Application of electric field fusion in plant tissue culture

Zachrisson, Anders; Bornman, Chris H.

doi:10.1111/j.1399-3054.1984.tb06333.x

Cited by 46 publications

(3 citation statements)

References 23 publications

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“…Although he observed microscopically visible loss of cytoplasm at some frequencies and voltages, he concluded that there was no loss of viability, based on observation of subsequent cell division. Zachrisson and Bornman (21) observed destruction of Brassica protoplasts subjected to a field of 40 V/cm at all sine wave frequencies below 500 kHz. Our investigation showed significant decreases in cell viability at very low and very high AC frequencies.…”

Section: Response Of Protoplasts To Electrofusion Parameters Conditiomentioning

confidence: 99%

Behavior and Viability of Tobacco Protoplasts in Response to Electrofusion Parameters

Saunders

Roskos²,

Mischke³

et al. 1986

Plant Physiol.

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This investigation examines responses of protoplasts in a systematic and quantitative way to the various electrical treatments used to achieve electrofusion and their individual and cumulative effect on protoplast viability. Mesophyll and cell suspension protoplasts from two species of the same genera, Nicotiana tabacum and N. rustica var brasilia were used in these experiments. Optimal frequencies for alignment of tobacco protoplasts were between 500 kilohertz and 2 megahertz at 100 volts per (22,24). For plant cells, typical procedures involve three steps; alignment of protoplasts with an AC field, disruption of contiguous membranes with a brief DC pulse, and fusion of the protoplasts (1,15,22,24). Much of the emphasis in previous research has been directed toward the modification of the DC pulse to achieve maximum fusion rates while little data has been presented on the effects of both the AC fields and the DC pulses on cell viability. Indeed, only recently have published reports appeared on the successful regeneration of shoots from hybrids of electrically fused protoplasts (2, 4). We report here the effects of AC and DC currents, separately and in combinaton, on the viability and integrity of protoplasts and on the movement of protoplasts.MATERIALS AND METHODS Plant Material. Nicotiana rustica var brasilia and N. tabacum cv MD 872 were grown under greenhouse conditions and harvested 70 to 90 d after germination. One week prior to harvest the plants were transferred to an environmental growth chamber that maintained a 12 h day length at 23°C. Shoots and cell suspensions of N. tabacum cv Connecticut 'John Williams Broadleaf' were cultured in vitro as described by Owens (11) and Uchimiya and Murashige (19), respectively.Preparation of Protoplasts. Protoplasts from greenhouse grown plants were isolated from mature mesophyll leaf tissue with 1% (w/v) Macerase3 (300 units/g) and 2% (w/v) Cellulysin (10,000 units/g) in 50 mm Mes-NaOH buffer (pH 5.7) with 0.6 M mannitol as described previously (16). Protoplasts were isolated from tissue culture by an adaptation of the methods of Owens (10) and Lin (6). One g of suspension cells in 40 ml of 0.15% Cellulysin, 1% rhozyme HP-150, and 0.05% Driselase was incubated for 4 h at 30°C with shaking at 50 rpm. All enzymes were desalted and dissolved in a solution of 0.7 M mannitol, 9.25 mM CaCl2, and 0.44 mm Ca(H2PO4)2 adjusted to pH 5.7. Protoplasts were harvested by centrifugation (100g for 3 min) and resuspended in 4 ml of 16% (w/v) Ficoll in 0.7 M mannitol. Four ml each of 14% (w/v) and 12% (w/v) Ficoll in 0.7 mannitol 3 Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the United States Department of Agriculture, and does not. imply its approval to the exclusion of other products or vendors that may also be suitable.

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Section: Response Of Protoplasts To Electrofusion Parameters Conditiomentioning

confidence: 99%

Behavior and Viability of Tobacco Protoplasts in Response to Electrofusion Parameters

Saunders

Roskos²,

Mischke³

et al. 1986

Plant Physiol.

