Experimental Study on Vascular Differentiation in the Shoot Apices of Herbaceous Dicotyledons

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

QUN XIA

Sprfng, 1997

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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 provascular 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 production 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 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.

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 . . 46 2.2.3.4 Inhibitors ...... -... 47 2.3 Results ...... 50 2.3.1 General external morphology and phyllotaxis ...... 50 2-3-2Organization of the shoot apex ...... 51 2.3.3 Early development in the shoot apex .... 53 2-3.3.1 Plastochron and divergence angle . . 54 2.3.3.2 Rhythmical changes in the shoot apex 55 2.3.3.3 Provascular tissue and procambium . . 57 2.3.3.3.1 Transverse view ...... 57 2.3.3.3.2 Longitudinal view ...... 60 2.3.3.4 Leaf traces and axial bundles .... 62 2.3.4 Maturation of primary vascular tissue ... 67 2.3.4.1 Phloem differentiation . 67 2.3.4.2 Xylem differentiation ...68 2.3.5 Esterases in the shoot apex ...... 71 2.3.5.1 Esterases in frozen sections .... 71 2.3.5.2 Esterases in paraffin sections ... 73 2.3.5.3 Carboxylesterases and inhibitors . . 75 2.4 Discussion: ...... 83 2.4.1 Provascular tissue cylinder ...... 83 2.4.1.1 Location of provascular tissue cylinder ...... 84 2.4.1.2 Histological features ...... 86 2.4.1.3 General conclusion ...... 89 2.4.2 The apical meristem of the shoot apex . . 89 2.4.3 Esterases in the shoot apex ...... 93 2.4.4 Hypothesis of early differentiation . . 98 2.5Figures ...... 100

Chapter 3 SURGICAL EXPE-NTS ON THE SHOOT APEX OF CARROT . 115 3.1Introduction ...... 115 3.2 Material and methods ...... 121 3.2.1 Material and growth condition ...... 121 3.2.2 Methods ...... 121 3.2.2.1 Surgical operations ...... 121 3.2.2.1.1 Preparing knives ...... 122 3.2.2.1.2 Preparing shoot apices ....122 3.2.2.1.3 Surgical operation techniques 122 3.2.2.1.4 Post-operative treatment . 124 3.2.2.2 Experirnents ...... 124 3.2.2.3 Histological and histochemical methods ...... 125 3.2.2.4 Measurements ...... 126 3.2.2.4.1 Vertical gxowth ...... 126 3.2.2.4.2 Apical meristem . 127 3.3 Results ...... 131 3.3.1 Puncturing only experiments ...... 131 3.3.1.1 Leaf primordia ...... 131 3.3.1.2 Vertical growth ...... 132 3.3.1.3 Apical meristem ...... 132 3.3.1.4 Provascular tissue ...... 133 3.3.1.5 Vascular differentiation .... 134

vii 3.3.2 Isolation experiments ...... 3.3,2.1Isolationonly ...... 3.3.2.1.1 Leaf primordia ...... 3.3.2.1.2 Vertical growth ...... 3.3.2.1.3 Apical meristem ...... 3.3.2.1.4 Provascular tissue ..... 3.3.2.1.5 Pith plug ...... 3.3-2.1.6 Vascular differentiation . . 3.3.2.2 Isolation plus partial puncturing . 3.3.2.2.1 Leaf primordia ...... 3.3.2.2.2 Vertical growth ..... 3.3.2.2.3 Apical meristem ..... 3.3.2.2.4 Provascular tissue ..... 3.3.2.2.5 Pith plug ...... -. 3.3.2.2.6 Vascular differentiation . . 3.3.2.3 Isolation plus complete puncturing 3.3.2.3.1 Leaf primordia ...... 3.3.2.3.2 Vertical growth ..... 3.3.2.3.3 Apical meristem ....-. 3.3.2.3.4 Provascular tissue ..... 3.3.2.3.5 Pith plug ...... 3.3.2.3.6 Esterase reaction tests . . 3.4 Discussion ...... 3.4.1 General effects of surgical operations . . 3.4.1.1 Apical rneristem ...... 3.4.1.2 Leaf primordia ...... 3.4.1.3Newgrowth ...... 3.4.2 Vascular differentiation in treated shoot apices ...... 3.4.2.1 Formation of provascular tissue . . 3.4.2.2 Esterase activity ...... 3.4.2.3 Control of vascular differentiation

viii 3.4.2.3.1 Control by the apical meristem 154 3.4.2.3.2 Control by leaf primordia . . 155 3.4.3Conclusion...... 156 3.5Figures ...... -159

Chapter 4 AUXIN EFFECTS ON VASCULAR DIFFERENTIATION IN THE SHOOT APEX OF CARROT ...... 171 4.1 Introduction ...... 171 4.2 Material and methods ...... 174 4.2.1 Plant material and growth conditions ....174 4.2.2 Preparation of IAA carriers ...... 174 4.2.2.1Lanolinemulsion ...... 175 4.2.2.2 Resin beads ...... 175 4.2.3 IAA application experirnents ...... 179 4.2.3.1 Experiments with lanolin ...... 180 4.2.3.2 Experiments with resin beads ....181 4.3 aesults ...... 183 4.3.1 IAA lanolin application experiments .... 184 4.3.1.1 Control shoot apices ...... 184 4.3.1.2 IAA in lanolin ...... 186 4.3.2 IAA bead application experiments ...... 189 4.3.2.1 Three low content IAA beads applied . 190 4.3.2.2 One high content IAA bead applied . . 192 4.4 Discussion ...... 195 4.4.1 IAA involvement in the early stages .... 195 4.4.2 IAA effects on maturation of vascuiar tissue ...... -199 4.4.3 IAA and leaf primordia ...... 200 4.4.4 Other substances in vascular differentiation ...... 203 4.5 Figures ...... 205 Chapter 5 EVIDENCE E'ROM OTHER HERBACEOUS

5.1 Introduction ...... 215 5.2 Material and rnethods ...... , ...... 218 5.2.1 Material and growth conditions . . . . . - . 218 5.2.1.1Potato ...... , ...... 218 5.2.1.2 Lupin ...... 218 5.2.2 Methods ...... 219 5.2.2.1 Light and scanning electron microscopy ...... 219 5.2.2.2Histochemistry ...... 220 5.2.2-3 Surgical operations . . . . . , . . . 220 5.2.2.3.1 Experirnents in potato . . . . 221 5.2.2.3.2 Experiments in lupin . . . . . 223 5.3Results. .., ...... 225 5.3.1 Potato . - ...... , . . . . * . 225 5.3.1.1 Normal development in the shoot apex 225 5.3.1.1.1 Organization of the shoot apex 225 5.3.1.1.2 Development in the shoot apex 226 5.3.1.1.3 Esterase activity in the shootapex ...... 228 5.3.1.2 Surgical operation experiments . . . 229 5.3.1.2.1 Isolation and puncturing . . . 229 5.3.1.2.2 Puncturing without isolation . 232 5.3.2 Lupin ...... , ...... 234 5.3.2.1 Normal development in the shoot apex 234 5.3.2.1.1 Organization of the shoot apex 234 5.3.2.1.2 Development in the shoot apex 236 5.3.2.1.3 Esterases in the shoot apex . 238 5.3.2.2 Surgical operation experirnents . . . 240 5.3.2.2.1 Isolation plus Puncturing . . 240 5.3.2.2.2 Esterases in treated shoot apices ...... 242 5.3.2-2-3 Puncturing without isolation 243 5.4 Discussion ...... 245 5.4.1 Developrnent of provascular tissue .....245 5.4.2 Histochernical evidence ...... 247 5.4.3 Surgical operations ...... 249 5.4.4 General conclusion ...... 251 5.5Figures ...... 252

Chapter 6 GENERAL DISCUSSION ...... 268 6.1 Control of primary vascular differentiation . . . 268 6.1.1 Initial vascular differentiation ...... 268 6.1-2 Differentiation of the eustelic primary vascular system ...... 272 6.1.3Thehormonedeterminationmodel ...... 274 6 2 Cornparison of vascular differentiation in ferns and angiosperms ...... 276 6-2-1Provascular tissue ...... 277 6.2.2 Control of vascular differentiation ....279 6.2.3 Hypothesis of shifting auxin source ....282 6.3 Figures ...... 285

REE'ERENCES CITED ...... O...... 287 LIST OF TABLES

2-1 Effects of inhibitors on the activities of esterases in the reaction in which naphthol AS-D acetate is used as substrate ......

2-2 The length of the youngest leaf primordium and its position, and the diameter of the apical dome at theleveloftheaxilofP, ...... 56

2-3 The vertical distance from the summit of the apical dome to the earliest appearance of a provascular ring compared with the position of the axil of the leaf primordia and the divergence point of their main leaf traces in the shoot apex ofadultplants ...... 59

2-4 The comparison of ce11 size among cells in provascular stage and in procambial stage in longitudinal view ...... 63

2-5 The earliest appearance of vascular bundles and leaf traces compared with the length of the youngest leaf primordium into which the trace will enter ...... 66

2-6 The summary of the time of vascular differentiation in relation to the length of P, ..... 69

2-7 Summary of esterase activity present in the shoot apex when shoot apex sections are incubated in

xii buffer without or containing different inhibitor for 45 min and full reaction medium without or containing each inhibitor for 60 min, al1 at 37°C 78

2-8 The results of esterase activity present in the shoot apex when sections of the shoot apex are pretreated in buffer or buffer containing various concentration of inhibitor DFP for 45 min at room temperature and then were reacted in the medium without substrate or full medium containing a corresponding concentration of inhibitor DFP or full reaction medium for 60 min; al1 at 37°C . . . 80

3-1 The cornparison of vertical growth of the shoot apices among different treatments ...... 128

3-2 Dimensional variation in the apical rneristem of shoot apices from surgical treatments and normal shoot apex of adult carrot ...... 130

xiii LIST OF FIGURES

Scanning electron micrographs of the dissected vegetative shoot apices of adult carrot . 100

The whole view of the dome of the shoot apex ofcarrot ...... IO0

A median longitudinal section of the shoot apex of a seedling, showing the organization of tunica-corpus ...... 101

A median longitudinal section of the shoot apex of an adult plant, showing the zonation organization ...... 101

A diagram of a median longitudinal section of the shoot apex of a carrot seedling, showing tunica - corpus organization ...102

A diagram of a longitudinal section through the median of the shoot apex of an adult carrot plant, showing the organization of zonation and relationship with other adjacent tissues and organs ...... 102

Transverse section through the level of 50 pm from the summit of the shoot apex, showing the Pl about to attach the apical dome . 103

Transverse section through the level of 90~ £rom the sunimit of the shoot apex, showing the appearance of a provascular tissue ring above the divergence point of the leaf trace of Pl . . 103

Transverse section through the level of the axil of the youngest leaf primordium, about 20

xiv un below the summit of the shoot apex, showing the apical dome and attached Pl ...... 104

2.10 Transverse section through the level of 70 pm from the summit of the shoot apex, showing the appearance of a provascular tissue ring above the divergence point of the main leaf trace (indicated by an arrow) of P, ...... 104

2.11 A median longitudinal section of the shoot apex of a seedling, showing the location of provascular tissue in the shoot apex ...... 105

2.12 A median longitudinal section of the shoot apex of an adult plant, showing continuum of leaf traces and axial vascular bundles . . . . . 105

2.13 Transverse section through the level of 70 pm from the summit of the shoot apex of a carrot seedling, showing detail of provascular tissue and an initial of an axial vascular bundle and a leaf trace ...... 106

2.14 Transverse section of the stem of an adult plant, showing leaf traces and axial bundles . . 107

2.15 Transverse section of the stem of an adult plant, showing axial bundles and leaf traces . . 107

2 -16 Median longitudinal section of a shoot apex, showing no reaction on the section ...... 108

2.17 Median longitudinal section of a shoot apex, showing positive reaction on the section . . . . 109

2.10 The fourth transverse section, 60 p below the summit of the shoot apex, showing the high reaction in the provascular ring ...... 109 Median longitudinal section of a shoot apex, showing esterase distribution in the shoot apex, note the graduated reduction of the esterase activity along the procambium to provascular tissue acropetally ...... 110

Transverse section of a shoot apex at the level of 90 pm from the summit of the apical dome, showing visible esterase reaction in the provascular tissue ring ..--...... 110

Median longitudinal section of a shoot apex, showing the esterase distribution in longitu- dinalview ...... 111

Transverse section of a shoot apex, showing esterase activity in provascular tissue ring after the section was treated by eserine ....111

Median longitudinal section of a shoot apex, showing the reaction on the section . . . . 112

Transverse section of a shoot apex, showing esterase activity in the provascular tissue ring after the section was treated by PCMPS . 112

Full medium controls: Frozen sections of the shoot apex of carrot were pretreated in buffer pH 6.5 without containing any inhibitor for 45 min at room temperature, followed by incubation in full medium for 1 h at 37" ...... 113

DFP tests: Frozen sections of the shoot apex of carrot were pretreated in buffer pH 6.5 containing 3x10'~ M DFP for 45 min at room temperature, followed by incubation in full medium for 1 h at 37" ...... -113 Transverse section of a shoot apex, showing no visible reactions when the DFP is present . .

Transverse section of a shoot apex under high magnification ......

Top view of an exposed shoot apex demonstrating a puncturing treated shoot apex in which four leaf primordia have been punctured during two weeks ......

Longitudinal section of a shoot apex with puncturing only treatment after two weeks of operations, showing newly f ormed provascular tissue......

Transverse section at 70 pm from the summit in an apex with puncturing treatment for 2 weeks showing provascular tissue ring ......

Transverse section at 120 pm from the summit in the same apex as in Fig. 3.3, showing the continuous provascular ring ......

Top view of an exposed shoot apex demonstrating isolation operation ......

Longitudinal section of a shoot apex with isolation treatment after two weeks of operations, showing a newly formed vascular cylinder extended in the isolated tissue plug ...

Transverse section at 30 pm from the summit of an isolation-only treated apex after 2 weeks showing a distinct apical meristem ......

Transverse section at 50 pm from the summit in the same isolation-only treated apex as in the Fig. 3.7 showing a distinct provascular ring .

xvii Transverse section at 390 pm from the summit in the same shoot apex as in Fige 3.7, showing a distinct vascular system in the pith plug . . 163

Portion of a transverse section at the base of the pith plug in the same isolated shoot apex as in the Fig, 3.7 showing distinct xylem withoutphloem...... 163

Transverse section at the base of the pith plug in an isolation-treated apex the same as in Fig, 3.7 showing a distinct vascular system independent from the original one ...... 164

Transverse section below the base of the pith plug in the same shoot apex as in Pig. 3.7 showing regenerated xylem elements . 164

Longitudinal section of a shoot apex with isolation treatment after 2 weeks of operations ...... 165

Transverse section at 70 prn from the summit in an isolation-treated apex with for 2 weeks with 1, allowed to develop, showing a distinct provascular ring

Transverse section at 90 p from the summit in an isolation-treated apex with for 2 weeks with 1, allowed to develop, showing a provascular ring and leaf traces of 1, . 166

Transverse section at the base of the pith plug in the same apex as in Fig. 3.16 showing distinct vascular bundles related to the unpuncturedI, ...... 167

Transverse section through the base of the pith plug in the same shoot apex as in Fig.

xviii 3.16, showing regenerated xylem elements which will establish connection with original vascu- larsystem ...... ,.167

3.18 Median longitudinal section of a shoot apex with isolation plus completely puncturing treatment after 2 weeks of operations . . + . . 168

3.19 Transverse section at 120 pm from the summit of a shoot apex with the fully operated treatment after 2 weeks of operations, showing provascular ring in the new growth ...... 168

3.20 Transverse section at 200 p from the summit of a fully operated shoot apex, showing provascular ring in the pith plug ...... 169

3.21 Portion of a longitudinal section of a shoot apex with isolation treatment after 2 weeks of operations, showing newly formed provascular tissue extended in the isolated tissue plug . . 169

4.1 Standard curve obtained by plotting the absorbency measurements at 278 nm of each concentration of IAA against the known IAA concentrations (O - 1 mM) ...... 205

4-2 Standard curve obtained by plotting the absorbency measurements at 278 nm of each concentration of IAA against the known IAA concentrations (O - 0.1 mM) ...... 206

4.3 Top view of an exposed shoot apex (indicated by an arrow head) showing al1 leaf primordia removed and 1 bead applied at the site of 1, . . 207

4.4 Top view of an exposed shoot apex (indicated by an arrow) showing al1 leaf prirnordia removed and 3 beads applied ...... 207

xix A median longitudinal section of a shoot apex with lanolin without IAA applied for 2 weeks, showing provascular tissue and its extension in the pith plug (below the two arrows) . 208

A transverse section at 150 pm from the summit of a shoot apex with lanolin without IAA applied for 2 weeks showing provascular ring . . 208

A median longitudinal section of an isolated shoot apex treated with IAA lanolin for two weeks, showing distinct provascular tissue and its extension in the pith plug (below the two arrows) O...... ,. ,209

A transverse section at 150 p.m from the summit in an isolation-treated apex with IAA-lanolin applied for 2 weeks showing distinct provas- cularring...... ,209

A transverse section at 280 p.m from the summit in an isolation-treated apex with IAA-lanolin applied for 2 weeks showing a vascular tissue ring...... O...,... 210

Top view of an isolated shoot apex which was treated with low content IAA beads for 3 weeks ...... 211

A transverse section of a shoot apex with low content IAA-containing beads applied as a cap for three weeks, showing a cylinder of mature vascular tissue extended into the pith plug .. . 211

A median longitudinal section of a shoot apex with 3 resin beads without IAA applied after two weeks, showing provascular tissue and its extension in the pith plug ...... 212 A rnedian longitudinal section of a shoot apex with 3 resin beads with IAA applied for two weeks, showing provascular tissue and its extension ...... 212

A transverse section at 100 pm from the summit in a shoot apex with 3 IAA-containing beads applied for 3 weeks, showing provascular tissue ...... O...... 213

A transverse section 340 pm below the summit of the shoot apex showing mature vascular tissue was formed under the influence of three IAA beads after 3 weeks ...... 213

A transverse section at 80 pm £rom the summit in a shoot apex with 1 IAA-containing bead applied for 3 weeks, showing provascular tissue...... 214

A transverse section below the site of 1, in a shoot apex with one high content IAA-containing bead applied after 3 weeks showing distinct phloem and xylern elements formed . . 214

The basic apparatus used in the experiments on potato ...... 252

Seedlings of lupin used in experiments .....252

The shoot apex at the early stage of plastochron; showing a layered organization ...253

The shoot apex at the late stage of plastochron; showing zonation organization . . . . 253

The first section through the summit of a shootapex...... ,. 254

xxi A section at the level of 60 pm from the apical summit showing the first appearance of the provascular ring ...... 254

A section at the level of 70 pm from the apical summit showing the provascular ring ...255

A section at the level of 90 pm from the apical sununit showing the leaf traces of P, and P, joined to the provascular ring ...... 255

A median longitudinal fro zen section showing the distribution of esterase activity in the shootapex ...... 256

A paraffin embedded transverse section of an isolated shoot apex of potato, 110 pm from the apical summit, showing provascular tissue after suppression of leaf primordia . 257

A paraffin embedded transverse section of an isolated shoot apex of potato, 130 ym from the apical summit, showing a provascular tissue ring after suppression of leaf prirnordia ....257

A paraffin embedded transverse section of a shoot apex of potato, 60 p from the apical summit, showing provascular tissue after suppression of leaf primordia ...... 258

A paraffin embedded transverse section of a shoot apex of potato, 100 pm from the apical summit, showing provascular tissue ring after suppression of leaf primordia ...... 258

The whole view of a dissected vegetative shoot apex of lupin ...... 259

A shoot apex split in a median plane ...... 259

xxii A median longitudinal paraffin-section of the shoot apex of a seedling of Lupinus albus L . . 260

A median longitudinal section of a vegetative shoot apex of lupin . .

A transverse section at the level of 40 p from the apical swnmit ...... 261

A transverse section at the level of 60 pn from the apical summit ...... 261

A transverse section at the level of 70 p from the apical summit ...... 262

A transverse section at the level of 90 p from the apical summit ...... 262

A transverse section at the level of 120 pm from the apical summit showing the first appearance of the provascular ring ...... 263

A transverse section at the level of 150 pm from the apical summit showing the provascular ring ...... 263

A rnedian longitudinal frozen section of the shoot apex of a lupin seedling shows esterase activity in the leaf traces ...... 264

A transverse section of a frozen shoot apex of a lupine seedling, 100 pn from the apical summit, shows esterase activity in the leaf traces ...... 264

A top view of an isolated shoot apex of which leaf prirnordia were punctured for three weeks ...... 265

xxiii A transverse plastic thin section of the isolated shoot apex of a lupin seedling, at the level of the 1, showing a provascular tissue ring (indicated by arrows) after suppression of leaf primordia ...... 265

A median longitudinal frozen section of the isolated shoot apex of a lupin seedling of which the leaf prirnordia were suppressed, showing esterase activity in the provascular tissue and its extension as well as in origi- na1 vascular tissue ...... 266

A traverse frozen section of a surgically treated shoot apex of a lupin seedling, 100 pm from the apical summit, showing esterase activity in the region of provascular tissue and an axial vascular bundle ...... 266

A transverse paraffin embedded section of the shoot apex of a lupin seedling, 300 p from the apical summit, showing provascular tissue after suppression of leaf primordia . - . . , . 267

A transverse paraffin embedded section of the shoot apex of lupine seedling, 500 p.m from the apical summit, showing a continuous provascular tissue ring after suppression of leaf primordia ...... 267

Parameters of the hormone determination model in polar (transverse view) ...... 285

Simplified diagram of the development of a shoot apical meristem, viewed from the side, showing terminology employed in the hormone determination mode1 described in the text ...286

xxiv LIST OF ABBREVIATIONS

micromoles of photons per square meter per second Fi9 microgram Fim micrometer 4-chloro IAA 4-chloro-indole-acetic acid Abs O.D. absorbency optical density DFP diisopropyl f luorophosphate the number of the Enzyme Commission classification systern E6OO diethyl p-nitrophenyl phosphate FAA formalin : acetic acid : 50% alcohol = 5 : 6 : 89 by volume hour high content IAA-containing bead (s) hydrogen fluoride successive newly formed leaf primordia after first surgical operation IAA indole acetic acid 1 BA indole butyric acid L/W ratio the greatest dimension / the least dimension LB(s) low content IAA-containing beads M mole mM millimole mo 1 mole ng nanogram nm nanomet re O.D. optical density ocs organizing centres Pl - P6 successive leaf primordium from the youngest to older PAA phenylacetic acid PCMPS parachloromercuriphenylsulphate tl - t4 successive time intervals TIBA 2,3,5-tri-iodo-benzoic acid Tris 2-amino-2 (hydroxy-methyl)-1,3-propanediol w/w WO the window of opportunity Chapter 1 IN!CRODUCTION

The development of a complex vascular system was one of the great advances in terrestrial plants. The complexity of the vascular system raises several questions regarding both its phylogeny and its ontogeny. Based on the morphology of fossil and extant plants, the evolution of the vascular system has been interpreted by systematic and palaeobotanical studies (Jeffrey 1899, 1902; Bower 1923, 1926, 1935; Ogura

1938, 1972; Beck et al. 1982; Slade 1971). Studies of vascular differentiation are also an important subject in the

field of plant development (Aloni 1987a, 1988; Roberts 1988a; Sachs 1981; Shininger 1979) . However, studies of vascular differentiation have generally paid little or no attention to

the evolution of the vascular system and vice versa- The integration of information from fossil and developmental studies could result in important interpretations of this complex system. In other words, understanding developmental differences in extant plants may help us interpret the long history of the vascular system and at the same time, knowledge of stelar evolution could shed light on some 2 aspects of vascular differentiation. A few workers have realized the importance of such an integration (Rothwell 1987, Steeves 1989) and have made cornparisons of vascular dif f erentiation in f erns and angiosperms (Wardlaw 1968, Steeves 1989). Aithough the evolution of the vascular system cannot be duplicated by experiments, these authors have brought forth some important dif ferences in vascular differentiation between ferns and seed plants. Recently, a series of papers have been published on initial vascular differentiation in a fern (Ma and Steeves 1992, 1994, 1995a, b) and a mode1 of stelar evolution in seed plants, integrat- ing fossil architecture and hormonal signals has been proposed (Stein 1993). These investigations have established a basis for a comparative study of vascular development in ferns and angiosperms .

