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Young leaves of Kabuli chickpea as well as soybean Xiangdou No.3, which are the current plants that studied in our laboratory were selected as materials. Effects on protoplasts yield and survival rate of different enzyme combination, concentration of D-Mannitol in enzyme combinations, pH of enzyme combinations and enzymolysis time are detected. The results showed that, the best condition for Xiangdou No.3 leaf protoplasts isolation is to rotate the cut materials for 6 hours in enyzme solution under temperature of 27 ℃ and rotate speed of 45 r/min for 6 h. Onozuka R-10 (0.5%), Hemicellulase (0.8%), Macerozyme R-10 (0.8%) in combination with Pectolyase Y-23 (0.4%) dissolving in CPW solution with MES (0.1%) and Mannitol (10%), pH 6.0 was found best for protoplasts isolation of Xiangdou No.3 leaves.The best condition for protoplasts isolation of Kabuli chickpea is to put the cut materials into enzymatic hydrolysate enzymolyse for 7 to 8 hours under temperature of 27 ℃ and rotate speed of 45 r/min on water bath shaker, the optimum combination of enzyme consists of Onozuka R-10 (0.5%), Hemicellulase (0.8%), Macerozyme R-10 (0.8%), MES (0.1%) and Mannitol (10%) dissolved in CPW solution with pH 4.8. The protoplasts prepared with the methods above are used in subcellular location and the effects show well.

Nanoparticles (NPs) in agricultural systems can potentially be used as appropriate candidate for change in growth, development, productivity, and quality of plants. In the present study, we assessed the effect of TiO2 NP concentrations (0, 2, 5, and 10 ppm) on changes of membrane damage indexes like electrolyte leakage index (ELI) and malondialdehyde (MDA) during cold stress (CS) 4 °C in sensitive (ILC 533) and tolerant (Sel 11439) chickpea (Cicer arietinum L.) genotypes. Aggregation of NPs within the vacuole and chloroplast indicated absorbed NPs in seedlings. Bioaccumulation of NPs showed that, under thermal treatments, the sensitive genotype had more permeability to NPs compared to the tolerant one, and TiO2 content was higher during CS compared to optimum temperature. Physiological indexes were positively affected by NP treatments during thermal treatments. TiO2 NP treatments (especially 5 ppm) caused a decrease in ELI during thermal treatments, whereas ELI content under CS treatment increased at 0 ppm TiO2 in both genotypes. Under thermal treatments, although the genotype 11439 showed lower accumulation of MDA than ILC 533 genotype, a significant decrease was observed in MDA content at 5 ppm TiO2. Results showed that TiO2 treatments not only did not induce oxidative damage in sensitive and tolerant chickpea genotypes but also alleviated membrane damage indexes under CS treatment. It was suggested for the first time that TiO2 NPs improved redox status of the genotypes under thermal treatments. New findings possibly would reveal the use of NPs generally or TiO2 NPs especially for increase of cold tolerance in crops.

Changes in bioactive carbohydrates, functional, and microstructural characteristics that occurred in chickpea under soaking, cooking, and industrial dehydration processing were evaluated. Raw chickpea exhibited important levels of raffinose family of oligosaccharides (RFOs), resistant starch (RS) and total dietary fibre (TDF), being insoluble dietary fibre (IDF) the main fraction (94%). The dehydration process increased RFOs (43%), RS (47%) and soluble dietary fiber (SDF) (59%) levels significantly. In addition, a noticeable increase in both fibre fractions was observed, being higher in soluble fibre in (SDF) (59%). The minimum nitrogen solubility of raw flours was at pH 4, and a high degree of protein insolubilization (80%) was observed in dehydrated flours. The raw and processed flours exhibited low oil-holding capacities (1.10 mg/ml), and did not show any change by thermal processing, whereas water-holding capacities rose to 5.50 mg/ml of sample. Cooking and industrial dehydration process reduced emulsifying activity and foaming capacity of chickpea flour. The microstructural observations were consistent with the chemical results. Thus, the significant occurrence of these bioactive carbohydrate compounds along with the interesting functional properties of the dehydrated flours could be considered useful as functional ingredients for food formulation.

Changes in starch, functional, and microstructural characteristics that occurred in chickpea and lentil under soaking, cooking, and industrial dehydration processing were evaluated. Available starch in raw legumes represented 57-64%, and resistant starch (RS) is a significant component. As a result of cooking, available starch contents of soaked chickpea and lentil were significantly increased (21 and 12%, respectively) and RS decreased (65 and 49%, respectively) compared to raw flours. A similar trend was exhibited by dehydration, being more relevant in lentil (73% of RS decrease). The minimum nitrogen solubility of raw flours was at pH 3, and a high degree of protein insolubilization (80%) was observed in dehydrated flours. The raw legume flours exhibited low oil-holding capacities, 0.95-1.10 mL/g, and did not show any change by thermal processing, whereas water-holding capacities rose to 4.80-4.90 mL/g of sample. Emulsifying activity and foam capacity exhibited reductions as a result of cooking and industrial dehydration processing. The microstructural observations were consistent with the chemical results. Thus, the obtained cooked and dehydrated legume flours could be considered as functional ingredients for food formulation.

The frost hardiness of many plants such as chickpea can be increased by exposure to low non-freezing temperatures and/or the application of abscisic acid (ABA), a process known as frost acclimation. Experiments were conducted to study the response over a 14 d period of enriched plasma membrane fractions isolated from chickpea plants exposed to low temperature and sprayed with exogenous ABA. Measurement of the temperatures inducing 50% foliar cell death (LT50), and subsequent statistical analysis suggest that, like many plants, exposure to low temperatures (5/-2 degrees C; day/night) induces a significant level (P <0.05) of frost acclimation in chickpea when compared with control plants (20/7 degrees C; day/night). Spraying plants with exogenous ABA also increased frost tolerance (P <0.05), but was not as effective as low temperature-induced frost acclimation. Both pre-exposure to low temperatures and pre-treatment with ABA increased the levels of fatty acid desaturation in the plasma membrane (measured as the double bond index, DBI). Exposure of chickpea plants to low temperatures increased the DBI by 15% at day 4 and 19% at day 14 when compared with untreated control plants. Application of ABA alone did not increase the DBI by more than 6% at any time; the effects of both treatments applied together was more than additive, inducing a DBI increase of 27% at day 14 when compared with controls. There was a good correlation (P <0.05) between the DBI and LT50, suggesting that the presence of more unsaturated lipid in the plasma membrane may prevent cell lysis at low temperatures. Both pre-exposure to low, non-freezing temperatures and pre-treatment with ABA induced measurable changes in membrane fluidity, but these changes did not correlate with changes in LT50, suggesting that physical properties of the plasma membrane other than fluidity are involved in frost acclimation in chickpea.