Electrofusion of mesophyll protoplasts from two varieties of sugar beet, (Beta vulgaris L.)

Somatic hybridization between different plants through protoplast fusion represent an efficient experimental approach to produce genetically transformed plant species. Electrofution of mesophyll protoplasts in sugar beet was occurred to overcome the barriers faced breeding program of this economically industrial crop Protoplasts were successfully isolated from leave's mesophyll of two varieties of sugar beet (Beta vulgaris L.). Various enzyme solutions were assessed for the cell wall degrading ability. They express different efficiency in isolation of mesophyll protoplasts of var. Baraka. The protoplasts yield was 18 × 104 cell ml-1 using the mixture consisting of 0.5% Cellulase RS, 1.0% Hemicellulase and 0.1% Pectolyase Y-23 with 13% mannitol. A total of 16 hrs. for cell wall digestion, and protoplast viability approached 93%. Protoplasts were isolated from leaf mesophyll of var. Carola using the same enzymatic mixtures. High protoplasts yield 20 × 104 cell ml-1 was obtained, requiring the same period 16 hrs. to approach viability 96%. The protoplasts were spherical in shape, varied in chloroplast distribution, having size ranged 12 – 52 µm. The present study succeeded in electrofusion between Baraka × Carola mesophyll protoplasts, producing somatic hybrid cells under conditions of 1MHz, 1000 Vcm-1, 2 pulses, 1.5 msec./pulse with fusion percent of 73%.

Effect of nanosilver on potato plant growth and protoplast viability

Potato tissue culture is sensitive to ethylene accumulation in the culture vessel. Ag inhibits ethylene action but no information on nanosilver application in potato tissue culture has been published so far. In our study, potato cv. White Desiree was treated with nanosilver (0, 1.0, 1.5, and 2.0 ppm) in vitro. Leaf surface was increased, while stem length and root length decreased. Nanosilver caused also a decrease in the number of isolated protoplasts and in the viability of isolated protoplasts when applied either directly or indirectly.

Effect of Different Cellulase and Pectinase Enzyme Treatments on Protoplast Isolation and Viability in Lilium ledebeourii Bioss.

For overcoming interspecific incompatibility, protoplast combination method is a proper procedure for making a new plant withdesired traits. For this purpose, protoplast preparation is a first and important step. Hence, experiments were conducted to evaluatevarious combinations of cellulose, pectinase and their treatment times on protoplast production and protoplast viability in Liliumledebeourii Bioss. The results of experiment revealed that the protoplast yield was significantly affected by different treatment levels.Cellulase at 4% gave the highest numbers of protoplasts at 3.71×105 protoplast/g FW. Pectinase at 1% gave the highest numbers ofprotoplast. For treatment times, the highest yield of protoplast was with leaf explants treated for 24 h. Analysis of variance indicated thatconcentration, time and three-way interaction of cellulase, pectinase and time were significant at p<0.01. Cellulase at 4% and pectinase at0.2% for 24 h gave the highest viability. Interactions of cellulase × pectinase, cellulase × time, pectinase × time and cellulase × pectinase× treatment time were significant at P≤0.05 for protoplast number. The highest and lowest protoplast numbers were produced in mediacontaining 4% cellulase and 1% pectinase for 24 h (6.65×105 protoplast/g FW) and 1% cellulase and 0.2% pectinase for 12 h, respectively.It’s concluded that, the best treatment for isolation of Lilium protoplast was 4% cellulase and 1% pectinase for 24 h.

Effect of biotic factors on the isolation of Lupinus albus protoplasts

Several tissue types of Lupinus albus L. were investigated as sources for the isolation of protoplasts. Cotyledons from in vitro seedlings were found to yield the highest number of protoplasts compared with leaves, hypocotyls and roots. A combination of the protoplast isolation enzymes, cellulase and Pectolyase Y23, was capable of releasing the highest number of protoplasts compared with a combination of cellulase and Macerase. Protoplast yield increased with increasing cotyledon age but was accompanied by a progressive decline in protoplast viability. The optimal combination of protoplast yield and viability occurred when the protoplasts were isolated from 14- to 18-day-old cotyledons. The ratio between the volume of enzyme solution and the tissue biomass did not affect the protoplast production significantly. This is the first report of the isolation of protoplasts from a lupin cotyledon and, following the procedure described in this paper, an average yield of 1.2 × 106 protoplasts per gram of fresh tissue was obtainable.

Improved electroporation buffer enhances transient gene expression in Arachis hypogaea protoplasts

An electroporation medium containing 50 mM glycine or 10 mM glycylglycine (glygly), 70 mM potassium glutamate, and 0.4 M mannitol was evaluated for its ability to improve transient β-glucuronidase (GUS) expression in immature cotyledonary protoplasts of Arachis hypogaea L. GUS activity in electroporated protoplasts was 8- to 430-fold greater than that obtained using any of other four commonly employed poration media. Analysis of viability and histochemical staining of protoplasts indicated that electroporation using the glycine- or glygly-based poration medium resulted in increased protoplast viability and GUS expression when compared with other poration media. Replacement of glygly with MES or HEPES buffers significantly reduced the level of GUS expression in electroporated protoplasts.Key words: transient expression, electroporation, Arachis, protoplasts, GUS.