Effect of proline and salt stress on organogenesis, growth, proline and carbohydrate content of regenerated plantlets in Citrus sinensis (L.) Osbeck cv. Local orange

The effect of exogenous proline and NaCl stress on organogenesis, growth and certain chemical constituents (soluble carbohydrates and free proline) in Citrus sinensis (L.) Osbeck cv. Local orange in vitro was investigated. Callus cultures of nucleus tissues derived from under developed ovule of immature fruits were used. Primary callus were cultured on different levels of NaCl in the culture media (0, 10, 20, 30, 40 and 50 mM). For interaction experiments, proline was used at 0, 25, 50 and 75 mg L-1, whereas NaCl concentrations were 0, 40 and 50 mM. Organogenic callus was obtained, when this primary callus was cultured on MS medium supplemented with 5.0 mg L-1 BA, adventitious shoots were obtained when the organogenic callus was incubated on MS medium supplemented with BA at 1.0 mg L-1 and NAA at 0.1 mg L-1. The addition of NaCl at 10 mM significantly increased the number and length of adventitious shoots, but high concentration of NaCl significantly reduced the number and length, this effect was alleviated by the exogenous application of proline, in particular at 25mg L-1. Complete plantlets were developed when the adventitious shoots were transferred to half strength MS medium supplemented with 0.1mg L-1 NAA. The addition of NaCl at 10 mM significantly increased plantlet height, leaf number per plantlet, fresh weight of the shoot and root system. However at higher concentration (20 – 50 mM), NaCl significantly decreased all the growth parameters of the plantlets. Proline on its own significantly increased organogenesis, and growth parameters of the plantlets. NaCl significantly decreased soluble carbohydrates of the plantlets and this effect was alleviated by the addition of proline. Proline alone significantly increased carbohydrate levels. NaCl significantly increased free proline of the regenerated plants, and so does exogenous proline treatment. Salt-tolerant plantlets were successfully regenerated, which were transferred to a potting mix containing sand and peat moss (2:1) and grown for 8 months, with a survival rate of 100 %.