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Factors controlling shoot regeneration from Arabidopsis root explants

Hans Motte (UGent)
(2013)
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Abstract
In vitro shoot regeneration, or the development of shoots from non-meristematic tissue, is a widely applied process in plant biotechnology, including tissue culture and genetic transformation. Although it is a well-studied developmental process, it is poorly understood why tissues of some plant species can readily be induced to regenerate into whole plants, whereas others remain recalcitrant to do so. To study factors controlling regeneration capacity, we adopted a two-step protocol for shoot regeneration from Arabidopsis thaliana root explants. In this protocol, root explants are first incubated on an auxin-rich callus induction medium (CIM), which lead to organogenesis competence, and subsequently transferred to a cytokinin-rich shoot induction medium (SIM), resulting in morphogenesis and development of shoots. To better time and quantify shoot regeneration, several shoot expressed marker constructs were evaluated via live-imaging during the course of a shoot induction protocol. Although all tested markers were expressed in shoots, none of them were able to strictly predict shoot formation. These results indicated that there is a certain flexibility in the shoot organogenesis program and hence shoot formation may be interrupted at late stages of development. However, the marker CUP-SHAPED COTELYDON2 (CUC2), which is already expressed during CIM incubation, was useful as it predicted organogenesis competence and marked sites that could develop into shoots or lateral roots. On the other hand, expression of LIGHT-DEPENDENT SHORT HYPOCOTYLS4 (LSH4) highly correlated with sites of shoot formation. LHS4 was not expressed elsewhere in the root explant and was the most predictive regeneration marker. Therefore, we used an LSH4-GFP line in a high-throughput chemical screen to identify shoot inducing compounds. Among the 10,000 small molecules applied on the Arabidopsis regeneration protocol, one appeared to induce shoots: phenyl-adenine (Phe-Ade). Comprehensive molecular, enzymatic and chemical analyses revealed that Phe-Ade exhibits a dual mode of action: it is a weak activator of the cytokinin receptors and a strong competitive inhibitor of the cytokinin degrading CYTOKININ OXIDASE/DEHYDROGENASE (CKX) enzymes. Phe-Ade is a cytokinin-like molecule that in contrast to classic cytokinins does not exhibit cytotoxicity at high concentrations. This property, together with the strong stimulation of shoot induction qualify Phe-Ade as a promising compound for future biotechnological applications. In another approach, we used the allelic variance of Arabidopsis to determine the genetic requirements for shoot regeneration. 88 accessions were subjected to the two-step regeneration protocol and the regeneration capacity was evaluated. In doing so, we demonstrated that, among the different accessions, there is hardly any pair-wise correlation between shoot regeneration and accompanying traits, such as callus formation or greening of the explant. To further identify genetic correlations with regeneration, two strategies were followed: (i) a quantitative trait locus (QTL)-analysis with an inbred population of two divergent accessions, revealing five regeneration QTLs and (ii) a genome-wide association study with 215,000 single nucleotide polymorphisms (SNPs), revealing about 30 candidate genes associated with shoot regeneration. We further combined the results of the two studies with a local association mapping, using data of completely sequenced accessions. This approach narrowed down the number of candidate genes revealing the probable quantitative trait gene (QTG) and regeneration-associated gene RECEPTOR-LIKE PROTEIN KINASE1 (RPK1). Remarkably, RPK1 is related to abscisic acid (ABA), which is to our knowledge not reported to be involved in shoot regeneration. Mutant analysis finally corroborated the importance of this ABA-related gene in shoot regeneration. This result demonstrated that next-generation mapping is a useful technique to identify genes related to a complex trait as shoot regeneration. Altogether, we applied two sophisticated technologies to enhance our understanding of shoot regeneration capacity. These approaches allowed the identification of Phe-Ade as an inducer of regeneration through the inhibition of CKXs and revealed the importance of RPK1 and possibly ABA as new players in the shoot regeneration process.
Keywords
phenyl-adenine, RPK1, CUC2, Arabidopsis thaliana, adventitious shoot, association study, chemical screen

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Citation

Please use this url to cite or link to this publication:

