EXPERIMENTAL STUDY ON VASCULAR DIFFERENTIATION IN THE SHOOT APICES OF HERBACEOUS DICOTYLEDONS A Thesis Submitted tu the College of Graduate Studies and Research in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy in the Department of Biology University of Saskatchewan Saskatoon Bu QUN XIA Sprfng, 1997 O Copy right Qun Xia, 1997. Al1 rights reserved. National Library Bibliothèque nationale 1*1 of Canada du Canada Acquisitions and Acquisitions et Bibliographic Sewices services bibliographiques 395 Wellington Street 395. rue Wellington OttawaON U1AON4 Ottawa ON KlA ON4 Canada Canada Your Ne Votre nifdmce Our lSle Notre rdf6renca The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distniiute or sell reproduire, prêter, distribuer ou copies of this thesis in microfom, vendre des copies de cette thèse sous . paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. UNIVERSITY OF SASKATCHEWW College of Graduate Studies and Research SLùWARY OF DISSERTATION Submitted in partial fulfilment of the requirements for the DEGREE OF DOCTOR OF PHILûSOPHY by Xia, Qun Deparmnt of Biology University of Saskatchewan Spring 1997 Examining Cornni ttee : Dr. J. F. Basinger ~/~~$~X~/Deanls Designate, Chair College of Graduate Studies and Research Dr. L. C. Fowke Chair of Advisory Committee, Department of Biology Dr. T. A. Steeves Supervisor, Department of Biology Dr. W. M. Kulyk Department of Anatomy Dr. V. K. Sawhney Department of Biology Dr. M. W. Zink Department of Biology External Eluminet : Dr. Usher Posluszny Department of Botany University of Guelph Guelph, Ontario N1G 2Wl Experimental study on vaacular differentiatfon in the shoot apicea of herbaceoue dicotyledonrp Vascular differentiation has been experimentally investigated in the shoot apex of carrot (D~UCUScdl~ta L. ) . AS in f erns, there is initial vascular tissue---provascular tissue---in the shoot apex. A distinct provascular ring was observed in the shoot apex of carrot and extended above the attachent of the youngest leaf trace when this could be identified in the late plastochron. Histochemical tests indicate that carboxylesterase, which is mainly characteristic of vascular tissue, is present in this tissue. Surgical experiments further reveal that, as in fems, the formation of provascular tissue is independent of the leaf primordia, but that unlike ferns,! further maturation of the provascular tissue depends upon an influence from the leaf prixnordia. Finally, auxin replacement experiments reveal that IAA produced the developing leaf primordia is one of the influences that affect the maturation of provascular tissue. Exogenous IAA applied in lanolin or more effectively in resin beads, enhanced provascular tissue developrnent and promoted the final maturation of vascular tissue. These results suggest that there is insufficient IAA for vascular maturation in the shoot apex when leaf primordia are suppressed and support the hypothesis that auxin production has shifted £rom axial to lateral centres in the evolution of seed plants. Furthemore, the surgical experiments have been extended to two other dicotyledons, potato (Solarium tuberosum L.) and lupin (Lupinus albus L.). Although there are variations in the normal shoot apices in these two species, the result of suppression of leaf primordia is similar to that in carrot. In conclusion, the difference of the present results Erom comparable observations in ferns, in which the final maturation of vascular tissue from provascular tissue does not require the influence of leaf primordia, is interpreted as a reflection of the long separation of fern and seed plant evolutionary lines. PREMïSSION TO USE In presenting this thesis in partial fulfilment of the requirements for the Postgraduate degree from the University of Saskatchewan, 1 agree that the Libraries of this University may make it freely available for inspection. 1 further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work, or in their absence, by the Head of the Department or the Dean of the College in which the thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to rnake other use of material in this thesis in whole or in part should be addressed to: Head of the Department of Biology University of Saskatchewan 112 Science Place, Saskatoon Saskatchewan, CRNAClA S7N 5E2 ABSTRACT Early vascular differentiation has been experirnentally investigated in the shoot apex of carrot(Daucus carota L. var, sativa DC.) . As in ferns, there is initial vascular tissue--provascular tissue--in the shoot apex. A distinct provascular ring was observed in the shoot apex of carrot and extended