Protoplast Yield Research Articles

Influence of enzyme solution on protoplast culture and transient gene expression in maize (Zea mays L.)

The influence of two enzyme solutions, differing only in the presence or absence of Macerozyme, on protoplast yield, colony formation and transient GUS (β-glucuronidase) activity was studied. For all parameters tested the presence of Macerozyme during protoplast isolation had a negative influence. Using an enzyme solution without Macerozyme suspension aggregates gave up to 4.4 times higher protoplast yield and plating efficiencies were increased up to 10-fold. Further, protoplasts isolated without macerozyme showed a 5.2-fold higher GUS activity in transient gene expression. Apart from the presence of Macerozyme, longer incubation (3 compared with 1.5 h) of cell aggregates in the enzyme solution also had a negative effect on transient transformation efficiency. These data demonstrate that protoplast isolation conditions have a profound effect on transient gene expression and it is proposed that these factors will also influence stable transformation efficiency.

Optimization of formation and regeneration of protoplasts from biocontrol agents of Trichoderma species

The optimal conditions necessary for a large yield and a high frequency of regeneration of protoplasts isolated from the biocontrol agentsTrichoderma koningii andT. harzianum were investigated. Protoplast yields were 1.2×108/ml fromT. koningii and 6×107/ml fromT. harzianum when 20-h mycelial culture was treated with a lytic enzyme solution containing Novozym 234 (15 mg/ml), sucrose (0.6 M) and citrate phosphate buffer (0.02 M), pH 5.6 at 31°C. When the protoplasts were grown in the regeneration medium containing yeast extract (1.5%), 1 I of Mandel's salts, pH 5.6, and glucose (0.8 M), a high frequency of regeneration of the protoplast was obseved: 66% forT. koningii and 45% forT. harzianum. Two patterns of regeneration were observed. First, the hyphae arose directly from the regenerated protoplast mother cell. Second, a chain of bud cells developed from the protoplast and subsequently generating hyphae generally protruded from the terminal bud cells.

PROTOPLAST ISOLATION AND CULTURE OF KENAF (HIBISCUS CANNABINUS L.)

Protoplast isolation and culture protocols were developed for leaf tissue from 6 kenaf cultivars [Everglades 41 (E41), E71, Guatemala 4 (G4), G45, G51, and Tainung 1]. For protoplast isolation, the best combination of hydrolytic enzymes was cellulysin (1% w/v; Calbiochem) plus macerase (0.5% w/v; Calbiochem), with a 24 hour digestion at 30°C in the dark. Yields reached 7.2 (10)6 protoplasts/g leaf tissue. Protoplast viabilities ranged from 65% to 96%. Minor cultivar differences were observed related to protoplast yield, but all viability estimates were in an acceptable range. Greatest cell division frequencies and plating efficiencies were obtained when protoplasts were initially cultured in liquid medium at a density of 1.0 (10)5 protoplasts/ml. Electrofusion protocols were developed for kenaf protoplasts testing the range from 1200 to 3000 V/cm. A fusion voltage of 2000 V/cm yielded the highest fusion frequency and retained viability above 80%.

Plant Regeneration from Protoplast-Derived Somatic Embryos of Asparagus officinalis L.

Summary A method for regenerating plants from embryogenic calli derived from protoplasts of asparagus is described. The protoplasts were isolated from calli of a selected crown of the genotype G-203 of Asparagus officinalis L. Optimum protoplast yield was obtained from calli of 8 to 10 d of age after subculture following enzymatic digestion. Of the isolated protoplasts 80 % to 90 % were viable. The highest plating efficiency (12.45 %) was obtained when cells were cultured at a density of 1 × 10 5 protoplasts/mL in 1/2 strength of Murashige and Skoog (MS) medium with 1 mg/L NAA, 0.5 mg/L zeatin, 0.5mg/L folic acid, 0.05 mg/L biotin, 1 g/L glutamine, 0.2 g/L casein hydrolysate, 0.6 M glucose, 0.1 M mannitol, and 0.1 % (w/v) Gelrite. A remarkable decrease in plating efficiency was recorded in the presence of both spermine and spermidine in the culture medium. Colonies were visible after 30 d and grew consistently into slimy and friable embryogenic calli after transferring them onto MS medium containing 1 mg/L 2,4-D, 3 % (w/v) sucrose and 0.2 % (w/v) Gelrite. Somatic embryos were induced from such calli when cultured on 1/2 MS medium with only 1.% (w/v) sucrose devoid of any growth hormones. After transplanting on 1/2 MS medium with 1 % (w/v) sucrose and 1 mg/L GA3, 40–45 % of embryos germinated normally and grew into plantlets within 30 d. These plantlets from protoplast-derived somatic embryos were diploid revealing 2n = 20 chromosomes.

