Improving Vigor in shrunken-2 Corn Seedlings

The shrunken-2 (sh2) mutant of maize (Zea mays L.) increases sucrose and reduces starch in developing endosperm. An associated trait is poor seed and seedling vigor in seeds containing the mutation. The specific effects of sh2 mutant endosperm on embryo and seedling vigor were determined by analyzing seeds that contained either concordant wild-type or nonconcordant combinations of mutant and wild-type embryo and endosperm genotypes. The nonconcordant seed types that contained a wild-type embryo in association with a sh2 mutant endosperm or a sh2 mutant embryo in association with a wild-type endosperm were generated using the TB-3La translocation chromosome in which a wild-type Sh2 gene is attached to the centromeric portion of a B chromosome. Under stress conditions (complex stress vigor test), the seeds with mutant endosperm had lower germination, seedling fresh and dry weight, and index of conductivity than seeds with wild-type endosperm. Mutant endosperm and embryos excised from mutant endosperm imbibed more water than wild-type endosperm or embryos excised from wild-type endosperm. Because of the high concentration of osmotic solutes in the mutant endosperm, a rapid water uptake may induce a membrane disorganization. Leachate conductivities of seeds with mutant endosperm were higher than seeds with wild-type endosperm. In addition, a higher sucrose content and a lower raffinose to sucrose ratio were measured in the wild-type embryos associated with mutant endosperms than in the normal embryos excised from concordant wild-type seeds. These results suggest that a high rate of water uptake caused by the elevated concentration of osmotic solutes in seeds with mutant endosperms may affect membrane integrity during imbibition. Alternatively, the lower raffinose to sucrose ratio present in the mutant endosperm class might affect stabilization of cell membranes during seed desiccation. Embryos cultured in media containing 10% starch or no carbohydrate produced smaller seedlings than embryos cultured in 5% or 10% sucrose. Wild-type embryos excised from mutant endosperms exhibited lower germination in 0% and 5% sucrose media than embryos from concordant seed, indicating that reduced water uptake rates associated with lower external osmotic potential (10% sucrose) can improve vigor of embryos associated with sh2 mutant endosperm. The reduced vigor of embryos and seedlings that develop in association with sh2 mutant endosperm can be traced to the physiological and biochemical effects of the elevated sucrose levels present during seed formation and imbibition. reserves are noted changes in sh2 seeds, the direct effect of the mutation on germination and subsequent seedling vigor is not clearly understood. Increased seed leakage after imbibition has been associated with membrane damage in cotyledons of soybean (Schoettle and Leopold, 1984). Heat-killed embryos have greater water uptake due to changes in fluidity or ordering of the membranes (Murphy and Noland, 1982). Koster and Leopold (1988) proposed that raffinose may prevent crystallization of the sucrose during dehy- dration, improving membrane protection and stabilization. Seed vigor of maize embryos was correlated with raffinose concentra- tion (Bernal-Lugo and Leopold, 1992). A high raffinose to sucrose ratio in maize seeds was highly correlated with membrane stability during drying (Chen and Burris, 1990). The characteristics of the endosperm affect the embryo during germination and subsequent seedling growth. The structure and chemical composition of the endosperm may alter the quality and quantity of the metabolites available for the embryo, seed osmotic potential, and germination. The embryo is responsible for the initial growth of the axes and subsequent control of reserve mobilization (Bewley and Black, 1978). Poor germination and seedling vigor of sh2 seeds is an enigma. All current molecular and biochemical data show that the sh2 gene is expressed only in the endosperm. Although the embryo and seedling contain starch and have ADP-glucose pyrophosphorylase activity, this activity is independent of the sh2 mutation in seedlings. Clearly, seedling vigor is reduced as a consequence of the seed mutation. This may be explained by the association between the endosperm and embryo as discussed above. The question can be addressed by creating seeds that contain a mutant endosperm and wild-type embryo. If the poor germination of sh2 is due to endosperm, then the genetic constitution of the sh2 locus in the