Graduate Thesis Or Dissertation
 

Effect of auxins, cytokinins and nutrition on adventitious bud formation in tissues of Douglas-fir grown in culture

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  • Vegetative propagation is most effective when individuals of demonstrated superior characteristic can be propagated. For this reason, a method for the induction of adventitious buds on cultured Douglas-fir shoot tips from 20-25 year old trees was developed. Douglas-fir shoot tips failed to survive and grow on a full strength Murashige and Skoog (M&S) mineral salt medium without growth regulators but would if the KNO3 and NH4NO3 levels were reduced to onefourth original strength. On a medium containing 10-4M N6-benzylaminopurine (BAP) and 0.0 or 1O-7M napthaleneacetic acid (NM) adventitious buds formed on the needle primordia of the shoot tips. These adventitious buds formed only when the basal medium contained one-fourth strength NH4NO3 suggesting that the NH4 ion level in the medium affected adventitious bud formation. Other cytokinins, including kinetin and zeatin when applied at 10-4M would also induce adventitious bud formation, while N6-isopentenylpurine (2iP) even when applied at 10-3M did not. Only a two to three week culture period on the high cytokinin medium was necessary to induce adventitious bud formation. The regeneration of adventitious buds from unorganized callus cultures of Douglas-fir was also studied. Douglas-fir callus cultures were induced from aseptically grown seedlings placed on a one-half strength M&S (macro and microelements) containing 10-5M NAA and 10-6M BAP. Adventitious buds arose on subcultured callus placed on a medium containing 1O5M BAP and either 0.0 or 10-7M NAA. Other cytokinins including kinetin, zeatin and 6-benzyl-9-tetrahydropyrone adenine (SD 8339) were all able to induce adventitious bud formation when applied at 1O-5M, however, 2iP induced bud formation only when applied at 10-4M. NAA levels greater than 106M inhibited adventitious bud formation. Callus cultures formed adventitious buds on a one-half strength M&S medium, and again the NH4 ion concentration was found to affect bud formation. The ability to form buds declined the longer callus was maintained in culture and it was completely lost after one year in culture. In an attempt to understand the role of IAA in bud formation in Douglas-fir tissues in culture, a method for the rapid isolation and quantitation of endogenous IAA levels was developed. This involved extraction of the IAA in methanol, followed by purification of the extract by column chromatography and trace enrichment, separation on HPLC and detection by the natural fluorescence of IAA. The system is sensitive enough to detect picogram amounts of TM in milligram tissue samples. No large differences were found in the endogenous IAA levels in bud forming and non-bud forming cultures. Estimates for IAA in Douglas-fir shoot tips collected before, during and after bud break in the spring showed that endogenous TM levels increase dramatically just before bud break in the spring and then begin to decline slowly. There were also no large differences in the endogenous IAA levels in Douglas-fir shoot tips forming and not forming adventitious buds. Because no large differences were found in either the callus or the shoot tip cultures forming or not forming buds, the role of endogenous TM in regulating bud formation may be questioned. The results also show that this method of IAA analysis can be used to detect and quantify endogenous TM levels in tissues and tissue cultures of Douglas-fir to aid in our understanding of the role of IAA in plant development.
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