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“…A number of laboratories are engaged in one or more of the following aspects: (1) anther culture to produce haploids and establish homozygous lines (HOFFMANN et al 1982); (2) creation of new genetic variation by sexual (SNELL 1977;DOLSTRA 1982) or somatic (SCHENK and ROBBELEN 1982) resynthesis; (3) release of potentially useful somaclonal variation (LARKIN and SCOWCROFT 1981) by regenerating plants from protoplasts (GLIMELIUS 1984), cells or callus; and (4) development of the methodology for the production of hybrids (PELLETIER et al 1983). However, it would not be incorrect to assert that most of the efforts in the Nordic countries are currently directed towards anther culture and the mechanics of protoplast isolation, culture, regeneration and, recently, also fusion (ZACHRISSON and BORNMAN 1984).…”

Section: Brassicaceaementioning

confidence: 99%

Regeneration in vitro of economically important crop plants in the Nordic countries

Bornman

2008

Hereditas

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Economically important crop plants comprise agronomic, horticultural and silvicultural species, of which there are numerous examples in the Nordic countries. This review only considers some species representative of the Brassicaceae. Chenopodiaceae. Poaceae and Pinaceae. By regeneration in vitro is meant the reproducible regenesis of plants from their component parts, rather than mass micropropagation which for most crop plants is as yet either irrelevant or unpractical. As regards agronomic crops, the state of application of cell and tissue culture techniques has progressed farthest with oilseed rape (Brassico napus): with allowance made for genotypic effects, regeneration from protoplasts and anther-cultured microspores has almost become routine. Again, allowing for genotypic differences, haploidization of sugar beet (Beta vulgaris) via ovule culture and regeneration of plants with the aid of disarmed, shooty-mutant plasmids of Agrobacterium tumefaciens, often without intervening tumorigenesis, has been shown to be possible. Regeneration in vitro of both Picea abies and Pinus sylvestris can be accomplished, but only when embryonic or seedling tissues are used. The problem of rejuvenation of mature tissues to allow rooting of the adventitious buds that are inducible, within limits, is still unsolved. As elsewhere, lack of understanding or control of factors such as genotype, physiological history and pretreatment of donor plants, and optimal physical (light) and chemical (medium) conditions, still hampers optimization of techniques in the Nordic countries.

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“…Electrofusion will minimally yield comparable results to chemical fusions, at least in systems with robust protoplasts in good chemical fusion protocols [5], and will do better with fragile protoplasts [6]. In addition, the most valuable aspects of electrofusion techniques are the high fusion frequencies attained, often tenfold higher than analogous chemical systems [7]. The concept of cell-cell electrofusion was first reported by Senda et al [8] in 1979, and pioneering research in electrofusion was carried out by Zimmermann and his co-workers [9,10], who demonstrated the electrofusion of plant protoplasts from Vicia faba mesophyll cells in large numbers in a l mL fusion chamber with parallel electrodes.…”

Section: Introductionmentioning

confidence: 99%

An electrofusion chip with a cell delivery system driven by surface tension

Ju¹,

Ko²,

Cheol³

et al. 2008

J. Micromech. Microeng.

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We have fabricated an electric cell fusion chip with an embedded cell delivery function driven by surface tension and evaluated its performance with several types of plant cells. The chip consists of a polydimethylsiloxane-based microchannel with a fusion chamber and gold-titanium (Au-Ti) electrodes. The velocity profiles of the microfluid in the channel and fusion chamber were calculated to predict cell movement, and the electric field distribution between the electrodes was also calculated in order to determine the appropriate electrode shape. The range of the fluid velocity in the fusion chamber is 20-50 μm s −1 and the measured speed of the cells is approximately 45 μm s −1 , which is sufficiently slow for the motion of the cells in the fusion chamber to be monitored and controlled. We measured the variation of the pearl chain ratio with frequency for five kinds of plant cells, and determined that the optimal frequency for pearl chain formation is 1.5 MHz. The electrofusion of cells was successfully carried out under ac field (amplitude: 0.4-0.5 kV cm −1 , frequency: 1.5 MHz) and dc pulse (amplitude: 1.0 kV cm −1 , duration: 20 ms) conditions. M This article features online multimedia enhancements S This article has associated online supplementary data files

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Application of electric field fusion in plant tissue culture

Cited by 46 publications

References 23 publications

Behavior and Viability of Tobacco Protoplasts in Response to Electrofusion Parameters

Behavior and Viability of Tobacco Protoplasts in Response to Electrofusion Parameters

Regeneration in vitro of economically important crop plants in the Nordic countries

An electrofusion chip with a cell delivery system driven by surface tension

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