Enhanced anther culture efficiency of indica rice (Oryza sativa L.) through modification of the culture media

The production of microspore-derived green plants from anther culture of indica rice is generally very low compared with japonica cultivars. A modified anther culture medium, consisting of a higher KNO3 content (31 mM) and casein hydrolysate (CH, 500 mg/l) but without ammonium salts, was tested in comparison with a medium consisting of the widely-used N6 medium nitrogen background, using four indica x indica F-1 hybrids as lest materials. Green plant regeneration frequency was at least three-fold higher in the microspore-calli derived from the former medium than in those derived from the modified N6 medium. More than 700 microspore-derived plants were raised in the field. Another study was carried out using indica x japonica and indica x javanica F-1 hybrids. The results indicated that a medium with higher (3.5 mM) ammonium sulphate may induce a higher frequency of anthers with microspore-calli but not necessarily lead to a larger number of green-plant regenerating calli. Subsequently, using the indica cv. 'IR-43' as the test material, use of a lower level (1.75 mM) of (NH4)(2)SO4, in addition to KNO3 (31 mM), was found to be better than CH (500 mg/l) for anther-response as well as green plant regenerability of the derived microspore-calli. Nitrate-nitrogen or ammonium-nitrogen alone elicited poor response. Twenty-five media involving combinations of KNO3 (20-34 mM) and (NH4)(2)SO4 (1-3 mM) were tested for their effects on anther response. Combinations involving KNO3 (31-34 mM) and (NH4)(2)SO4 (2.0-2.5 mM) were found superior not only for achieving greater anther response but also, for subsequent green-plant regeneration. This contrasts with the 28 mM of KNO3 and 3.5 mM of (NH4)(2)SO4 in the widely-used N6 medium developed for japonica rice. Other than potassium nitrate and ammonium sulphate, and potassium phosphate to some extent, the levels of other inorganic salts tested did not make any significant difference to the process of anther response. Based on these results, modified media with three levels of ammonium sulphate were tested for anther culture efficiency of indica x japonica and indica x javanica derivatives (F(3)s). Microspore-calli derived from a medium of a lower (1.75 mM) level of (NH4)(2)SO4 showed a higher regeneration potential overall than those derived from a higher (3.5 mM) level. A revised medium has been suggested, on the basis of these results, for the realization of improved anther culture efficiency and, consequently, improved feasibility of using doubled haploids in genetic and breeding research with indica rice.