High Yields Of Protoplasts Research Articles

Preparation of High Yields of Algal Protoplasts Using Buccal Juice of Sea Hare and Commercial Cellulase.

; Buccal juice of the sea hare Aplysia juliana was found to degrade algal polysaccharides. The optimal enzyme composition for protoplast preparation from Undaria pinnatifida was protein at 48 µg/ml buccal juice from sea hare, 10 mg/ml cellulase Onozuka-RS, 0.4 M NaCl, 0.8 M sorbitol, 2 mg/ml dextran sulfate sodium salt, and 1 µl/ml 2-mercaptoethanol in 10 mM MES buffer (pH 6.0). Protoplasts of Eisenia bicyclis, Endarachne binghamiae (Phaeophyta), and Ulva pertusa (Chlorophyta) could also be prepared in a similar manner. Yields of these protoplasts were about 10(7) cells per gram of fresh weight alga.

Plant regeneration from protoplasts of Triticum aestivum L. cv. Nakasoushu

A fast-growing, small, granular, embryogenic callus was selected from primary calli induced from the Japanese wheat cultivar Nakasoushu and the Australian wheat cultivar Bodallin. Regenerable and fine suspension cultures were induced three to six months after liquid culture was initiated and were characterized by dense cytoplasm and active division. These suspension cultures routinely provided high yields of protoplasts with about 90% viability when incubated in a modified KMP (Kao and Michayluk, 1975) medium containing 1 mg l-1 2,4-D (2,4-dichlorophenoxyacetic acid), and 1 mg l-1 zeatin. Nakasoushu and Bodallin protoplasts divided at frequencies of 8.6% and 11.1%, respectively, in agarose-solidified media. When Nakasoushu protoplasts were cultured with effective nurse cells of sorghum and wheat, protoplast division increased to 16.9% and 12.6%, respectively. Plating efficiencies varied from 0.03% to 2.5%. After subculture, protocolonies yielded embryogenic calli and somatic embryos, from which green plants were eventually regenerated. Whole plants obtained from Nakasoushu protoplasts were fertile, demonstrating the first report of Japanese cultivars in wheat protoplast cultures.

Protoplast isolation, culture, and regeneration.

Protoplasts of Arabidopsis thaliana have been isolated from a variety of explant sources with varying degree of success (, , ), Most workers have faced problems in achieving a high frequency of sustainable division of protoplasts in liquid culture. The problem can be alleviated to a certain extent by embedding mesophyll protoplasts m calcium algmate (). We have developed a technique allowing the culture of protoplasts derived from roots or cell suspensions m llqurd medium and then regeneration to plantlets (). The yield of root-derived protoplasts capable of division and regeneration is considerably higher in comparison to protoplasts obtained from leaf mesophyll cells. The protoplast lsolatton requires a ready supply of root cultures marntamed m auxin containing medium. Alternatively, a high yield of protoplasts, suitable for transient gene expression studies using direct DNA transformatron, can also be obtained from cell suspension cultures, excluding any limitatton of available starting material. Because the methods may have to be tailored to the needs of different experiments, here we describe protocols for the rsolatlon and culture of protoplasts from leaf mesophyll tissue (), as well as from root and cell suspension cultures. These methods are equally effective for the Arabzdop- szs ecotypes Columbia, C24, RLD, and WaSSllJeWSkiJa.

Isolation, culture and plant regeneration from protoplasts of Sideritis angustifolia

Summary Factors promoting high yields of viable protoplasts from leaves of Sideritis angustifolia are reported. Isolated protoplasts divided and gave rise to callus when plated in modified liquid MS medium with growth regulators. Best plating efficiency was obtained when protoplast were cultured in the presence of 6 μM BA and 2 μM NAA. Calli were then transferred onto solidified regeneration media. Adventitious bud differentiation was only observed in calli grown on medium containing 8 μM BA and 2 μM NAA. These buds proliferated quickly when placed on hormone-free medium and subsequendy regenerated new buds and shoots. Once developed shoots were excised, the remaining tissues maintained this regenerative capability in successive subcultures. Regenerated shoots were easily rooted on MS medium without growth regulators.