Chicago
Motte, Hans. 2013. “Factors Controlling Shoot Regeneration from Arabidopsis Root Explants”. Ghent, Belgium: Ghent University. Faculty of Bioscience Engineering.
APA
Motte, H. (2013). Factors controlling shoot regeneration from Arabidopsis root explants. Ghent University. Faculty of Bioscience Engineering, Ghent, Belgium.
Vancouver
1.
Motte H. Factors controlling shoot regeneration from Arabidopsis root explants. [Ghent, Belgium]: Ghent University. Faculty of Bioscience Engineering; 2013.
MLA
Motte, Hans. “Factors Controlling Shoot Regeneration from Arabidopsis Root Explants.” 2013 : n. pag. Print.
@phdthesis{3155817,
  abstract     = {In vitro shoot regeneration, or the development of shoots from non-meristematic tissue, is a widely applied process in plant biotechnology, including tissue culture and genetic transformation. Although it is a well-studied developmental process, it is poorly understood why tissues of some plant species can readily be induced to regenerate into whole plants, whereas others remain recalcitrant to do so. To study factors controlling regeneration capacity, we adopted a two-step protocol for shoot regeneration from Arabidopsis thaliana root explants. In this protocol, root explants are first incubated on an auxin-rich callus induction medium (CIM), which lead to organogenesis competence, and subsequently transferred to a cytokinin-rich shoot induction medium (SIM), resulting in morphogenesis and development of shoots.
To better time and quantify shoot regeneration, several shoot expressed marker constructs were evaluated via live-imaging during the course of a shoot induction protocol. Although all tested markers were expressed in shoots, none of them were able to strictly predict shoot formation. These results indicated that there is a certain flexibility in the shoot organogenesis program and hence shoot formation may be interrupted at late stages of development. However, the marker CUP-SHAPED COTELYDON2 (CUC2), which is already expressed during CIM incubation, was useful as it predicted organogenesis competence and marked sites that could develop into shoots or lateral roots. On the other hand, expression of LIGHT-DEPENDENT SHORT HYPOCOTYLS4 (LSH4) highly correlated with sites of shoot formation. LHS4 was not expressed elsewhere in the root explant and was the most predictive regeneration marker. Therefore, we used an LSH4-GFP line in a high-throughput chemical screen to identify shoot inducing compounds. Among the 10,000 small molecules applied on the Arabidopsis regeneration protocol, one appeared to induce shoots: phenyl-adenine (Phe-Ade). Comprehensive molecular, enzymatic and chemical analyses revealed that Phe-Ade exhibits a dual mode of action: it is a weak activator of the cytokinin receptors and a strong competitive inhibitor of the cytokinin degrading CYTOKININ OXIDASE/DEHYDROGENASE (CKX) enzymes. Phe-Ade is a cytokinin-like molecule that in contrast to classic cytokinins does not exhibit cytotoxicity at high concentrations. This property, together with the strong stimulation of shoot induction qualify Phe-Ade as a promising compound for future biotechnological applications.
In another approach, we used the allelic variance of Arabidopsis to determine the genetic requirements for shoot regeneration. 88 accessions were subjected to the two-step regeneration protocol and the regeneration capacity was evaluated. In doing so, we demonstrated that, among the different accessions, there is hardly any pair-wise correlation between shoot regeneration and accompanying traits, such as callus formation or greening of the explant. To further identify genetic correlations with regeneration, two strategies were followed: (i) a quantitative trait locus (QTL)-analysis with an inbred population of two divergent accessions, revealing five regeneration QTLs and (ii) a genome-wide association study with 215,000 single nucleotide polymorphisms (SNPs), revealing about 30 candidate genes associated with shoot regeneration. We further combined the results of the two studies with a local association mapping, using data of completely sequenced accessions. This approach narrowed down the number of candidate genes revealing the probable quantitative trait gene (QTG) and regeneration-associated gene RECEPTOR-LIKE PROTEIN KINASE1 (RPK1). Remarkably, RPK1 is related to abscisic acid (ABA), which is to our knowledge not reported to be involved in shoot regeneration. Mutant analysis finally corroborated the importance of this ABA-related gene in shoot regeneration. This result demonstrated that next-generation mapping is a useful technique to identify genes related to a complex trait as shoot regeneration.
Altogether, we applied two sophisticated technologies to enhance our understanding of shoot regeneration capacity. These approaches allowed the identification of Phe-Ade as an inducer of regeneration through the inhibition of CKXs and revealed the importance of RPK1 and possibly ABA as new players in the shoot regeneration process.},
  author       = {Motte, Hans},
  isbn         = {9789059895928},
  keyword      = {phenyl-adenine,RPK1,CUC2,Arabidopsis thaliana,adventitious shoot,association study,chemical screen},
  language     = {eng},
  pages        = {174},
  publisher    = {Ghent University. Faculty of Bioscience Engineering},
  school       = {Ghent University},
  title        = {Factors controlling shoot regeneration from Arabidopsis root explants},
  year         = {2013},
}