Freeze-dried mouse spermatozoa are capable of participating in normal embryonic development after injection into oocytes. When the freeze-dried spermatozoa are used as a method for storage of genetic materials, however, it is essential to assure the relevance of long-term preservation over several decades or centuries. Thus, we applied the theory of accelerated degradation kinetics to freeze-dried mouse spermatozoa. Thermal denaturation kinetics were determined based on Arrhenius plots derived from transition-state theory analysis at three elevated temperatures: 30, 40, and 50 degrees C. Accelerated degradation kinetics were calculated by extrapolation of Arrhenius plots. This theory also is being applied to the long-term stability of drugs. The estimated rate of development to the blastocyst stage at 3 and 6 mo and at 1, 10, and 100 yr of sperm storage at 4 degrees C were 21.60%, 7.91%, 1.00%, 0%, and 0%, respectively. At -80 degrees C, estimated development rates to the blastocyst stage that would be expected after 100 yr of storage did not decline significantly. In addition, after 3 or 6 mo of storage at 4 or -80 degrees C, preimplantation development of the embryos derived from intracytoplasmic sperm injection (ICSI) was examined. The actual developmental rates to the blastocyst stage from ICSI by freeze-dried sperm stored for 3 mo at 4 and -80 degrees C were 21% and 62%, respectively, and the rates for such sperm stored for 6 mo were 13% and 59%, respectively. These results indicate that the determination of accelerated degradation kinetics can be applied to the preservation of freeze-dried mouse spermatozoa. Furthermore, for long-term preservation, freeze-dried mouse spermatozoa appear to require being kept at lower than -80 degrees C.

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SynopsisA genetic linkage map of guinea grass ( Panicum maximum Jacq.) was generated with nine of the AFLP markers found to be associated with apospory. These aposporyassociated markers were assigned to a linkage group having previous association with microsporogenesis of the aposporous guineagrass cultivar 'Natsukaze'. An aposporous linkage group was constructed utilizing 38 AFLP markers. Embryo sac analysis revealed that sexual and apomictic embryo sacs occurred at a frequency of 1 : 1, indicating simple inheritance of a single major gene controlling apospory in guineagrass. In addition, utilizing 56 AFLP primer combinations and 41 RAPD primers, 39 linkage groups and 360 simplex marker loci were assigned to the genetic map of the 'Natsukaze' cultivar. These markers covered 1703.5 cM of the autotetraploid guineagrass genome (2n = 4x = 32), with an average spacing of 4.7 cM. These tightly linked markers to apospory locus in guineagrass could be a powerful tool for marker-assisted selection of apospory and map-based cloning of the apospory gene.

We investigated whether or not lettuce growth was inhibited by diffused L-3-(3,4-dihydroxyphenyl)alanine (L-DOPA), an allelochemical exuded from the roots of velvetbean (Mucuna pruriens (L.) DC. var. utilis) cultivars using a modified plant-box bioassay. For all the cultivars and one accession examined L-DOPA diffused from the roots and caused radicle and hypocotyl growth inhibition. A high correlation co-efficient (r ¼ 0.838 to 0.982) was observed between L-DOPA concentration and lettuce seed sowing distance. L-DOPA diffused equally in all directions from roots at 0 mm position (close to root surface) in the plantbox, while the inhibition (%) of lettuce radicle growth gradually decreased with distance from the roots. For all cultivars the concentration of L-DOPA was significantly different at 0 mm position: being highest in cv. preta (167 lg/ml) and lowest in cv. jaspeada and cv. ana (13 lg/ml). The correlation between lettuce radicle growth inhibition and concentration of diffused L-DOPA was high (r ¼ 0.856 to 0.966) in all cultivars and accession examined. However, the concentration of diffused L-DOPA did not correlate with the fresh weight concentration of L-DOPA measured in roots. The lettuce radicle growth inhibition from mucuna diffused L-DOPA was very similar that induced by synthetic L-DOPA, suggesting that diffused L-DOPA was the allelochemical responsible for growth inhibition.

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