Fertile Indica rice plants regenerated from protoplasts isolated from scutellar tissue of immature embryos

We report on the regeneration of fertile Indica rice (Oryza sativa L.) plants from protoplasts isolated from scutellar tissue of immature embryos. The average yields of protoplasts after purification ranged from 2.8 × 10(5) to 3.5 × 10(5) protoplasts per fifty embryos. Protoplasts developed rapidly to colonies when cultured in maltose containing medium using the nurse culture method. Upto 146 or 39 visible colonies per 10(6) protoplasts were obtained for the varieties Basmati 370 and IR43 respectively. Of two basal culture media compared, R2 medium containing 3 mg l(-1) kinetin, 1 mg l(-1) naphthalene acetic acid (NAA), 30 g l(-1) maltose and 3.0 g l(-1) agarose was found to be more effective in producing green plants. All scutellum protoplast-derived plants that were transferred to the greenhouse survived and were fertile.

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.

Preparation of protoplasts from the cyanobacterium Spirulina platensis and a novel viability assay

A method is described for the isolation of protoplasts from the cyanobacterium Spirulina platensis using an elite strain (S.pl. FT 06) selected after screening. Trichomes of the cyanobacterium were treated with lysozyme (0.1%) for 28 h at room temperature in 0.03 mol l-1 phosphate buffer (pH 6.8) with 0.5 mol l-1 mannitol as the osmoticum. This resulted in an 80% yield of protoplasts. Cells were intact in their fine structure and viable as evidenced by a rapid and efficient viability assay using a novel mixture of fluorescein diacetate and calcofluor white.

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.

Highly efficlent plant regeneration from mesophyll protoplasts of Indian field cultivars of tomato (Lycopersicon esculentum)

Three Indian cultivars ofL. esculentum were assessed for shoot regeneration from protoplast-derived calli. Consistent yields of viable protoplasts (>9.0×106 g f.wt.-1) were obtained from leaflets of 14 days old cultured shoots. Protoplast viability (88–94%) and planting efficiency (55–70%) were recorded for the three cultivars. Up to 71% of the protoplast-derived tissues regenerated shoots.

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.

ブドウ葉肉プロトプラストの単離および培養に及ぼす酵素液の影響

Viable mesophyll protoplasts were isolated from in vitro grown leaves of grapes (Vitis labruscana cv. Kyoho and V. vinifera cv. Cabernet Sauvignon). The best yield of mesophyll protoplasts (1.8 × 107 and 2.4 × 107 • g -1 leaf fresh weight, 'Kyoho' and 'Cabernet Sauvignon', respectively) was obtained in a combined enzyme solution of 0.5% Cellulase YC, 0.2% Macerozyme R -10 and 0.1% Driselase. More than 85% of the isolated protoplasts were viable. Most protoplasts regenerated the cell wall within first 5 days of culture. Cell division occurred after 5 to 10 days of culture in Gamborg's B5 liquid medium supplemented with 5μM 2, 4 - D, 2.5μM BA and 0.6 M sorbitol. In 'Kyoho', 12% of cell division frequency was found in the protoplasts isolated with 0.5% Cellulase YC, 0.2% Macerozyme R -10, 0.1% Driselase and 0.01% Pectolyase Y -23. In 'Cabernet Sauvignon', the frequency of cell division was low (1.4%) when the same enzyme solution was used for the protoplast isolation.

Development of Protoplasts from Grateloupia sparsa and G. filicina (Halymeniaceae, Rhodophyta)

For isolation of protoplasts from Grateloupia sparsa and G. filicina, commercial enzymes of cellulase, macerozyme, agarase, abalone acetone powder and papain were used in seven combinations. A maximum yield of protoplasts of 7 × 10 8 g −1 (fw) was obtained with a mixture of 4% cellulase, 2% macerozyme, 50 U/mL or 1000 U/mL agarase and 4% papain. Very few protoplasts (10 3 g −1 fw) were isolated with mixtures of cellulase and macerozyme. After isolation, protoplasts were incubated in 0.7 M mannitol-PES medium for 10 days then transferred to PES medium to obtain the highest number of regenerated protoplasts. Protoplasts excreted amorphous matrix on their surface as soon as they were isolated, and deposited cell wall after 4 days of culture

Effect of silver nitrate on sugarcane cell suspension growth, protoplast isolation, ethylene production and shoot regeneration from cell suspension cultures

Silver nitrate (AgNO), an inhibitor of the physiological action of ethylene, reduced cell growth, promoted ethylene production, increased the yield of protoplasts and reduced shoot regeneration from sugarcane heterogeneous cell suspension cultures. The increase in the rate of protoplast isolation from cultures treated with AgNO (0 to 59 μM) correlate with an increase in endogenous ethylene production by the cells. The addition to the culture medium of chemicals that either inhibited (aminoethoxyvinylglycine, AVG) or promoted (aminocyclopropane-1-carboxylic acid, ACC) ethylene biosynthesis did not alter the number of protoplasts isolated from these cultures. However, protoplasts were isolated with AVG in combination with AgNO even though ethylene production was inhibited. These results suggested that AgNO may be having another more direct effect on protoplast release. One such site may be the cell wall or on cell metabolism conditioning cells to release protoplasts after enzyme treatment.

