ◽  
Vol 143 (3) ◽  
pp. 693-707
Author(s):  
Magdalena Klimek-Chodacka ◽  
Dariusz Kadluczka ◽  
Aneta Lukasiewicz ◽  
Aneta Malec-Pala ◽  
Rafal Baranski ◽  
...  
Keyword(s):  
Plant Regeneration ◽  
Callus Induction ◽  
Somatic Embryos ◽  
High Capacity ◽  
Protoplast Isolation ◽  
Callus Cultures ◽  
Naphthaleneacetic Acid ◽  
Regeneration System ◽  
Nigella Damascena

AbstractIn this study we report the development of effective in vitro systems for a medicinal plant Nigella damascena L. comprising: (1) callus induction, (2) somatic embryogenesis in callus cultures with subsequent plant regeneration, and (3) isolation and regeneration of callus-derived protoplasts. Callus development was achieved on 83–100% of hypocotyl and cotyledon explants, whereby Murashige and Skoog medium (MS) supplemented with 3 mg L−1 6-benzylaminopurine and 0.5 mg L−1α-naphthaleneacetic acid (NAA; BN medium) was more advantageous than MS with kinetin and NAA (KN medium). Histological observations of calli revealed the presence of embryogenic zones from which somatic embryos developed on the hormone-free medium. Plant regeneration was observed on 76–95% of calli. A high capacity to form somatic embryos and regeneration was maintained in long-lasting cultures, i.e. even in 2 year old callus.The obtained callus was also a good source tissue for protoplast isolation. By applying a mixture of cellulase and pectolyase, the acceptable yield of viable protoplasts was achieved, especially from hypocotyl-derived callus maintained on BN medium. Protoplasts embedded in an alginate matrix and cultured in modified Kao and Michayluk media re-constructed their cell wall and re-entered mitotic divisions. About 30% of small cell aggregates formed microcalli, which, after the release from alginate, proliferated continuously on KN and BN media, irrespective of the tissue variant used as the protoplast source. Somatic embryo formation and plant regeneration were successful on hormone-free media. An effective plant regeneration system of N. damascena protoplast cultures has been developed and is being reported for the first time.

Observations of Frozen-Hydrated Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 504-505
Author(s):  
D. Chrétien ◽  
D. Job ◽  
R.H. Wade
Keyword(s):  
Fourier Transforms ◽  
Structural Complexity ◽  
Image Contrast ◽  
Phase Behaviour ◽  
Experimental Conditions ◽  
Thin Sections ◽  
Surface Lattice ◽  
Cilia And Flagella ◽  
Outstanding Question

Microtubules are filamentary structures found in the cytoplasm of eukaryotic cells, where, together with actin and intermediate filaments, they form the components of the cytoskeleton. They have many functions and show various levels of structural complexity as witnessed by the singlet, doublet and triplet structures involved in the architecture of centrioles, basal bodies, cilia and flagella. The accepted microtubule model consists of a 25 nm diameter hollow tube with a wall made up of 13 paraxial protofilaments (pf). Each pf is a string of aligned tubulin dimers. Some results have suggested that the pfs follow a superhelix. To understand how microtubules function in the cell an accurate model of the surface lattice is one of the requirements. For example the 9x2 architecture of the axoneme will depend on the organisation of its component microtubules. We should also note that microtubules with different numbers of pfs have been observed in thin sections of cellular and of in-vitro material. An outstanding question is how does the surface lattice adjust to these different pf numbers?We have been using cryo-electron microscopy of frozen-hydrated samples to study in-vitro assembled microtubules. The experimental conditions are described in detail in this reference. The results obtained in conjunction with thin sections of similar specimens and with axoneme outer doublet fragments have already allowed us to characterise the image contrast of 13, 14 and 15 pf microtubules on the basis of the measured image widths, of the the image contrast symmetry and of the amplitude and phase behaviour along the equator in the computed Fourier transforms. The contrast variations along individual microtubule images can be interpreted in terms of the geometry of the microtubule surface lattice. We can extend these results and make some reasonable predictions about the probable surface lattices in the case of other pf numbers, see Table 1. Figure 1 shows observed images with which these predictions can be compared.

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