1.1 Evolution of the vascular system The vascular system is one of the important tissue systems in the body of a vascular plant. The bundles of vascular tissue were originally interpreted as fundamental units by early botanists such as De Bary, Geyler and Nageli (cited in Beck et al. 1982). Later, Sachs (cited in Beck et al. 1982) recognized that al1 of the vascular tissue in the plant body, from root to stem, constitutes a continuous 3 system, This vascular system was named the stele by van Tieghem and Douliot in 1886 (cited in Beck et al. 1982) . Evolution of the vascular system has generally been interpreted according to the "stelar theory" (Gifford and Foster

1989). In general, three types of stele, based on the morphology of the sporophyte of vascular plants, have been recog-

nized: protostele, siphonostele (Smith 1955, Gifford and Foster 1989) and eustele (Schmid 1982). The protostele is a type of stele with a solid central core of xylern surrounded by phloem and lacking pith. The siphonostele includes al1 types of steles in the lower vascular plants or non-seed plants that have a hollow cylinder of xylem with pith in the

centre. In siphonosteles, the xylem and phloem forin a cylinder around the pith. Based on the positions of the phloem and xylem, two subtypes of siphonostele are distin-

guished: (1) ectophloic siphonostele and (2) amphiphloic siphonostele. The former has only external phloem surrounding the xylem, whereas the latter has both exterior and interior phloem surrounding the xylem, The endodermis appears both outside and inside the vascular tissue on the borders of cortex and pith, respectively. Most lower vascular plants contain plth and interior phloern, therefore the hollow cylinder of the siphonostele exhibits amphiphloic 4

structure. Based on the number and size of leaf gaps, amphiphloic siphonosteles can be distinguished in two categories. The solenostele is a continuous cylinder with successive leaf gaps distant from one another, so that only one leaf gap appears in a cross section, The dictyostele shows a network of vascular tissue with overlapping gaps (the lower part of one gap is parallel with the upper part of another gap), The eustele is only used to describe the vascular system in seed plants. The xylem and phloem of the eustele are arranged in distinct strands separated by parenchyma tissue. In gymnosperms and dicots, the vascular strands are arranged in a cylinder. In monocots, the vascular strands are arranged throughout the ground tissue; such a eustele is called an atactostele. In a superficial view, the protostele, siphonostele, dictyostele and eustele could appear to constitute a sequence of evolution. However, only the proto-siphono-dictyostefic evolutionary series can be observed in lower vascular plants. In lower vascular plants, the protostele is primitive, the siphonostele is derived, and the dictyostele is more advanced than the siphonostele and is derived from it. In a living Eern, a young sporophyte stem has an initial protostele, which is succeeded by a siphonostele. The dictyostele occurs in the adult stem. This series of changes of the vascular configuration at different ontogenetic stages in ferns is 5 parallel to the stelar evolutionary line of lower vascular plants and was cited as evidence to support the evolutionary interpretation. The proto-siphono-dictyo-eustelic evolutionary series

was advanced by Jeffrey (1917) who had a major influence

among American botanists. Jeffrey believed that ferns are ancestral to the seed plants and that the two groups are thus closely related in the vascular pattern of their steles- He suggested that the eustele of the seed plants represents a

greatly modified, reduced siphonostele and that the leaf gap in seed plants is homologous or morphologically equivalent to the leaf gap in siphonostelic ferns. Although the proto-siphono-dictyo-eustelic evolutionary series was widely accepted, other views were also advanced. The concept that the eustele had a different evolutionary derivation through protostelic dissection was proposed in the 1920s (Posthumus 1924) and received strong support from

fossil evidence in the 1960s (Beck 1960af b, 1964, 1970;

Namboodiri and Beck 1968af b, c). Based on palaeobotanical discoveries, Beck ( 1964 ) proposed that seed plants evolved directly frorn a protostelic ancestor in an independent evolutionary line. Later Namboodiri and Beck (1968a, b, c) described eustele evolution in gymnosperms and progymnosperms (extinct vascular plants that possessed gymnospermous anatorny 6 but reproduced by free-sporing methods) . Later, Slade (1971) extended this concept to the angiosperms . The Posthumus view of eustelar evolution had a different interpretation of the origin of leaf gaps from Jeffrey's view and questioned the homology of leaf gaps in ferns and the interfascicular regions in seed plants. After comparing the steles of leptosporangiate ferns and of the pteridosperms,

Lyginopteris and Heterangi um, Posthumus ( 1924) concluded that the interruption in vascular tissue of pteridosperms, resulting from the insertion of leaf traces, is different from the leaf gap present in the ferns. The protostelic dissection concept suggested that the eustele in seed plants evolved directly from the protostele through longitudinal dissection without the formation of leaf gaps. This concept of eustelar evolution put the ernphasis on the pattern of the primary vascular bundles in the stem instead of the whole mature vascular architecture. Analysis of eustelic prirnary vascular systems requires that a clear distinction be made between 1) the axial bundles and the leaf or branch traces and 2) primaxy and secondary vascular tissue. As to axial vs leaf or branch bundles, Devadas and Beck (1971) claimed that there is a difference in bundle size and tracheary element number between axial bundles and leaf or branch traces. As to prirnary vs secon- 7 dary vascular tissues, Beck et al. (1982) proposed thac it was necessary to use "provascular strands" and / or protoxylem strands rather than mature primary vascular bundles as the basis for determining stelar patterns (Beck et al. 1932).

The provascular strands used by Beck et al. (1982) refer to young primary vascular bundles which may include only proc~ium. This is not the sense of provascular tissue defined by McArthur and Steeves (1972). The use of "provascular bundles" elirninates the problems of tissue identification associated with secondary growth. Beck et al.

(1982) proposed that structures such as "provascular bundles" and protoxylem strands, being developmentally closer to the organizing centres in the plant, might reflect more ac- curately the basic stelar patterns than mature vascular bundles - "Provascular strands", protoxylem strands and mature vascular bundles are related developmentally. Mature primary vascular bundles develop from llprovascularstrands", metaxylem develops in relation to protoxylem and there is evidence of a one-to-one relationship between protoxylem s trands and "provascular strands" in seed plants (Larson 1975) . Arnong lower vascular plants, Chau (1981) found a one- to-one relationship between lrprovascular bundles" and protoxylem strands in the eusporangiate ferns Ophioglos- saceae. However, these eusporangiate ferns are a very 8 curious group which is thought by some to be a derivative of

aneurophytalean progymnosperms (Bierhorst 1971). Moreover, this relationship has not been reported in leptosporangiate ferns .

1.2 Development of the vascular system

Developmental changes play an important role in the

evolution of vascular plants (Rothwell 1987, Steeves 1989).

Wight (1987) pointed out that stelar evolution initially resulted from a change in the factors which control the growth and developrnent of plants and was only subsequently affected by natural selection on stelar morphology. In other words, physiological changes take place prior to structural changes, and changes in controlling mechanisms are fundamen- ta1 to evolution. Sachs (1993) also stressed that the evolution of structure is the consequence of the evolution of developmental processes, and that developmental processes constrain morphological evolutionary change. The mature structure of the stele results from specific patterns of growth and development; therefore, evolutionary trans formation in stelar rnorphology must be examined against the background of development. Thus, examination of changes in ontogeny might provide a key to understanding or interpreting the observed variation in mature structure (Wight 1987). Al1 9 these views have emphasized that developmental information is important to an understanding of the morphological changes in evolution, Since fossil data have provided evidence that the

eustele was derived directly from the protostele (Beck et al.

1982) and that ferns and plants with eusteles are separate evolutionary lines, it is reasonable to expect that there will be some developmental differences in vascular differentiation between ferns and seed plants. There is an extensive literature which deals with vascular differentiation and vascular system developrnent . These studies include experimental analyses, particularly with regard to the influence of different plant growth

substances (Roberts et al. 1988)- Most of these studies have

considered questions regarding seed plants, and are generally concerned with rnechanisms of differentiation at the cellular

or rnolecular level (Aioni 1987a, Northcote 1995), rather than the development of the patterned vascular system. Moreover, much of this work has dealt with the formation of xylem or phloem from cambium or mature parenchyma and with the later stages of vascular differentiation, Little attention has been paid to the initial stage of vascular differentiation, The eustelar evolution concept places more emphasis on the pattern of the primary vascular system than on the whole mature vascular architecture. Therefore, investigation on 10 the early developmental process, rather than the later stages, may be significant in interpreting the evolution of the vascular system. Because shoot apices are the source of al1 tissues, including the vascular tissue, and because the initial events of differentiation occur in the shoot apex, the following review will focus on the differences in the initial stage of vascular differentiation between ferns and seed plants, particularly angiosperms.

1.2.1 Shoot apex and vascular differentiation

Experimental studies have shown that there is a difference in the autonomy of the shoot apex of ferns and angiosperms. The shoot apex in both groups has an important function in the initiation of the stele, as well as in the later development of the stele. This has been demonstrated by several experimental studies. One experimental approach was the surgical isolation of the apical meristem in the shoot apex from young leaf primordia and partially from mature parts underneath. After the operation, the apex reconstructed leaf primordia and developed a vascular system in the new shoot (Bal1 1948, 1952; Wardlaw 1950) . When the isolation experiment was done in ferns, similar results were reported (Wardlaw 1947, 1950; Soe 1959; Ma and Steeves 1995b). These isolation experirnents demonstrated that the 11 basic function of shoot apices in both groups is the same.

A new shoot with a vascular system can be developed from the partially isolated shoot apex. Further studies on the autonorny of shoot apices however, showed a different requirement among lower vascular plants and angiosperms in studies on the growth of the isolated shoot apex in vitro. In angiosperms, the culture of isolated shoot apices required leaf primordia to be present (Bal1

1946; Wetmore 1954; Morel and Martin 1952; Morel 1963, 1964), or a medium with growth substances such as auxin (Smith and

Murashige 1970; Rivière l973), both indole acetic acid (IAA) and kinetin (Shabde and Murashige 1977) or coconut milk and gibberellic acid (Ball 1960). Al1 of these studies except that of Ball (19601, show that auin is a necessary component for shoot autonomy. Later studies have proved that auxin- like substances are also present in coconut milk (Deangkinay and Ramirez 1971; Dix and van Staden 1982). Therefore, it rnay be concluded that auxin is a critical factor affecting shoot autonorny in angiosperms. In lower vascular plants, auxin was not shown to be important for the autonomy of the shoot apex. Wetmore (1954) pointed out that the apical meristem, removed from certain lower vascular plants with or without leaf primordia, could grow into a new plant in vitro on a sterile agar medium containing mineral salts, minor 12 elements and sucrose. This has been shown with apices of

Adiantum pedatum (Wetmore and More1 1949) and Selaginella willdenovii, Lycopodium cernuum and Equisetum hymale (Wetmore 1954). Similarly the central portion of the shoot apex of the fern Pteris cretica, a cube c.300-500 pm3 containing the apical cell, its derivatives and some subjacent tissue but no leaf primordia, was also grown in culture with normal organogenesis (Michaux-Ferrière 1973 ) . The difference in auxin requirement of excised apical meristems of angiosperms and ferns in culture suggests a fundamental difference in meristem autonomy between these two groups of plants (Steeves and Sussex 1989) . The culture of the excised shoot apex of angiosperms requires auxin or leaf primordia. This suggests that auxin can replace the leaf primordia which are the probable source of auxin in the shoot apex of angiosperms. In ferns, the isolated shoot apex can develop into a mature plant with or without leaf primordia and exogenous auxin is not necessary. One possible interpretation is that endogenous auxin in the Eern apical meristem is sufficient to maintain shoot development.

1.2 -2 Defoliation and vascular differentiation Another dif ference between ferns and angiosperms was demonstrated by defoliation experiments. In the fern, 13 Dryopteris, by systematic suppression of leaf prirnordia in their early development, or by isolating the central portion of meristem on a plug of pith tissue, Wardlaw (1944a, b,

1947, 1949b) obtained a functional vascular system from the shoot apex without leaves. Since, in the absence of leaves, the vascular system lacked leaf gaps, a compact solenostele, or even a protostele, replaced the normal highly leaf- influenced dictyostele, This indicates that the shoot apical meristem establishes the basic pattern of the vascular system and that the leaves subsequently affect the development of the basic pattern so that a modified vascular system is obtained. A similar result was reported in surgical experiments on the fern, Onoclea sensibilis (Soe 1959).

Onoclea is a fern with a dictyostelic vascular system with large vertically elongated leaf gaps. When al1 leaf primordia were suppressed and the next incipient (1,) position was punctured so that no primordia developed at all, a perfect solenostele was present in the portion of the axis formed during the experimental treatrnent.

Recently, in a similar experiment on Matteuccia struthiopteris, a solenostele instead of a dictyostele was produced by rernoval of leaf primordia and isolation of the shoot apex. Ma (1994) reported that "when the shoot apex was isolated by vertical incision and incipient leaf primor- 14

dia were suppressed systematically for three weeks, a mature solenostele with reduced diameter and uninterrupted by leaf gaps replaced the normal dictyostele" . This result fbrther confirmed that the provascular tissue which was produced by

the shoot spical meristem can become a mature siphonostelic vascular system in the absence of leaves even though reduced

in diameter (Ma and Steeves 1992). In angiosperms, the leaf primordia appear to play a more important role in initial vascular differentiation and the

response of the shoot apex to similar surgical operations is

quite different from that in ferns. In an early report, Heh (1932) discovered that defoliation has a strong effect on the upper part of the procambial tissue, causing it to differen-

tiate as parenchyma. In Lupinus, Young (1954) observed that

if P, was rernoved, parenchyma appeared at the base of the rernoved P,. When he removed al1 leaves from the shoot apex, however, a meristematic ce11 ring in cross section persisted down to 560 pm below the apical dome. Although his experiment lasted 21 days, the meristematic tissue did not advance beyond this stage. More recent defoliation experiments were reported in Geum. The shoot apices of Geum were treated with a combination of isolation and puncturing (McArthur and Steeves

1972). In these surgically treated shoot apices, a substan- 15 tial provascular tissue cylinder occurred throughout the region of new growth. This cylinder also developed basipetally into the supporting pith plug, presumably by the rediiferentiation of existing pith cells. However, further differentiation was not obtained in the absence of leaves (McArthur and Steeves 1972). Although the results of defoliation in seed plants have been reported by others with some variations (Helrn 1932, Young 1954), one thing is sirnilar: they have never obtained mature vascular tissue without leaves . In ferns, provascular cells differentiate into vascular elements whether or not leaf primordia are present. This difference in the vascular differentiation related to leaf influence indicates that the shoot apical meristem in the two groups, ferns and angiosperms, plays a different role in the development of the stele. In ferns, the shoot apical meristem alone may directly control the vascular differentiation process, although leaf participation modifies the pattern of the stele; while in seed plants the same process needs the participation of leaf primordia as well as the apical meristem. Considering the results from the culture of the shoot apical meristem, the leaf primordia may be the main source of auxin in the angiosperms. Therefore the difference could be interpreted in terms of the relative auxin-pzoducing 16 capacity of the apical meristem and the leaf primordia in f erns and angiosperms .

1.2.3 Auxin and vascular differentiation

1.2.3.1 Auxin control of vascular differentiation Several plant growth substances may be involved in controlling the mechanisms of vascular differentiation (Aioni 1987a), but auxins are thought to be one of main factors (Aloni 1987b, Moore 1989, Lyndon 1994, Sachs 1991). Before discussing the difference between ferns and angiosperms, it is worthwhile to review briefly the previous studies of auxin control of vascular differentiation in both groups.

1 .2.3.1.1 Studies in angiosperms Auxin in higher vascular plants is synthesized in the shoot apex, young leaves and fruit (Salisbury and Ross 1985, Mohr and Schopfer 1995). Auxin is involved in ce11 elongation and ce11 division and probably takes part in many other growth activities (Salisbury and Ross 1985) including effects on lateral bud development (Hillrnan 1984) and root formation (Batra et al. 1975). There is ample evidence that auxin is essential for vascular differentiation. There are several systems in which the mechanism of auxin control of vascular 17 differentiation has been investigated in angiosperms- Some of thern are discussed below-

1.2.3-1.1-1 Intact plant system Auxins have been considered as the main factor controlling vascular differentiation in angiosperms (Aloni 1987b, Moore 1989, Sachs 1981, 1991) . Following the initial works of Vochting, Simon and Freundlich (cited in Fukuda 1992), Sinnott and Bloch (1944, 1945) demonstrated that, when a vascular bundle of a young internode of Coleus was disrupted by a cut, new vascular tissue could be formed around the wound and connect the discontinuous strands. Jacobs (1952, 1954) further showed that the formation of new vascular tissue in Coleus depended on the presence of an adjacent leaf above the wound and auxin from the leaf played a major role in regeneration of xylem around the wound. These experiments demonstrate that auxin prornotes vascular differentiation.

In intact nodes of pea stem (Pisum sativum), Sachs

(1968) demonstrated that auxin plays an important role in vascular strand connections. In pea stem, leaf traces are not connected to the axial vascular bundles until they reach a certain distance from the apex. However, if the shoot apex is removed from a pea seedling, a lateral bud will form and the trace of the lateral bud connects to the axial vascular 18 bundles. If both the leaf blade and the apex are removed, the trace of the newly formed lateral bud connects to both the leaf trace and axial. bundles. This experiment is consistent with the interpretation that the leaf, axillary bud and apex are al1 sources of induction for vascular strands. It also indicates that a vascular strand inhibits newer strands from fusing with it, Further experiments

(Sachs 1969) were launched by using IAA in lanolin to mimic the endogenous auxin sources in a decapitated epicotyl sturnp. Two IAA sources can induce two parallel strands that do not fuse but a strand induced by one auxin source can fuse with a preexistent strand. Sachs concluded that IAA polar transport was a limiting factor for the formation of xylem strands. Through a series of studies, Sachs (1969, 1981,

1986) proposed the hypothesis that auxin flux determines the orderly pattern of vascular differentiation in intact plants.

He found that auxin can increase the auxin transport capacity of cells and that the direction of auxin flow determines the polarity when vascular strands are regenerated. He concluded that it is the flow of auxin through the cells and its direction that causes differentiation of vascular tissue.

1.2.3.1.1.2 Callus system

Vascular tissue can be induced in a callus by implanting a shoot bud into the surface of the callus (Camus 1949, 19 Wetmore and Sorokin 1955). Wetmore and Rier (1963) further showed that when a bud was grafted into Syringa callus, scattered nodules of vascular tissue formed below the bud. When auxin and sucrose were substituted for the bud, vascular tissue could be induced and continuous application of auxin could induce a continuous ring of vascular tissue. Jeffs and Northcote (1967) found that tracheary element differentiation occurred in the region of a Phaseolus callus where the auxin and sucrose concentrations became optimum. Al1 of these suggested that auxin was a limiting factor of xylem differentiation in callus tissues.

1.2.3.1.1.3 Single - ce11 culture system The cultured ce11 system has advantages over intact plants and callus in the study of control of vascular differentiation because cells are more homogeneous and conditions can be controlled more easily. In the single-ce11 culture system, studies of auxin induction of vascular dif ferentiation have been progressing (Fukuda 1994) , Ce11 suspensions of Zinnia mesophyll can be induced to differentiate into tracheary elements in a medium containing auxin, cytokinin, basal salts, cofactors and sucrose (Demura and Fukuda 1994). Cells can directly differentiate into tracheary ce11 elements without ce11 division (Fukuda and Komamine 1980). Protoplasts were also shown to be trans- 20 formed to tracheary elements without ce11 division in the

presence of auxin and cytokinin (Kohlenbach et al. 1982, Kohlenbach and Schopke 1981) . The culture system confirms that auxin is an important factor in vascular differentiation although other factors may also be involved.

1.2.3.1.1.4 Molecular mechanism of auxin action Studies of the molecular mechanism of auxin action in plants have been progressing since the 1980s. Many genes which are expressed rapidly in response to auxin treatment have been isolated (Guilfoyle et al. 19921 . Auxin is proposed to act first with a membrane-bound auxin-binding

protein (ABP) which is called an auxin receptor. The gene of auxin-binding protein 1 (ABPI) produces the protein which first responds to auxin treatment and has been isolated and

characterized from maize (Lazarus et al. 1991, Yu and Lazarus

1991, Schwob et al. 1993) . Evidence from physiological studies with crop plants and transgenic plants supports the

conclusion that ABP1 does play an important role in prirnary auxin perception (Venis 1995). However, the possible role of ABP1 as a receptor which recognizes auxins and transmits the signal to promote cell elongation is argued, Using data from crop species, it is argued that ABPl does not fulfil this role (Hertel 1995) . Many studies (Theologis 1986, Fukuda 1992) showed that a variety of tissues and ce11 types express auxin-responsive transcripts and that diff erent tissues respond rapidly to exogenous auxin by expressing different hormone-responsive genes .

Three vascular cell-specific genes (TED2, TED3 and TED4) have been reported to be related to vascular system development (Demura and Fukuda 1994). TED2 gene is expressed at a very early stage of differentiation of procambium and the

TED2 protein may play some essential role in the basic processes of differentiation. TED3 and TED4 gene expression is later than TED2 gene expression. The TED3 protein is a type of secondary wall protein specific for tracheary elements. TED4 transcripts were present mainly in the immature primary xylem. This work was carried out in a single-ce11 culture system. The possible relationship between these auxin-related proteins and vascular differentiation in intact plants has not been demonstrated.

