Use of tissue culture for the improvement of salt tolerance in pasture legumes

Michael Kevin Smith

1. Conditions were identified for the establishment of callus and cell suspension cultures for cultivars of three species of pasture legumes (Medioago sativa L. cw. Hunter River, Cuf 101, Falkiner, Regen S; Tvdfoliimi vepens L. cw. Ladino, Louisiana, Haifa; and T. fragiferum L. cv. Palestine). Regeneration of whole plants from culture was accomplished for Hunter River, Cuf 101, Regen S, Ladino and Louisiana, and factors influencing differentiation and regeneration were identified. The genotype of the material was a key factor in regeneration which involved a two-step procedure. Callus was first grown on an induction medium with 10-30 µM 2, 4-D and 5-10 uM kinetin and then transferred to a regeneration medium lacking these hormones and containing instead 550 uM inositol and 2 gl"1 yeast extract. The age of callus and origin of explant were also important factors influencing regeneration. 2. The effect of NaCl on growth was examined for whole plants and callus cultures of a salt-sensitive glycophyte (Phaseolus vulgaris L.), a salt-tolerant glycophyte (Beta vulgaris L.) and two halophytes (Striplex undulata D. Dietr., which has salt glands, and Suaeda austvalis (R-Br.) Moq., a succulent). Whole plants were grown in nutrient solution culture at NaCl concentrations of 0.1-250 mM. Callus cultures were initiated from the same seed stock, and similar saline regimes were imposed. Whole plant responses were characteristic for the various types of plants-. P. Vulgaris showed a decrease in growth with increasing salinity; B. vulgaris showed a slight increase in growth at the intermediate salt level and a decrease at higher levels; A. undulata and S. australis showed well-defined growth optima at 62.5 mM and 125 mM. NaCl, respectively. Callus cultures of P, vulgaris and the two halophytes grew very poorly when salinity was increased. Callus of B. vulgaris showed the same tolerance to salt as did the whole plants. Thus salt tolerance of the halophytes depends on the anatomical and physiological complexity of the intact plant while callus from B. vulgaris appears to have a mechanismCs) of salt tolerance which operates at the cellular level. 3. The effect of NaCl was also examined on the growth of callus from M. sativa cvs. Hunter River, Cuf 101, Hasawi and line W75RS, and T. repens cv. Ladino, all of which are regarded as salt sensitive pasture legumes, and T. fragiferum cv. Palestine which is considered a moderately salt tolerant pasture legume. Only M. sativa line W75RS showed some tolerance to NaCl, as callus growth was comparable at 0.1 and 62.5 mM NaCl. Whole plants in nutrient solution culture were grown from the same seed stocks and exposed to the same levels of NaCl as the callus. The responses were very similar to those shown by the callus cultures with the W75RS line showing some tolerance at 62.5 mM NaCl which indicates the validity of using callus cultures to screen for tolerance to NaCl. However, on a yield basis, W75RS grew less than the other cultivars of M. sativa. 4. M. sativa lines with a high incidence of regeneration were established as suspension cultures and used to select for NaCl tolerant lines. Attempts were then made to regenerate plants from these NaCl tolerant calli. Regeneration was severely depressed by screening against NaCl and the only plants'-that' were successfully regenerated were from one W75RS callus exposed to 62.5 mM NaCl. Callus subcultured from the selected line, and the plants regenerated from it, have shown a tolerance to NaCl comparable to the initial population response rather than a markedly improved level of tolerance.

Use of tissue culture for the improvement of salt tolerance in pasture legumes

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