Structural studies on cell wall biogenesis-I: A method to produce high-quality protoplasts from alga Micrasterias denticulata, an emerging model in plant biology

Micrasterias denticulate is a freshwater unicellular green alga emerging as a model system in plant cell biology. This is an algae that has been examined in the context of cell wall research from early 1970s. Protoplast production from such a model system is important for many downstream physiological and cell biological studies. The algae produce intact protoplast in a straight two-step protocol involving 5% mannitol, 2% cellulysin, 4mM calcium chloride under a temperature ramping strategy. The process of protoplast induction and behavior of protoplast was examined by light microscopy and reported in this study.

Efficient production of Aschersonia placenta protoplasts for transformation using optimization algorithms

The insect pathogenic fungus Aschersonia placenta is a highly effective pathogen of whiteflies and scale insects. However, few genetic tools are currently available for studying this organism. Here we report on the conditions for the production of transformable A. placenta protoplasts using an optimized protocol based on the response surface method (RSM). Critical parameters for protoplast production were modelled by using a Box–Behnken design (BBD) involving 3 levels of 3 variables that was subsequently tested to verify its ability to predict protoplast production (R2 = 0.9465). The optimized conditions resulted in the highest yield of protoplasts ((4.41 ± 0.02) × 107 cells/mL of culture, mean ± SE) when fungal cells were treated with 26.1 mg/mL of lywallzyme for 4 h of digestion, and subsequently allowed to recover for 64.6 h in 0.7 mol/L NaCl–Tris buffer. The latter was used as an osmotic stabilizer. The yield of protoplasts was approximately 10-fold higher than that of the nonoptimized conditions. Generated protoplasts were transformed with vector PbarGPE containing the bar gene as the selection marker. Transformation efficiency was 300 colonies/(μg DNA·107 protoplasts), and integration of the vector DNA was confirmed by PCR. The results show that rational design strategies (RSM and BBD methods) are useful to increase the production of fungal protoplasts for a variety of downstream applications.

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.

Protoplast production from napier grass and pearl millet triploid hybrids

The objective of this work was to obtain protoplasts from napier grass and pearl millet triploid hybrids as a basis for future studies on chromosomal duplication. Explants were taken from mesophyll of in vitro- and in vivo-cultured plants or from calli of two triploid hybrids (H1 and H2), which were treated with enzymatic solutions containing different concentrations of cellulase R-10 (0.5, 1.0, 1.5 and 2.0%) with an additional 0.2% macerozyme and 0.1% driselase or 1.0% pectolyase Y-23 and 0.5% hemicellulase. Enzymatic digestion was monitored once every hour for five hours. Protoplasts were obtained from in vitro and in vivo leaflets of both triploid hybrids, and in vitro leaflets were the best explant sources. The quantity of produced protoplasts varied according to the hybrid, the enzymatic solution and the treatment time.

Factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum

The present work reports factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum. The usefulness of protoplast isolation is relevant for many different applications and has been principally used in procedures involving genetic manipulation. Osmotic stabilizers, lytic enzymes, incubation time and mycelial age were evaluated in terms of their effects on protoplast yield. The optimal condition for protoplast production included the incubation of young mycelia (48 h) in 0.6 mol l-1 NaCl as the osmotic stabilizer, with 30 mg ml-1 Lysing Enzymes from Trichoderma harzianum for 3 h of incubation. In these conditions protoplasts production was higher than 10(6) protoplatos ml-1 in the digestion mixture, number suitable enough for experiments of transformation in fungi. Sucrose concentrations of 1.2 mol l-1 and 1 mol l-1 were the most suitable osmotic stabilizers for the regeneration after 48 h, with rates of 16.35% and 14.54%, respectively. This study produced an efficient method for protoplast production and reverted them into a typical mycelial morphology using a Colletotrichum lindemuthianum LV115 isolate.

Aspects of isolation underpinning mitotic behaviour in lupin protoplasts

This study reports on the influence of critical isolation factors on the subsequent culture of protoplasts of Lupinus albus L. Protoplasts were isolated from in vitro seedling cotyledons of five early maturing accessions in which protoplast yields and division frequencies appeared to be correlated as a high protoplast yield corresponded with a high division frequency. The overall difference among the accessions for mitosis was non-significant, although the highest yield and division frequency were observed in accession LA132, with Alban giving a significantly lower level. Accession Lucrop produced the lowest number of protoplasts, all of which collapsed during culture. Of the enzyme types used for tissue maceration, Pectolyase Y23, was significantly inferior to Macerase in terms of giving way to mitosis. The extent of division in Macerase-isolated protoplast population was 266% higher than that in the Pectolyase Y23-isolated one. The physiological maturity level of the explant, expressed in terms of developmental age, was optimal when 14–18-day-old seedling cotyledons were used for protoplast production and culture, rather than more mature ones, despite higher protoplast yields in the latter. On K8p medium, the protoplast division frequency was 129% greater when 18-day-old seedling cotyledons were used, than that with any other treatment. This work on protoplast culture of the potentially important lupin species, which is a pulse rich in dietary protein, oil and fibre, allows a further understanding of the biology, with an aim to advance lupin biotechnology.