A general method for the high-yield isolation of mesophyll protoplasts from deciduous tree species

Butt, Adrian D.

doi:10.1016/0168-9452(85)90028-7

Cited by 26 publications

(17 citation statements)

References 7 publications

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“…The release of compounds that inhibit enzymatic cell wall digestion and consequent poor protoplast release from chopped explants (leaves etc.) has been reported (Butt 1985). In our experiments with the digestive enzymes used, chopped explants yielded as many protoplasts as leaves with epidermal layers peeled off would do.…”

Section: Discussionmentioning

confidence: 57%

A simple method for the isolation of plant protoplasts

Rao

Prakash

1995

J. Biosci.

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A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described• The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplastreleasing enzymes that are stable and efficient at higher temperatures• Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO 4), and a protectant (CaCl 2 2H 2 O), all dissolved in distilled water• The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division• Plant regeneration was demonstrated for Nicotiana tabacum cv• Thompson from protoplasts isolated by this method• Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.

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Section: Discussionmentioning

confidence: 57%

A simple method for the isolation of plant protoplasts

Rao

Prakash

1995

J. Biosci.

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“…Some clues about the nature of this phenomenon were provided by Butt [ 22 ], who reported that thoroughly washing chopped leaf material in water prior to enzymatic digestion significantly increased protoplast yields in four woody plant species. Further, when the washed leaves were incubated in their own wash water, the tissues regain their resistance to enzymatic digestion.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana)

et al. 2012

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BackgroundProtoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division.ResultsThis study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27) in controls to 65.3% (±4.60). Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose.ConclusionsThis study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived electrofusion and viable heterokaryons were produced. Together, these results provide the first evidence of sustained cell division, callus regeneration, and potential application of somatic cell fusion in American elm, suggesting that this source of protoplasts may be ideal for genetic manipulation of this species. The technological advance made with American elm in this study has potential implications in other woody species for fundamental and applied research which require availability of viable protoplasts.

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“…It seems that the choice of a suitable basal culture medium was the most crucial factor in the development of a protoplast culture method for willow, and that the variations in the quality and the concentration of sugars and plant growth regulators were of minor importance, even if these latter modifications could improve the recovery of calli from the protoplasts. Butt (1985) discussed the need for washing the starting material to remove leachate that inhibits the cell wall degrading enzymes when isolating protoplasts from leaves of deciduous trees. Our procedure of cutting, incubating and rinsing the shoot material in W5 wash solution did not improve protoplast yields from in vitro-grown shoots.…”

Section: Discussionmentioning

confidence: 99%

“…The large number of dying cells may have had a negative effect on the surviving colonies, but did not inhibit their growth completely. Butt (1985) isolated viable protoplasts from shoots of S. caprea and S. alba, but did not discuss the culture of these protoplasts. Our attempts to culture mesophyll protoplasts failed, even in the clone (78-0-90) that could be regenerated to callus via cell suspension protoplasts.…”

Section: Discussionmentioning

confidence: 99%

“…Isolation and culture of protoplasts of angiosperm forest trees are difficult, but progress has been made during the last years. Thus, viable protoplasts have been isolated from several species, like Populus (Butt 1985;Russell & McCown 1986a), Ulmus (Redenbauch et al 1980(Redenbauch et al , 1981Sticklen et al 1985;Butt 1985), Alnus (Huhtinen et al 1982(Huhtinen et al /1983Tremblay et al 1985;Butt 1985), Sorbus (J6rgensen & Binding 1984), Fagus (Lang & Kohlenbach 1988), Betula (Butt 1985;Tremblay 1988) and Salix (Butt 1985). Nevertheless, plant regeneration has only been reported for Populus (Russell & McCown 1986b, 1988.…”

Section: Introductionmentioning

confidence: 99%

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Callus production from willow (Salix viminalis L.) protoplasts

Vahala

Eriksson

1991

Plant Cell Tiss Organ Cult

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Protoplasts were isolated from cell suspensions of Sal& viminal& basket willow) clone 78-0-90 and S. schwerinii clone 77-0-77, using cellulysin and macerase in modified Woody Plant medium. For clone 78-0-90, 6.3.106 -+ 1.9.106 protoplasts were obtained per gram flesh weight. Cell divisions started two days after protoplast isolation and gave rise to callus which has been maintained in culture for up to four years. Protoplast yield from the clone 77-0-77 was lower (less than 106 protoplasts per gram cells), cell division was infrequent and no callus was obtained. Protoplasts were also isolated from the leaves of willow shoot cultures using cellulysin and pectolyase, but these did not show cell divisions.

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A general method for the high-yield isolation of mesophyll protoplasts from deciduous tree species

Cited by 26 publications

References 7 publications

A simple method for the isolation of plant protoplasts

A simple method for the isolation of plant protoplasts

Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana)

Callus production from willow (Salix viminalis L.) protoplasts

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