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Mangoes (Mangifera indica L.) are an important kind of perennial fruit tree, but their biochemical testing method and transformation technology were insufficient and had not been rigorously explored. The protoplast technology is an excellent method for creating a rapid and effective tool for transient expression and transformation assays, particularly in plants that lack an Agrobacterium-mediated plant transformation system. This study optimized the conditions of the protoplast isolation and transformation system, which can provide a lot of help in the gene expression regulation study of mango. The most beneficial protoplast isolation conditions were 150 mg/mL of cellulase R-10 and 180 mg/mL of macerozyme R-10 in the digestion solution at pH 5.6 and 12 h of digestion time. The 0.16 M and 0.08 M mannitol in wash solution (WI) and suspension for counting (MMG), respectively, were optimal for the protoplast isolation yield. The isolated leaf protoplasts (~5.4 × 105 cells/10 mL) were transfected for 30 min mediated by 40% calcium-chloride-based polyethylene glycol (PEG)-4000-CaCl2, from which 84.38% of the protoplasts were transformed. About 0.08 M and 0.12 M of mannitol concentration in MMG and transfection solutions, respectively, were optimal for protoplast viability. Under the florescence signal, GFP was seen in the transformed protoplasts. This showed that the target gene was successfully induced into the protoplast and that it can be transcribed and translated. Experimental results in this paper show that our high-efficiency protoplast isolation and PEG-mediated transformation protocols can provide excellent new methods for creating a rapid and effective tool for the molecular mechanism study of mangoes.

Ananas comosus var. bracteatus (Ac. bracteatus) is a typical leaf-chimeric ornamental plant. The chimeric leaves are composed of central green photosynthetic tissue (GT) and marginal albino tissue (AT). The mosaic existence of GT and AT makes the chimeric leaves an ideal material for the study of the synergistic mechanism of photosynthesis and antioxidant metabolism. The daily changes in net photosynthetic rate (NPR) and stomatal conductance (SCT) of the leaves indicated the typical crassulacean acid metabolism (CAM) characteristic of Ac. bracteatus. Both the GT and AT of chimeric leaves fixed CO2 during the night and released CO2 from malic acid for photosynthesis during the daytime. The malic acid content and NADPH-ME activity of the AT during the night was significantly higher than that of GT, which suggests that the AT may work as a CO2 pool to store CO2 during the night and supply CO2 for photosynthesis in the GT during the daytime. Furthermore, the soluble sugar content (SSC) in the AT was significantly lower than that of GT, while the starch content (SC) of the AT was apparently higher than that of GT, indicating that AT was inefficient in photosynthesis but may function as a photosynthate sink to help the GT maintain high photosynthesis activity. Additionally, the AT maintained peroxide balance by enhancing the non-enzymatic antioxidant system and antioxidant enzyme system to avoid antioxidant damage. The enzyme activities of reductive ascorbic acid (AsA) and the glutathione (GSH) cycle (except DHAR) and superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were enhanced, apparently to make the AT grow normally. This study indicates that, although the AT of the chimeric leaves was inefficient at photosynthesis because of the lack of chlorophyll, it can cooperate with the GT by working as a CO2 supplier and photosynthate store to enhance the photosynthetic ability of GT to help chimeric plants grow well. Additionally, the AT can avoid peroxide damage caused by the lack of chlorophyll by enhancing the activity of the antioxidant system. The AT plays an active role in the normal growth of the chimeric leaves.