Control of cytokinin autonomy in Phaseolus lunatus callus cultures Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/sx61dp71k

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  • The ability of N,N'-diphenylurea (DPU) to substitute for cytokininactive adenine derivatives in promoting callus growth of Phaseolus lunatus and Nicotiana tabacum has been examined. In general, DPU stimulated callus growth of P. lunatus at high concentrations and the growth of most callus tissues was irregular, while the response of tobacco callus tissue to DPU was more uniform. Genotypic variations in sensitivity and uniformity of callus growth in response to DPU were found among different genotypes of P. lunatus. Tissues cultured on DPU-containing medium for one passage acquired the ability to proliferate in subsequent passages in the absence of either DPU or cytokinin-active adenine derivatives. Corresponding P. lunatus callus tissues maintained on optimal concentration of kinetin and tobacco callus tissue grown on DPU-containing medium remained cytokinin- dependent. However, P. lunatus callus tissues grown on media containing suboptimal or supraoptimal concentrations of kinetin also exhibited cytokinin-autonomous growth when transferred to cytokinin-free media. In studies of the interaction of kinetin and DPU in promoting the growth of P. lunatus callus tissues, it was observed that the effects of kinetin and DPU in promoting callus growth were not additive and that the effect of increasing kinetin concentrations in suppressing the development of the cytokinin-autonomous growth habit was overcome to some extent by the presence of DPU in the culture medium. N⁶-ureidopurines also stimulated vigorous and uniform callus growth of P. lunatus genotypes and the behavior of these callus tissues when transferred to cytokinin-free media was similar to that of callus tissues grown on either benzylaminopurine or kinetin. Optimal concentrations of these compounds suppressed the development of cytokinin autonomy. The use of stronger auxin than indole-3-acetic acid in the tobacco tissue culture media did not significantly alter the response of tobacco callus tissue to DPU. Methodology for the detection of cytokinin biosynthesis (from adenine-8-¹⁴C) in callus tissue has also been examined. Model experiments using Amberlite XAD-2 columns demonstrated the effectiveness and simplicity of the use of this material in preliminary purifications of naturally occurring cytokinins and in the reduction of background label in experiments to detect cytokinin biosynthesis from adenine-8-¹⁴C. The recovery of cytokinin activity from tRNA hydrolysates and from standard solutions applied to Amberlite XAD-2 column has been tested. In addition, the effectiveness of this material in the initial purification of cytokinins from Agrobacterium tumefaciens culture filtrate and from callus tissue of P. lunatus has been examined.
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