Abstract
BackgroundOur aim is to improve knowledge of gene regulatory circuits important to dedifferentiation, redifferentiation, and adventitious meristem organization during in vitro regeneration of plants. Regeneration of transgenic cells remains a major obstacle to research and commercial deployment of most taxa of transgenic plants, and woody species are particularly recalcitrant. The model woody species Populus, due to its genome sequence and amenability to in vitro manipulation, is an excellent species for study in this area. The genes recognized may help to guide the development of new tools for improving the efficiency of plant regeneration and transformation.ResultsWe analyzed gene expression during poplar in vitro dedifferentiation and shoot regeneration using an Affymetrix array representing over 56,000 poplar transcripts. We focused on callus induction and shoot formation, thus we sampled RNAs from tissues: prior to callus induction, 3 days and 15 days after callus induction, and 3 days and 8 days after the start of shoot induction. We used a female hybrid white poplar clone (INRA 717-1 B4, Populus tremula × P. alba) that is used widely as a model transgenic genotype. Approximately 15% of the monitored genes were significantly up-or down-regulated when controlling the false discovery rate (FDR) at 0.01; over 3,000 genes had a 5-fold or greater change in expression. We found a large initial change in expression after the beginning of hormone treatment (at the earliest stage of callus induction), and then a much smaller number of additional differentially expressed genes at subsequent regeneration stages. A total of 588 transcription factors that were distributed in 45 gene families were differentially regulated. Genes that showed strong differential expression included components of auxin and cytokinin signaling, selected cell division genes, and genes related to plastid development and photosynthesis. When compared with data on in vitro callogenesis in Arabidopsis, 25% (1,260) of up-regulated and 22% (748) of down-regulated genes were in common with the genes regulated in poplar during callus induction.ConclusionThe major regulatory events during plant cell organogenesis occur at early stages of dedifferentiation. The regulatory circuits reflect the combinational effects of transcriptional control and hormone signaling, and associated changes in light environment imposed during dedifferentiation.
Highlights
Our aim is to improve knowledge of gene regulatory circuits important to dedifferentiation, redifferentiation, and adventitious meristem organization during in vitro regeneration of plants
We studied gene expression during dedifferentiation and regeneration of shoots via organogenesis
Callus and shoot development during regeneration To determine the time points for taking tissue samples during in vitro shoot organogenesis, we carried out a preliminary regeneration experiment where 3 to 4 mm internodal stem segments (Figure 1A) were placed on auxinrich callus induction medium (CIM) in dark for 15 days, transferred them to cytokinin-rich shoot induction medium (SIM) following our optimized transformation protocol
Summary
Our aim is to improve knowledge of gene regulatory circuits important to dedifferentiation, redifferentiation, and adventitious meristem organization during in vitro regeneration of plants. In vitro regeneration is a common research tool and important method for plant propagation. It is essential for most forms of genetic transformation, which require the regeneration of single transgenic cells into non-chimeric organisms [1,2]. Both embryogenic and organogenic regeneration pathways are widely employed, with the system of choice varying among species and research or propagation goal. When cytokinin is high relative to auxin, shoots are induced; when the reverse is true, roots are induced. There has been a great deal of progress in identification of key genes that regulate embryogenesis and organogenesis [46], as well as genome scale studies of in vitro regeneration [7,8,9], the studies have focused on only a few species and specific regeneration systems
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