Evaluation of salt tolerance in hybrid progeny of apple rootstock Malus prunifolia × R3

【Objective】With the influence of global climate change and human production activities, the scope of soil salinization in China is expanding. Soil salinization creates problems in apple growth and fruit yield and quality formation and has become one of the important factors restricting the development of apple industry in China. In the actual production of apples, the salt tolerance of seedlings is weak, and rootstocks can provide salt-tolerant roots for apples. The quality of rootstocks directly affects the absorption of nutrients and the adaptability of fruit trees to adversities. Therefore, rootstocks are the main determinant of salt tolerance in grafted plants. China’s apple production model used to be arborization cultivation. With the continuous advancement of technology, arborization cultivation has exposed many problems and has gradually been replaced by dwarfing and dense planting. However, most of the common dwarfing rootstocks commercially available are introduced from abroad. Due to the climatic limitations of the birthplace, there are certain limitations in introducing them into domestic production, such as poor adaptability and weak stress resistance. Therefore, it is urgent to breed excellent dwarfing rootstock varieties in China. In this study, the salt tolerance of the hybrid offspring of Malus prunifolia × R3 was evaluated and its salt tolerance was comprehensively analyzed. The study will provide valuable reference for the breeding of dwarf rootstocks with strong salt tolerance.【Methods】In this study, tissue cultured seedlings of five asexual reproduced rootstock materials were used. They were k15 and m2, which were selected from the hybrid offsprings of apple rootstock Malus prunifolia × R3 by drought resistance, M. prunifolia, M9-T337 and M26. The seedlings were transferred to the rooting medium for growth for 40 days, and transplanted into an 8 × 8 cm2 nutrient pot after hardening seedlings, and potted plants were placed in incubator with a constant temperature and light condition. After the seedlings grew about 7-8 fully expanded true leaves, they were moved to a hydroponic pot containing 6.5 L of 1/2 concentration of Hoagland nutrient solution, and the nutrient solution was refreshed every 3 days. After one week of pre-adaptation, the even-sized healthy plants were selected for hydroponic salt treatment. The treatment was divided into two groups: (1) Control (CK), with 1/2 Hoagland nutrient solution; (2) Salt treatment (ST), with 150 mmol·L-1 NaCl added in the nutrition solution. Nutrient solution was refreshed every 3 days over a period of 15 days. During the treatment, the photosynthetic rate was measured every 3 days. At the end of the treatment, 10 plants of each strain in the control group and the treatment group were taken to determine the plant height, leaf number, dry and fresh weight. Membrane leakage, chlorophyll, chlorophyll fluorescence, of fresh functional leaves were measured and NBT and DAB staining was observed. Fresh roots were taken to determine root activity and analyze root architecture. The fully mature leaf samples were wrapped in tin paper, immediately frozen in liquid nitrogen and stored at -80 ℃. The contents of MDA, H2O2, O2 - , activities of antioxidant enzymes and amino acid content in each strain were determined. The contents of Na+ and K+ in roots, stems and leaves were determined with dry samples after dry weight determination.【Results】In the control group, there was no significant difference between the five rootstocks through the phenotype. In the salt treatment group, the phenotype of M26 appeared earliest, with severe wilting and necrosis of the leaves on the 9th day, followed by the m2 with brown spots of the leaves on the 10th day. Subsequently, on the 12th day of treatment, salt stress phenotype appeared in Malus prunifolia, k15, and M9-T337. The roots of the salt treatment group were brown, and the root area decreased compared with the control group. Compared with the control group, plant height and leaf number in each strain of the treatment group decreased, and the dry and fresh weights in the treatment group were significantly reduced. Membrane leakage and MDA content in each strain in the salt treatment group increased, while root activity decreased. Results of the NBT and DAB staining and O2 - and H2O2 determination suggested that each strain under salt treatment accumulated a certain amount of reactive oxygen species. Activities of SOD, POD and CAT in each strain under salt treatment increased. At day 3 of salt stress treatment, Pn, Tr, Gs and Ci in each strain were decreased significantly by salt treatment. Total chlorophyll content in all strains in the salt stress group decreased, among which M. prunifolia had the smallest decrease and M26 had the largest decrease. Fv/Fm value in k15, M. prunifolia and M9-T337 decreased less than the other two lines after salt treatment, suggesting smaller degree of photosystem damage in the former two strains. Compared with the control group, the Na+ content in roots, stems and leaves of M26 had the highest increase, and the increase was relatively small in M. prunifolia and k15; the K+ content in each part showed different degrees of decrease after salt treatment. Among the strains, M26 had the largest decrease, while M. prunifolia and k15 had the smallest change. Salt stress significantly increased Na+ /K+ ratio in each strain, and the increase in M. prunifolia and k15 was relatively small. The contents of Pro, Tyr and Phe increased significantly after salt treatment, while the contents of Gly, Leu and Asp decreased. By comprehensive analysis of growth, photosynthetic parameters, antioxidant enzyme activities, mineral element and amino acid contents and other 16 related indicators, the average membership function value of each strainwas calculated. The results showed that the average membership function value of k15 was the highest, indicating that k15 was least affected by salt stress, indicating its salt tolerance was the strongest among the five apple strains. The average membership function value of M26 was the lowest, indicating that its salt tolerance was the weakest.【Conclusion】The results showed that k15 showed strongest salt tolerance among the five strains and M26 had the poorest salt tolerance. Under salt stress, plant growth, root area, root activity and dry and fresh weights decreased. Membrane leakage and MDA content increased, with the accumulation of reactive oxygen species and the increase of antioxidant enzyme activities. Under salt stress, photosynthesis was significantly inhibited with decrease in chlorophylls, while Na+ content increased significantly, K+ content decreased, causing Na + /K+ imbalance; the contents of Pro, Tyr and Phe increased significantly, while the contents of Gly, Leu and Asp decreased. However, due to the differences in resistance among strains, the range of changes in different indicators differed. Through the comprehensive analysis of membership function, the order of salt tolerance of the 5 strains was: k15 ＞ M. prunifolia ＞ M9-T337 ＞ m2 ＞ M26. Therefore, the hybrid k15 of M. prunifolia × R3 has strong salt tolerance and is expected to become a new high-quality rootstock resource.