Screening of IAA content in jackpine (Pinus banksiana Lamb.) seedlings for purposes of early selection: Development and application of an indole -3 -acetic acid immunoassay.

摘要

The general objective of this dissertation was to examine variation of cambial indole-3-acetic acid (IAA, auxin) concentrations in relation to tree stem growth performance and the possibility of screening IAA for early selection of related inherently superior stem growth in tree improvement by development and application of IAA immunoassay techniques.;Using IAA linked through the C-5 position to a protein carrier, and 5-hydroxy-IAA linked through the C-6 position to a protein carrier as novel immunogens in Balb/c mice, 28 monoclonal antibodies (McAb) against IAA were developed. Eight McAbs possessed the capability to inhibit in vitro oxidation of IAA by peroxidase. One of them elicited against IAA C-5 was selected following measurement of cross-reactivity which indicated high specificity for IAA.;The selected McAb was used to develop an enzyme-linked immunosorbent assay (ELISA) for IAA quantification. The detection range was from 35 fg to 350 pg/assay with an IC50 value of 4.37 +/- 1.08 pg. The detection limit (LOD) (B0 -2 SD) was 0.059 +/- 0.019 pg/assay. The effects of assay conditions, including pH, salt content and sample purification, on IAA ELISA were evaluated and optimized. IAA ELISA was confirmed by combined gas chromatography-mass spectrometry (GC-MS) using 13C6-IAA isotope dilution to give accurate and precise estimates of endogenous IAA content.;Using both ELISA and GC-MS cambial IAA concentration was retrospectively compared with stem growth potential in three half-sib families of jackpine (Pinus banksiana Lamb.) differing in height and diameter growth (slow, intermediate, fast). Endogenous IAA levels were correlated with stem height growth (r2 = 0.562) in all 3-month-old greenhouse-grown seedlings (n = 168), but not with root-collar diameter or seedling fresh weight. The mean IAA content in the fast-growing family (130 ng · g -1 FW) was higher than in the intermediate (110 ng · g-1 FW) and slow-growing (80 ng · g-1 FW) families. Correlations were also obtained for stem height growth (r2 = 0.920), diameter growth (r2 = 0.950) and the volume growth (r2 = 0.985) for 18-year-old field-grown mature trees (9--11 m in height) of the same three half-sib families. The slow-growing family contained the lowest IAA content (600 ng · g-1 FW), and the intermediate height family (1400 ng · g-1 FW) had higher IAA content than the fast-growing family (1050 ng · g-1 FW), in accord with the family of intermediate height-growth potential having the strongest capacity of diameter growth. A close correlation was found between mean family IAA content and mean family tree volume (r2 = 0.965). These studies indicate that endogenous cambial IAA content in growing stems is an indicator of both height and diameter growth potential, thus that cambial IAA content can be screened as an early selection criterion to predict growth performance in jackpine.;The selected McAb for ELISA was found to inhibit peroxidase-catalysed aerobic oxidation of IAA competitively and to promote IAA-dependent elongation in jackpine hypocotyls, an indication that the IAA epitope is the same site recognised by peroxidase when it oxidises the molecule. Peroxidase may be responsive to the variation of IAA levels in the growing stem.