Plant regeneration from protoplasts of Dimorphotheca and Rudbeckia

Protoplasts were isolated from leaves, shoots, cotyledons, ray florets and callus cultures of Dimorphotheca aurantiaca (syn. D. sinuata) (Cape Marigold, Star of the Veldt) and Rudbeckia hirta, R. laciniata and R. purpurea; species of ornamental value. For Dimorphotheca, plants were regenerated from protoplasts of all sources apart from the ray floret, whilst for the Rudbeckia species, although protoplast division was induced in most cases, only leaf mesophyll protoplasts of R. hirta c.v. Marmalade gave plants. The establishment of plant regeneration for these ornamental species, from protoplasts, now provides a basis for their somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - K kinetin - GA₃ gibberellic acid - MS Murashige and Skoog (1962) - f.wt. fresh weight     

Plant regeneration from protoplasts of Panax ginseng (C.A. Meyer) through somatic embryogenesis

Summary Protoplasts of Panax ginseng were isolated from embryos obtained from the 4-year old embryogenic cell line KCTC PCL 49031 which was derived from a zygotic embryo. High protoplast yields of 22–25 × 10⁶ protoplast / g tissue were obtained following 5–6 h digestion with 2% Cellulysin, 1% Pectinase and 1% Macerasae in half strength Murashige and Skoog's medium containing 12% mannitol. A plating density of 1×10⁵ protoplasts /ml was found optimal for protoplast culture. An initial division frequency of 10% was obtained in an agarosegelled defined medium. Myo-inositol (6%) was found to be the most suitable osmoticum. Somatic embryos were formed from protoplast derived embryogenic callus, which regenerated into plantlets.Abbreviations NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kn kinetin - BA benzyladenine - GA₃ gibberellic acid - MS Murashige and Skoog medium     

Highly Efficient Isolation of Populus Mesophyll Protoplasts and Its Application in Transient Expression Assays

Background

Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function.

Methodology/Principal Findings

We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events.

Conclusions/Significance

This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.     

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

High yields (1.2–8.0 × 10⁶ g fresh wt.^?1) of viable leaf mesophyll protoplasts have been isolated from a range of mature deciduous woody-plant species (Betula pendula, Alnus glutinosa, Salix caprea var. Kuroyanagi, S. alba var. Tristis, Populus Tacatricho (= P. tacamahacca × trichocarpa 32), Ulmus glabra var. camperdown), and juvenile glasshouse-grown material (B. pendula, B. pubescens, A. glutinosa). Protoplasts are only released if chopped leaf tissue is thoroughly washed prior to digestive enzyme addition. The nature of the washing requirement has been investigated and it has been demonstrated that water soluble compounds are released from chopped leaves which modify their cell-wall structure rendering them resistant to enzymic digestion. When analyzed by paper chromatography the leachate from B. pendula leaves was found to contain the hydroxycinnamic acids p-coumaric acid (PCA) and o-coumaric acid (OCA). Pre-incubation of B. pendula tissue (which is normally susceptible to enzymic digestion) in authentic samples of PCA and OCA prior to enzymic incubation, completely suppressed protoplast yields. The relevance of hydroxycinnamic acids to protoplast isolation and plant tissue culture is discussed.     

Protoplast production in Chondrus crispus gametophytes (gigartinales,rhodophyta)

Protoplasts were isolated from female gametophytes of Chondrus crispus (Stackh.) using commercial cellulase and various carrageenases prepared from marine bacteria. Depending on the nature of the donor tissue (apices or whole thallus, wild or cultivated strains), yields ranged from 1.0–8.5×10⁸ protoplasts per gram of fresh tissue. Preincubating the tissue with a potassium chelator, Kryptofix 222, enhanced protoplast yields by 30–50 %. Based on staining with fluorescein diacetate most protoplasts were viable. A few protoplasts regenerated a cell wall and divided.     

