| Abstract |
- Vegetative propagation allows the amplification of selected genotypes for research,
breeding, and commercial planting. However, efficient in vitro regeneration and
genetic transformation remains a major obstacle to research and commercial
application in many plant species. Our aims are to improve knowledge of gene
regulatory circuits important to meristem organization, and to identify genes that
might be useful for improving the efficiency of in vitro regeneration. In this thesis, we
have approached these goals in two ways. First, we analyzed gene expression during
poplar (Populus) regeneration using an AffymetrixGeneChip® array representing
over 56,000 poplar transcripts. We have produced a catalog of regulated genes that can
be used to inform studies of gene function and biotechnology. Second, we developed a
GUS reporter system for monitoring meristem initiation using promoters of poplar
homologs to the meristem-active regulatory genes WUSCHEL (WUS) and
SHOOTMERISTEMLESS (STM). This provides plant materials whose developmental
state can be assayed with improved speed and sensitivity.
For the microarray study, we hybridized cDNAs derived from tissues of a
female hybrid poplar clone (INRA 717-1 B4, Populus tremula x P. alba) at five
sequential time points during organogenesis. Samples were taken from stems prior to callus induction, at 3 days and 5 days after callus induction, and at 3 and 8 days after
the start of shoot induction. Approximately 15% of the monitored genes were
significantly up-or down-regulated based on both Extraction and Analysis of
Differentially Expressed Gene Expression (EDGE) and Linear Models for Microarray
Data (LIMMA, FDR<0.01). Of these, over 3,000 genes had a 5-fold or greater change
in expression. We found a very strong and rapid change in gene expression at the first
time point after callus induction, prior to detectable morphological changes.
Subsequent changes in gene expression at later regeneration stages were more than an
order of magnitude smaller. A total of 588 transcription factors that were distributed in
45 gene families were differentially regulated. Genes that showed strong differential
expression encoded proteins active in auxin and cytokinin signaling, cell division, and
plastid development. When compared with data on in vitro callogenesis from root
explants in Arabidopsis, 25% (1,260) of up-regulated and 22% (748) of down-
regulated genes were in common with the genes that we found regulated in poplar
during callus induction.
When ~3kb of the 5' flanking regions of close homologs were used to drive
expression of the GUSPlus gene, 50 to 60% of the transgenic events showed
expression in apical and axillary meristems. However, expression was also common in
other organs, including in leaf veins (40% and 46% of WUS and STM transgenic
events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical
GUS staining of explants during callogenesis and shoot regeneration using in vitro
stems as explants showed that expression was detectable prior to visible shoot
development, starting 3 to 15 days after explants were placed onto callus inducing medium. Based on microarray gene expression data, a paralog of poplar WUS was
detectably up-regulated during shoot initiation, but the other paralog was not.
Surprisingly, both paralogs of poplar STM were down-regulated 3- to 6-fold during
early callus initiation, a possible consequence of its stronger expression in the
secondary meristem (cambium) than in shoot tissues. We identified 15 to 35 copies of
cytokinin response regulator binding motifs (ARR1AT) and one copy of the auxin
response element (AuxRE) in both promoters. Several of the WUS and STM transgenic
events produced should be useful for monitoring the timing and location of meristem
development during natural and in vitro shoot regeneration.
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