Protoplast Isolation from Shoots of Asparagus Cultures

Experiments to maximize the isolation and purification of viable protoplasts from shoot cultures of asparagus (Asparagus oficitzalis L.) were conducted. Important factors for high yield of viable protoplasts included: the use of in vitro etiolated shoots as source material; 0.6 M glucose as an osmoticum in a modified KM medium; a combination of pectinase, cellulase, and hemicellulase, each at 1% (wlv) for enzymatic digestion of cell walls; and physical factors such as the volume of enzyme solution and speed of gyratory shaking. Protoplasts were purified by suspending digested etiolated shoot tissue in 0.6 M sucrose, overlaid with KMG medium and centrifugation at 650 g. The asparagus genotype had a marked influence on protoplast yield, with some genotypes yielding up to 18.4 X 10h protoplastslg fresh etiolated shoot tissue with 90% viability.

Protoplast production and transformation of morphological mutants of the Quorn ® myco-protein fungus, Fusarium graminearum A3/5, using the hygromycin B resistance plasmid pAN7–1

A protocol for the generation of high yields of viable protoplasts has been developed for several highly branched (colonial) strains of the Quorn ® myco-protein fungus, Fusarium graminearum A3/5. Driselase was found to produce higher protoplast yields ( ca 10 9 g -1 wet weight) than the other lytic enzymes tested (Glucanex, Novozyme, β-glucuronidase, Sigma lytic enzyme, or ICN lytic enzyme), although yields differed for the various strains. Protoplast regeneration frequencies of 25–50% were observed when glucose (1.0 M) or sucrose (1.0 M) was used as the osmotic stabilizer. A highly branched strain of F. graminearum CC1–5, which grows better in submerged culture than the more sparsely branched wild-type strain (A3/5) was transformed using the hygromycin B resistance plasmid pAN7–1.

Plant regeneration from protoplasts isolated from callus ofGentiana crassicaulis

Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1-2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4-5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA3, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.

EFFICIENT CALLUS INDUCTION AND PLANT REGENERATION VIA SOMATIC EMBRYOGENESIS FROM IMMATURE LEAF-DERIVED PROTOPLASTS OF GROUNDNUT (ARACHIS HYPOGAEA L.)

High yields of protoplasts were obtained from immature leaves of aseptically grown plants ofArachis hypogaeausing an enzyme solution containing cellulase 2.0% (w/v) and Macerozyme 1.0% (w/v) in 0.6 M mannitol. Isolated protoplasts were cultured in Kao's medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The protoplasts started to divide after 3–5 days of culture. Sustained divisions resulted in mass production of cell colonies and mini calli in 4 weeks. After 4 weeks, protoplast colonies were transferred to the Murashige and Skoog (MS) medium supplemented with a-naphthalene acetic acid (NAA) and BAP. Colonies proliferated into actively growing calli. Further attempts to regenerate plants from such calli were not successful. However, protoclones differentiated roots on the same medium. Alternative methods for plant regeneration from protoplast derived callus cultures were tried through somatic embryogenesis. Protoplast-derived calli treated with 2,4-D and BAP formed somatic embryos. Somatic embryogenesis began in the proembryo stage and proceeded from globular to dicotyledonary stage. Embryos were then transferred onto hormone-free MS medium for germination. Five to ten percent of these embryoids germinated and grew to plantlets. Regenerated plants were transferred to plastic cups and grown to maturity.

Isolation, culture and regeneration of protoplasts from birdsfoot trefoil (Lotus corniculatus)

A method was developed for rapid plant regeneration from protoplasts of birdsfoot trefoil (Lotus corniculatus L. cv. Leo). Green cotyledons from in vitro grown seedlings were preplasmolyzed in CPW salts containing 13% mannitol (CPW 13 M) for 1 h prior to the enzyme treatment. The enzyme formula consisted of 2% (w/v) Onozuka Cellulase R-10, 1% (w/v) Macerase and 0.1% (w/v) Pectolyase Y-23 in CPW 13 M. This method produced high yields of viable protoplasts after purification. The procedure is reproducible and takes approximately 2.5 months from protoplast isolation to plantlet establishment in a greenhouse. More than 100 plantlets were grown in soil. Two somaclonal variants, a chimeric plant for chlorophyll production and an albino cell line, have been obtained by this procedure.

Cryopreserved callus: a source of protoplasts for rice transformation

We cryopreserved whole rice calli (Oryza sativa L cv Taipei 309) to investigate the ability of the surviving cells to regenerate plants and yield protoplasts competent for genetic transformation. Four out of six callus lines cryopreserved after four months in culture contained small sectors able to continue cell division and subsequently regenerate fertile plants. Both cryopreservation efficiency and regeneration ability decreased when using eight month old cultures. High yields of protoplasts were obtained from different cryopreserved callus lines. Protoplasts were transfected with chimeric genes consisting of the maize ubiquitin 1 promoter, first exon and first intron fused to the coding region of either the GUS or BAR marker genes. Levels of transient gene expression from both marker genes were similar to those previously obtained using protoplasts derived from callus that had not been frozen. Stable transformants were selected by their resistance to Bialaphos and could be identified with the pH indicator chlorophenol red. Southern blot analysis confirmed the integration of the BAR gene into the rice genome. Therefore, cryopreservation does not affect the ability of rice cells to integrate and express foreign genes.

