Characterization of an endochitinase able to rescue the carrot somatic embryo variant ts11

A.J. de Jong

Research output: Thesisinternal PhD, WU

Abstract

<p>Cultured carrot cells secrete proteins, many of which are glycosylated, into the culture medium. A correlation has been found between somatic embryogenesis and the presence or absence of some of these secreted proteins, and evidence has been obtained that one or more secreted glycoproteins are actually essential for somatic embryo formation. The starting point of the experiments described in this thesis was the temperature-sensitive carrot cell line ts11, originally identified on the basis of the temperature- sensitive arrest in the transition of globular to heart stage somatic embryos. The arrest in ts11 embryo development at the nonpermissive temperature could be lifted by addition of medium proteins, secreted by wildtype cells, to the culture medium. The major goal of the study presented in this thesis was to identify the secreted proteins, that were able to rescue the arrested ts11 embryos.<p>In chapter 1 a brief introduction in zygotic and somatic embryogenesis is presented, followed by an overview of what is currently known about the first essential steps of the development of the zygotic embryo and of the formation of embryogenic cells and somatic embryos in vitro. Based on these studies, it is discussed whether analogous cellular mechanisms control early zygotic embryogenesis and the formation of embryogenic cells in tissue culture.<p>In chapter 2 the experiments are described that demonstrate that is 11 embryos can be rescued by a single secreted protein of 32 kD. The amino acid sequences of two tryptic peptides of this protein shared homology with several plant endochitinases. Biochemical analysis showed that the 32-kD protein is an acidic endochitinase.<p>In chapter 3 the results of a search for putative products of endochitinase activity effective in ts l 1 rescue, are presented. A molecule produced by <em>Rhizobium,</em> the N-acetylglucosamine-containing lipo-oligosaccharide, NodR1v-V(Ac, C18:4), appeared to be effective in stimulating the formation of ts11 embryos with a similar efficiency as the 32-kD endochitinase.<p>In chapter 4 evidence is presented that a decreased amount of an otherwise fully functional endochitinase is closely correlated with the window of sensitivity of ts1 1 cells to addition of the 32-kD endochitinase. Morphological observations suggest that the original ts l 1 mutation is quite pleiotropic and does not only affect embryogenesis in this line.<p>In chapter 5 experiments are described to identify a 32-kD endochitinase cDNA. The deduced amino acid sequence of the isolated cDNAs was found to be nearly identical to the amino acid sequences of the 32-kD endochitinase-derived peptides. The EP3 cDNA sequences suggested that the 32-kD endochitinse is a class IV chitinase.<p>Finally, in chapter 6 the significance of chitinases and lipo-oligosaccharides for plant development in general is discussed.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • van Kammen, A., Promotor, External person
  • de Vries, Sacco, Promotor
Award date6 May 1994
Place of PublicationS.l.
Publisher
Print ISBNs9789054852483
Publication statusPublished - 1994

Keywords

  • somatic embryogenesis
  • tissue culture
  • embryo culture
  • plants
  • embryology
  • growth
  • plant development
  • enzymes
  • enzymology
  • fermentation

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