1.2.3.1.2 Studies in ferns In contrast to extensive information on angiosperms, the knowledge of auxin induction of vascular differentiation in lower vascular plants is relatively limited (Johri 1990).

Lower vascular plants have an independent garnetophyte and the control of vascular differentiation is complicated. In the 22 gametophyte of ferns, there is no vascular tissue. Albaum

(1938) demonstrated that the apical region of the prothallus of the fern Pteris longifolia is able to produce auxin. In the fern Todea barbara, no vascular tissue was found in wild and cultured fern prothalli, but it was possible to induce the formation of tracheids in cultured prothalli by adding auxin and sugar to the medium (DeMaggio, Wetmore and More1 1963) . However, when sucrose was omitted from the medium, the addition of auxin alone failed to induce tracheary element differentiation (Wetmore, DeMaggio and Rier 1964). Further experiments revealed that sucrose was the most effective single supplement in inducing tracheary element dif ferentiation but IAA did stimulate the induction if sucrose was present (DeMaggio 1972). These results suggest that the vascular differentiation in the fern gametophyte may be suppressed under normal conditions. In the sporophyte of ferns, the control of vascular differentiation was believed to be similar to that in higher vascular plants or seed plants. Auxin was demonstrated in fern sporophytes promoting ce11 elongation and ce11 division

(Steeves and Briggs 1960) and inhibiting lateral bud growth

(Wardlaw 1946). In al1 of these aspects, auxin works in the same ways as in higher vascular plants. For example, as in leaves of angiosperms, expanding fronds of the fern Osmunda 23 cinnamomea were able to produce considerable quantities of

auxin (Briggs et al. 1955a, b; Steeves and Briggs 1960) . Steeves and Briggs (1960) dernonstrated that removal of the pinnae of the fern Osmunda cinnamomea prevented xylem maturation in the frond rachis, Using 0.5 8 IAA lanolin paste to replace excised pinnae, final maturation of vascular elements in the frond rachis was reached, They proved that auxin is not only involved in the elongation of the rachis but also in the final differentiation of xylem in the rachis. This study dealt only with the final stage of xylem maturation, namely secondary wall formation and Lignification and no information is available as to the early stage of differentiation.

1.2.3.2 Auxin in early vascular differentiation

1.2.3.2.1 Studies in angiosperms

The review of shoot apical autonomy in angiosperms showed that auxin plays an important role in this phenornenon, and that leaf primordia appear to be the source of auxin. Defoliation experiments further demonstrated that in the absence of leaf primordia vascular differentiation in the shoot apex of dicots is limited to the initial stages. If this is the case, then if auxin is applied to surgically treated apices of angiosperms, it should promote vascular 24 differentiation. However, the results from different systems are contradictory. The most commonly cited experiment in auxin application to the shoot apex is Young's work in 1954.

In Lupinus, if P, was removed, parenchyma formed below the stump of the P, position. If auxin was applied to the place where P, was removed, parenchyma did not appear; instead, meristematic cells remained below the stump of P,. After 21 days no procambium was observed at the site below the removed

leaf primordium. Young's interpretation was that auxin prevents the meristematic cells from differentiating into parenchyma and that vascular differentiation depends on other factors as we11 as auxin. An interesting observation is that, when al1 leaf primordia were removed, a ring of meristematic tissue instead of parenchyma was observed in the shoot apex (Young 1954) . It is difficult to interpret Young's results precisely because the role of auxin in controlling initial vascular differentiation is far from clear. Young's work (1954) did not show that application of auxin has a promotional effect on vascular differentiation in a defoliated shoot apex. In contrast, exogenous auxin does promote vascular differentiation in certain other dicots. In the rhizome of Geum chiloense (McArthur and Steeves lWZ), after the isolation of the shoot apex and removal of leaf primordia, 25

IAA application in a lanolin cap alone caused the appear-lnce of "more densely stained provascular" tissue. Because IAA alone did not demonstrate strong evidence of leaf-primordial replacement, sucrose as a source of nutrition was added to the non-sterile nutrient solution of the rhizome. In an IAA plus 2% sucrose treatment, the isolated apex showed ''a complete procambial cylinder in tissue directly related to the provascular cylinder". In this case, the auxin prornoting function was enhanced by addition of sucrose. Auxin increased the distinctive character of the provascular tissue in an apex in which leaf primordia had been suppressed and, in combination with sucrose, it promoted the further differentiation of provascular tissue to procambium, but not to mature vascular tissue. In this case, auxin affecte ! the early stage of vascular differentiation. Another study suggests auxin affects a relatively later stage or final maturation of vascular tissue. Bruck and

Paolillo (1984) demonstrated that in Coleus, applied ciuxin could replace an excised leaf primordium in promoting normal development of procambium as well as its subsequent differentiation as xylem and phloern. In Coleus, they noted that when young leaf primordia "were excised soon after their inception, no xylem or phloem differentiated in the corner procambial traces of the excised P, in the subjacent inter- 26 node for up to four weeks. Procambium differentiation also failed to occur in the position of the side bundle and phloem-only bundles in the subj acent internode" . However, they also stated that "it was not possible to obtain internodes lacking (young) vascular tissues in al1 the bundle locations positionally related to a leaf". Using IAA loaded beads applied to the place where the leaf primordium was removed, after 2-4 weeks, they obtained normal quantities of vascular tissue. Under the influence of IAA, the plants restored normal structure. They believed that IAA is "the likely leaf-lirniting factor for vascular differentiation in situ". Because young vascular tissue existed in the internodes below the removed leaf prirnordium, those mature vascular bundles, which were believed to be restored by IAA beads, probably arose frorn pre-existing vascular tissue at an early stage.

Al1 these cases suggest a complicated mechanism of auxin regulation of vascular system development in dicots. Auxin may not simply turn a ce11 into xylem elements but may act in combination with other factors which may be substances produced by the developing leaves. In angiosperms, the fact that auxin takes part in vascular differentiation has been demonstrated by a number of experiments, There is also evidence that it is auxin flow rather than auxin per se that induces vascular differentiation (Sachs 198 6). However, the role of auxin in the shoot apex of angiosperms is far from

clear ,

1.2.3.2.2 Studies in ferns

There is very little evidence indicating how auxin acts on vascular differentiation in ferns. An experiment of IAA application to the shoot apex by resin beads was done

recently in a fern species, Matteuccia. In the shoot apex of

Matteuccia, after removal of al1 leaves, which are considered

to be a lateral auxin source, provascular tissue still

develops into a mature siphonostele (Ma and Steeves 1995b). This may indicate that there is enough endogenous auxin in

the apex alone to maintain vascular tissue developrnent. In contrast to the general view, an exogenous auxin source in the apices of ferns seems not to act prirnarily as a promoter of vascular differentiation. Leaf primordia were punctured before they appeared and IAA loaded beads were placed on the

sites of puncture. Because beads can release absorbed IAA to the punctured sites, IAA could influence the development of cells underneath. In the fern with continued puncturing of leaf prirnordia for a period of six weeks, exogenous auxin application restored the overall expansion of the stele, but the provascular tissue confronting the auxin sources dif- 28 ferentiated as parenchyma cells, that is, it simulated leaf gaps (Ma and Steeves 1992). The authors stated that "5 auxin loaded beads make 5 parenchymatous gaps, one bead makes one gap" in the defoliated shoot apices of this fern. The relation between the auxin bead number and gap number is so close that the auxin effect appears to be the only cause. It is also important that the auxin did not induce a simulated leaf trace. Ma's experiments suggest that there is a high endogenous auxin in the fern shoot apical meristem. This may help explain why there is a different response to exogenous auxin in the fern shoot apex. It is particularly interesting that auxin induced parenchyma gaps in the developing stele, and it is notable that there was no vascular leaf trace induced even with auxin present. It was speculated that the exogenous auxin rnay cause a localized supraoptimal concentration for vascular differentiation (Ma and Steeves 1992). This response to auxin should be considered as the essential difference from that in angiosperms. A comparison of the difference in response to exogenous auxin between ferns and angiosperms, favours the hypothesis that a shift in auxin source from the shoot apex to leaves may have occurred during the early evohtion of seed plants (McArthur and Steeves 1972, Scheckler 1978, Stein 1993) . 1 .3 Objectives

From the above review, it is clear that there are still significant questions that need to be answered regarding the early stage of vascular dif ferentiation of angiosperms . These questions include the relative roles of shoot apical meristem and leaf primordia in vascular differentiation in angiosperms, and the role of auxin in early vascular dif-

ferentiation. Using carrot (Daucus carota L.) and other dicotyledons as experimental plant systems, the present study is an attempt to obtain developmental information on vascular differentiation through the application of a variety of techniques including light microscopy, histochemistry, and surgical and hormonal experiments,

In the present study, carrot was used as the primary material for experimentation. Other plants such as potato

(Solanum tuberosum L.) and lupin (Lupinus albus L.), developed from stem tuber or seeds, have also been employed in these studies. The reasons that these species were chosen are the following: 1) They represent three different orders in the angiosperms; 2) they are economic plants common in Saskatchewan so that materials are easily obtained; 3) the apices are relatively easy to manipulate by surgical operations; 4) the species are dicotyledons and are comparable to the species which have been used by previous workers (Wardlaw 3 0 1947; Bal1 1952; Young 1954; Sussex 1955a) so that the results could be easily compared. The present study was undertaken in order to clarify: 1) the respective role of the apical meristem and developing leaves in controlling the pattern of initial differentiation of vascular tissue and its subsequential maturation in angiosperms and 2) the participation of auxin in the control of these processes. These developmental and experimental investigations will be interpreted, as far as possible, in the context of the current understanding of stelar evolution. This study involved the following three aspects: 1) a study of normal vascular differentiation in the shoot apex including normal developmental anatomy and a histochemical study to examine the localization of esterase activity as a marker of initial vascular differentiation; 2) a study of the effects of developing leaves on vascular differentiation by surgical suppression of leaf prirnordia and examination of the resulting pattern of vascular tissue development; 3) a study of exogenous auxin effects on vascular differentiation without the influence of developing leaves by using different IAA carriers. Chapter 2 INITIAL WCULAR DIFE'ERENTIATION IN

THE SHOOT APEX OF CARROT

2.1 Introduction

2.1.1 Initial vascular differentiation In recent studies on the initiation of vascular differentiation in ferns, it has been shown that there is provascular tissue in the shoot apex above the youngest leaf primordia and that provascular tissue, rather than procambium, is the initial phase of vascular differentiation (Ma and Steeves 1994, 1995a). This investigation has further shown that the apical meristern plays a key role in the development of the stele (vascular system) in ferns, although the stele is subsequently modified by leaves (Ma and Steeves

1992, 1995b) ,

The developrnental processes at the shoot apex in angiosperms have been extensively studied (see reviews by

Clowes 1961, Allsopp 1964, Lyndon 1976, 1994, Halperin 1978, Medford 1992)- It has long been noted that there is a recognizable tissue just under the apical meristem and in the path of differentiation of vascular tissue to leaf primordia in 32 angiosperms (see Clowes 1961) . Sorne workers thought that this is a pro- or prevascular tissue, which precedes leaf primordia, because the procambium of leaf traces arises from

it (e.g. Sussex 195Sa, Bowes 1963, McArthur and Steeves

1972). Because in general histological studies, this tissue shows no major difference from the meristernatic cells of the apical meristem, others called it the residual meristem with an emphasis on its meristematic character and the fact that

only part of it develops into procambium (e-g. Esau 1977). Sorne authors neglected the difference between the procambium and the earlier initial stage, and used the term procambium to refer to the entire initial tissue (e.g. Bal1 1952) . Although there is general agreement that this tissue is the

precursor of the vascular system of the shoot axis (e.g.

Esau 1977, Sussex 1955a), procambium, which is positionally related to leaf primordia, is generally considered to be the initial stage of vascular differentiation in the shoot of seed plants (e.g. Esau 1977) . The main disagreement is on the initial stage of vascular differentiation. The vascular bundles of the stele are continuous with leaf traces and the relationship between leaf traces and axial vascular bundles is very close. Since

Esau (1943b) published a review on the development of primary vascular tissue, the procambium which is related to the leaf 33 primordia has generally been regarded as the earliest

vascular tissue which can be identified in the shoot apex (Clowes 1961). The role of an apical rneristern on provascular

tissue differentiation has not drawn much attention. If the immediate derivative tissue of an apical meristem is regarded as the early stage of the vascular system, it suggests that the stem vascular system is directly related to the apical meristem. Although provascular tissue in angiosperms, which is believed to be independent from leaf primordia, was proposed again by McArthur and Steeves (1972), the concept has not generally been accepted.

2-1.2 Esterases and early differentiation Esterase activity in plant tissues has been studied specifically in experiments using naphthol AS-D acetate as a substrate (Gahan 1981, Rana and Gahan 1982, Gahan and Bellani 1984, Gahan and Carmignac 1989) . In roots, esterases were

present in al1 tissues such as stele, cortex and root cap, but had high activity in vascular tissue (McLean and Gahan

1970, Gahan 1981, Rana and Gahan 1982, Gahan and Carmignac

1989) . In roots of Pisum satiwm, high esterase activity could be induced in the cortical parenchyma cells by wounding the stele and a vascular bridge was correlated with cells with high esterase activity (Rana and Gahan 1983). Auxin or 34 cytokinin, or a combination of the two, could also increase the activity of esterases in root segments in culture (Gahan and Rana 1983). The esterase activity induced by wounding, or by auxin and cytokinin in root culture experiments has been identified as due to carboxylesterases (Rana and Gahan 1982, Gahan and Rana 1983). Thus, carboxylesterase activity has a strong relationship with vascular differentiation in roots. In shoot apices, the situation is complex because of the presence of leaf appendages. Nevertheless, studies have revealed that esterases or carboxylesterases are involved in vascular tissue differentiation in the shoot (Gahan and Bellani 1984, Ma and Steeves 1995a, Mueller 1995). In a dicot Trifolium, Mueller (1995) reported that esterase activity "only appeared in relation to discrete sites in continuity with proximal procambial strands". In the shoot apices of two dicotyledons, Pisum satiwm and Vicia faba, carboxylesterases were identified in vascular tissue and the meristem cells which are already comrnitted to form vascular elements (Gahan and Bellani 1984) . Carboxylesterases were also reported to be a good marker to identify early vascular differentiation in the shoot apices of the fern Osmunda (Ma and Steeves 1995a) . 35 From these studies, carried out in both roots and

shoots, it may be concluded that naphthol AS-D esterase activity is involved in vascular differentiation or in early commitment to vascular tissue, and that carboxylesterase activity is a precocious marker of meristematic cells which are committed to form the vascular tissue (Gahan and McLean 1969, Gahan and Bellani 1984, Mueller 1991, Ma and Steeves 1995a) .

2.1.3 Terminology in the study of the shoot apex The study of the shoot apex has a long history. Dif- ferent authors have used different terms to describe the shoot apex and sorne terms are not objective or readily definable. The confusion of terms often causes the study of the shoot apex to be unnecessarily complicated. Thus, some tems related to the shoot apex and used in the present study are clarified here.

2.1.3.1 Leaf prirnordia Labelling leaves can be from oldest to youngest or vice versa. In the present work, al1 leaves are numbered following Snow and Snow (l93l), the starting point being the smallest recognizable primordium at the apex, The youngest visible leaf primordium is designated Pl, the second is P, and 36 so on, while the next incipient primordium position beyond P, is designated 1, (Snow and Snow 1931, 1947, Ma and Steeves 1994). Other terms related to a leaf primordium are the axil, the length and the base of the leaf primordiwn. The angle on the upper side between the leaf primordium and the shoot apex is referred to as the axil of the leaf primordium. The vertical distance from the tip of the leaf primordium to the level of the lowest point of its axil is defined as the length of the leaf prirnordium. The part below the level of the axil which rnerges with the shoot is referred to as the base of the leaf primordium.

2.1.3.2 Plastochron The leaf primordia arise from the shoot apex at regular tirne intervals. The time interval between two succussive primordium initiations is referred to as a plastochron

(Schmidt 1924, Esau 1960, Fahn 1990, Ma 1994). During a plastochron, the apical dome has a minimal and a maximal area (Schmidt 1924) . In the present study, the plastochron is roughly divided into two stages. The early plastochron refers to the stage at which P, has just appeared at the periphery of the apical dome. The late plastochron refers to the stage at which P, is well developed and encloses the 37 apical dome with P, but 1, has yet to appear. These two stages of a plastochron will facilitate the description of events in the developmental process in the present study.

2.1.3-3 Shoot apex and apical dome The term "shoot apex", following Ma's definition (Ma

1994), is used here to describe the distal end of the shoot including several young leaf primordia. The "apical dome" is defined as the part of the apex above the youngest leaf prinordium (McArthur and Steeves 1972). Both the height of the dome and the diameter of the dome depend on the position of the youngest leaf primordium which delimits it. Therefore it is necessary to point out the status when the size of an apical dome is described. Because the new leaf primordium arises so close to the smit of the apical dome, the apical dome in the early plastochron stage will be much smaller than in the late stage. Gifford (1954) has realized this problem in his review of the shoot apex in angiosperms and has suggested that the apical dome should refer to the size at the maximal phase of the apex- In the present study, the maximal size of an apical dome refers to the size at the late stage and the minimal size refers to the size at the early plastochron. 2-1-3.4Provascular tissue and procambium Provascular tissue in the shoot apices of fems has been characterized (Wardlaw 1949b, Ma and Steeves 1994, 1995a) . It is a stage of vascular differentiation before or prior to the procambium. There is also a transitional state between provascular tissue and procambium (Ma and Steeves 1994). In angiospexms the term provascular tissue has sometimes been used in a very broad sense as the equivalent of procambium (Esau 1943a, 1954, 1960) . In a strict sense, the term provascular tissue is used to describe what is also called residual meristem (McArthur and Steeves 1972). Residual meristem has been described as a ring of less vacuolate, more densely staining cells remaining between the lightly staining, inner and outer ground meristem regions, observed in transverse sections of the shoot apex of some angiosperms (e.g. Esau 1977) . In the present study, the terrn "provascular tissue" will be used to describe the proposed early stage of vascular tissue, following the interpretation of McArthur and Steeves (1972) Because the term "provascular tissue" is used as a description of a stage of vascular differentiation, it can be present in leaf traces (Ma and Steeves 1994) as well as in the axis of the shoot. In order to avoid confusion, the provascular tissue present at the axis of the shoot is 39 referred to as the provascular tissue cylinder or a ring as observed in transverse sections, The tenn "procambium" is also defined as a stage of vascular differentiation. Procambium can be in leaf traces and axial vascular bundles. In other words, leaf traces can be at the stage of procambium, but procambium does not refer to leaf traces only. Because the provascular tissue gradually differentiates into procambium, a transitional state is present (Ma and Steeves 1995a). Therefore, it is difficult to indicate a point which separates procambium from provascular tissue,

2.1.3.5 Leaf traces and axial vascular bundles The term "leaf trace" is defined by Fahn (1990) as that part of a vascular bundle in the stem from the point at which it enters the leaf to the point at which it joins the vascular system of the stem. In the present study it refers to a vascular bundle in the stem that diverges from the ring of provascular tissue and extends to a leaf primordium* Axial vascular bundles or axial bundles refer to main bundles of a shoot, which comprise the stem vascular system (Fahn 1990). An axial bundle in early differentiation consists of

a few cells and is located in the ring of provascular tissue, while a leaf trace is observed out of the ring. 40 The point where a leaf trace diverges from an axial bundle is defined as the divergence point or attachent of the leaf trace. Because the influence of the leaf on the vascular system of the stem is probably carried by leaf traces, the point at which a leaf trace diverges from the stele may be an important reference point,

2.1.4 Aim of the study

The primary aim of this study is to determine the pat.tern of vascular differentiation in the stem of carrot, It is necessary to first examine the shoot apex and its organization, because the apical meristem is the source of al1 cells in the shoot. The present work will focus on two aspects. First, the morphology and organization of the shoot apex and the normal vascular system development in the shoot will be described. The emphasis will be placed on the location of the provascular tissue ring and its relationship to the development of leaf primordia and leaf traces, Second, the nature and distribution pattern of esterases will be exarnined in the shoot apex by histochemical methods.

Carrot (Daucus carota L, var. sativa DC, CV, Little Finger) was selected for this study because there are some similarities between carrot and Geum chiloense, the species in which an extensive experimental study was done on vascular 41 differentiation in the shoot apex (McArthur and Steeves

1972). Carrot is similar to Geum in its spiral phyllotaxis

and short stem. The sirnilar phyllotaxis facilitates a comparison of surgical experiments between carrot and Geum.

Because the stem is short, the analysis of developmental

processes can be achieved in a few transverse sections. The

short stem is especially advantageous in surgical experiments, because the effect of stem elongation will not be a critical factor when cornparison is made between normal and

leaf suppressed shoots. Carrot is also a favourable material for experimental purposes in that it has a large fleshy root

which is a source of nutrition during experiments, Also, the shoot apex of carrot is large, is easily exposed, and remains viable after dissection.

The morphology and anatomy of the vegetative organs of carrot have been studied by Havis (1939) and Esau (1940) and

the inflorescence by Borthwick et al. (1931). However, these studies emphasised root, hypocotyl and inflorescence development. Vascular differentiation in the shoot of carrot has not been investigated in detail. In addition, the distribution of esterase activity in the shoot apex of carrot has not been reported. This study will provide a developmental basis for further analysis of the shoot apex by surgical and hormonal experiments. 2.2 Materials and methods

2.2.1 Materials and growth condition

The seeds of carrot (Daucus carota L. var. sativa DC, CV. Little Finger) were obtained from Speers Seeds, Sas- katoon, Saskatchewan. Seedlings were grown in a growth chamber under fluorescent and incandescent lights at 215 -

305 p mol rn'' s-' with a photoperiod of 8 h day and 16 h night. Temperature was set at 23O~day / 19"~night with relative humidity at 90 - 95 %. After germination, seedlings with 5 - 7 true leaves were used in the present study. Adult material was harvested during the months of June to August frorn plants which were sown in May in the Biology Department garden in Saskatoon. Plants with roots of ca. 10 mm diameter were chosen for the study.