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.

Production and regeneration of protoplasts from the Y-phase of the human pathogenic fungus Paracoccidioides brasiliensis.

Protoplasts of Y-cells and partially converted M-cells from several human isolates of Paracoccidioides brasiliensis were obtained with a combined enzyme system containing Novozym 234 and a chitinase. A laboratory made extract from Trichoderma harzianum CBS-345-33 supplemented with chitinase induced the release of protoplasts from partially converted M-cells but not from established Y-cells. A similar yield of protoplasts (1-2 x 10(5) ml-1 after 16 h) was produced by using either enzymatic system. Protoplasts regenerated on nutrient gelatin at 23 degrees C at a frequency of 0.1%.

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.

Factors influencing protoplast isolation from Coffea arabica cells

Cultured plant cells such as Coffea arabica L. cells, accumulate low concentration of secondary metabolites. One way to obtain high-producing plant cell cultures is to prepare single cell clones by using protoplast systems. Identification of limiting factors should facilitate the development of an isolation procedure that can generate adequate yields of intact and viable protoplasts Coffea arabica L. suspension cells. The most suitable conditions for protoplasting were as follows: 6 g of fresh tissue were plasmolysed in 100 ml of K3 salts (Nagy & Maliga 1976) containing 0.5 M sucrose for 1 h at 24°C. Then, 1 g of preplasmolysed cells were incubated in 10 ml of cellulase R10 (1%), macerozyme R10 (0.8%) and driselase (0.5%) in preplasmolysis medium. The protoplasts were collected and purified after 15 h of lytic reaction in the dark, at 28°C. More than 75% and 95% of the cells were converted into protoplasts when 5 and 8 day-old suspensions respectively were used for the release step. A number of viable protoplasts ranging from 3.5×106 to 4.6×106 P g-1 fresh weight was obtained corresponding to an increase by a factor 10 to 15 of the protoplast yield obtained by Acuna & De Pena (1991).

Genetic transformation ofStreptomyces caespitosus

Genetic transformation ofStreptomyces caespitosus by plasmid pIj 702 was carried out. Optimal conditions for the protoplast preparation ofStreptomyces caespitosus, its regeneration, and its transformation by pIj 702 were evaluated. Addition of 2% glycine to the culture broth was optimal for protoplast yield. Formation and regeneration of protoplasts were most efficient when the mycelium were harvested at between late log and stationary growth phase. The regeneration frequency of the protoplasts was 15% when the protoplasts were regenerated on R2YE agar media containing 0.5M sucrose. Under the best condition for protoplats regeneration, the optimal transformation frequency was achieved with 40% polyethylene glycol (M.W. 4,000) treatment for 2 minutes.

Production and characterization of guluronate lyase from Klebsiella pneumoniae for applications in seaweed biotechnology

Cultures of Klebsiella pneumoniae fermenting sodium alginate produce an extracellular guluronate-specific alginate lyase. This enzyme production was studied in stirred-tank fermentors. Different alginate substrates gave moderate differences in growth and enzyme yield. Alginates with guluronic content gave reduced biomass but favored enzyme production. Low molecular weight (down to DP n ≈ 270) also favored enzyme production. Excessive depolymerization of substrates occured during heat sterilization of culture media. The enzyme was characterized by its specificity and sensitivity to pH, salt, and calcium. Improved yields of viable protoplasts were documented for Laminaria digitata (Huds.) Lamour.

Factors affecting protoplast yield of the carrageenophyte Solieria filiformis (Gigartinales, Rhodophyta)

The effect of age, pH of the culture medium, pre-treatment of tissues, enzymes sources and enzymatic adaptability of phycophages fed with a monospecific diet were analyzed on the protoplast yields of the red seaweed Solieria filiformis (Kützing) Gabrielson. New apices from fast growing plants showed the highest protoplast yields. The protoplast yield decreased when the pH of the culture medium increased from 6.0 to 9.0. Crude extracts from the abalone Haliotis coccinea canariensis Nordsieck, fed with Solieria filiformis thalli for three months in combination with cellulysin, released the highest number of viable cells and protoplasts. Yields ranged from 1.0 to 8.5 x 10(6) protoplasts per gram of fresh weight.