ABSTRACTtions suggests that both traits are controlled by relatively few genes. To our knowledge this is the first study demonstrating independent genetic control of the two main traits associated with frost or winter survival. Our results show that it should be possible to incorporate these traits from wild germ plasm into cultivated crop plants by independent selection. These results help explain the lack of progress in improving winter survival through field selection. Furthermore, our study demonstrates relative simplicity of the inheritance of cold acclimation, thus providing avenues for understanding the link between biochemical and genetic aspects of low-temperature stress in crop plants.

Micropropagation of three Echinacea species, E. angustifolia DC., E. pallida Nutt., and E. purpurea Moench., was investigated as a potential means of germplasm preservation of species faced with overcollection in the wild and rapid clonal propagation of elite individuals with unique medicinal or ornamental properties. Very high contamination rates occurred with shoot-tip explants but not with nodal segments. Contamination rates for seed explants were inversely related to the number of seedcoat layers removed, ranging from 100% contamination from intact seeds to near 0% contamination from excised embryos. Dormancy of seed explants was also eliminated when the pericarp and integument were removed. Addition of benzyladenine (BA) to the culture medium induced shoot multiplication and inhibited root formation in all three species. Shoot multiplication rates were low (1-3 shoots per culture) when seed explants were placed on a medium with BA levels low enough to avoid adventitious shoot formation (0.45 μm). Shoot count was higher on half-strength Murashige and Skoog (MS) minerals, while leaf size was greater on full-strength MS minerals. Cultures did not perform well in Woody Plant Medium. Reducing subculture frequency from 4 to 2 weeks increased shoot multiplication from 1.4 to 1.8 shoots per subculture and total shoots produced per subculture after 12 weeks from 2.8 to 23.9. Rooting occurred readily on shoots isolated from E. purpurea cultures and was not promoted by addition of IBA to the rooting medium. Rooting was low and nil on shoots from cultures of E. angustifolia and E. pallida, respectively. Methods described in this study allow rapid multiplication of three Echinacea species and subsequent rooting of E. purpurea. Future improvements in root induction treatments will allow these methods to be used effectively for micropropagation and maintenance of disease-free germplasm of Echinacea species. Chemical names used: N-(phenylmethyl)-1H-purine-6-amine (BA); 1H-indole-3-butyric acid (IBA).

Anatomical events of adventitious root formation in response to root induction medium, observing changes during induction and post-induction stages, were made with microcuttings of 'Gala' apples. Shoot explants on root induction medium containing water, 1.5 µM IBA, 44 mM sucrose, or 1.5 µM IBA + 44 mM sucrose after 4 days of treatment averaged 0, 0.2, 2.2, and 11.9 meristemoids per microcutting, respectively. Meristemoids formed in response to sucrose were confined to leaf gaps and traces. Time-course analysis of root induction with 1.5 µM IBA + 44 mM sucrose over 4 days revealed that some phloem parenchyma cells became densely cytoplasmic, having nuclei with prominent nucleoli within 1 day; meristematic activity in the phloem was widespread by 2 days; continued division of phloem parenchyma cells advanced into the cortex by 3 days; and that identifiable root primordia were present by 4 days. Cell division of pith, vascular cambium, and cortex did not lead to primordia formation. Meristematic activity was confined to the basal 1 mm of microcuttings. Timecourse analysis of post-induction treatment revealed differentiation of distinct cell layers at the distal end of primordia by 1 day; primordia with a conical shape and several cell layers at the distal end by 2 to 3 days; roots with organized tissue systems emerging from the stem by 4 days; and numerous emerged roots by 6 days. Root initiation was detectable within 24 hours and completed by day 4 of the root induction treatment and involved only phloem parenchyma cells. Chemical names used: 1 H -indole3-butryic acid (IBA).

Involvement of pH and IBA on adventitious root initiation was investigated with Malus domestica Borkh. microcuttings. The pH of unbuffered root initiation medium (RIM) increased from 5.6 to 7 within 2 days. Buffering with 2[N-morpholino] ethanesulfonic acid (MES) adjusted to specific pHs with potassium hydroxide prevented pH changes and resulted in a 2-fold higher root count at pH 5.5 compared to pH 7 or unbuffered medium. As pH decreased, lower concentrations of IBA were required to increase root counts. Colorimetric measurement of IBA in buffered RIM showed greater IBA loss and higher root count were associated with lower pH levels in all cultivars. This suggests that IBA loss from RIM depends on medium pH, which affects root count. Root count differences between easy-to-root through difficult-to-root cultivars were not consistent with amount of IBA loss from RIM. Cultivar differences in root count could not be explained solely by IBA loss from RIM.

The influence of root initiation medium pH on root formation was investigated in relation to uptake and metabolism of applied IBA in microcuttings of Malus ×domestica Borkh. `Gala' and `Triple Red Delicious'. Root formation and uptake of H3-IBA were related inversely to root initiation medium pH. Maximum root count (10.3 roots) and IBA uptake were observed at pH 4.0. Regardless of pH, overall root count of `Gala' was higher (13.5 roots) than `Triple Red Delicious' (4 roots). Uptake of IBA was highest at pH 4.0 for `Gala' (1.7% uptake) and at pH 4 and 5 for `Triple Red Delicious' (0.75% uptake). Metabolism of IBA was the same regardless of root initiation medium pH or cultivar examined. One-half of the IBA taken up was converted to a compound that coeluted with IBAsp during high-performance liquid chromatography. Apparently, pH regulates root formation by affecting IBA uptake but not metabolism. The level of auxin in tissue appeared unrelated to root formation between genotypes. Chemical names used: 1H-indole-3-butyric acid (IBA); 5-H3-indole-3-butyric acid (H3-IBA); indole-3-butrylaspartic acid (IBAsp).