2.2.2 Light and scanning electron microscopy The shoot apices used in the developrnental study were fixed in formalin-acetic acid-alcohol solution (FAA, formalin

: acetic acid : 50% alcohol = 5 : 6 : 89 by volume, Johansen

1940) . Fixation was carried out at a temperature of 4°C for at least 24 h, because at low temperature the rate of both autolysis and fixation would be reduced (Berlyn and Miksche 1976). After the fixative had been washed out with two changes of 50% alcohol, samples were dehydrated in a series 43 of ethyl and butyl alcohols and embedded in paraffin (Para- plast Plus tissue embedding medium, Oxford Labware, melting point 62°C). Serial sections were cut at 6 - 10 pm thickness on a rotary microtome (Leitz Wetzlar), attached to glass slides (Fisher Scientific Co. ) by Mayer's solution (Johansen

1940) and dried on a warming tray at 42°C. Staining was carried out by a non-deparaffinized or a deparaffinized method. Sections were stained without prior removal of paraffin by toluidine blue O (Sakai 1973) or safranin and fast green (Ma, Sawhney and Steeves 1993) . Alternatively, sections were stained after paraffin was removed in safranin and fast green (Johansen 1940) or in safranin-tannic acid- iron alum (Shannan 1943). Safranin-tannic acid-iron alun was chosen because it is a useiul stain for identifying phloem. Slides were mounted in permount (Fisher Scientific Co., New Jersey) . Shoot apices of both the seedlings and adult plants of carrot were studied. Twenty shoot apices of seedlings and nineteen shoot apices of adult plants were prepared for transverse and longitudinal sections. Observations were carried out under the light microscope (Wild Heerbrugg, Swit zerland or Olympus, Japan) . Differentiated xylem was identified with the aid of polarizing plates (Nikon, Co. Japan) on an Olympus microscope. Diagrams were drawn from transverse sections of the shoot apex by using a drawing tube 44

(Zeichentubus) fixed on the Wild microscope. Photography was carried out under a Nikon MicroFlexHE'M (M-35fA) camera on a Nikon Optiphot Microscope or a MC63 Pho tomicrographic camera on a Universal Larges Research Microscope (Zeiss, West Germany) . Thirty-five mm Fuji colour film (Super HG, ASA 100) was used with a blue filter when sections were photo- graphed and films were developed and printed in a commercial shop (Ma 1994). For scanning electron microscopy (SEM), the samples were fixed in glutaraldehyde, dehydrated in an acetone series to 100%, critical point dried, coated with gold and observed under SEM. Photographs were taken on polaroid type 665 positivehegative black and white instant pack film.

2.2.3 Histochemistry

To prepare sections for testing esterase activity, both freezing and non-freezing sectioning can be used. In the freezing method fresh material can be embedded in a water soluble embedding medium (Gahan et al. 1967). In the non- freezing method, either paraffin or Glycol methacrylate (GMA, trade name Polyscience's JB-4) can be used as embedding material. The paraffin embedding method has been reported for animal tissues but there are few reports for plant tissues 45

(Higuchi et al. 1979). In the present work, the fresh frozen method and paraffin embedding methods were used.

2.2.3.1 Ftesh frozen sections The preparation of unfixed frozen sections followed the methods of Gahan et al. (1967). Shoot apices were irnmersed in embedding medium (O.C.T. compound, Tissue-tek) , and were sectioned transversely or longitudinally using a cryostat

(Arnes Cryostat II Miles Inc. USA) at a temperature of -22'~.

The cutting thickness was set at 20 pm. The sections were floated on distilled water and incubated in the appropriate reagent medium in a 5 cm petri dish. The nwnber of shoot apices used in each experiment was 10 - 12. Two or four shoot apices were used for control, others for esterase or inhibitor tests. Using fresh frozen sections, esterase tests were repeated at least four times and inhibitor tests at least twice.

2.2-3.2 Paraff in embedded sections The preparation of paraffin embedded sections followed the methods of Higuchi et al. (1979). The preparation of rnaterial and the procedure for sectioning were the same as described in the section on Histology. Twenty shoot apices of adult plants were fixed, dehydrated and embedded in paraf- 46 fin. The embedded material was stored in a refrigerator at 4"~. Sectioning was carried out on a rotary microtome- The ribbons were mounted on precleaned microscope slides with Mayer' s solution . The slides with paraf fin ribbons were dried at room temperature to avoid the probable reduction of enzyme activity by using a warming tray. Paraffin was removed before incubation. Esterase activity tests were repeated twice.

2.2.3.3 Incubation for esterase activity

For histochemical identification of esterases, the azo dye method (Gahan 1984) was followed. Sections were incubated in a medium which consists of naphthol AS-D acetate

(sigmaa) used as a substrate, and fast blue BB (Sigmaœ) as the

------

pppppppp------aiazoniui sait in O. 1 M Tris (2-amino-2 (hydroxy-methyl)-l,3-

propanediol, rizm ma@ Base, sigma? - HCI buffer at pH 6.5 (Rana and Gahan 1983, Gahan and Bellani 1984). Esterase

activity was tested at 37°C for varying periods of the up to

1 h (Gahan 1981). The enzyme in the plant tissue cleaves the acetate radical; then colourless naphthol couples with the diazonium to form a blue colour cornplex. Controls consisted of; 1) omitting the substrate (McLean and Gahan 1970, Gahan

1981), 2) omitting the diazonium salt in the incubation medium, or 3) using heat-inactivated sections which were 47 boiled 5 minutes before putting into the full incubation medium (Rana and Gahan 1982, 1983). For identification of esterases in the unfixed frozen

material, sections of the shoot apex were incubated in the

incubation medium for 1 h at 37°C. For microscopie examination and photography, sections were mounted on glass slides in glycerin, sealed with nail polish, exarained and photo- graphed immediately through a Nikon Optiphot microscope with an attached Microf lex canera. For incubation of paraff in embedded sections, procedure and solutions were the same as for the frozen sections except for the incubation time. The optimal incubation time for paraffin embedded sections was 3 -8 h. After incubation, sections on the slide were dehydrated in an alcohol series and mounted in permount.

2.2.3.4 Inhibitors

The inhibitor tests followed the procedure of Gahan and Bellani (1984) with sorne modification. Frozen sections were pre-incubated in an inhibitor solution which contained either

M eserine (sigmam)or IO-' M PCMPS (Sigmam) or IOe6 - M DFP (sigmam) for 45 min at room temperature (Table 2-1).

As controls, sections were preincubated in the buffer without any inhibitor. Sections were then transferred to the Table 2-1. Effects of inhibitors on the activi ties of esterases in the reaction in whf ch naphthol AS-D acetate is used a8 substrate*

I Inhibitor Enzyme Sulphydryl Organo- Eserine reagent s phosphates ( PCMPS ) (DFP)

- Carboxylesterases - - + Aryles terases - + -

Acetylesterases - - f

t sensitive; - resistant; + resistant only in very low concentration; * Informatiron according to Gahan (1981, 1984) and Gahan & Carmignac (1989). 49 complete incubation medium or the medium without substrate, both of which contained the corresponding inhibitor. After incubation, sections were mounted on slides and examined by light microscope. Because neither a microdensitometer nor a microspectrophotometer was available for this study, the results of the reaction were evaluated by visual observation only . Inhibitor tests were not carried out with paraffin embedded sections, because the incubation time for paraffin sections is very long. During long incubation, the main inhibitor DFP can undergo slow hydrolysis in water, yielding hydrogen fluoride (HF, Techinical Information Bulletin -422,

Aldrich Chernical Company Inc. 1993). The hydrolysis causes the DFP to lose its - inhibitory function. Therefore, the

------results of=nhibitions in long time incubation are ques- tionable - 2 .3 Resul ts

2.3.1 General external morphology and phyllotaxis

Carrot is a biennial plant with a large tap mot and a very short stem produced during the first year. The next year, a floral axis forms and seeds are produced. In this study under greenhouse and growth chamber conditions, plants rarely produced inflorescences. A plant has been grown for four years and did not elongate its stem or form an inflorescence before it died. The "root" is conical in shape, plump or fleshy, and develops from the primary mot and the hypocotyl of the seedling. The stem is vertical and short.

In seedlings, the stem is 500 - 1000 pm in height, and in adult plants, it is 1750 - 2500 p. The leaves are separated by very short internodes. Each leaf has a long stalk that encircles the stem with a wide base. Cotyledons are simple and elongate obovate, and al1 other leaves are lobed or more deeply divided.

In seedlings, the two cotyledon and first pair of true leaves are opposite, while al1 subsequent leaves are helically arranged. In adult plants, the short stem bears its helically arranged leaves in a rosette. In a dissected shoot apex, P, contacts with P, and P,, P, contacts with P, and P,

(Fig. 2. la) and the number of contact parastichies is 2 + 3

(Cutter 1959). Phyllotaxis of carrot was determined by the method of directly counting from living plants (Cutter 1959, 51 Mohr and Schopfer 1995). From a given leaf in the stem of carrot, five leaves must be passed along a helix, that encircles the stem twice, before arriving at a leaf inserted approximately on the same vertical line (orthostichy) as the one with which the count was started. Therefore, the phyllotaxis of carrot is determined as 2/5.

2-3.2 Organization of the shoot apex The apical dome of a shoot apex of carrot is hemis- pherical (Fig. 2.2af b) and extends 50 - 60 p above the axil of Pl at the late plastochron (Fig. 2. lb) . The apical dome in seedlings and adult plants may Vary in size but the basic organization is not very different. In the following description, the shoot apex will refer to the apex of either seedlings or adult plants at the late stage of a plastochron. Only significant differences between them will be pointed out.

and corpus

In median longitudinal section, the dome of the shoot apex has a convex shape and shows four to five layers of small isodiametric cells with large round nuclei (Fig. 2.3). Based on the tunica-corpus concept (Schmidt 1924), the small isodiametric cells in the apical dome of carrot can be divided into two regions (Fig. 2.5) . The tunica comprises 52 two outer peripheral layers of the apical dome and is characterized by predominantly anticlinal division (Fig. 2.3). In most shoot apices examined, the cells in these two layers are similar in size (Fig. 2.3) . In a few cases, cells in the second layer are larger than those in the surface layer, The surface layer of the apical dome is continuous with the protoderm which will develop into epidermis. A cuticle can be seen on the outer surface of the tunica and the protoderm. The corpus is a group of cells which lie under the tunica (Fig. 2.5) . With different stains, the cells in the corpus are observed to be nonuniform in ternis of the degree of staining- Therefore, a zonation pattern may also be used to describe the characteristics of the apical dome of carrot .

2.3.2.2 Zonation The apical dome can be recognized as including several zones based on cytohistological characteristics in longitudinal section (Fig. 2-4)- The central zone is in the centre of the dome and is surrounded by the peripheral zone (Fig. 2.6). Cells in the central zone are isodiametric and larger than those in the peripheral zone (Fig. 2-41, Cells in the peripheral zone are smaller and are darkly stained by several dyes (Fig, 2.4). Using toluidine blue staining, the central zone is less stained than the peripheral zone (Fig. 53

2.4 ) . Using saf ranin-fast green staining, the peripheral zone is densely stained with safranin. Although with safranin-tarinic acid-iron alun staining the zonation is not very evident, the peripheral zone is somewhat darker than the central zone (Fig. 2.3). Rib meristem is located under the central zone (Fig. 2.6) . Although transverse divisions predominate in the rib meristem, a few longitudinal divisions occur which result in an increase of pith diameter (Fig. 2.4). Zonation is more distinct in the shoot apex at the late plastochron because the apical dome has reached maximum size.

2.3.3 Early development in the shoot apex In order to trace the process of early development in

the shoot apex, serial transverse sections 10 pm in thickness were examined and particular attention was given to the provascular tissue ring in relation to the position of the

youngest leaf primordium. The uppermost section showing the

tip of the apex will be used as a reference point in a vertical scale measured from the tip of the shoot dome downward toward the root. Nine shoot apices of seedlings and twelve shoot apices of adult plants have been examined in transverse sectional view. In addition, longitudinal sections of eighteen shoot apices were examined. 2.3.3.1 Plastochron and divergence angle

2.3-3.1.1 Plastochron

The initiation of successive leaf primordia occurred periodically with the time interval of a plastochron. The plastochron was determined by comparing the number of leaves and leaf primordia in shoot apices of nine seedlings fixed before and after a nine day interval. After nine days, the total number of leaves and ieaf primordia increased by three. Therefore, the plastochron of carrot seedlings is judged to be three days. The plastochron interval was also determined by direct observation of every new leaf emerging from the shoots of five adult plants in the growth chamber. Leaves present before the observation began were marked with a marker pen. The new leaves arise from the centre and can be identified by the absence of a mark, Usually, a new leaf was found every third day. Thus, in the growth conditions used in the present study the interval was approximately three days

2.3.3.1-2 Divergence angle

In the shoot apex of carrot, the base of a leaf primordium looks like a crescent enclosing the apex. The shoot apex is seen to be surrounded by several leaf primordia (Fig. 2.la, b). Since the leaf has a wide base, P, is often in contact with P, and P3, while P, is enclosed by P, and P,, P, 55 by P, and P,, and so on (Fig. 2.la). Each successive leaf primordium arises on the shoot apex near the ventral face of P,. The divergence angle from the last leaf primordium was measured from photographs and diagrams drawn from transverse sections of shoot apices of two seedlings and two adult plants. Rays were drawn from the axis centre to each main leaf trace. The angle of divergence between the rays of two successive primordia, P, and P,, on the apical dome was about

137" on average. The position of a new leaf primordium (1,) can be estimated by visually projecting the phyllotactic spiral around the apical meristem through an arc of ca. 137" beyond the last-forrned primordium (Pl).

2.3.3.2 Rhythmical changes in the shoot apex The rhythmical change in the shoot apex is reflected in the location of the axil of P,. Examination of serial transverse sections of shoot apices showed that a new leaf primordium is initiated very close to the summit of the apical dome. The shortest vertical distance recorded from the summit of the apex to the axil of the P, was about 10 pm because of the limit of the 10 pm section thickness (Table 2-

2). As the shoot apex becomes elevated through the activity of the apical meristem, the location of the youngest leaf primordium is displaced downward £rom the summit. The maximum distance attained before the next new leaf initial Table 2-2. The langth of the youngest leaf primordium and ite position, and the diameter of the apical dame at the level of the axil of Pl

------Shoot Length of Position of Diameter of the shoot apex Pl (Firn) the axil of apex at the level of P,4 (ml the axil of Pl (m) Seedlings

-- - Adult plants

The vertical distance £rom the summit of the shoot apex to the axil of the leaf primordiurn. 57

was elevated was 60 prn (Table 2-2). There was no difference between seedlings and adult carrot plants in terms of the minimum or maximum distance of the axil of P, from the summit (Table 2-2).

Still other rhythmical changes were observed in the shoot apex. For example, the rhythmical change affects the

diameter of the apical dome which is smaller in the early plastochron than in late plastochron, because the P, is close to the sumit. There was a difference in the maximum and minimum diameters of the dome between the seedling and adults- In seedlings the diameter of the dome ranged from

62.5 p to 112.5 pxn depending upon the stages of the plas-

tochron, while in adult plants the range was 80 to 200 pn (Table 2-2) . Table 2-2 shows that the diameter of apical dome is related to the position of P, but not to its length.

2.3.3.3 Provascular tissue and procambium

2.3.3-3.1 Transverse view

When transverse sections were basipetally traced from the summit, it was found that the earliest appearance of a provascular tissue ring is related to the vacuolation of surrounding cells. The earliest ring probably results from the differentiation of the pith and cortex rather than of the ring itself. In carrots, the earliest pith differentiation started as high as the level of 50 below the tip where rib 58 meristem was located (Fig. 2.7) . At first only 1 - 3 large cells could be seen in the centre of the rib meristem.

Juvenile pith cells could be identified by large size and vacuolation (Fig. 2.8) . Cortex development occurred later than pith formation and was at the level of 70 - 110 p depending on the plastochron status (Table 2-3).

At the late plastochron, the shoot apical dome extends

60 pm above the axil of Pl. No vascular differentiation was observed above this level. A section through the 50 pm level showed that the central part is less stained than the periphery, so that there is a ring of meristernatic cells (Fig. 2.7). This ring is equivalent to the peripheral zone of the apical meristem in a longitudinal section, which is the source of both the cortex and provascular tissue. At the 90 pm level, since the cortex was not well differentiated, a provascular ring was present but not distinct (Fig. 2.8). At the early plastochron stage, no evidence of vascular differentiation could be recognized in this region above or at the level of the axil of the youngest leaf primordium (Fig. 2.9) . At the 70 pm level, when the cortex was well developed, this together with pith development blocked out a distinct provascular ring (Fig. 2.10) , The shape of cells in the provascular ring is nearly isodiametric in transverse view (Fig. 2.10) . The cells in the ring are smaller than the cells of the pith and cortex. Table 2-3. The vertical diatance from the srlamiit of the apical dome to the earliest appearance of a provascular ring compared with the position of the dlof the leaf prfmardia and the divergence point of their main leaf traces in the shoot apex of adult plants

The The Diver- The 1 evel gence level of gence level of Shoot of the point of the axil point of provas - apex axi l leaf of P, leaf cular of P1O trace of (w) trace of ring (iun) pi (Pm) pz (lm (Frrn)

Aver- age 60 They have dense cytoplasm, are less vacuolate and, therefore, are densely stained (Fig. 2-10). The nuclei are round and large and a nucleolus is distinct in each cell- The main feature in which provascular tissue differs from surrounding

tissue is that the cells are not distinctly vacuolate. The location of a provascular ring in relation to Pl and P, was also examined following the series of transverse sections. Among twelve adult plants examined, the level of the earliest appearance of the provascular ring was often below the axil of Pl and occasionally below the axil of P , (Table 2-3) However, the ring could be above or at the sarne

level as the divergence point of the P, leaf trace if the P,

leaf trace is present (Table 2-3). The ring clearly exists above the divergence point of P, (Table 2-3) .

2.3.3.3-2 Longitudinal view In longitudinal sections, the provascular cells are present below the peripheral zone of the apical dome (Fig, 2.4) and are continuous with the procambium underneath (Fig. 2 1 The cells in the position which corresponds to the level of the appearance of the provascular ring are al1 nearly rectangular in shape but not longer than the surrounding parenchymatous cells in cortex and pith (Fig. 2.3). They are distinct from surrounding parenchyma cells by the 61 densely stained cytoplasm and the nucleus is large and round (Fig. 2 -3). In longitudinal view, provascular tissue has some similarity to cells in the peripheral zone, especially in being darkly stained (Fig. 2 .1 . The cells in the peripheral zone are isodiametric to rectangular in shape and look like the cells of provascular tissue (Fig. 2.3) . The nuclei of cells in the peripheral zone are large and round and the nucleoli are distinct (Fig. 2.3). There is no sharp boundary between them and provascular cells, In contrast to the similarity to provascular tissue, cells in the peripheral zone show some differences from the cells in the central zone which are less stained and larger in size (Fig. 2.4) . In spite of similarity, the tissue in the peripheral zone is not considered as provascular tissue because it is the source of both cortex and provascular tissue. The provascular tissue is also similar to procambium in longitudinal view. The cells of procambium are densely stained (Fig. 2.11) and their cells have a large nucleus and dense cytoplasm. The main difference is that procambium evidently connects to the older vascular bundles below (Fig. 2.11). The procambium cells near the mature vascular tissue have an apparent elongate shape and the nucleus is also elongate (Fig. 2.11) . In order to demonstrate this objec- tively, cells in provascular tissue and in procambium were 62 compared. Cells from provascular tissue at the level of 90 - 110 p were measured because this corresponds to the level of the earliest appearance of the provascular ring. The cells in procambium were selected from those near mature vascular tissue. The greatest dimension, L, and the least dimension, W, were recorded and the ratio was calculated (Table 2-4) . The ratio does show that there is a significant difference. However, there is no sharp boundary between procambium and provascular tissue in longitudinal view, The gradua1 change in ce11 shape and nucleus can be followed,

2.3-3.4 Leaf traces and axial bundles In seedlings, each cotyledon has three traces and the first pair of true leaves each has three to five leaf traces, In both seedlings and adult plants, the youngest leaf primordium has no or one leaf trace as it emerges from the apical dome, but the number of leaf traces in it increases as the leaf primordium develops, The second leaf primordium has one to three traces, the third has five, and the fourth has seven traces. When a leaf primordium is fully developed, the leaf bas nine to thirteen leaf traces in the growth conditions of this study. As defined in the Introduction a leaf trace and an axial vascular bundle form a continuous vascular strand but the former bends at the divergence point and extends to a leaf Table 2-4. The cornparison of ce11 size among cells in provascular stage and in procambial stage in longitudinal view

Cells in the Cells in Cells in leaf traces procambium the in near the provascular provascular mature ring stage vascular tissue

Width (p)1 5.19 (17) 1 5.2 (10) 1 5.5 (7) L/W ratio 1 2.65:l 1 2.58:l 1 7.1:l

Al1 values in brackets () are sample size; L/W ratio: the greatest and least dimension ratio. 64 (Fig, 2.12) . In longitudinal view, the leaf trace-axial

vascular bundle is not uniform in ce11 shape (Fig. 2.12). At the tip of a leaf primordium there is a group of meristematic cells. The cells along the leaf trace are gradually elongate

basipetally (Fig- 2.12)- In other words, the leaf traces are not immediately at the procambial stage. In order to determine the status of the cells of leaf traces, the L/W ratio of cells was measured. The cells under the tip of a leaf primordium do not have as high a ratio as the procambium

cells near the mature vascular tissue (Table 2-4). The cells in leaf traces at the early stage are similar to provascular tissue and should be considered as the provascular stage instead of procambial. In other words, the provascular stage may be identified in leaf traces as well as in the axis. Similarly, in a study of the fern Matteuccia, the provascular

stage was recognized in describing the leaf traces as well as the transitional, procambial and mature stages (Ma and Steeves 1994). No discontinuities were observed in the procambial and provascular strands. Leaf traces and axial vascular bundles at a very early stage have a similar appearance in transverse sectional view. The initial of an axial vascular bundle consists of a group of cells which are smaller than other provascular tissue cells (Fig. 2.13) . The srnall size of cells suggests that longitudinal divisions have occurred in these initial cells. 65 The initials of axial vascular bundles which are found in the provascular ring (Fig. 2.13) will differentiate into the stem vascular system. The earliest leaf traces appear as several darkly stained cells arranged in a group and located outside of the ring in the stem (Fig. 2.13). The location of leaf traces indicates that they have diverged from the stem vascular system. In the leaf trace, a few small cells were also observed which had resulted from longitudinal divisions (Fig. 2-13}. When the timing of vascular differentiation in leaf traces of P, was followed, it was show11 that the number of the leaf traces was related to the length of Pl (Table 2-5).

