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

Abstract

For overcoming interspecific incompatibility, protoplast combination method is a proper procedure for making a new plant with desired traits. For this purpose, protoplast preparation is a first and important step. Hence, experiments were conducted to evaluate various combinations of cellulose, pectinase and their treatment times on protoplast production and protoplast viability in Lilium ledebeourii 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 3.71×10 5 protoplast/g FW. Pectinase at 1% gave the highest numbers of protoplast. For treatment times, the highest yield of protoplast was with leaf explants treated for 24 h. Analysis of variance indicated that concentration, time and three-way interaction of cellulase, pectinase and time were significant at p<0.01. Cellulase at 4% and pectinase at 0.2% 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 media containing 4% cellulase and 1% pectinase for 24 h (6.65×10 5 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 Enzyme Treatments on Protoplast Isolation from Leaves of Vetiver (Vetiveria spp.)

“…It is possible that the higher cellulase concentration used as over-digestion of explants resulted in the decreasing of protoplast yield (Zhu et al, 2005). Moreover, higher concentrations of enzymes might negatively influence the viable protoplasts (Chamani et al, 2012). Similar observations were obtained by Yao et al (2016) who reported that 1.0% cellulase R-10, 0.5% pectolyase Y-23 and 0.6 M mannitol (pH 5.8) was the optimum concentration used to obtain high yield of protoplast for sweet cherry (Prunus avium L.), 4.3x10 6 protoplasts/g FW of explants and 0.5% celluase onozuka RS10 and 0.5% macerozyme R-10 in combination was the suitable enzyme concentration for obtaining high protoplast yields of Citrus reticulata L. (Wulandari et al, 2018).…”

“…They found that 0.5% macerase and 1% cellulysin with 24 h of incubation time was the suitable condition. Chamani et al (2012) reported that a digestion period of 24 h resulted in the highest yield of protoplasts (6.65x10 5 protoplasts/g FW) from Lilium ledebeourii. In addition, incubation time of 24 h was the appropriate for isolation of protoplast from pine (Pinus Iambertiana) when treated with the combination of 0.15% onozuka, 0.05% rhozyme and 0.08% pectinase (David and David, 1979).…”

“…Large amount of viable protoplast can be obtained with enzymatic methods. Less cell breakage and osmotic shrinkage occur compared with mechanical method (Chamani et al, 2012). It is often found more effective to apply a combination of enzymes, such as cellulase, hemicellulase and pectinase, to obtain optimum isolation.…”

“…Cellulase and hemicellulase generally used to break the plant cell wall, while pectinase used for the separation of cell aggregates (Cove, 1979;Sija et al, 2016). The success of protoplast isolation depends on several factors that affect the release of protoplast in plants, such as the concentration and combination of enzyme, duration of enzyme incubation, pH and osmoticum of the enzyme solution, temperature and the extent of cell wall thickening (Chamani et al, 2012). These factors, especially enzyme treatments, influence different species and genotypes differently.…”

Biochemical Prospects of Various Microbial Pectinase and Pectin: An Approachable Concept in Pharmaceutical Bioprocessing

“…Enzymatic preparations of pectin are also implemented in various pharma-based industries for the development of low-methoxy pectin that is beneficial for diabetic patients ( 14 , 16 ). Purified forms of pectinase, which are commercially isolated from different fungal species, have a great role in plant protoplast culture studies as they help to generate good yields of viable protoplast when treated with cellulose ( 17 ). However, like other enzymes, this one is also more activated during its commercial production at 30 to 50°C and a pH of 4.5 to 8.5, but the effectiveness is more specific to the source of particular microorganisms and other environmental factors ( 10 ).…”