Optimization of macro-element composition in the medium for in vitro propagation of apple dwarfing

Abstract:【Objective】‘SH6', a representative Chinese apple dwarfing rootstock, was used in this study to investigate the effect of macro-elements in tissue culture medium on in vitro subculture, in order to improve the efficiency of tissue culture. Growth regulators and carbohydrates have been widely studied in tissue culture research. However, macro-elements have also an important effect on tissue culture. Macro-elements play indispensable roles in the rapid propagation of apple dwarfing rootstock. Reasonable dosage and composition of macro-elements are important for rapid propagation system.【Methods】A five-factor quadratic regression orthogonal rotation design(1/2) was used to construct the experiment. Concentrations of macro-elements in MS medium including KNO3, NH4NO3, MgSO4· 7 H2O,KH2PO4 and CaCl2· 2 H2O were set as independent variables. The amounts of trace-element followed the standard MS medium. 6-BA at 1.0 mg · L-1 and IBA at 0.1 mg · L-1 were used as growth regulators, and the pH value was adjusted to 5.8. The temperature of the culture chamber was controlled at(232) ℃;the photoperiod was 16 h/8 h(day/night); the light intensity was 30 µmol · m-2· s-1. Statistical analysis was carried out after 35 days of culture. The subculture coefficient and the average height of seedlings as dependent variables were recorded. The regression models of subculture coefficient and average seedling height were built based on the experiment results. Principal component analysis was carried out for studying the order of the effect of independent variables on the dependent variables. Single-factor analysis was carried out for studying the effects of each independent variable on subculture coefficient and seedling height. Two-factor interaction analysis was carried out for studying the combined effect of the two independent variables on the dependent variables. Frequency analysis was carried out with 3 125 coded combinations for obtaining the possible change area scope of the dependent variables. The optimized proposal of macro elements was predicted based on frequency analysis under a given target objective value. The verification experiment was performed to test the predicted results.【Results】The reliable models(p < 0.01) of subculture coefficient and average height of SH6 seedlings were constructed based on the five variables. During the culture process, the subculture coefficient and the average height of seedlings were affected by macro elements significantly. The results of principal component analysis show that the order of degree of the effect on subculture coefficient was KNO3> NH4NO3> MgSO4·7 H2O >KH2PO4> CaCl2· 2 H2O, and the order of degree of effect on average seedling height was KNO3>NH4NO3> CaCl2· 2 H2O > MgSO4· 7 H2O > KH2PO4. The results of single-factor analysis show that the subculture coefficient decreased with the increase in the amount of KNO3, NH4NO3 and MgSO4· 7 H2O,whereas it decreased at first and then increased with the increase in the amount of KH2PO4 and CaCl2·2 H2O. The average height decreased with the increase in the amount of KNO3 and NH4NO3, whereas it decreased at first and then increased with the increase in the amount of MgSO4· 7 H2O, KH2PO4 and CaCl2· 2 H2O. The results of two-factor interaction analysis show that the regeneration coefficient was significantly influenced by the interaction of NH4NO3and CaCl2· 2 H2O. The subculture coefficient increased slowly with the increase in the amount of NH4NO3 and CaCl2· 2 H2O, but with increases in the amounts of CaCl2· 2 H2O and NH4NO3, the subculture coefficient increased greatly. The average height was significantly affected by three groups of two-factor interactions. In the interaction of KNO3 and NH4NO3, the average height decreased with the increase in the both factors. In the interaction of KNO3 and KH2PO4, irrespective of changes in KH2PO4, the average height decreased with the increase in the amount of KNO3. In the interaction of KNO3 and CaCl2· 2 H2O, the average height decreased with the increase in KNO3 but increased with the increase in CaCl2· 2 H2O. Two optimized proposals were predicted based on the subculture coefficient > 4 and average height > 3 cm. Except KH2PO4, there was no overlap between the same factor in different proposals. Depended on the optimized proposal of subculture coefficient > 4, the result of the verified experiment was subculture coefficient of 4.2 and average height of 2.6 cm. It showed that there was no significant difference between theoretical prediction and experimental verification, but one proposal hardly met two or more target values.【Conclusion】The in vitro regeneration culture of apple dwarfing rootstock SH6 was significantly influenced by the amount of macro-elements in MS medium. The main factors affecting the efficiency of tissue culture were KNO3 and NH4NO3. In vitro subculture of SH6 during 35 days with the result of subculture coefficient of 4.2 and the average height of 2.6 cm was obtained by the optimized macro-element proposal composed of KNO31 535 mg · L-1, NH4NO31 430 mg · L-1, MgSO4· 7 H2O 310 mg · L-1, KH2PO4172 mg · L-1 and CaCl2·2 H2O 445 mg·L-1.