In observations of nine seedlings and twelve adult plants, P, varied in length from less than 10 ym to 210 pn, and might or might not have leaf traces. A Pl with a length greater than

80 prn had a leaf trace, while in a Pl with a length less than

80 pm there was no evidence of a leaf trace in the primordium above the level of the axil, although the trace might be identified in the base. Even when the length of P, was 10 pm or less, however, an axial bundle has been observed in the provascular ring below the leaf primordium. This rnay suggest that the vascular bundle, which is positionally related to a leaf primordium above, exists prior the protrusion of the leaf prirnordium. Although the length of Pl could approach

200 pm, no mature vascular tissue, phloem and xylem, was Table 2-5. The number of va~cularbundles and leaf traces corqpared with the length of the PI grimordium to which the bundles and trace8 are related

Shoot Length of Number of leaf Number of vascular apex I Pl (W) I traces in PT bundles' related to P, Seedlings

Adult plants

O Observed at or above the level of axil of leaf primordium; * Vascular bundles in the provascular ring or leaf traces below the level of the mil of the leaf primordium. 67 found in the main leaf trace. The axial vascular bundle connected to the main leaf trace was more differentiated but nothing indicated that it had passed beyond the procambial stage,

2-3.4 Maturation of prixnary vascular tissue The following description is concerned with the later stages in primary vascular differentiation in the apices of carrot. The pattern of primary vascular differentiation in the shoot apices of carrot was followed both in serial transverse and longitudinal sections. Al1 shoot apices examined in serial sections had the same basic pattern of vascular differentiation. However, there were variations, such as varied distances from the apical summit for the first occurrence of mature protophloem sieve tubes or protoxylem elements at certain developrnental stages. This variation is interpreted as a reflection of the fact that shoot apices are at different stages of the plastochron,

2.3.4.1 Phloem differentiation Phloem was found associated with the third or forth leaf primordium (Table 2-6) but was not found in relation to P, or P,. The youngest leaf primordium to have phloem associated with it was P, and the oldest leaf primordium without phloem was also P,, Phloem in the stem vascular system could be 68 found about 150 - 250 p below the tip of the apical dome beneath P, or P,, In a given vascular bundle, the first sieve element matured before the first xylem element (Fig. 2-14). The phloem elements appear first in the median trace then in the lateral traces. No discontinuities were detected in the differentiating phloem strands and mature phloem was observed first in the lowermost parts of traces. In other words, the young phloem was always at the top of mature phloem indicating acropetal differentiation. The protophloem was observed at the outer boundary of a procambium strand as a single sieve element with or without a companion cell. The metaphloem sieve elements were associated with companion cells. Both proto-a and metaphloem sieve elements can be identified by brightly green stained ce11 walls and angular shape (Fig. 2.14). The ce11 walls of sieve elements showed weak birefringence under polarized light.

2.3.4.2 Xylem differentiation The first xylem element occurs in a procambial strand related to P,, P, or P,. The youngest leaf trace or axial vascular bundle with xylem is related to P,, the oldest without xylem is related to P, (Table 2-6)- As in the case of phloern, no xylem was found in relationship with P, or P,. Xylem differentiates later than phloem because phloem alone Table 2-6. The summazy of the the of vascular differentiation in relation to the length of Pl

Vascular tissue in stem related Status to the distance from the tip of The of apex (pm) and to the leaf Shoot length leaf prirnordium (P) apex of P, trace (ml of Plf Phloem Xylem

94080 30 mer 200 (5) 280 (P4) 4-A2 94080 50 mer 220 (P4) 230 ( P4) 4-A6

* At the level of the axil of leaf primordia; mer: Meristematic stage; pvt: Provascular tissue stage. 70 (Fig. 2.14) or both phloem and xylem could be found in a leaf trace (Fig. 2.15) but no xylem was found without phloem.

Xylem in the stem vascular system was observed at the level of about 230 - 380 pm below the tip of the apical dome beneath P,, P, or P,. The appearance of protoxylem begins at the point where

vascular bundles diverge to leaf traces of P, to P,. At first, a single protoxylem element is located on the inner boundary of the procarnbium strand in transverse section (Fig. 2.15). This could be a single isolated element representing the longitudinal extent of xylem differentiation or several xylem elements in a linear sequence, The ce11 walls of protoxylem were stained red by safranin and showed birefringence (Fig. 2.15). In longitudinal view, the pattern of the thickening was in the form of rings or a simple long single spiral. The continuity of protoxylem could not easily be followed in longitudinal view. Following transverse sections basipetally, in a vascular bundle two protoxylem elements at one level could be replaced by one and then by none. This indicates that protoxylem differentiation occurs basipetally and discontinuously. In its downward course the protoxylem eventually becornes connected with that of the older vascular bundles. In its upward development, the protoxylem quickly extends along the protophloern to the leaf traces. Therefore, 71 the maturation of the protoxylem within a given trace is discontinuous and proceeds in two directions. One is upward to leaves and the other is downward to the stem vascular system. The discontinuity of protoxylern differentiation is observed in the adult plant, but in seedlings this discontinuity of protoxylem was not found. The metaxylem occurs between procambium and protoxylem in the vascular bundle. The ce11 walls of metaxylem were stained red colour by safranin and showed a strong birefringence. In longitudinal view, they showed different thickening patterns. They often had a short spiral thickening and double or more complex spirals.

2.3.5 Esterases in the shoot apex

2.3-5.1 Esterases in frozen sections Esterase activity has been tested under several conditions. No enzyme activity was observed when sections were incubated for I h at 37°C in the incubation medium from which the substrate or diazonium was omitted (Fig. 2.16). Similar- ly, when sections pre-treated in boiling water for 5 min (heat-inactivated) were incubated in full incubation medium for 1 h at 37"C, no colour reaction was found.

When frozen fresh sections were incubated in a full medium for 1 h at 37"C, esterase activity was identified in al1 plant tissues. Highest activity was clearly observed in 72 the vascular tissue of the shoot apex but weak activity was also observed in other tissues, such as the developing cortex and pith (Fig. 2-17). Under high magnification, esterase activity was shown by a blue deposit suspended in the cytoplasm. Most of the blue deposits in cells were observed to be adjacent to ce11 walls. In longitudinal frozen sections of the shoot apex, heaviest esterase activity was found in vascular bundles and in leaf traces (Fig. 2.17). The high esterase reaction could be seen to extend from the procambium to provascular tissue. The heavy reaction approached but did not clearly enter the peripheral zone of the shoot apex. In addition to vascular tissue, high esterase reaction was also identified in the zone of the developing pith cells (rib meristem) . In the zone of high reaction which included the provascular ring and developing young pith, ce11 division and differentiation were rapidly occurring. In the apical dome, there was a positive reaction in the surface layer and the central zone. The peripheral zone showed only a very weak esterase reaction.

Esterase activity in mature tissue was low. There was a weak reaction in the mature pith and cortex but the epidermis showed higher esterase activity than the cortex. In transverse sections, the highest reaction was observed in the leaf traces and vascular bundles (Fig. 2.18). At a level where the provascular ring was observed in a 73 normal shoot apex, a high positive reaction ring could be seen (Fig. 2.18). This ring included both provascular tissue and developing pith which is just inner to (inside) the provascular tissue. At this level, the reaction in the provascular ring was the same as that in these developing pith cells and obviously higher than that in the cortex. This indicates that rapid ce11 division was occurring inside the provascular tissue contributing to pith lateral expansion. Since pith developrnent is earlier than provascular tissue, the high reaction related to pith differentiation in the centre of the apex was observed above the provascular ring. When the second serial section from the summit of the shoot apex was observed, it clearly showed that a higher esterase reaction was present in the central than in the peripheral zone and the buttress of a leaf primordium.

2.3.5.2 Esterases in paraffin sections Paraffin embedding takes tirne and enzyme activity is reduced by the high temperature of the embedding process, compared with fresh frozen sectioning. However, the distribution of esterases in paraffin sections was the same as that in frozen fresh sections. In addition, the paraffin embedded rnaterial can be stored at a cool temperature (5°C) for months- After six months storage, esterase tests showed that enzyme activity was not apparently reduced. 74 When slides with sections were incubated at 37°C in a medium with the substrate ornitted, no positive reaction was observed at a time when sections in full medium started to show blue colour. Even if the sections were incubated for 10 hours, no blue colour was detected under high magnification under these conditions, With a full incubation medium, the reaction could be observed after 1 h and the maximum reaction could be reached

at 5 h. Eight hours incubation did not evidently increase the blue colour. The distribution of esterase activity could be more precisely determined but there was no large difference from the fresh frozen sections. Heaviest reaction was observed in the vascular region and weak activity was detected in other tissues. In longitudinal section, the esterase reaction was observed from procambium to provascular tissue, both of which

could be identified by ce11 shape (Fig. 2.19). Activity in provascular tissue gradually became weaker acropetally toward the peripheral zone of the apical dome (Fig, 2-19). In the apex, al1 parts of apical meristem showed some weak reaction except the buttresses of leaf primordia near the peripheral zone (Fig. 2.19) . Only weak activity was detected in the surface layer of the apical dome, but in epidermis activity was higher. Under the apical meristem the rapidly developing pith cells showed a higher reaction which was more intense 75 than the reaction in the provascular tissue at the same

level. When cells in the centre of the pith were well developed, the activity in them became reduced. Activity in developing cortex was also high. As cells in the cortex vacuolated and enlarged, the activity was reduced.

In transverse sections, a similar result was observed.

At the level of 20 or 30 pxn from the summit, the central zone close to the top of the rib meristem showed a higher positive esterase reaction than the cells in the peripheral zone. From the level of 40 p to 50 m, the rib meristem showed higher positive reaction than the surrounding tissue. From

the 60 pm to 80 pm level, the enzyme reaction in the provascular ring gradually became stronger as the sections were

Eollowed basipetally. At 90 prn from the summit, where a provascular ring is often observed in general histological methods, the provascular ring showed a relatively high positive reaction (Fig. 2 -20).

2-3.5.3 Carboxylesterases and inhibitors The above tests demonstrated that enzyme activity in the shoot apices of carrot is relatively high in the vascular tissue. Whether or not this activity in vascular tissue is mainly contributed by carboxylesterases can be answered by inhibitor tests. In order to identify the esterases, inhibitors were added to the full incubation medium. The 76 effect of each inhibitor is described in Table 2-1. Adding eserine eliminates other enzyme reactions, Le. only esterase activity is present when eserine is added to the buffer. After the eserine tests, only aryl-, acetyl- and carboxylesterases are possible candidates. A PCMPS test is necessary to demonstrate if arylesterases are present. The DFP test determines if carboxylesterases are present. Because a low concentration of DFP can inhibit the activity of carboxylesterases, and acetylesterases are sensitive only to a high concentration of DFP, the results depend on the concentration of DFP in the buffer. When a high concentration of DFP is used to treat the sections, the reaction in tissues results from aryl esterases. When a low concentration of DFP is used to treat the sections, the reaction in tissues is the result of both aryl and acetylesterases. The following description was based on tests of frozen sections.

Controls Two controls were carried out with each inhibitor test. One consisted of omitting substrate, No enzyme activity was observed when sections pretreated in the buffer without the substrate and inhibitors, were incubated for 1 h at 37"~in the incubation medium. The other control, in which sections were pretreated in the buffer without inhibitors followed by 77 incubation in the full medium, showed that a positive reaction occurred in the provascular tissue ring (Fig. 2.25).

2.3.5.3.2 Eserine

Using M eserine as an inhibitor, a heavy reaction was observed in the vascular tissue including axial vascular bundles and provascular tissue (Fig. 2.21). Thus reaction in the vascular region was not apparently reduced compared to the sections pretreated in buffer followed by full medium without inhibitor (Table 2-7)- In a transverse section, the axial vascular bundles and provascular tissue between them showed high positive reaction (Fig. 2.22). The fact that the reaction in the provascular tissue has not disappeared indicates that the enzymes causing the reaction in both the provascular tissue and the axial bundles are the same-

Because only esterases are not sensitive to eserine, this test demonstrated that the reactions observed in sections are produced by esterases .

2.3.5.3.3 PCMPS

The results using 10-~M PCMPS as an inhibitor are shown in Table 2-7. The reaction should be somewhat reduced because arylesterases were inhibited but the reduction in vascular tissue including provascular tissue was not very evident (Fig- 2.23) compared to the controls pretreated in Table 2-7. Smmazy of esterase activity present ia the shoot apex when 8hoot apex sections are incubated in buffer without or containfng different inhibitor for 45 min and full reaction medium without or contaiaing each inhibitor for 60 min, crll ut 37°C

Full Fu11 Full medium + medium + medium + Full 10-~M IO-*M - 10-~M medium Eserine PCMPS DFP Vascular +++++ +++++ tissue* provascular tissueu

Developing cortex I

Developing pith celfs 1 *+++ 1 * Not including provascular tissue Q At the level of divergence point of the leaf trace of P, Act ivi ty (arbitrary blue colour density estima ted under light microscopy) : + lowest but can be identified under high magnification; ++ low but can be observed under low rnagnification; +++ median; ++++ high; +++++ heavy or highest colour. 79 buffer (Fig. 2.25). Without a microdensitometer, the reduction could not be evaluated quantitatively. A heavy reaction was still present in the vascular tissue region and developing pith and weak reactions were present in other tissues (Fig. 2.24). This indicates that the reaction of esterases in the vascular tissue including provascular tissue is probably not mainly contributed by arylesterase activity. Although the colour reduction was not quantitatively measured, the weak reduction indicates that the activity of arylesterases in the tissues is not high.

2.3.5.3.4 DFP

DFP tests have been summarized in Tables 2-7 and 2-8.

In controls, without substrate in the incubation medium, no reaction was detected (test 1-3). In full medium controls, pretreated in buffer without DFP, al1 sections showed the reaction (test 4-8, Fig, 2.25) . When sections were pretreated in a buffer containing 3 x or 6 x IOe6 M DFP, the reaction was not evidently inhibited (test 9-12) compared to full medium controls. When 3 x M DFP was tested, colour was reduced but still visible (test

13-17) , It was found that the colour reduction depends on the tissue. The greatest reduction of blue colour was found to be in vascular tissue including provascular tissue (Fig.

2.26). Heavy blue colour was found in the vascular region in Table 2-8. The resulta of esterase activity present in the 8hoot -ex when section^ are pretreated in buffet or buffw containing various concentrationa of inhibitor DPP for 45 min at room teerature and then were reacted in the medium without substrate or full medium con- tainf ng a corresponding concentration of inhibitor DFP or full reactfon medium for 60 min; a21 at 37*C

Incubation Pre-treated DFP Result Test medium in solution concent rat ion colour (Ml

1-3 omit buf fer substrate

4-8 full buf f er heavy blue

9 full DFP heavy blue

10-12 full DFP heavy blue

- -- 13-17 full DFP 3x10-' light blue

18 full DFP 7x10-' weak blue

19 full DFP lx10-~ weak blue

20-21 full DFP 1. TXIO-~ weak blue 22-23 full DFP ZXIO-~ weak blue

24-25 full DFP 3x10-~ vev weak blue

26 full DFP

27 full DFP 3x10-= vew weak blue 81 full medium controls (Fig. 2-25), but this reaction was reduced in DFP treated sections (Fig. 2-26). The reaction in other tissues showed overall reduction. The reaction in the centre of the apical dome and in the developing pith cells was reduced in the DFP treated sections compared with the control. In the provascular ring, in which the reaction was observed in full medium controls (Fig. 2.25) , the activity was much weaker when DFP was present (Fig. 2.26). In contrast to the overall reduction, a few pith cells still showed a high reaction (Fig. 2.26). This indicates that part of reaction in the developing pith was not caused by carboxylesterases.

When sections were tested in buffers containing 7 x or 1 x, 1.5 x, 2 x 1W4 M DFP, the strong reaction which is usually related to the vascular tissue had disappeared; only weak but still visible blue colour could be observed in al1 tissues directly under the microscope (test 18-23 in Table 2-

8). This blue colour was so weak that it was difficult to demonstrate in the photograph (Fig. 2.27) and appeared similar to the control without substrate. Under high magnification, a number of fine blue particles were present in M DFP treated sections (Fig. 2.28) but not in controls without substrate. The weak reaction blue colour and fine blue particles may be the product of the reactions of both acetyl and arylesterases . 82

When the sections were in the buffers containing 3 x 1.0'~ or 1.5 x, 3 x IO-" DFP, the reaction distribution pattern was completely changed because both carboxylesterases and acetylesterases might be completely inhibited, There was no blue colour observed directly under the microscope (test 24- 27 in Table 2-8) . However, fine blue deposits were found in al1 tissues under high magnification, This indicates that al1 reactions contributed by carboxyl and acetyl esterases were inhibited. Because acetylesterases are sensitive to a high concentration of DFP, only arylesterases cannot be inhibited by DFP. This blue deposit is interpreted as the contribution of arylesterases. 2.4 Discussion: A study of normal development was undertaken to re- examine critically the early stages of vascular differentiation in the shoot apex of carrot. This was also a necessary first step for experimental investigations on early vascular differentiation. In addition to histological study, the activity of esterases which are believed to be related to vascular differentiation was examined. The vascular system of carrots poses problems because, with many trace per leaf, it is complex but the plant has advantages for experimental work. Because of its complexity, no attempt has been made to study the full development of the vascular system. Rather the study has been concentrated on the early stages where the ma j or unsolved problems remain.

2.4.1 Provascular tissue cylinder

Provascular tissue has been demonstrated in the fern Matteuccia to be the early stage of differentiation of vascular tissue (Ma and Steeves 1994, 1995a) . In angiosperms, as mentioned in the introduction, the presence of provascular tissue has been questioned because there is no sharp distinction between the provascular tissue and the apical meristem (Mcllrthur and Steeves 1972) . 2 - 4 -1.1 Location of provascular tissue cylinder In ferns, the provascular tissue is located immediately under the one layer of promeristem and above the youngest leaf primordium (Wardlaw 1944a, Ma and Steeves 1994, 1995a) . The presence of provascular tissue in angiosperms has only been reported in Primula and Geum (Wardlaw 1950, McArthur and

Steeves 1972). The provascular tissue described in these two species is very close to the residual meristem which is on the pathway from procambium to the apical meristern in several angiosperms (Helm 1931, Louis 1935, Kaplan 1937, Sussex

1955a, Esau 1977) The present work shows that there is a provascular cylinder in the shoot apex of carrot which can be observed in both seedling and adult plants. This provascular tissue region is outlined by the initial differentiation of pith and cortical tissue- At first, the developrnent of cortex and pith is more advanced than that of the provascular tissue, so that the ring appears to result from the development of cortex and pith, rather than from its own differentiation. From this tissue region axial vascular bundles are initiated. In most situations, axial vascular bundles are observed in this ring but these early bundles are just groups of cells with a little darker staining than other provascular tissue 85 cells- This is consistent with other observations in other dicots (McArthur and Steeves 1972). Examination of serial transverse sections of the carrot shoot apex showed a ring like provascular region above or at the level of the divergence point of the youngest leaf trace. However, the provascular ring was below the axil of the youngest leaf primordium. The present study reveals that leaf initiation in carrot occurs very close to the summit of the apical dome. At this initial stage, the whole leaf primordium is in the meristematic stage and no cortex can be identified in the axis. At the late plastochron, the cortex is well developed in relation to Pl, but no cortex is developed above the axil of P,. Because the visible ring is the result of the development of cortex and pith, it was not possible to recognize it above the axil of the youngest leaf primordium in any of the examined shoot apices of seedling and adult plants, in early or the late plastochron. This result supports the observation in another dicot, lupin, in which the ring was not found above the youngest leaf primordia (Bal1 1949) . However, as noted in the introduction, it is probably of greater significance that the provascular tissue is present at, or often above, the level at which the trace of the youngest leaf diverges from the stele. 86

2 .4.1.2 Histological features In ferns, the provascular tissue has cytohistological features distinct from those of the promeristem (Ma and Steeves 1994, 1995a). In angiosperms, the general histological study showed that provascular tissue is relatively densely stained with safranin and is not very distinguishable from the peripheral zone of the apical meristem. In the dicot species Geum chiloense, McArthur and Steeves (1972) found that at the base of the apical meristem there is a provascular cylinder which is apparent because the cells of the pith and cortex enlarge and vacuolate. It was observed that the cells of provascular tissue are similar to those of the apical meristem, very densely stained with large nuclei which nearly fil1 the ce11 lumina and contain many large safranin-stained nucleoli which are characteristic of meristematic tissue. In general, the histological features of provascular tissue in Geum are closer to meristematic tissue than to vascular tissue. In carrot, the shape of cells in the provascular ring is nearly isodiametric in transverse view. The cells in the ring are smaller than the cells of the developing pith and cortex. They have dense cytoplasm, are less vacuolate and, therefore, are densely stained. The nuclei are round and large and a nucleolus is distinct in each. The main feature 87 in which provascular tissue differs from surrounding tissue is that the cells are not distinctly vacuolate. In longitudinal sections of carrot shoot apices, the provascular cells are present below the peripheral zone of

the apical dome and are continuous with the procambium underneath. The cells in the position which corresponds to the level of the appearance of the provascular ring are al1 nearly rectangular in shape but not longer than the surrounding parenchymatous cells in cortex and pith. They are distinct from surrounding immature parenchyma cells by the densely stained nucleus and cytoplasrn. Although the shape of provascular cells is nearly rectangular, the nucleus is large and round. In angiosperms, the first stage of vascular differentiation is often described as procambium (e.g. Esau 1977). Both procambium and provascular tissue are densely stained by safranin or toluidine blue. Procambium is evidently continuous with the older vascular bundles below. The procambium cells near the mature vascular tissue have an elongate shape and the nucleus is also elongate. The length - width ce11 ratio shows that there is a large difference between provascular tissue and advanced procambium. However, there is no sharp boundary between procambium and provascular tissue in longitudinal view. The gradua1 change in ce11 88 shape and nucleus from provascular tissue to procambium can be followed. The present work also demonstrated that the provascular stage not only is present in the axial vascular systern but also is present in the leaf traces. In longitudinal view, the leaf trace-axial vascular bundle is not uniform in ce11 shape. At the tip of a leaf primordium there is a group of meristematic cells. The cells along the leaf trace are gradually elongate basipetally. In other words, the leaf traces are not immediately at the procambial stage, The L/W ratio of cells showed that the cells under the tip of a leaf prirnordium do not have as high a ratio as the procambium cells near the mature vascular tissue. The cells in leaf traces at the early stage are similar to provascular tissue in the L/W ratio and should be considered as provascular instead of procambial. In other words, the provascular stage may be identified in leaf traces as well as in the axis. Similarly, in a study of the fern Matteuccia, the provascular stage was recognized in describing the leaf traces as well as the transitional, procambial and mature stages (Ma and

Steeves 1994)- 2-4.1.3 General conclusion In summary, the present general histological study provided evidence to support the concept that early vascular differentiation takes place in the provascular tissue of angiosperms. The provascular tissue cylinder is located between the apical meristem and prxambium and occurs prior to the appearance of leaf traces and axial vascular bundles. Provascular tissue also is present in the leaf traces before they advance to the procambium stage. Provascular tissue has similarity to procambium in histological features but is

different from procambium in ce11 L/W ratio. This evidence supports the interpretation that the provascular tissue is the early stage of the vascular differentiation in angiosperms .

