Author

Fuyu Xu

Date of Award

2002

Level of Access

Open-Access Thesis

Degree Name

Master of Engineering (ME)

Department

Forest Resources

Advisor

Michael S. Greenwood

Second Committee Member

Katherine K. Carter

Third Committee Member

Keith W. Hutchison

Abstract

Loblolly Pine is the most important and widely cultivated timber species in the southern United States. Due to its fast growth, it is extensively planted for lumber and pulpwood. Vegetative propagation will enhance gains from genetic improvement of tree species. Rooted-cutting is at present the most reliable non-somatic embryogenesis method for cloning specific genotypes. However, an abrupt decline of adventitious rooting capacity has hindered the application of vegetative propagation in loblolly pine. Unraveling the rooting mechanism may facilitate a way to overcome this barrier. Regulation of lateral and adventitious root formation by auxin has been demonstrated through the application of exogenous auxin to roots. Natural lateral root initiation may depend on the localization and redistribution of IAA at the root tip. We studied spatial and temporal responses of lateral root formation to exogenous NAA (1- naphthaleneacetic acid) on the primary roots of loblolly pine seedlings. A significant increase in the frequency and the growth of lateral root primordium (LRP) in response to NAA pulses could be detected at 24 and 48 h. The region 0.5-2.0 cm behind from root tip responds to auxin treatments, but the most responsive region is 0.5-1.5 cm with the largest number of LRPs at both 24 and 48 h. Four poles of primary xylem are often seen in loblolly pine roots. Positioning of successive LRP formation in a given vascular pole is not random. The probability that the next LRP will develop in the same file is nearly 0. Treatment with an exogenous pulse of NAA increases the probability that the next primordium will form in the same file, which suggests that LRP formation may deplete the local auxin concentration in the vascular pole, lowering the probability of successive LRP forming close by. In loblolly pine, there exists a stepwise decrease in auxin-induced rooting response from root, hypocotyl, to epicotyl in young seedlings. Competence to organize root meristems is normally confined to cells in pericycle or vascular parenchyma located centrifbgal to primary xylem poles. Auxin can induce cellular reorganization and cell division in all parts of the seedling, but does not always promote the organization of root meristems in epicotyls. Expansins were found to be auxin-inducible while searching for auxin-induced genes specific to adventitious rooting. To investigate the localization and time course of expansin expression during adventitious and lateral root formation, 25- day-old hypocotyls, 50-day-old hypocotyls and epicotyls, and 10-day-old primary roots were treated with auxin at different concentrations. Non-radioactive in situ localization of expansin mRNA using digoxigenin-labeled probes was used to compare expansin expression at the cellular level in different parts of the seedling. Expansin expression was observed in the auxin-treated hypocotyl and epicotyl tissue, but no or a very weak signal was observed in the untreated tissue. In addition, the auxin-induced increase of expansin mRNA in 25-day-old hypocotyls and primary roots was highly localized to the region of the parenchyma from which adventitious roots will form. In the lateral rooting zone of primary root, strong expression, without auxin treatment, occurred in the pericycle cells prior to lateral root primordium organization. Different patterns of expansin gene expression in response to auxin were found between hypocotyl and epicotyl cuttings. Relatively strong and localized expression in vascular parenchyma of hypocotyls contrasts with relatively weak and diffuse expression in cortex cells in epicotyl cuttings. A preliminary Western blot, detected the expansin protein in hypocotyls at both 24 and 48 h after auxin treatment but not in epicotyls. Collective results suggest auxin-induced expansin expression may play a role in both lateral and adventitious root formation in loblolly pine seedlings.

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