Purification of enzymatically isolated mesophyll protoplasts from c(3), c(4), and crassulacean Acid metabolism plants using an aqueous dextran-polyethylene glycol two-phase system

Enzymatic digestion of leaf segments with 2% cellulase, in combination with a pectinase in some species, yields intact protoplasts mixed with epidermal tissue, vascular tissue, broken protoplasts, and chloroplasts. Epidermal and vascular tissue are removed with sieves of various porosity. Intact protoplasts in the filtrate are separated from other components by an aqueous two-phase system which consists of dextran-polyethylene glycol, with sorbitol and sodium phosphate. Intact protoplasts partition at the interphase, while chloroplasts and broken protoplasts partition in the lower phase when the separation is facilitated by low speed centrifugation. The optimum conditions for purification of maize mesophyll protoplasts with high yields are centrifugation of the two-phase system at 300g for 6 minutes at 2 C with a mixture including 0.46 m sorbitol, 10 mm sodium phosphate, 5.5% polyethylene glycol 6000, and 10% dextran of average molecular weight of 20,000 to 40,000. The collection of protoplasts at the inter-phase was proportional to the amount of chlorophyll added over a wide range of concentrations regardless of the initial contamination of the preparation by other cellular debris. The two-phase system is applicable for protoplast purification from a wide variety of species, including C₃, C₄, and Crassulacean acid metabolism plants, regardless of protoplast size.     

Tissue degradation and enzymatic activity observed during protoplast isolation in two ornamental <Emphasis Type="Italic">Grevillea</Emphasis> species

Summary Degradative changes in tissue during protoplast isolation were a contributing factor to low protoplast yields in the saltsensitive Grevillea arenaria (R. Brown) and the salt-tolerant Grevillea ilicifolia (R. Brown). Protein and malondialdehyde content decreased significantly during the protoplast isolation procedure. Acid and neutral proteases were identified, and high acid protease activities were correlated to low protoplast yields. Acid phosphatase, catalase, polyphenol oxidase and lipoxygenase activities increased in both Grevillea species with cell wall digestion. High activities of catalase and low levels of polyphenol oxidase were correlated with high protoplast yields. Levels of acid phosphatase and lipoxygenase were not good indicators of final protoplast yields. The addition of the anti-oxidant, reduced glutathione, and the acid protease inhibitor, pepstatin A, significantly increased protoplast yields. Strategies were identified to minimize deleterious degradative effects during the isolation of protoplasts, including strict pH control, testing a number of cell wall digestion enzymes, and the addition of anti-oxidative metabolites and protease inhibitors.     

Uptake and retention of external solutes from the digest medium during preparation of protoplasts

The extent to which solutes present in the digest medium enter cells and are retained during preparation of protoplasts was investigated. When barley (Hordeum vulgare, L. cv. Clipper) leaf slices were incubated in sorbitol there was considerable uptake of sorbitol into the tissue, which continued for up to 6 h and was dependent on the sorbitol concentration in the external medium. Protoplasts prepared by digesting leaf slices in a medium containing [¹⁴C]sorbitol but isolated and purified in media with unlabelled sorbitol contained significant amounts of [¹⁴C]sorbitol. From measurements of the protoplast volume, the internal sorbitol concentration was calculated to be 100 mM, assuming uniform distribution of the sorbitol throughout the protoplasm. The uptake of sorbitol during digestion and its retention by protoplasts was confirmed by measuring sugars in protoplast extracts by gas sucrose or inositol. Vacuoles prepared from the protoplasts contained 83% of the sorbitol present in protoplasts. It is concluded that considerable uptake of solutes from the external medium occurs during digestion of leaf tissue and that these solutes are retained within the protoplasts during isolation and purification. The solutes appear to be uniformly distributed throughout the subcellular compartments of the protoplast.     

A simple method for the isolation of plant protoplasts

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 protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO₄), and a protectant (CaCl₂ 2H₂O), 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 forNicotiana 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.     

Isolation of protoplasts from female gametophytes of Torenia fournieri

Protoplasts from the cells of mature embryo sacs (ES-protoplasts) of Torenia fournieri were obtained during incubation of ovules in an enzyme solution. Four protoplasts which arose from each embryo sac were connected together after isolation, or aggregates of the egg cell protoplast and two synergide protoplasts dissociated from the protoplast of the central cell. The ES-protoplasts stayed viable for 2 weeks in culture, but they did not regenerate cell walls.Abbreviations ES embryo sac - FAA fixative (formalin : acetic acid : alcohol = 1 : 1 : 18) - FDA fluorescein diacetate - PAS periodic acid Schiff reaction - 2,4-D 2,4-dichlorophenoxyacetic acid     