2.4.2 The apical meristem of the shoot apex The apical meristem is the source of al1 cells in the shoot, and tissue differentiation in the shoot apex could provide a basis for interpreting further developnent. Previous studies have shown that the apical meristem has three basic functions: initiating tissue and organs, corn- municating signals and maintaining itself as a formative region (Medford 1992) . However, what important role the apical meristem plays in the pattern formation of the prirnary 90 vascular system in the stem is little known. Since the general histological features and morphological evidence show that provascular tissue has a strong relationship to the peripheral zone, it is reasonable to expect that certain changes related to early vascular differentiation might occur in the peripheral zone. When the organization of the apical meristem is studied comparatively, significant differences between ferns and angiosperms may be recognized. In ferns, the existence of an

apical ce11 is a nearly constant feature (Bierhorst 1977). The apical meristem, or promeristem, consists of a single layer of distinctive cells immediately overlying tissue in the initial stage of differentiation (Steeves and Sussex

1989). Zonation patterns are ccmmon among angiosperm apical

meristems (Wardlaw 1957, Steeves and Sussex 1989) . Histolo- gical zonation has been supported by many studies of the

vegetative shoot apex of angiosperms (review by Hara 1995) including supporting evidence from ultrastructural studies.

Electron microscopie studies on shoot apices have revealed that zonation is present but is not distinctive at this level. In dicotyledons, ultrastructural differences between

tunica and corpus cells are mainly quantitative (Lyndon and Robertson 1976, Mauseth 1980) . Morphometric ultrastructural studies also have shown that each zone has its own distinct ultrastructure, but that the differences among the zones are 91 srna11 (Mauseth 1981a, 1982, Berggren 1984). These small dif ferences, however, may establish a basis for further dif ferentiation. The present histological study showed that the apical dome of carrot can be divided into several zcnes, The peripheral zone is located at the top of the provascular tissue and the earliest changes could take place there. Periclinal ce11 division in the peripheral zone, which was commonly explained as an early indication of the initiation of a leaf primordium, may be an indication of early differentiation, After the leaf primordium has emerged, a vascular bundle or leaf trace extends into it. However before the leaf primordium emerges, no other morphological evidence for vascular differentiation was found in this zone. The present study also showed that the distinctive features of the peripheral zone are closer to those of the tissue below it than to those of the central zone. In histological characteristics, the cells of the peripheral zone are relatively darker staining than cells in the centre of the apical meristem and in this respect are very similar to the cells in the provascular tissue. The differences between peripheral zone and central zone are demonstrated by different staining methods, such as safranin-fast green, toluidine blue and safranin-tannic acid-iron alum. Safranin alone does not make the central and peripheral zones very 92 distinct, but the difference between them still can be identified. The central zone is less stained in toluidine blue, while tannic acid-iron alun makes the peripheral zone more distinct. In other cytological features, however, whereas in the provascular tissue the ce11 division plane is predominantly longitudinal, sirnilar to that of procambium, in the peripheral zone this ce11 division plane is not predominant. The similarity between provascular tissue and peripheral zone not only reflects their close location, but also suggests an inherent relationship between them. Because a rhythrnical change occurs in the shoot apex, the peripheral zone is always present but the cells in this zone are not permanent. The tissue in the peripheral zone in one plastochron will become provascular tissue and cortex in the next plastochron. The peripheral zone is a source of both cortex and provascular tissue and has been suggested to be a place where early dif ferentiation might occur (Sussex 1955a) . Although the morphological evidence is sparse, early differentiation in the peripheral zone has been suggested by histochemical and molecular studies. Histochemical study on the shoot apices of Pisum and Vicia showed that carboxylesterase activity is present continuously from the stele through the procambial zone to the outermost layer of the 93 tunica (Gahan and Bellini 1984). From the procambium zone to the tunica, the peripheral zone must be traversed. In other words, carboxylesterase activity is reported to be present in the peripheral zone. Carboxylesterase was reported as an indicator of vascular dif ferentiation (Gahan 1981, Ma and Steeves 1995a) . Therefore, histochemical evidence indicated that vascular differentiation had occurred in the peripheral zone. However, in the present study of carrot, esterase activity was not detected in the peripheral zone. In conclusion, the present histological study of carrot did not provide clear morphological evidence to support the occurrence of early vascular differentiation in the peripheral zone, although there are indications that some initial steps may occur in this region.

2.4.3 Esterases in the shoot apex Although the histological study provided clear evidence for the existence of a provascular stage in the differentiation of vascular tissue in carrot, the argument could be made that this tissue is no more than an extension of the apical meristem, a residual meristem. For this reason, it was important to obtain histochemical evidence for the initiation of differentiation in the provascular tissue. Esterases, particularly carboxylesterase, have been recognized as a 94 marker of vascular differentiation in a number of previous

studies (Gahan and Bellani 1984, Ma and Steeves 1995a). Standard methods for the recognition of these enzymes were applied to both fresh frozen sections and sections prepared by the paraffin material in the present investigation. The paraffin method has advantages in that serial sections allow

developmental events to be traced and tissue localization can be more precise. However, the reaction is less intense and it is not possible to carry out the inhibitor tests which are required to identify carboxylesterase specifically. The present work showed that enzyme activity may be detected in al1 tissues in the shoot apex and semi-quantitative differences could be detected by visual observation between the vascular and non-vascular tissues. Vascular tissue showed the highest reaction and the peripheral zone

and leaf buttresses of the apical meristem showed the lowest reaction. In other tissues, the activity was different among

ce11 types. The reaction in epidermis, for example, was observed to be higher than that in cortex. The difference between low activity and a negative reaction depends on the conditions selected. The conditions of a histochemical localization test are usually established with the object of differentiating regions of high activity from those of low activity or none, and the reaction times and conditions are selected that produce the clearest 95 contrast (Vanden Born 1963) . Under those circumstances a negative reaction indicates relatively low activity or none, and no distinction is made between these two conditions (Vanden Born 1963) . Because the conditions of histochemical tests in the present investigation were set to detect low activity, certain tissues such as cortex in which no reaction has been reported by others (Gahan and Bellani 1984, Ma and Steeves 1995a) showed a reaction in the present work. In both fresh and paraffin sections, esterase activity was identified in the provascular region. Since esterases are considered to be early markers of vascular differentiation, this evidence supports the interpretation that provascular tissue is a differentiating tissue. Because the provascular tissue is still in the early stage of vascular differentiation, the esterase activity might not be expected to be as high as in the more differentiated vascular region. Although the peripheral zone is the source of both cortex and provascular tissue, this zone showed no or very low activity. This indicates the meristematic status of the peripheral zone. At the base of apical meristem, three basic tissue systerns are differentiating. Since pith differentiation is earlier than that in cortex, in the rib meristem relatively high esterase activity was observed. As the cortex developed, an increase in esterase activity appeared in this tissue. In contrast to the cortex and rib meristem, 96 the activity in the provascular ring was clearly low. In

this situation, the ring could be interpreted as residual meristern if consideration is given to the delay in differentiation behind the cortex and pith. Although at the immediate base of the apical meristem

the esterase activity in the provascular tissue ring was low, at lower levels in the provascular cylinder and in procambium, the activity was much increased. This basipetal increase of esterase activity is consistent with the idea that procambium differentiates acropetally (Esau 1977, Sussex 195%) . There are other enzyme systems which might yield a false-positive reaction for esterases (Gahan 1981), but this problem can be overcome by adding eserine which cannot inhibit the activity of esterases, to the incubation medium (Gahan 1984)- In the eserine pre-treated sections of carrot, there was no apparent reduction in the activity of the enzymes. This suggests that the heavy colour in vascular tissue and the weak colour in non-vascular tissues occurs because of the presence of esterases, The wide distribution pattern of esterase in the shoot apex of carrot indicates the participation of esterases in tissue development and ce11 differentiation. Although a broad spectrum of esterases is present in plant tissues (Gahan 1984), only carboxylesterase activity is 97 believed to be related specifically to vascufar dif ferentiation (Gahan 1981). Using naphthol AS-D acetate as a substrate, three main groups of esterases, namely carboxyl-, acetyl- and arylesterases can be demonstrated at the same the (Gahan 1984, Pearse 1972). PCMPS tests determine if the reaction results from the activity of aryles terase (Gahan 1984). In the present experiments, a reduction in the reaction was not evident when the sections were pre-treated with PCMPS. This indicates that if arylesterases are present they are not present in high quantity. Carboxylesterases are sensitive to a low concentration of the inhibitor DFP, while acetylesterases are not (Gahan 1984). It is difficult to demonstrate the activity of acetylesterases in sections in which carboxylesterases are completely inhibited. The concentration of DFP which can totally suppress the carboxylesterases, also inhibits acetyl esterases. The concentration of DFP, which does not affect the acetylesterases, cannot fully suppress the activity of carboxylesterase. In order to distinguish between them, a range of concentrations of DFP was tested in the present work. When the concentration of DFP was in a range of 10-6~, the reaction in sections was not evidently reduced. When the concentration of DFP was higher than 10-4~,the reaction appeared to be completely inhibited, but under high magnification, reaction still could be observed. This test is 98 significant and indicates that the reaction was not completely inhibited and suggests that acetylesterases are present. When the concentration of DFP was at the 3 x 10%, the reaction in the sections was generally reduced but compared with the control, the reduction in vascular tissue and provascular tissue was much greater. The reaction in a few developing pith cells was not evidently reduced. This suggests that carboxylesterases were inhibited but acetylesterases were not. It is difficult to conclude from one test, but analysis of results from these tests indicated that carboxylesterases are present in the vascular tissue including the tissue that has been designated provascular. In conclusion, the present work did not show that carboxylesterases are strictly restricted to vascular tissue and provascular tissue, but it did show that the enzyme is present in the vascular tissue including provascular tissue.

Thus, provascular tissue is not a residual meristem but has begun to differentiate.

2.4.4 Hypothesis of early differentiation The present developmental work demonstrated that early vascular differentiation in the shoot apex of carrot occurs in the provascular tissue, From this point, a hypothesis of early vascular differentiation can be proposed. The apical 99 meristem gives rise to the provascular tissue within which the axial vascular bundles differentiate. The leaf traces diverge from the axial vascular bundles and extend into leaf primordia. The development of the leaf prirnordia promotes the leaf traces and axial vascular bundles to further dif ferentiation and f inally a mature vascular system is formed. If the leaf controls further differentiation of provascular tissue into mature vascular tissue, the suppression of leaf primordia should prevent this process. If the influence from leaves is artificially replaced it would be expected that the provascular tissue in the ring would differentiate into mature vascular tissue, In the following chapters, experirnents will be carried out to investigate the influence of leaf primordia on the provascular differentiation. 1. Scanning el ectron micrographs of the dissected vegetati ve

shoot apices of adul t carrot.

Figure 2.1 a) The top view of a shoot apex. 1 to 3: the

first to third leaf primordia; 4: the base of the

fourth leaf primordium. Scale bar = 100 pm.

b) The lateral view of a dissected shoot apex.

A: the dome of the shoot apex; I and 2: the first

and second leaf prirnordia. Scale bar = 100 p.

Figure 2.2 a, b) The whole view of the dorne of the shoot

apex of carrot. A: Apical meristem; Pl: The

youngest leaf primordium. Scale bar = 100 m.

2. Organization of the shoot apex of carrot

Figure 2.3 A median longitudinal section of the shoot apex

of a seedling, showing the organization of tunica- corpus. LM micrograph, section was stained in sa-

franin-tannic acid-iron alum after paraffin was removed (Sharman 1943). T: tunica; C: corpus;

pvt: provascular tissue; Pi: pith; Cx: cortex; LP:

leaf primordium. Scale bar = 18 m.

Figure 2.4 A median longitudinal section of the shoot apex

of an adult plant, showing the zonation organization. Section was stained by toluidine blue O without prior removal of paraffin (Sakai

1973) . LM micrograph, Cz: central zone; Pz:

peripheral zone; RM: rib meristem; double arrows :

provascular tissue; LP: leaf primordium. Scale

bar = 18 pm. Fig. 2.3

Fig. 2.4 3. Diagrams of the organization of shoot apices

Figure 2.5 A diagram of a median longitudinal section of

the shoot apex of a carrot seedling, showing tunica - corpus organization. T: tunica; C:

corpus ; PVT: provascular tissue; PC : procambium;

Pi: pith; Cx: cortex; LP: leaf primordium.

Figure 2.6 A diagram of a longitudinal section through the rnedian of the shoot apex of an adult carrot plant, showing the organization of zonation and relationship with other adjacent tissues and organs. Cz: central zone; Pz: peripheral zone;

RM: rib meristem; PVT: provascular tissue; PC:

procambium; LP: leaf primordium. Fig. 2.6 4. The later plastochron: Transverse sections from a shoot

apex of a seedling in the later plastochron, were

stained in safranin-tannic acid-iron al um af ter paraffin

was removed (Shaman 1943). The section is 10 pm in

thickness .

Figure 2.7 Transverse section through the level of 50 pn

from the smit of the shoot apex, showing the P, about to attach the apical dome. A: the apical dome; Pl: the first leaf primordium; P,: the second leaf primordium; P,: the third leaf primordium;

arrow: a leaf trace. Scale bar = 39 m.

Figure 2.8 Transverse section through the level of 90~

from the summit of the shoot apex, showing the appearance of a provascular tissue ring above the

divergence point of the leaf trace of P,. Pl: the first leaf primordium; P,: the second leaf

primordium; P3: the third leaf primordium; pvt : provascular tissue ring; arrow: a leaf trace.

Scale bar = 39 W. Fig. 2.7 5. The early plastochron: Transverse sections, from a shoot

apex of a seedling in the early plastochron, were

stained in safranin-tannic acid-iron alum af ter paraffin

was removed (Shaman 1943). The thickness of the

section is 10 pm.

Figure 2.9 Transverse section through the level of the axil

of the youngest leaf primordium, about 20 pm below the sdtof the shoot apex, showing the apical dome and attached Pl. There are three leaf traces

(indicated by arrows ) in the P,, but only one in

Pl A: the apical dome; Pl: the first leaf

primordium; P, : the second leaf primordium; arrow:

a leaf trace. x565. Scale bar = 18 W.

Figure 2.10 Transverse section through the level of 70 prn

from the summit of the shoot apex, showing the appearance of a provascular tissue ring above the divergence point of the main leaf trace (indicated

by an arrow) of P,. pvt: provascular ring; Pi: pith; P,: the second leaf primordium; P,: the third leaf primordium; single arrow: a leaf trace; double arrows: the location of the axial vascular

bundle related Pl. Scale bar 35 p.

104 Fig. 2.9

Fie. 2.10 6. Longitudinal view of provascular tissue and procambium

Figure 2.11 A median longitudinal section of the shoot apex

of a seedling, showing the location of provascular

tissue in the shoot apex. The section was stained

in safranin-tannic acid-iron alum after paraff in

was removed (Sharman 1943). LM micrograph, A:

apical dome; Cx: cortex; pvt: provascular tissue;

Pc: procambium; Pi: pith; LP: leaf primordium.

Scale bar = 78 p.

Figure 2.12 A median longitudinal section of the shoot apex

of an adult plant, showing continuum of leaf traces and axial vascular bundles. The section

was stained in safranin and fast green after

paraffin was removed (Johansen 1940) , LM

micrograph, A: apical dome; LP: leaf primordiurn;

single arrow: a leaf trace; double arrows: axial vascular bundle; hollow arrow: the divergence

point of a leaf trace. Scale bar = 43 pm. Fig. 2.12 7. The axial bundles and leaf traces in relation with the

provascular ring: Transverse sections of the shoot apex are 10 pn in thickness.

Figure 2.13 Transverse section through the level of 70 pm

from the summit of the shoot apex of a carrot seedling, showing detail of provascular tissue and an initial of an axial vascular bundle and a leaf trace. LM micrograph, section was stained in sa-

franin-tannic acid-iron alum after paraffin was removed (Sharman 1943). Solid arrow: leaf trace; hollow arrow: axial bundle in the provascular

tissue ring. Scale bar = 23 m. Fin. 2.13 8. Primary vascul ar tissue: LM micrographs. Sections were

stained in safranin and fast green after paraffin was

removed (Johansen 1940). The sections are 10 pm in

thickness.

Figure 2.14 Transverse section of the stem of an adult

plant, showing leaf traces and axial bundles.

Single arrow: protophloem element; double arrows: metaphloem element; LT: leaf trace; a: axial

vascular bundle; Pi: pith. Scale bar = 53 pm.

Figure 2.15 Transverse section of the stem of an adult

plant, showing axial bundles and leaf traces.

Single arrow: protoxylem element; ph: phloem

element; LT: leaf trace; a: axial vascular bundle;

Cx: cortex. Scale bar = 53 pm. Fig. 2.14

Fig. 2.15 9. Omitting substrate controls: showing no esterase reaction

when the frozen sections were incubated for 1 h at 37 OC

in the medium from which the substrate was omitted. The

thickness of sections is 20

Figure 2-16 Median longitudinal section of a shoot apex, showing no reaction on the section, LM micrograph; A: apical meristem; LP: leaf

primordium; LT: leaf trace; vt: vascular tissue;

single arrow: procambium; double arrows :

provascular tissue. Scale bar = 71 pm. Fig. 2.16 10. Esterase activity in fresh frozen sections: Unfixed

frozen sections of the shoot apex of carrot reacted in

full medium at 37°C for 1 h for esterase activity using

AS-D acetate as substrate and fast blue

salt. The thickness of sections is 20 W. Note

actlvi ty in vascular tissue.

Figure 2.17 Median longitudinal section of a shoot apex,

showing positive reaction on the section. LM

photograph. A: apical meristem; LP: leaf

primordium; LT: leaf trace; vt : vascular tissue;

single arrow: procarnbiuzn; double arrows : provascular tissue; arrow head: wlern. Scale bar

= 74 pm.

Figure 2.18 The fourth transverse section, 60 pm below the

summit of the shoot apex, showing the high reaction in the provascular ring. LM micrograph. Single arrow: vascular bundle; double arrows:

provascular tissue. Scale bar = 81 pm. Fia. 2.27

Fig. 2.18 11. Esterase activi ty in paraffin embedded sections: Sections

of the shoot apex of carrot were incubated in full

medium for 8 h at 37OC. The thickness of sections is ./O

Pm. Note heavy activity in vascular tissue.

Figure 2.19 Median longitudinal section of a shoot apex,

showing esterase distribution in the shoot apex, note the gradua1 reduction of the esterase activity along the procambium to provascufar tissue acropetally. LM photograph. A: apical meristem; LP: leaf primordium; LT: leaf trace; arrow head: peripheral zone; single arrow:

procambium; double arrows : provascular tissue.

Scale bar = 32 p.

Figure 2.20 Transverse section of a shoot apex at the level

of 90 pm from the summit of the apical dome, showing visible esterase reaction in the provascular tissue ring. LM photograph. LT: leaf trace; VT: vascular tissue; Pi: pith; double

arrows: provascular tissue. Scale bar = 32 p. Fig. 2.19

Fig. 2.20 Eserine inhibi tor tests: sections the shoot

apex of carrot were pretreated in buffer pH 6.5

containing 10-~ M eserine for 45 min at room

temperature, followed by incubation in full medium for

1 h at 37". The thickness of sections is 20 pm. Note

heavy activity in sections was not inhibited.

Figure 2.21 Median longitudinal section of a shoot apex,

showing the esterase distribution in longitudinal

view. LM photograph. A: apical meristem; LP: leaf primordium; LT: leaf trace; vt: vascular

tissue; single arrow: procambium; double arrows :

provascular tissue. Scale bar = 92 m.

Figure 2.22 Transverse section of a shoot apex, showing

esterase activity in provascular tissue ring after

the section was treated by eserine. Single arrow: vascular bundle; double arrows: provascular tissue

region, Scale bar = 92 W.

13. PW!S inhibitor tests: Frozen sections of the shoot apex

of carrot were pretreated in buffer pH 6.5 containing

lu-' M PCMPS for 45 min at room temperature followed by

incubation in full medium for 1 h at 37". The thickness

of sections is 20 m. Note heavy activity in sections was not evidently inhibited.

Figure 2.23 Median longitudinal section of a shoot apex,

showing the reaction on the section. LM photograph. A: apical meristem; LP: leaf primordium; LT: leaf trace; vt: vascular tissue;

single arrows : procambium; double arrows :

provascular tissue. Scale bar = 92 m.

Figure 2.24 Transverse section of a shoot apex, showing esterase activity in the provascular tissue ring after the section was treated by PCMPS. Single

arrows : axial vascular bundle; double arrows :

provascular tissue region. Scale bar = 92 pm. Fie. 2.23

Fig. 2.24 14. DFP inhibitor test and its full medium controls: The

thickness of sections is 20 m.

Figure 2.25 Full medium controls: Frozen sections of the

shoot apex of carrot were pretreated in buffer pH 6.5 without containing any inhibitor for 45 min at room temperature, followed by incubation in full medium for I h at 37". The fourth transverse section, 60 pm below the summit of the shoot apex, showing the high reaction in the provascular ring. LP: leaf primordium; LT: leaf trace; single

arrows : vascular bundles; double arrows :

provascular tissue. Scale bar = 85 p.m.

Figure 2.26 DFP tests: Frozen sections of the shoot apex of carrot were pretreated in buffer pH 6.5 containing

3x10-~M DFP for 45 min at room temperature, followed by incubation in full medium for 1 h at 37". Note the proportion reduction of esterase activity in different tissue by DFP. The fourth transverse section, 60 pm below the summit of the

shoot apex, showing the reaction in the

provascular ring reduced. LP : leaf primordium;

LT: leaf trace; single arrows : vascular bundles; double arrows: provascular tissue. Scale bar = 92

W. Fig. 2.25

Fig. 2.26 15. Frozen sections of the shoot apex of carrot were

pretreated in buffer pH 6.5 containing 1x1 M DFP for

45 min at rok temperature, followed by incubation in

full medium for 1 h at 37 O. The thickness of sections

20 p. Note almost complete inhibition of activi ty

DFP.

Figure 2.27 Transverse section of a shoot apex, showing no

visible reactions when the DFP is present. Single

arrow: leaf trace; double arrows: provascular ring. Scale bar = 237 m.

