Masters Theses

Date of Award

3-1988

Degree Type

Thesis

Degree Name

Master of Science

Major

Life Sciences

Major Professor

Bob V. Conger

Committee Members

R. N. Trigiano, C. E. Sams, O. J. Schwarz

Abstract

The purpose of this study was to determine if indole-3-acetic acid (lAA) and cytokinins were related to genotypic and gradient responses of orchardgrass (Dactylis glomerata L.) leaf sections in vitro to produce somatic embryos. Plant growth regulator (PGR) concentrations of leaves were analyzed by high performance liquid chromatography (HPLC) and compared in basal sections (lower 3 cm) of the two innermost leaves of two embryogenic subclones and two nonembryogenic genotypes. Plant growth regulator concentrations were also determined and compared between basal and distal (3 cm to 6 cm) leaf sections of embryogenic plants. The ratio of cytokinins/IAA was also compared.

The cytokinins, zeatin riboside (ZR), dihydrozeatin riboside (DHZR), zeatin (Z) and dihydrozeatin (DHZ) were identified and quantified in leaves and summed to yield a value for total cytokinins. Leaves of the two nonembryogenic genotypes contained significantly greater amounts of total cytokinins than those from the two embryogenic subclones on a μg g-1 dry weight basis (10.74 and 8.06 vs. 2.08 and 3.05 respectively). The concentrations of ZR and DHZ, were greater in the nonembryogenic genotypes (7.13 and 4.96 vs. 1.05 and 1.74 for ZR; 0.92 and 0.86 vs. 0.19 and 0.08 for DHZ). Levels of Z and DHZ were not significantly different between nonembryogenic and embryogenic genotypes (0.89 and 0.88 vs. 0.33 and 0.81 for DHZR; 1.81 and 1.28 vs. 0.51 and 0.36 for Z). Cytokinin concentrations in basal leaf sections of the embryogenic genotype were not significantly different from those found in distal sections. Cytokinin concentrations for distal sections (μg g-1 dry weight) were 1.07 and 1.68 of ZR, 1.12 and 0.17 of DHZR, 2.15 and 0.17 of Z and 0.19 and 0.03 of DHZ respectively for the two subclones. There were no significant differences between the two embryogenic subclones or between the two nonembryogenic genotypes. One embryogenic subclone and one nonembryogenic genotype were analyzed on a whole tiller basis and contained 2.83 vs. 4.26 of ZR, 0.45 vs. 0.74 of DHZR, 0.24 vs. 0.75 of Z, 0.01 vs. 0.45 of DHZ, and 3.53 vs. 7.35, respectively of total cytokinins. Concentrations of total cytokinins was significantly higher in the nonembryogenic genotype than in the embryogenic subclone.

Indole-3-acetic acid levels (ng g-1) were not significantly different between basal leaf sections of the embryogenic subclones and the nonembryogenic genotypes (18.32 and 20.93 vs. 22.91 and 26.25 respectively). Distal leaf sections of the embryogenic subclones contained significantly less lAA (9.47 and 2.66) than basal sections. Tillers of the embryogenic genotype contained 16.39 ng g-1 of lAA; whereas, the nonembryogenic genotype contained 12.11 ng g-1. This difference was not significant. The ratio of cytokinins/IAA was significantly different both between basal and distal sections and between basal sections of leaves of embryogenic and nonembryogenic genotypes.

In the embryogenic genotype, embryogenesis was inhibited by exogenously added cyotkinins. Zeatin levels as low as 0.001 μM reduced somatic embryo formation from cultured leaf segments.

These results suggest possible relationships between PGR levels and genotypic and gradient specific responses of orchardgrass leaves in in vitro culture. In particular, cytokinin levels seem to influence genotypic specific responses.

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