above the attachent of the youngest leaf trace when this could be identified in the late plastochron. Histo- chernical tests indicate that carboxylesterase, which is mainly characteristic of vascular tissue, is present in this tissue. Surgical experiments further reveal that, as in ferns, the formation of provascular tissue is independent of the leaf primordia, but that unlike ferns, further maturation of the provascular tissue depends upon an influence from the leaf primordia. Finally, auxin replacement experiments reveal that IAA produced by the developing leaf primordia is one of the influences that affect the maturation of provas- cular tissue. Exogenous IAA applied in lanolin or more ef fectively in resin beads, enhanced provascular tissue development and promoted the final maturation of vascular tissue. These results suggest that there is insufficient IAA for vascular maturation in the shoot apex when leaf primordia are suppressed and support the hypothesis that auxin produc- tion has shifted from axial to lateral centres in the ev-o- lution of seed plants. Furthemore, the surgical experiments have been extended to two other dicotyledons, potato (Solarium tuberosum L. ) and lupin (Lupinus albus L. ) . Aithough there are variations in the normal shoot apices in these two species, the result of suppression of leaf prirnordia is similar to that in carrot. In conclusion, the difference of the present results from comparable observations in ferns, in which the final maturation of vascular tissue from provas- cular tissue does not require the influence of leaf primordia, is interpreted as a reflection of the long separation of fern and seed plant evolutionary lines. iii ACKNOWLEDGEMENTS 1 would like to express rny gratitude and many thanks to Dr. Taylor A. Steeves for his guidance, encouragement, support, and patience throughout the course of this program. I wish to thank the members of my advisory codttee, Drs. L. C. Fowke, W. M. Kulyk, V, K. Sawhey and M. W. Zink for their valuable comments and guidance on rny thesis. 1 sincerely acknowledge the financial support of a scholarship from the University of Saskatchewan. I thank Dr. A. Davis for providing his laboratory equipment to continue my seemingly endless work after the retirement of Dr. T. A. Steeves. 1 also thank the following people for technical assistance: Mr. Dennis Dyck for his help with photography, Mr. Yukio Yano for the technical assistance in electron microscopy, Mrs. Jeaniene Smith for the culture of plants, and Mrs. Yang, Xiuli for providing technical assistance with microscopy and histochernical tests. 1 also would like to thank my colleagues, Mr. Fawzi Razem and Mr. Prakash Venglat for their help, encouragement and stimulating discussions. 1 am also indebted to Mr & Mrs George Goetz, Mr & Mrs Reg Pope and Dr & Mrs Craig Campbell for their friendship. A special acknowledgement goes to Mr. Ivan CS. McArthur and Dr. Yilun Ma whose work on a dicityledous Geum chiloense and a fern Matteuccia struthiopteris respectively provided a base for the present comparative study. TABLE OF CONTENTS PERMISSIONTO USE ................i ABSTRACT ....................... ii ACKNOWLEDGEMENTS ................... iv TABLE OF CONTENTS ..................v LISTOFTABLES .................... xii LIST OF FIGURES ...................xiv LIST OF ABBREVIATIONS ....m........ ...XXV Chapter 1 INTRODUCTION ............... 1 1.1 Evolution of the vascular system ..........2 1.2 Development of the vascular system .........8 1.2.1 Shoot apex and vascular differentiation . 10 1.2.2 Defoliation and vascular differentiation . 12 1.2.3 Auxin and vascular differentiation ..... 16 1.2.3-1 Auxin control of vascular differentiation ............ 16 1.2.3.1.1 Studies in angiosperms .... 16 1.2.3.1.2 Studies in ferns ....... 21 1.2.3.2 Auxin in early vascular differentiation ............ 23 1.2.3.2.1 Studies in angiosperms .... 23 1.2.3.2.2 Studies in ferns ....... 27 1.3 Objectives .................... 29 Chapter 2 INITIAL VASCULAR DIFFERENTIATION IN THE SHOOT APEX OF CARROT ...... 31 2.1 Introduction ................... 31 2.1.1 Initial vascular differentiation ...... 31 2.1.2 Esterases and early differentiation .... 33 2.1.3 Terminology in the study of the shoot apex . 35 2.1.4 Aim of the study .............. 40 2.2 Materials and methods ............... 42 2.2.1 Materials and growth condition ....... 42 2.2.2 Light and scanning electron rnicroscopy ... 42 2.2.3Histochemistry. .............. 44 2.2.3.1 Fresh frozen sections ........ 44 2.2.3.2 Paraffin embedded sections ..... 45 2.2.3.3 Incubation for esterase activity .