Figure 2.28 Transverse section of a shoot apex under high

magni f ication. Products £rom arylesterase

reactions can be observed as fine particles al1

over the section. Arrow: fine particle. Scale

bar = 23 W. Fig. 2.27

Fig. 2.28 Chapter 3 SURGICAL EXPERIMZNTS ON

THE SHûûT APEX OF CARROT

3.1 Introduction Provascular tissue, as defined and described in the previous chapter, is a tissue at the stage between apical meristematic cells and procambium, In the shoot apex of carrot Daucus carota L. var. sativa DC (Little finger), under the apical meristem there is a short provascular tissue cylinder which is not related to leaf prirnordia. During development of the shoot apex, axial vascular bundles arise from the provascular tissue cylinder and connect to leaf primordia by leaf traces which extend from the provascular cylinder. Since the provascular tissue is independent from leaf primordia, the vascular system in the stem can be interpreted as initially the product of the apical meristem. However, since leaf traces connect leaf prirnordia and axial bundles, the vascular systems in leaves and the stem constitute a continuum, The development of leaves, thus, may have a direct influence on the process of vascular differentiation. 116 What is the relative contribution of axis apical meristem and lateral leaf primordia to the vascular system in the stem? The simplest way to test any influence from the development of leaves on vascular differentiation is to remove the leaf primordia at a very young stage. A number of surgical experiments have demonstrated that the suppression of leaf development has a strong effect on vascular differentiation. Some early surgical experiments involving isolation of the shoot apex demonstrated that the formation of a new vascular systern from the isolated shoot apex is independent from the original leaf traces and mature vascular system. In Dryopteris and several other ferns, the apical meristem isolated on a plug of parenchyma continued to grow and formed a short shoot (Wardlaw 1944a,b, 1947, 1949a,b, 1950, 195233) . In the angiosperm Primula, a new shoot with a complete vascular system could be obtained from the isolated apex

(Wardlaw 1950). In another angiosperm, Lupinus, Ball (1952) isolated the shoot apex by vertical cuts and found that after seven days, "contemporaneously with the first foliar primordium, the isolated shoot apex differentiated a cylinder of procambium". After 30 days, the isolated apex gave rise to a new shoot (Ball 1948, 1952) . The involvement of leaf effect on vascular differentiation was explored further by defoliation experiments. In Dryopteris and several other ferns, Wardlaw (l944a,b, 1946,

1949a,b, 1950) found that if the leaf primordia of these selected ferns were removed at an early stage, the rhizome stele still developed, but it was lacking leaf gaps. Soe

( 1959) experimentally worked on the fern Onocl ea sensibilis and by systematic suppression of leaf primordia he obtained a mature stele without gaps. He concluded that there is an evident effect of ieaves in modifying the stem vascular system, In seed plants, Wardlaw (1950) reported that closely comparable results were obtained when the same treatments were applied to a very differently constituted shoot apex.

When the apex of Primula was continuously defoliated for some tirne (but not isolated) an uninterrupted cylinder of vascular tissue was obtained (Wardlaw 1950) . However, Helm (1932) found that the rernoval of the leaf effect could cause procambium "to convert or redifferentiate into parenchyma" (cited in Allsopp 1964) . Young (1954) found that in lupin the removal of P, resulted in the formation of parenchymatous cells in the axial vascular system under the P, position, while removal of al1 leaves caused a "meristematic ring" present dom to 570 of a defoliated plant, at which level 118 procambial strands were well differentiated in untreated plants, Although some of these pioneer surgical experiments did not completely limit leaf development, they did give rise to a suggestion that the leaf primordia play an important role in vascular differentiation. Based on his surgical experiments on fern and angiosperm species, Wardlaw proposed that the formation of the vascular tissue is independent of the leaves (Wardlaw 1950, 1952a) and the leaves only rnodify the vascular system by the addition of leaf gaps (Wardlaw, 1947,

1950, 1952b). The inception of vascular tissue at the apex was thought probably to be due to the action of a basipetally moving auxin (Hegedus 1949 cited in Wardlaw 1950). In addition, in al1 those pioneer surgical experiments in which a mature vascular system was obtained, leaf primordia were allowed to develop early or later. Although later developed leaves could not change the pattern of vascular tissue which had formed earlier, later developed leaves rnay have promoted the maturation of vascular tissue (Ma 1994). Wardlaw

( l944a, b) speculated that certain chernical substances produced in the leaves and transported downward into the shoot or stem could induce the maturation of vascular tissue. This chernical substance was also thought to be related to auxin which is synthesized in and exported from leaf primordia (reviewed in Sachs 1981). 119 Surgical isolation experiments with complete suppression of leaf primordia have been carried out by Soe (l959), but the results were not published in journals. More elaborate studies of vascular differentiation, using both isolation and puncturing techniques and focusing on complete suppression of leaf primordia, have been reported since the 1970s (McArthur and Steeves 1972, Ma and Steeves 1995b). Isolation separates the apex from the influence of mature tissues and continuous puncture of leaf prirnordia eliminates any possible ef fect from the leaves. By this combination, the role of leaf primordia on vascular differentiation has been more clearly demonstrated. The experiments of complete suppression of leaf primordia in angiosperms showed similar results with Young's

(1954). When the shoot apex was isolated and the leaf primordia punctured in Geum, the shoot apex gave rise to indistinct provascular tissue without mature vascular tissue (Mmthur and Steeves 1972) . The designation "provascular tissue" was used to emphasize the view that this tissue is in the process of early difiexentiation. In contrast, using the same technique, Ma and Steeves (1995b) obtained a fully mature siphonostele with reduced diameter from the new growth of the apices of fern Matteuccia after six weeks or more of operations in either puncture or puncture combined with 12 0 isolation treatment. With continuous suppression of leaf primordia in angiosperms no mature stele was obtained, but in ferns it was. Studies of vascular differentiation by surgical operation experiraents have a long history, but the species used in such studies in the angiosperms are few in number (Lupinus,

Primula, Geum) . In order to clarify the situation with other examples among angiosperms, surgical experiments with an emphasis on complete suppression of leai prirnordia were carried out in carrot in the present work. 3 -2 Material and methods

3.2.1 Material and growth condition The plant material used in the surgical studies has been

described in Chapter 2. Only the shoot apices of adult plants of carrot were employed in the surgical experiments.

Adult plant material was harvested from the Biology Depart- ment garden. Carrot roots, 15-20 mm in diameter with al1 mature leaves removed, were sterilized in a 1-28 solution of Javex for about 10 minutes. The mots were then planted in clean pots with sand for experiments. Photoperiod was set at 16 hours under fluorescent and incandescent lights at 215 - 305 pol m-2 s-' and 8 hours in dark. Temperature was 25°C in

the light, 22°C in the dark.

3.2.2 Methods

3.2.2.1 Surgical operations The method of surgical operation used in the present work followed McArthur and Steeves (1972). The surgical manipulations including isolation of the shoot apex and puncturing of leaf primordia were carried out under a Wild M5 stereo-microscope, illuminated by a cool light lamp (Fibre

Optic Illuminator, Cambridge Instruments). Al1 operations and observations were carried out in a Class II laminar flow cabinet to reduce contamination. 3.2.2.1.1 Preparing knives

Fine knives for isolation and puncturing operations were fabricated using Gillette blades. Following McArthur (1967), a Gillette blade was cut into pieces with garden shears and pieces having an edge 0.15-0.3 mm wide were selected and carefully polished and thinned just above the factory edge. The knives were held in a vise grip holder and sterilized in 70% alcohol for five minutes before using.

3.2.2.1.2 Preparing shoot apices

The shoot apex was exposed by careful removal of the overlying young leaves with a dissecting needle. The leaf primordia often broke at their bases. Usually, a great deal of liquid escaped from the wounded leaf bases and the loss of water could affect the subsequent growth of the plant. In order to protect the shoot apex from such effects, after rernoval of the younger leaves, plants with the two or three youngest leaf primordia intact were left one - two days before starting surgical operation experiments so that they could recover from the wounds.

3.2.2.1.3 Surgical operation techniques

After recovery of one - two days, either of two basic surgical techniques was used. 3.2.2-1.3.1 Isolation of the shoot apex The purpose of isolation of a shoot apex is to separate it from the influence of young organs and mature tissue which surrounded it using a fine knife. Four vertical cuts were made about 200 pm deep downwards into the pith. Vertical cutting was just inside the youngest existing leaf primordia. The isolation separated the apex from the original vascular system and from the remaining leaf primordia, The apex was supported by the pith plug underneath so that some nutrition would be available to the apex,

3.2.2.1.3.2 Puncturing leaf primordia The purpose of puncturing leaf primordia is to suppress their development. Under a binocular dissecting microscope a fine knife or a needle was used to pierce the site on the apical dome where the next primordium was expected to arise. The position of a new leaf primordium could be estimated by visually projecting the phyllotactic spiral around the apical meristem through an arc of ca. 137" beyond the last-formed prirnordium (Pl) or the site of the last punctured primordium

(1, 1, etc. ), so that leaf primordia were punctured as or just before they appeared, The arc of 137" is based on the average measurements of the divergence angle between successive primordia P, and P, on the apical dome (in chapter 2) . 3.2.2.1.4 Post-operative treatment After the operation, the shoot apex was covered with a small wet filter paper cap to keep moisture around the surgically treated apex. The pot of experimental plants was covered with two sheets of Kleenex tissue or Scott towels (Scott Paper Company, Canada) and one sheet of clear plastic (Ma 1994) tightened with a rubber band after each operation. After manipulations, the treated plants were returned to the growth chamber with the same conditions under which they had been growing .

3.2.2.2 Experiments

Puncturing of leaf primordia or isolation can be used

alone or the two may b'e combined. Four groups of plants were treated as follows: (1) puncturing without isolation; (2)

isolation without puncturing; (3) isolation with one leaf primordium remaining; (4) isolation plus complete puncturing of leaf primordia. Another group was untreated as a control. In treatment 1, al1 the leaf primordia on each shoot apex were removed and successive incipient leaf primordia were punctured just before or as they ernerged. The experimental plants were checked every day and any leaf primordia that appeared were punctured. In this way, the new leaf primordia were suppressed before or just after they 125 appcared. In treatment 2, the shoot apex was isolated but newly formed leaf primordia within the isolated shoot apex were allowed to develop. In treatment 3, the shoot apex was isolated as above, and al1 subsequent primordia except 1, on the isolated apex were punctured continuously. In treatment

4, the shoot apex was isolated and al1 subsequent primordia on the isolated apex were punctured continuously. For the design of the surgical experiments, preliminary trials were carried out to determine the best operation and conditions. Because the process from provascular tissue to mature vascular tissue needs four plastochrons (12 days) in normal plants, the experiments were desig-ned for two - three weeks to ensure that there was enough time for vascular tissue differentiation. Twelve plants were used in each treatment and each experiment was repeated twice. Because the survival rate in treatments with complete suppression of leaf primordia was low, in order to obtain enough valid results, additional experiments were carried out. One hundred and twenty six plants were used, but only sixty five shoot apices survived to the end of the experiments.

3.2.2.3 Histological and histochemical methods For general histological study the same methods were used as described in Chapter 2. Shoot apices were harvested at the end of experiments. Each shoot apex was excised on a tissue plug approximately 4 x 4 mm on a side and 5 mm deep for histological and histochernical examination. In some cases, shoot apices were fixed at different stages of an experiment to assist in tracing the time of development. For histochemical examination (esterase test), shoot apices at the end of treatment 4 were collected and frozen directly in the cryostat . The principles and procedures relating to the esterase test have been given in Chapter 2. The results of the inhibitor tests in normal carrots were used as a reference for evaluating the present results.

3.2.2.4 Measurements

3.2.2.4.1 Vertical growth

The vertical growth could be measured in two ways: indirect and direct. The indirect measurernent of the new growth was carried out by marking a toothpick beside the shoot apex of the rhizome before and after experiments (Ma 1994). Using epidermis as a mark, the direct measurernent was carried out after the shoot apex was fixed and sectioned (McArthur and Steeves 1972). For measurement of the growth of an apex of an angiosperm, the indirect method was difficult because the shoot apex could not survive long enough and the growth of shoot 127 apices is very limited when leaf primordia are suppressed. In the present study, the measurement was carried out by measuring directly from the sections of the experirnental shoot apex on slides. The vertical growth was estimated by the distance from the summit of the shoot apex to the punctured Pl in the puncturing only shoot apices. In isolated shoot apices, because the pith plug has no epidermis, the distance could be judged from the summit to the lowest level of epidermis- For cornparison, the distance from the sunmiit of a shoot apex to the axil of P, in controls was measured as the vertical growth of shoot apices after five plastochrons. The data were statistically analyzed and the

means 4 standard deviations (SD) are given in Table 3-1.

3.2-2.4.2 Apical meristem

The size of the apical meristem or shoot apical dome was determined by two measurements. One was the maximum diameter of the apical dome. The other was its height. Shoot apices from each experimental group were examineci using median longitudinal sections for these two measurements . In untreated control plants, apical dome height and diameter were measured with respect to the first visible primordium.

Height was defined as the distance from apical summit to the Table 3-1. The camparison of vertical growth of the shoot apf ces among different treatments

Treatment Vertical growth (pm)

-- Al1 values are means + SD pm (~amplessize of shoot apices in the bracket).

P: Puncturing without isolation;

1: Isolation only;

1 + 1LP: Isolation followed by puncturing but one leaf primordium allowed to develop; 1 + P: Isolation followed by complete puncturing; C: Controls. 129 axil of this primordium, while diameter was the distance between the axil and the opposite flanking surface of the

shoot apex. In surgically treated apices, the diameter was measured at the base of the apical dome or at the level where

the last leaf primordium was punctured. The height of the apical meristem, was measured from the top of the rib meristem to the summit of the shoot apex. The data were statistically analyzed and are presented as rneans 2 standard deviations (SD) in Table 3-2. Table 3 -2. Dimensional varfatf on in the apical meristem of ~hootapices fram eurgical treatments and normal mhoot apex of adult carrot

Apical meristem Treatment Height (p) Diameter (-1

Al1 values are means i SD pm (samples size of shoot apices in the bracket).

P : Complete puncturing without isolation; 1 : Isolation only; 1 + 1LP: Isolation followed by puncturing but one leaf prirnordium allowed to develop; 1 i P : Isolation followed by complete puncturing; C: Controls. 3.3 Results

3.3.1 Puncturing only experiments Before this treatment, al1 the leaves were removed so that the shoot apex was exposed. Successive incipient leaf positions were punctured but the apex was not isolated. A shoot apex with the puncturing only treatrnent is demonstrated in Fig. 3.1 in which Il, 1,, I, and I, were punctured.

3.3.1.1 Leaf primordia

In general, the leaf primordia in this treatment were not observed because they were punctured before or as they were emerging. However, the swelling of the remaining tissue still could be identified. The recorded number of leaf primordia punctured could be confimed by counting these swellings of leaf bases; four or five leaf primordia were punctured during the experimental time. Although the puncturing did not interrupt the formation of new leaf primordia, after I, was punctured, the continued emergence of new leaf primordia was rare. About two or three days after removal of al1 existing leaf primordia, the 1, emerged on the shoot apex if puncturing was not applied. This could be identified at the shoot apex based on the occasional emergence or the swelling of the punctured sites. The length of a plastochron may be shorter 132 than the average length of a normal plastochron because the occasional emergence of leaf primordia earlier than expected was observed during experirnents.

3.3.1.2 Vertical growth The vertical growth of the shoot apex after removal of

leaf primordia was lirnited if continuous puncturing of leaf primordia was applied. The distance from the summit to the

site of the punctured P, (after 5 plastochrons, it is

equivalent to P, in the normal intact shoot) is 195 i 29 pm (Table 3-1). Although the vertical growth in normal shoots

is limited, the vertical distance from the summit of the shoot apex to the P, in controls is 426 + 67 pm (Table 3-1). Compared with the untreated plants, the vertical growth in the shoot apices receiving puncturing treatment showed a

statistically significant reduction (t=8.0417; P<0.01).

3.3.1.3 Apical meristem

The effect of the puncture of leaf primordia on the shoot apex itself was not apparent. In median longisection

of treated plants, there was a densely stained apical meristem at the top of the shoot (Fig. 3.2). It consisted of about three - four layers of cells under LM observation- The peripheral zone of the apical meristem was darker stained 133 than the central zone. The average diameter of a treated apical meristem was 133 +, 49 pm (Table 3-Z), and was not statistically different from the untreated ones (t=0.6912;

P>0.4), The height of the apical meristem receiving puncturing treatment was about 33 4 6 pm (Table 3-2) , and showed no statistically significant difference from the controls (t=0.8413; D0.4) .

3 .3.1.4 Provascular tissue Removal of leaf primordia appeared to have no effect on the formation of provascular tissue compared with untreated shoot apices. The provascular tissue first appeared at the

50 pm level from the summit of the shoot apex but the ring was clearly observed at 70 - 80 pm from the summit of the shoot apex (Fig. 3.3). This is sirnilar to untreated shoots. However, suppression of leaf prirnordia did have a strong effect on the further development of provascular tissue, In normal development, axial vascular bundles differentiated into procambial strands among the provascular tissue and diverged as leaf traces, The divergence of leaf traces resulted in the provascular ring being broken. With puncturing of leaf primordia, no axial vascular bundles were developed and the complete provascular tissue ring was observed, so that this further differentiation was prevented 134 (Fig. 3 -4). The complete provascular tissue ring could be

traced dom to 250 p.m below the tip of shoot apex. Compared with untreated shoot apices, mature xylem and phloem elements would be observed at this level, In a longitudinal view, provascular tissue extended to the peripheral zone of the apical meristem. Cells of provascular tissue were darkly

stained and had large nuclei and dense cytoplasm (Fig. 3 -2) but they were not evidently elongated.

3.3.1.5 Vascular differentiation

In al1 puncture treated shoots, no phloem or xylern was found in the new growth portion above the punctured P,, In cases where leaf primordia were not completely punctured in time, the bases of leaf primordia could develop. In these cases, procambium and even phloem and xylem, could be found in the bases of leaf primordia. When the leaf primordia were punctured before or as they were emerging so that the bases could be kept to minium size, no vascular tissue was developed in such leaf bases.

3.3.2 Isolation experiments

Al1 rernaining treatments involved isolation. As emphasized above, the purpose of isolation was to separate the mature tissues, especially the vascular system, from the 135 shoot apex. One or two days after isolation, gaps resulting from the vertical cuts surrounded the isolated shoot apex. These gaps separated the shoot apex from surrounding leaf primordia and cortex, An isolated shoot apex showing the four vertical cuts is illustrated in Fig, 3.5. The primordia outside the isolated square were removed before the incisions and new primordia were not formed on this isolated apex. Although isolation of a shoot apex separated mature vascular tissue and procambium from the shoot apex, some provascular tissue could be included because it was very close to the apical meristem. In the following description, an isolation treatment with al1 new leaf primordia allowed to develop is designated "isolation only", An isolation treatment with one leaf primordium allowed to develop is considered "partially operated". An isolation treatment followed by continuous puncturing of leaf primordia is referred to as "fully operatedf'.

3-3.2-1 Isolation only

3.3.2.1.1 Leaf primordia

In this treatment, because no leaf prirnordia were punctured, the number of leaf primordia could be traced easily during the experirnental period, Isolation alone did not affect the formation of leaf primordia, In two week ex- 136 periments, five leaf primordia were formed. The average

length of the plastochron was about 2.8 days which was a little shorter than the average plastochron length of three days in normal plants.

3.3.2.1.2 Vertical growth

The vertical growth in the isolation only treated shoot apices appeared to be not affected by isolation treatment. In two week experiments, the total height of the isolated shoot apex was 500 + 283 pm (Table 3-1). Compared with untreated plants, the vertical growth of the experimental treated shoot apex was not significantly affected by isolation treatment

(t=O.9549; DO.2) .

3 -3.2.1.3 Apical meristem

Because leaf primordia developed on the isolated shoot apices, the apical dorne was expected to resemble an untreated one. In longitudinal section, a group of densely stained cells at the top of the dome of the isolated shoot apex comprise the apical meristem (Fig. 3.6). In cross sections stained with safranin-fast green, the peripheral zone was more densely stained by the safranin than the central zone (Fig. 3.7). The diameter of the treated apical dome at the level of the I, was 123 + 4 p (Table 3-2). Compared to untreated plantsf the difference in diameter between the 137 isolation only plants and untreated plants is not statistically significant (t=0.7647; P>0.4). The average height of the apical dome above the axil of the last leaf primordium,

I5 (equivalent to P, in a normal shoot apex) was 33 2 4 p (Table 3-2) and showed no significant reduction for isolation treatment (t=O-7061; P>O.4).

3.3.2.1.4 Provascular tissue

The first appearance of a provascular ring in the isolated shoot apex was observed at the level of 50 - 60 p from the sumrnit of the shoot apex and above the divergence point of the leaf trace of I, (equivalent to Pl, Fig- 3.8) . This level appears to be higher than in the untreated shoot apex. The provascular tissue was darkly stained by safranin and the provascular ring was not uniform in its thickness in transverse view. The wider areas are the sites for the development of the further axial vascular bundles (Fig. 3.8). As in the untreated shoot apex, the ring was broken by the divergence of the leaf traces.

3.3-2.1.5 Pith plug The most distinct reaction in the pith plug observed after the operation was that the outer cells were arranged periclinally parallel to the cutting edge and the cells from these divisions formed an outer sheath (Fig. 3.9) . This sheath functioned as a protective layer and was apparently a 138 wounding reaction, Inside the outer sheath, parenchyma cells periclinally divided or dedifferentiated, corresponding to the formation of leaf prirnordia, and became a basipetal provascular extension. This extension could establish a base for the vascular tissue differentiation (Fig. 3.9).

3.3-2.1.6 Vascular differentiation Isolation appears to promote the maturation of vascular tissue. In the isolated shoot apex, the phloem and xylem were first found in the leaf trace of I, (Fig. 3.8) which was equivalent to the P, in normal plants. The appearance of phloem and xylem was thus earlier than in the normal plants in which the earliest maturation was found in the leaf trace of P,, Phloem was found at the outer side of the xylem in the

leaf traces or axial vascular bundles. Phloem was stained green in colour, cornpanion cells with nucleus were darkly stained by safranin. The elements of phloem were continuous from the leaf traces to the pith plug (Fig. 3.9) . The protoxylem first formed near the base of a leaf trace below the divergence point because at this level the nurnber of tracheary elements was 2-3; upward and downward the number in this bundle was one. The metaxylem was sirnilar; the transverse sectional area of a bundle was reduced as sections were followed downward as was the number of the tracheary elements. Final maturation occurred acropetally throughout the 139 new growth part and the top of the isolated pith plug and basipetally extended into the pith plug. At the base of the plug, there was xylern but no phloem (Fig. 3.10).

The vascular system in the pith plug was independent

f rom the original vascular system (Fig. 3 .1 . The xylern elements at the base of the pith plug were similar to the parenchyma cells of the pith in shape and size (Fig. 3.12). This indicates that they were converted or regenerated from the parenchyma cells instead of procambium. The secondary walls of these xylem elements were scalariform thickened with simple perforation plates (Fig. 3.12). These regenerated xylem elements could establish a connection between the new vascular system and the original vascular system (Fig. 3.12).

3.3.2.2 Isolation plus partial puncturing

3.3.2.2.1 Leaf primordia As in the treatrnents above, isolation did not prevent the formation of leaf primordia. In the two week experiments, there were two to three leaf primordia punctured, in addition to the leaf primordium which was allowed to develop. The response to the puncturing of leaf primordia was similar to that in the punctured only experiments.