An enzyme system to liberate spore and mycelial protoplasts from a dimorphic fungal plant pathogen Ophiostoma ulmi (Buism.) Nannf.

Viable protoplasts were successfully liberated from both the spore (yeast) and mycelial growth phases of a dimorphic fungal plant pathogen Ophiostoma ulmi (Buism.) Nannf. A high yield of spore protoplasts was obtained through treatment of spores with the lysing enzyme driselase prepared in 0·6 m mannitol, 0·1 m sodium citrate, 0·01 m EDTA, pH 58 and a high yield of mycelial protoplasts was obtained through treatment of mycelia with driselase prepared in 0·6 m mannitol, pH 58. When the enzyme treatments were interchanged the protoplast yield for each fungal growth phase was reduced approximately 50%. All protoplasts fluoresced in the presence of fluorescein diacetate when observed by fluorescence microscopy indicating that the protoplasts were viable. Approximately 50% of the spore and mycelial protoplast yield regenerated into actively growing fungal colonies when incubated on agar medium. The enzyme system developed provides a means whereby viable protoplasts from both the spore (yeast) and mycelial growth phases of O. ulmi can be obtained for studies focused on the elucidation of mechanism (s) that regulate dimorphism in O. ulmi.

Changes in the cell wall accompanying drying and maturation determine the ease of isolation of protoplasts from wheat aleurone layers

Cell walls of aleurone tissue of developing wheat grains (25 days post anthesis) are degraded by cellulase to give a good yield of protoplasts. As grains become older the aleurone cell walls become increasingly resistant to cellulase, which is completely ineffective on 45-day old material. Resistance to cellulase is provoked even in aleurone cells of young grains by enforced drying of the grains to 12% water content. This suggests that the decreasing effectiveness of cellulase that occurs as grains develop and mature may be caused partly by the natural loss of water.Resistance to degradation by the aleurone cell walls of older or dried grains is to a large extent overcome by the addition of hemicellulase and xylanase to the digestion mixture but completely if acetyl esterase is also included. Changes involving hemicelluloses and xylans are therefore implicated in the effects of drying or maturation, including acetylation processes especially in grains which are almost mature. Arising from these findings a method has been developed for obtaining a high yield of protoplasts from aleurone layers of mature wheat grains, having a high viability and response to gibberellic 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.

Cell aggregates in wheat suspension cultures and their effects on isolation and culture of protoplasts

Fine embryogenic suspension cultures of wheat (Triticum aestivum cv Hartog and Timmo, and T. durum cv D6962) tend to grow into large cell clumps (1-3 mm), resulting in the formation of mixed suspension cultures consisting of both fine and large cell clumps. The cell clumps were separated according to their sizes and cultured as new lines to investigate their growth rate and differentiation potential and the effects of cell aggregate size on protoplast culture. The results showed that the fine clusters (<310 μm) had a higher growth rate but a lower differentiation frequency than the large cell aggregates (310-2000 μm). After 2-4 weeks incubation, all the new lines reformed mixed suspension cultures again. The large clumps (>1100 μm) released fine cell clusters into the medium so it was possible to initiate fine embryogenic suspension cultures from the large clumps. With regard to the isolation and culture of protoplasts, although the highest yield of protoplasts was obtained from the fine cell clusters, the protoplasts isolated from different sized cell aggregates all had similar potential for sustained cell division and plant regeneration.

An Efficient Method for Callus Formation of Protoplasts from Peanut(Arachis hypogaea L.) Leaf Mesophyll.

High yields of viable protoplasts were obtained from leaf mesophyll of 10-day-old peanut seedlings (Arachis hypogaea L. var. Lokal). An enzyme solution containing 1% Cellulase YC, 2% Meicelase P-1, 0.15% Pectolyase Y-23 and 0.5% Macerozyme R-10 was found to be the best for protoplast isolation. The protoplasts were then cultured in liquid or in agarose-gel modified KM8P medium. Cultured protoplasts divided 2 days after culture. In solidified media, protoplasts divided continually and formed many colonies 1 month after culture. The best viability and colony formation was obtained in a culture medium solidified with 1.2% agarose.