Factors affecting isolation of protoplasts from leaves of grape (Vitis vinifera)

A method of isolating grape mesophyll protoplasts was developed to facilitate the eventual use of genetic engineering techniques in this species. The effects of several factors influencing protoplast isolation could be evaluated quickly by using leaf disks 1 cm in diameter and known volumes of maceration and wash media. The best yields of mesophyll protoplasts were obtained using medium sized leaves of grapevines kept in the dark for 24 hours prior to maceration in 1% Cellulysin, 0.5% Macerase, 0.7 M mannitol, 5 ppm 2, 4 D, 0.1 ppm BAP, 1/10 strength Murashige and Skoog medium, and incubated at 22°C in cool-white fluorescent light (70–100 E m^-2 s^-1) for 24 hours. Over 30×10⁶ protoplasts per cm² of leaf were produced using these conditions. This method of screening factors affecting protoplast isolation could be applicable to other species.     

Investigation of the enzymatic digestion of plant cell walls using reflectance Fourier Transform Infrared spectroscopy

A method has been developed to determine the reflectance Fourier Transform Infrared spectra of plant cells grown in vitro and of the protoplasts released from such cells by enzymatic digestion. It is demonstrated that there is a smooth and reproducible transition in spectral detail as enzymatic digestion procedes. Reflectance Fourier Transform Infrared spectroscopy has been used to monitor the progress of protoplast release during enzymatic digestion of cell wall material.Abbreviations FTIR Fourier Transform Infrared - SEM Scanning Electron Microscope     

Plant regeneration from mesophyll protoplasts of Diplotaxis muralis,a wild crucifer

Mesophyll protoplasts from leaves of aseptically grown shoot tips of Diplotaxis muralis were isolated (6.2–7.1×10⁵ protoplasts/g fresh weight of tissue) using one step enzyme digestion. The protoplasts (71% viability) underwent divisions (4.2+0.1%) on plating in M8PS₂ medium and ultimately formed calli with 0.45+0.03% plating efficiency. Plant regeneration could be achieved both through embryogenesis and organogenesis. The efficiency of plant regeneration through organogenesis was 9 times higher than embryogenesis. Forty eight out of 52 plants regenerated so far from 3 independent experiments were normal with respect to fertility and meiotic chromosomal behavior.Abbreviations BAP 6-benzylaminopurine - GA₃ Gibberellic acid - A Kao and Michayluk, 1981 - KM Kao and Michayluk, 1975 - MK₃ Modified K₃ - M8P Modified 8P - MS Murashige and Skoog, 1962 - NAA 1-naphthalene acetic acid - PE Plating efficiency     

Condensed tannins in the tissue culture of sainfoin (Onobrychis viciifolia Scop.) and birdsfoot trefoil (Lotus corniculatus L.)

Two forage legumes, birdsfoot trefoil (Lotus corniculatus L.) and sainfoin (Onobrychis viciifolia Scop.), containing condensed tannins in their leaves and stems were used as source material to study condensed tannins in tissue culture. More protoplasts were isolated from mesophyll tissue of a low tannin-containing strain of birdsfoot trefoil than from a high tannin-containing strain, but more tannin-filled protoplasts were observed in the latter. Growth rates of leaf explant-derived callus tissue were greater for the high-tannin than for the low-tannin strain. In sainfoin, callus cultures from leaf explants produced numerous tannin-filled cells by 21 days. Explants from sainfoin cotyledons and roots, tissues which normally do not contain tannins, also formed callus with tannin-filled cells in 21 days but in almost every case, a cytokinin was required for tannin formation to occur. The occurrence of tannin-filled cells in callus from root and cotyledon explants was variable and genotype specific. These results show that endogenous tannins can affect protoplast isolation and possibly callus growth in birds-foot trefoil, and that the formation of condensed tannins in sainfoin callus culture can be influenced by a growth regulator.Abbreviations BAP benzylaminopurine - KIN kinetin - NAA naphthaleneacetic acid - PAR photosynthetically active radiation Contribution no. 920 of Agriculture Canada Research Station, 107 Science Cres., Saskatoon, Saskatchewan, Canada S7N OX2     

