Effect of Enzyme Treatments on Protoplast Isolation from Leaves of Vetiver (Vetiveria spp.)

Protoplast isolation is a first and important step for establishing a new plant with desired traits through protoplast fusion technology. This experiments were conducted to evaluate various concentration of enzymes and incubation time on protoplast yield and viability in two vetiver ecotypes, Kamphaeng Phet 2 (Vetiveria zizanioides Nash) and Prachuap Khiri Khan (V. nemoralis A.Camus). The results revealed that protoplast yields were significantly affected by different enzyme treatments. The highest protoplast yield (6.12x105 protoplasts/ml) and high viability (98.61%) in Kamphaeng Phet 2 was obtained through the process of cell wall digestion when treated with enzyme solution containing 0.5% (w/v) cellulase onozuka R-10 and 0.5% (w/v) macerozyme R-10 in combination. While, the optimal enzyme solution for protoplast isolation from leaves of Prachuap Khiri Khan was the combination of 1.0% (w/v) cellulase onozuka R-10 and 0.4% (w/v) macerozyme R-10, resulting in the highest yield (6.80x105 protoplasts/ml) and viability (96.56%) of protoplasts. Meanwhile, incubation time of 24 h with the optimal enzyme solution resulted in the highest protoplast yields of both ecotypes. Our findings have the potential to generate an efficient protocol to isolate the protoplast from leaves of vetiver which can be used for further research studies in protoplast culture and fusion for vetiver improvement. Keywords: Cellulase onozuka R-10, Macerozyme R-10, Protoplast isolation, Vetiver

Isolation of Protoplasts from Nepenthes - A Plant Carnivore

Protoplasts were isolated from the lamina of greenhouse grown Nepenthes ampullaria and the hybrid N. ‘Rokko’ Exotica in order to develop a protocol for protoplast isolation suitable for wild species of Nepenthes. Various molarities utilizing mannitol or sorbitol and different enzyme mixtures and concentrations as well as incubation times were evaluated to maximize protoplast yield and viability. The most effective treatment, a 4 hrs incubation at 40 rpm and 25°C in a solution consisting of 0.5 M sorbitol, 5% cellulase ‘Onozuka’ R-10, 0.5% macerozyme R-10, and 0.3% pectolyase Y-23, generated 4.35 × 106 protoplasts/ gfw of which 62.1% were viable. Culture was attempted in respect of regeneration of the cell wall, however, no cell division was observed. Plant Tissue Cult. & Biotech. 24(1): 93-100, 2014 (June) D. O. I. http://dx.doi.org/10.3329/ptcb.v24i1.19217

Effects of environmental preconditioning, donor tissue and isolation conditions on tomato (Lycopersicon esculentum Mill.) protoplast yield

The effects of soil or <em>in vitro</em> grown plants, pretreatment conditions, donor tissue and isolation procedure on protoplast yield from cotyledons and leaves of tomato cv. 'Perkoz' and 'Zorza' were studied. The highest protoplast yield of 1.5 x 10⁷/g FW was obtained from leaves of <em>in vitro</em> grown plants. Low light intensity during donor plants <em>in vitro</em> culture and dark pretreatment were essential for successful protoplast isolation while cold pretreatment was not. Tissue preplasmolysis prior to transfer to enzyme mixture increased 4-fold the number of isolated protoplasts. Glycine and bovine serum albumin in the isolation medium did not significantly influence the protoplast yield.

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.

The Transformation in Trichoderma viride Protoplasts by Restriction Enzyme Mediated Integration (REMI)

The transformation conditions of the protoplasts from Trichoderma viride mediated by restriction enzyme were studied in this paper. The optimum generation conditions of protoplasts were as followed: 8 mg/ml glucanex was added into the phosphate buffer (pH 6.98), the mycelial that cultured for 24 hr was hydrolyzed for 4 hr at 30°C under 40 r/min shaking speed. The protoplast yield was 4.7×107 cfu/mg. The regeneneration rate of protoplast was 14.5% on CM medium contained 0.3 mol/L KCl and 0.3 mol/L Inositol. Transformants were obtained by transfering hygromycin B resistance gene into T. viride by restriction enzyme mediated integration (REMI), The preliminary identification of the transformants indicated that the exogenous gene had been integrated into T. viride genome.

Isolation and transformation ofTrichoderma virideprotoplasts

The conditions for protoplast isolation and regeneration fromTrichoderma viridewere studied. Protoplasts were optimally isolated when mycelia ofT. viridethat had been cultured for 24 h were digested with 4 mg/ml Glucanex in phosphate buffer (pH 6.98) for 4 h at 30°C, resulting in a protoplast yield of 4.7×107cfu/mg. The maximum regeneration ratio (14.5%) was obtained in mycelia culture medium containing 0.3 mol/l KCl and 0.3 mol/l inositol. In addition, a plasmid pCSSNCC1 carrying a hygromycin resistance gene and an elicitor-producing gene was transformed intoT. virideprotoplasts, with an efficiency of 1–2 transformants/μg DNA. The hygromycin-resistant transformants were determined by PCR and the elicitor protein was detected by ELISA. The results indicate that the elicitor protein was expressed stably inT. viride.

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.