Requirement of thidiazuron for healthy protoplast development to efficient tree regeneration of a hybrid poplar ( Populus tremula ß p. alba)

Summary Methods were developed for obtaining high yields of leaf protoplasts of a hybrid clone of white poplar, Populus tremula × P. alba (717-1-B4), as well as rapid and efficient plant regeneration. Protoplasts were isolated from leaves of shoot culture. A precise adaptation of liquid media for preparation and culture of protoplasts led to high rates of cell colony formation, on the average for 30% of initially plated protoplasts. The procedures employed avoided the formation of any exudate. Glucose (9%) as an osmoticum and carbon source was the only sugar leading to high viability. A precise balance of low concentrations of nitrate and ammonium in the culture medium was beneficial for development of healthy cell colonies. A combination of thidiazuron (0.05 µM) as the only cytokinin and 2,4-D (14 µM) as auxin was essential for sustained development of protoplast derived cell colonies. After dilution in liquid medium, about 10% of the colonies formed calli. Plants regenerated through bud formation on practically all protoplast-derived calli. Plants regenerated from 3% of the initially plated protoplasts. In vitro rooting and transfer to soil were also achieved with good efficiency.

A protoplasting technique with general applicability for molecular karyotyping of hymenomycetes

Pulsed-field gel electrophoresis (PFGE) has been used to facilitate genetic fingerprinting within a natural hymenomycete population. A protoplasting technique has been developed which shows that the basidial cells of the commercially important mushrooms Agaricus bisporus, A. bitorquis and Lentinus edodes (Shiitake), as well as from many wild growing Agarics, are sensitive to commercially available fungal cell wall degrading enzymes. The technique produces a high yield of intact protoplasts within short time periods. Using the basidial cell protoplasting technique, chromosomal-sized DNA from a wild fruit body of Coprinus comatus has been prepared and resolved into distinct bands using PFGE. This protoplasting technique will allow the application of PFGE to provide electrophoretic karyotypes within natural and commercial populations of hymenomycetes.

Protoplast formation and DNA-mediated transformation of Fusarium culmorum to hygromycin B resistance

This work involved firstly optimizing the protoplast yields for F. culmorum 159026, then setting up a system for DNA-mediated transformation. Higher protoplast yields and more rapid regeneration were obtained when the organic stabilizers sucrose and sorbitol were used rather than NH4Cl. Successful transformation was achieved using the plasmids pAN7-1 and pHRC containing the E. coli hygromycin B resistance gene (hygromycin B phosphotransferase, hpt) fused to Aspergillus nidulans expression signals.

Isolation of intact proplastids from suspension cultured cells of Theobroma cacao L.: de novo long chain fatty acid biosynthesis

Proplastids were located in the cytoplasm of Theobroma cacao L. suspension cells and in the developing cotyledons of maturing zygotic embryos of T. cacao as demonstrated using transmission electron microscopy. A method has been developed for the preparation of protoplasts from T. cacao suspension cells. The highest protoplast yields were obtained using suspension cells harvested during the exponential phase of growth. Proplastids were isolated from T. cacao suspension cells on discontinuous sucrose density gradients after osmotic lysis of their protoplasts. Proplastids were localized within the gradients by assaying for organelle marker enzymes and their identity and purity confirmed using electron microscopy. Intact proplastid preparations were shown to incorporate radiolabelled [1- 14C]acetate into de novo synthesised fatty acids, confirming both the biochemical integrity of the proplastid isolates and demonstrating this organelle to be a site of long chain fatty acid biosynthesis.

The toxicity of antibiotics to protoplast cultures of Triticum aestivum L.

A cell suspension culture was established from an immature embryo-derived callus culture of Triticum aestivum cv Fundulea. This culture provided high yields of protoplasts which, at low plating densities (2.5–5.0 × 10 4/ml), could reestablish cell cultures. Low density cultures were required to measure reproducibly responses to a range of antibiotics. Cell division and plating frequencies were used to evaluate phytotoxic responses. The aminoglycoside antibiotics hygromycin, G418, bekanamycin and kanamycin were extremely toxic to the wheat cells (50% inhibition at 15 μg/ml). The least toxic antibiotics were vancomycin, spectinomycin and cefotaxime. Vancomycin (1000–2000 μg/ml) enhanced cell division and plating efficiencies two-fold indicating its potential for establishing protoplast cultures of recalcitrant species.