Recovery of plants from leaf protoplasts of hybrid-poplar and aspen clones

Leaf protoplasts were isolated from shoot cultures of two hybrid poplar clones (Populus alba × P. grandidentata Crandon, NC-5339 and P. nigra Betulifolia × P. trichocarpa, NC-5331) and the Upright European Aspen (P. tremula Erecta) and were cultured in contact with screen discs floated in liquid medium. Protoplast culture was influenced by the growth medium of the source shoot cultures, the protoplast purification procedure, the plating density, and the presence or absence of a coconut water and casein hydrolysate supplement added to the culture medium. The protoplast-derived cells divided more quickly and with higher incidence than previously reported for hybrid poplars. Shoots were regenerated from the protoplast-derived calli and were maintained as shoot cultures. Plants were developed from microcuttings rooted ex vitro and were grown-on in the greenhouse and field.Abbreviations BA benzyladenine - NAA 1-naphthaleneacetic acid - TDZ thidiazuron - WPM Woody Plant Medium, Lloyd and McCown (1980) - MS Murashige and Skoog Medium (1962) - NC-XXXX North Central Forest Experiment Station accession number assigned to hybrid poplar clones.     

Callus culture as substrate for the production of specific cell wall degrading enzymes for derived protoplast isolation

Callus culture of spruce (Picea excelsa LINK) appears to be a suitable substrate for the fungusTrichoderma reesei to produce an efficient extracellular lytic system for protoplast isolation. In comparison with Onozuka R-10 cellulase, a yield of protoplasts from the spruce callus 2·5 higher was obtained. Another testea commercial cellulase DK was less efficient. The addition of Macerozyme R–10 significantly enhanced release of protoplasts within all tested enzyme preparations. No difference in the viability of protoplasts has been observed.     

An improved protocol for plant regeneration from leaf- and hypocotyl-derived protoplasts of carrot

An easy and effective regeneration system from leaf- and hypocotyl-derived protoplasts was established for carrot. The protoplast isolation efficiency after preplasmolytic treatment and digestion of source material in enzyme mixture consisted of 1% cellulase Onozuka R-10 and 0.1% pectolyase Y-23 reached on average 3 × 10⁶ and 10⁶ protoplasts per g of leaf and hypocotyl tissue, respectively. A modified thin alginate layer technique was applied for the protoplast culture. Direct somatic embryogenesis on a simplified Kao and Michayluk medium in the presence of 2,4-D and zeatin occurred during cultivation of both leaf- and hypocotyl-derived protoplasts for all accessions used. Morphologically normal plants were produced at very high efficiency within two months after initiation of the protoplast culture. Ninety three percent of in vitro derived plants were diploids. Pollen viability and seed set after self-pollination were similar to those of plants obtained from seeds.     

Isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato

Optimum conditions for the isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato (Ipomoea batatas) were investigated. Growth phase of callus and the ratio of callus-enzyme solution affected the yield of protoplasts. Composition of the medium and protoplast density were examined for protoplast culture.Small colonies were regenerated from the protoplasts. Upon transfer to light a high amount of anthocyanin was accumulated in these colonies.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-(N-morpholino)-ethanesulfonic acid     

Isolation of cells and protoplasts from leaves of in vitro propagated peach (Prunus persica) plants

Yields of 10⁶–10⁸ peach mesophyll cells and protoplasts · gfw^-1 were obtained depending on factors such as digesting enzymes, and leaf size. Onozuka R-10 (2%) in combination with Macerase (0.5%) was found best for protoplast isolation and mediocre for cell isolation among several enzyme combinations tested. Viability was 90% for protoplasts and 60% for cells. Pectolyase Y23 was found to be ineffective in our investigation. Small leaves, 4–10 mm in length, were a superior source for protoplast isolation than medium or big expanded leaves, 22–30 mm in length. The high yields of protoplasts could be obtained only when keeping the ratio of leaf biomass to volume of digesting enzyme solution under 20 mg ml^-1. Purification of protoplasts on a sucrose gradient yielded about 10⁷ protoplasts · gfw^-1, however, the preparation was still contaminated by intact cells. Protoplasts were cultured under different growth regulators and physical conditions. Limited growth and division of protoplasts embedded in agarose drops were observed.Abbreviations BA 6-benzyladenine - IBA indolebutyric acid - FDA fluorescein diacetate - MES 2-M-morpholinoethane sulphonic acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid - PVP polyvinylpyrrolidone