Knockdown of NON-YELLOW COLORING 1 (NYC1)-like gene or chlorophyllin application enhanced chlorophyll accumulation with antioxidant roles in suppressing heat-induced leaf senescence in perennial ryegrass

Loss of chlorophyll (Chl) and oxidative damages co-occur during heat-induced leaf senescence. This study aimed to determine the functions of Chl catabolic gene, NON-YELLOW COLORING 1 (NYC1)-like (NOL) in regulating heat-induced leaf senescence and to characterize antioxidant roles of a Chl derivative, sodium copper chlorophyllin (SCC), in suppressing heat-induced leaf senescence. In two separate experiments, one by comparing NOL RNAi transgenic and wild-type plants, and the other by analyzing the effects of sodium copper chlorophyllin (SCC, 1 mM) treatment, perennial ryegrass (Lolium perenne) were exposed to heat stress (38/35 oC, day/night) or optimal temperature (25/20 oC). Results showed that both knockdown of LpNOL and application of SCC suppressed heat-induced leaf senescence, as manifested by increased Chl content, reduced electrolyte leakage, and down-regulation of Chl-catabolic genes and senescence-related genes, as well as enhanced antioxidant capacity in the peroxidase (POD) pathway for H2O2 scavenging. Ex vivo SCC incubation protected membranes from H2O2 damage onto mesophyll protoplasts of perennial ryegrass. The suppression of leaf senescence by knockdown of NOL or chlorophyllin application was associated with enhanced chlorophyll accumulation playing antioxidant roles in protecting leaves from heat-induced oxidative damages.

Development of a robust transient expression screening system in protoplasts of Cannabis

Transient expression systems in mesophyll protoplasts have been utilised in many plant species as an indispensable tool for gene function analysis and efficacious genome editing constructs. However, such a system has not been developed in Cannabis due to the recalcitrant nature of the plant to tissue culture as well as its illegal status for many years. In this study, young expanding leaves from aseptic in vitro Cannabis explants were used for protoplast isolation. Factorial designs were used to optimise variables in viable protoplast isolation and transient expression of GFP, with a range analyses performed to determine, and quantify, significantly impacting variables. Viable protoplast yields as high as 5.7 × 106 were achieved with 2.5% (w/v) Cellulase R-10, 0.3% (w/v) Macerozyme R-10 and 0.7 M mannitol, incubated for 16 h. As indicated by the transient expression of GFP, efficiency reached 23.2% with 30 μg plasmid, 50% PEG, 1 × 106 protoplasts and a transfection duration of 20 min. Application of the optimised protocol for protoplast isolation was successfully evaluated on three subsequent unrelated genotypes to highlight the robustness and broad applicability of the developed technique.

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%.