3.3.2.2-2 Vertical growth The vertical growth of the isolated shoot apex appeared to suffer from the puncturing of leaf primordia. In the 140 experiment where one leaf primordium was allowed to develop,

the vertical growth was 193 +_ 12 pxn (Table 3-1). This treatment is similar to the puncturing only because only one leaf primordium was allowed to develop. Cornpared with the controls, the vertical growth was significantly affected by puncturing ( t=7.6332; PeO. 01) .

3.3.2.2.3 Apical meristem The apical meristem in this treatment looks like the normal one (Fig. 3.13) + It was darkly stained with safranin- fast green, the peripheral zone was more darkly stained by safranin than the central zone. The apical dome extended 36 2 3 p above the axil of the latest leaf primordium, and the diameter at this level was 117 t 22 prn (Table 3-2) . Compared with the untreated one, there was no significant influence of treatment either on the diameter (t=l.4201; P>0.1) or on the height (t=O.4370; P>O.5).

3.3.2.2- 4 Provascular tissue The provascular tissue ring first appeared at the level of 50 p but at the level of 70 - 80 pm it becarne clearer (Fig. 3.14). The provascular ring persisted dom and was independent from the leaf traces of the unpunctured leaf primordium at the 90 prn level (Fig. 3.15). Probably because of the development of the unpunctured leaf primordium, the 141 provascular ring was broken by the divergence of its median leaf trace at the level of 110 pm and became faint basipetally. No provascular extension was observed in the base of the pith plug (Fig. 3.16) . The development of a provascular extension appeared to be affected by the presence of the unpunctured leaf prirnordium.

3.3.2.2-5 Pith plug

Changes in the pith plug were similar to those in the isolation only treatment. A protective sheath was observed in the pith plug. Provascular extension was not well developed and was not observed in the base of the pith plug; the vascular bundles formed were related to the unpunctured leaf primordiun (Fig. 3.16).

3.3.2.2.6 Vascular differentiation Vascular differentiation was similar to the isolation only treatment. Xylern and phloem were found in the main trace of the unpunctured leaf prirnordium. Similar to the isolation only treatment, xylem differentiation was observed in the vascular bundles which were related to the unpunctured leaf prirnordium. The protoxylem first started in the axial bundle near the base of the leaf trace and acropetally and basipetally developed in the leaf primordium and in the pith plug. At the base of the pith plug there was xylem related to the unpunctured leaf primordium but without phloem. 142 Regenerated xylem elements were present at the base of the pith plug. These elements had characteristic scalariform thickened celf walls with simple perforation plates (Fig. 3.17).

3-3.2.3 Isolation plus complete puncturing

3.3.2.3.1 Leaf primordia There was no apparent difference between the puncturing only and isolation plus complete puncturing treatments in terms of the resulting new leaf primordia. The isolation plus continuous puncturing did not prevent the formation of leaf primordia In the two week experiments, four leaf primordia were punctured. However, after the fourth leaf primordium was punctured, rarely did the fifth leaf primordium emerge.

3.3.2.3.2 Vertical growth The growth of the shoot apex was limited if after isolation continuous puncturing of leaf primordia was applied. The vertical distance from epidermis to the summit of the shoot apex in this treatment group was 177 + 14 p.m (Table 3-1) and this indicates that vertical growth was limited, Compared with the shoot apices in the untreated group, the eff ect of puncturing leaf primordia was significantly evident ( t=10.9295; P

meristem. In longitudinal section, there was a group of

meristematic cells at the tip of the dome of the isolated shoot apex (Fig. 3.18). They were densely stained and can be identified as four - five layers under high magnification. The average height of the apical dome above the axil of leaf

primordia was 38 i 10 p, and the average diameter at this

level was 145 f 42 pm (Table 3-2). The apical dome was not

significantly reduced either in diameter (t=0.2367; D0.5) or

in height (t=0.9371; P>O.2), compared with the average size of the shoot apices of untreated plants,

3.3.2.3.4 Provascular tissue

In the new growth, a provascular ring appeared at the level of 50 - 70 p. The provascular cylinder was persistent in the new growth portion (Fig, 3.19) and extended basi-

petally from the new growth part into the pith plug.

Provascular tissue was poorly developed but could be seen in the pith plug (Fig. 3.20)- There was a basipetal extension of this provascular tissue, so that it was suspended in the pith plug but established no connection with the mature vascular system (Fig. 3.21). 3.3.2.3.5 Pith plug Similar to isolation only treatments, wounding reactions in the pith plug could be observed in a few days after the operation. First, the outer cells were periclinally divided and the cells from these divisions formed an outer sheath

(Fig. 3.21). This sheath functioned as a protective layer. Unlike the isolation only treatments, there was no xylem and phloem in the pith plug when the leaf primordia were completely suppressed.

3.3.2.3.6 Esterase reaction tests The experiments described above demonstrated that after suppression of leaf primordial development, further dif ferentiation of the provascular tissue has been affected. Esterase activity was tested in fully operated shoot apices of carrot to observe any effect on esterase activity by suppression of the leaf prirnordia. Longitudinal sections (Fig. 3.22) showed that provascular tissue, demonstrated by heavy blue colour, extended upward to the apical meristem and downward into the pith plug. In contrast to the reaction in provascular tissue which was demonstrated by a dark blue colour, a weak positive reaction occurred in the cells of cortex and pith probably due to other esterases. In cross section, a ring of provascular tissue showed a darker blue colour, Although inhibitor tests were not done in the surgically treated material, based 145 on the results of esterase tests in Chapter 2, this heavy blue colour was interpreted as prirnarily the reaction of carboxylesterases. The esterase tests of surgically treated shoot apices showed that esterase activity was present in the provascular tissue and was not affected by suppression of leaf primordia. 3.4 Discussion Surgical operations employed in the present study include apical isolation and leaf primordium puncturing. The aim of isolation experiments is to prevent influence from the mature tissues on the apical meristem, while the puncturing is intended to eliminate the influence of leaf primordia. Surgical operations may disturb the normal development processes, but they provide important information which cannot be obtained from simple observation. Analysis of the effects of these surgical operations on the shoot apex should contribute to an understanding of the relationship between early vascular differentiation and the apical meristem.

3.4.1 General effects of surgical operations

In previous isolation studies of the shoot apex of

Lupinus, apices were separated from leaf primordia by four longitudinal cuts 1-2 mm deep. Al1 leaf primordia and the mature vascular system were excluded with only the pith plug connecting the meristem to the subjacent tissue. After the operation, the apex reconstructed leaf primordia and procambial tissue developed basipetally through the pith plug to connect to the vascular system of the stem (Bal1 1948, 1952).

In another angiosperm, Primula, Wardlaw (1950) obtained the same result. Although the isolation work done by Wardlaw 147 (1950) and Bal1 (1948, 1952) did not eliminate the involvement of leaves, it demonstrated that the isolation treatment does not affect the initiation of provascular tissue and the development of a new vascular systern. The present work confirmed these pioneer results.

3.4,l.l Apical meristem The present results clearly showed that the apical meristem was not seriously affected either by isolation or by cornplete suppression of leaf primordia in terms of height or diameter during the two weeks of experiments. In the isolation only treatment, the apical meristem of the newly generated shoot did not differ from the normal, The data of the present study also showed no statistically significant effect on the size of the apical rneristem during the two weeks of puncturing only or isolation plus puncturing treatments.

3.4 .1.2 Leaf primordia In the isolation only treated shoot apices, the new leaf primordia were initiated normally and were not different from those in untreated plants, In addition, the continued initiation of leaf primordia was not prevented if the experimental time was extended, However, in the puncturing 148 experiments where the leaf primordia were suppressed, the ernergence of new leaf primordia was observed only during the first two weeks. After that, the influence of puncturing affected the initiation of new leaf primordia so that beyond I, or , the occurrence of additional leaf primordia was rare. The prevention of further leaf initiation beyond I, may be caused by either the wounding of the apical dome or suppression of leaf primordia. The former explanation is reasonable. Wounding scars on the surface of the apical dome

block the initiation of new leaf primordia. The latter explanation suggests that leaf primordia are necessary for continued growth of the meristem and new leaf primordial initiation and is supported by experiments with cultures of angiosperm apices where a few leaf primordia are necessary

for the survival of explanted apices (Bal1 1946, 1960;

Wetmore 1954; More1 1963, 1964) . In an in situ apex with isolation, there may be sufficient residual influence to permit continued development for a time. With leaf primordia allowed to develop, the vascular system is established in the stem and the developing leaf primordia can give rise to additional influence to maintain the development of the shoot apex, so that, new leaf primordial initiation continues. With al1 leaf primordia continuously suppressed, this 149 influence ultimately is depleted, no functional vascular tissue is built up in the stem, and no new influence cornes from the leaves, with the result that new leaf primordium initiation ceases. Experiments with cultured shoot apices suggest that the

leaf influence may be replaced by exogenous IAA (Bal1 1946,

1960; Wetmore 1954; More1 1963, 1964). Therefore, the influence from the leaf primordia may be hypothesized as auxin flow or auxin concentration, Auxin from leaf primordia maintains the vascular differentiation and the vascular tissue is a necessary factor for apical meristem survival which in turn produces additional leaf prirnordia, Although the apical meristem is one source of auxin, the production of auxin in the apical rneristem in seed plants may be so low that the influence on the vascular tissue maturation is not sufficient in the absence of leaf primordia, However, the cumulative number of leaf primordia

initiated during the two week experiments did not show a significant difference between isolation plus puncturing and puncturing only. Furthemore, the numbers of leaf primordia initiated during the two experimental weeks in al1 treatments were not significantly different from the normal. Al1 these results suggest that the function of the apical meristem was not adversely affected by the surgical operations during the experimental period. 150 The length of the plastochron between the isolation only and the normal was not different . However, there was an indication that the puncturing of a primordium promotes the next one in sequence to emerge earlier. In other words, the length of the plastochron became shorter during the suppression experiment. A similar result was reported in the fern

Matteuccia (Ma 1994). The significance of shortening the

plastochron is not clear, but it suggests that leaf primordia interact with one another.

3.4.1.3 New growth In the normal carrot plants, the short stem indicates that the vertical growth of the shoot apex is limited, The vertical growth in experimentally treated shoot apices was limited too. This limited vertical growth did not represent a major difference from normal shoot apices of carrot, where vertical growth is also of lirnited extent. Thus, in the study of vascular differentiation the possible effect of vertical elongation was minimized by the choice of plant material (McALthur and Steeves 1972) . Although limited vertical growth occurred in both treated and untreated carrot, statistical analysis showed that there were significant quantitative differences among these treatments. In the isolation only treated shoot apex, 151 the vertical growth was similar to the untreated ones because leaf primordia were allowed to develop, The vertical growth, however, was limited in shoot apices with puncturing only or isolation plus complete or partial puncturing treatment. The vertical growth in experiments was mainly affected by the suppression of leaf primordia.

3.4.2 Vascular differentiation in treated shoot apices

3.4.2.1 Formation of provascular tissue In the previous chapter, early vascular differentiation has been identified as provascular differentiation- The apical meristem produces a provascular cylinder within which axial vascular bundles differentiate, Leaf traces diverge from these vascular' bundles and enter leaves. Thus a provascular cylinder is present in the shoot apex under the apical dome. However, in normal plants, the provascular ring is not persistent because cells start to differentiation under the influence from leaf developrnent (Ma and Steeves 199Sb). When the leaf primordia develop, the ring is broken by leaf trace divergence from the provascular ring. In the normal shoot apex of some angiosperms, such as carrot, this provascular ring can be demonstrated. However, because of the presence 152 of leaf primordia, the initiation of provascular tissue cannot be attributed to the apical meristem alone, Surgical operations on shoot apices provide evidence for the existence of the provascular stage of vascular differentiation. A provascular cylinder has been demonstrated in the shoot apices of angiosperms by isolation only treatrnents

(Wardlaw 1950, Ball 1952), puncturing only (Young 1954) or isolation combined with puncturing (McArthur and Steeves 1972). In ferns, a provascular cylinder demonstrated by surgical experiments was also reported (Wardlaw 1947, Soe 1959, Ma and Steeves 1995b) . Although some of the earlier workers allowed leaf prirnordia to develop (Ball 1950), the presence of a provascular cylinder was confirmed by experiments in Geum where no leaf primordia were developed (McArthur and Steeves 1972). In the puncturing only treatment, the provascular cylinder could be traced downward to the level where in normal plants, mature vascular tissue is formed, In the present study, a provascular ring was observed in the shoot apices with isolation and complete suppression of leaf primordia. In the suppression of leaf primordia, with or without isolation, no incipient leaf achieved more than an observable primordial stage (P,) before being suppressed, Most of the leaf primordia were suppressed in the incipient 153 stage. Moreover, in the isolation plus puncturing treated apices, the isolation of the apical meristem by vertical incisions ruled out possible influences from mature tissue.

Thus, the observed formation of provascular tissue is

believed to be under the control of the apical meristem alone .

3.4.2.2 Esterase activity

In the experiments with Primula (Wardlaw 1950) and lupin (Bal1 NSO), the provascular tissue produced by the apical meristem was claimed to be vascular tissue because this tissue could differentiate into mature vascular tissue when

leaves were later allowed to develop (Wardlaw 1950, Bal1 1950). With continuous puncturing, since there was no mature

vascular tissue obtained, the status of provascular tissue in lupin (Young 1954) and in Geum (McArthur and Steeves 1972) remained unclear.

Provascular tissue as described in Chapter 2, showed meristematic features but it was considered as a differen-

tiating tissue because of the presence of carboxylesterases. In the present surgical experiments, it was found that provascular tissue showed a positive reaction for esterase activity. Furthemore, the provascular tissue extension in the pith plug showed a denser colour than surrounding tissue. 154 This high density, although not analyzed by a microdensitometer, indicates that vascular differentiation was initiated in the provascular tissue. Because a large sample size could not be collected in one experiment, and the treated shoot apices were dif f icult for frozen sectioning, no inhibitor tests were carried out in the experimental material. The conclusion in fully operated shoot apices relied on the tests in the normal shoot apex to indicate that the reaction was mainly produced by carboxyl esterases instead of the other esterases. Based on the esterase test results, it is reasonable to conclude that the provascular tissue is differentiating vascular tissue.

3.4.2.3 Control of vascular differentiation

3.4.2.3.1 Control by the apical meristem

Provascular extension was noted in surgical operation experiments of Geum (McArthur and Steeves 1972). This extension was interpreted as evidence that the apical meristem controls initial vascular differentiation. In the present study where the shoot apex was isolated and puncturing elirninated the leaf prirnordia at a very early stage, the shoot apex was supported entirely by pith, it had no leaf primordia, and there was no subjacent vascular system. The provascular ring was not only observed under the apical dorne but also was found basipetally extending into the pith plug. The provascular extension resulted from redifferentiation of parenchyma cells. Without leaf influence, provascular tissue not only was formed under the apical meristem, but also was produced by redifferentiation of parenchyma under the control of the apical meristem. However, this dif ferentiation was limited. Esterase activity was observed in this tissue but there were no morphological changes indicating further differentiation.

3.4.2.3.2 Control by leaf primordia In the present work, with continuous puncturing of leaf primordia, the initiation of provascular tissue continued in the shoot apex, but further differentiation was prevented. However, in the isolation experiments, a new shoot with a complete mature vascular systern was formed if leaf development was allowed. If one leaf prirnordium was allowed to develop, a part of the mature vascular system was found in the isolated shoot apex. This supports the conclusion that, although initiation of provascular tissue was controlled by the apical meristem, further development of vascular tissue in the isolated shoot apex was under the influence of leaf primordia. 156 Other evidence from isolation only treated shoot apices indicates that leaf development has a strong effect on further vascular differentiation. Under the isolation only condition, the maturation of vascular tissue was found earlier than in normal plants. In normal plants, the earliest xylem was found in the traces of P, or P,, but in an isolated shoot apex, the xylem was found in the trace of the

equivalent of P,. Furthemore, at the base of the pith plug there was no phloem outside of xylem and some xylem elements had scalariform ce11 walls and were similar to the parenchyma cells in size. These elements were directly regenerated £rom parenchyma cells instead of procambium. Where one leaf primordium was allowed to develop, the regeneration of xylem elements at the base was under the remaining leaf primordium. The regeneration of xylem elements was not found in the normal pith or the whole pith plug without leaf development. The regeneration of xylem elements suggests that leaf development was the source of auxin, Further vascular differentiation may thus be regulated by endogenous auxin.

3-4.3 Conclusion

Surgical operation represents a technique for obtaining considerable information about the growth of the shoot apex, including the initiation and development of vascular tissue. The surgical experiments demonstrated conclusively the ability of the shoot apical meristem of angiosperms to initiate provascular tissue without the participation of developing leaves and the mature vascular system. However, further differentiation and development of vascular tissue needs some factors from the developing leaves. The present work strongly supported McArthur and Steevesr (1972) conclusion. The most critical question arising from surgical operations is the function of apical meristem and leaf primordia in relation to the maturation of vascular tissue.

Leaf primordia play an important role in angiosperms while in ferns they are not necessary for maturation of the vascular system. Why is the leaf important in maturation of the vascular tissue in angiosperms? In vitro culture of the shoot apex suggests an answer to this question. In cultures of the shoot apex of angiosperms without leaf primordia, auxin was necessary for survival and growth (Smith and Mura- shige 1970, Murashige 1974, Bal1 1980). In cultures of the shoot apex of ferns, additional auxin was not necessary (Wetmore 1954, Wetmore and More1 1949, Michaux-Ferrière

1973). Therefore, the difference may be interpreted by the involvernent of auxin. 158 There are two possible explanations, One is that the cells in the shoot apex of ferns are more sensitive to auxin

than cells in angiosperms. If the level of auxin is the same, cells in fern shoot apices could be differentiated into mature vascular tissue but cells in angiosperm shoot apices could not. Alternatively, the shoot apex of ferns may produce more auxin than that of the angiosperms, If this is

true, in the defoliated shoot apex of ferns, there rnay be enough auxin to maintain the maturation of the stele. In the

defoliated shoot apex of angiosperms, there would appear not to be enough auxin in the apical meristem, so that the maturation of the vascular system depends on auxin from the leaves. The most plausible interpretation is that auxin produced by leaves is transported to the provascular tissue and causes it to undergo further differentiation, If leaf primordia are suppressed, no mature vascular system can be obtained. If this hypothesis is true, application of auxin should provide verif ication. The next experirnent was designed to explore what role auxin plays in vascular differentiation. 1. Puncturing only

Figure 3.1 Top view of an exposed shoot apex demonstrat ing

a puncturing treated shoot apex in which four leaf primordia have been punctured during two weeks.

Hollow arrow: apical dome; 1,: the first punctured

leaf prirnordium; : the second punctured leaf

primordium; 13: the third punctured leaf

primordium; I, : the fourth punctured leaf

primordium. Scale bar = 320 p.m.

Figure 3.2 Longitudinal section of a shoot apex with

puncturing only treatment after two weeks of

operations, showing newly f ormed provascular

tissue. Section was stained by safranin-fast green

with prior removal of paraffin. LM rnicrograph. A:

apical meristem; pvt : provascular tissue; Pi : pith; arrows: the sites of the punctured leaf

primordia. Scale bar = 71 pm. Fin. 3.1 2. Puncturing only (continueci)

Figure 3.3 Transverse section at 70 p from the summit in

an apex with puncturing treatment for 2 weeks

showing provascular tissue ring. LM micrograph, stained by safranin-fast green with prior removal

of paraffin. pvt: provascular tissue; 1,: the

second punctured leaf primordium; 1: the third

punctured leaf primordium. Scale bar = 71 m.

Figure 3.4 Transverse section at 120 p f rom the summit in

the same apex as in Figure 3.3, showing the

continuous provascular ring. pvt: provascular

tissue; 1, : the f irs t punctured leaf primordium;

1,: the second punctured leaf primordium. Scale bar

= 142 Fig. 3.3 3. Isolation

Figure 3.5 Top view of an exposed shoot apex demonstrating

isolation operation. The apical dome was isolated from leaf primordia by vertical incisions. Hollow arrow: apical dome; solid arrow: cutting mark.

Scale bar = 200 p.

Figure 3.6 Longitudinal section of a shoot apex with

isolation treatment after two weeks of operations,

showing a newly formed vascular cylinder extended in the isolated tissue plug. LM micrograph. Section was stained in safranin-tannic acid-iron

alun after paraf fin was removed (Sharman 1943 ) , A: apical meristem; double arrows: provascular

tissue; VT: vascular tissue; Pi: pith. Scale bar =

142 W. Fig. 3.5

Fig. 3.6 4. Isolation without puncturing

Figure 3.7 Transverse section at 30 pm from the summit of

an isolation-only treated apex after 2 weeks showing a distinct apical meris t em. LM micrograph,

stained by safranin-fast green with prior removal

of paraffin. Pz: peripheral zone; Cz: central

zone; LP: the youngest leaf primordium. Scale bar

= 35 pm.

Figure 3.8 Transverse section at 50 p from the sumit in

the sarne isolation-only treated apex as in the

Figure 3.7 showing a distinct provascuLar ring. LM

micrograph, stained by safranin-fast green with

prior removal of paraffin. pvt: provascular ring;

arrows: leaf traces, Scale bar = 71 m. Fig. 3.8 5. Isolation wi thout puncturing (continued)

Figure 3.9 Transverse section at 390 pm from the summit in

the same shoot apex as in Figure 3.7, showing a

distinct vascular system in the pith plug. LM

micrograph under polarized light, stained by safranin-fast green with prior remova1 of paraf fin. Arrow: xylem element; arrow head:

phloem element. Scale bar = 142 m.

Figure 3.10 Portion of a transverse section at the base of

the pith plug in the same isolated shoot apex as in Figure 3.7 showing distinct xylem without

phloem. LM micrograph under polarized light, stained by safranin-fast green with prior removal of paraffin. Arrow: xylern element; m: cutting

mark; Pg: pith plug. Scale bar = 35 p. Fin. 3.9

Fig. 3.10 6. Isolation wi thout puncturing (continued)

Figure 3.11 Transverse section at the base of the pith plug

in an isolation-treated apex the same as in Figure

3.7 showing a distinct vascular system independent

f rom the original one. LM micrograph, stained by safranin-fast green with prior removal of

paraf f in. Pg: pith plug; arrow heads: newly formed vascular system in pith plug; m: cutting mark; double arrows: original vascular system.

Scale bar = 350 W.

Figure 3.12 Transverse section below the base of the pith

plug in the same shoot apex as in Figure 3.7

showing regenerated xylem elernents. LM micrograph,

stained by safranin-fast green with prior removal of paraffin. Single arrows: regenerated tracheary elements; double arrows: vesse1 element; VT: original vascular tissue. Scale bar = 35 m. Fig. 3.11

Fig. 3.12 7. Isolation pl us partial puncturing

Figure 3.13 Longitudinal section of a shoot apex with

isolation treatment after 2 weeks of operations.

The 1, was not punctured. Newly fomed provascular cylinder extended into the isolated tissue plug.

LM micrograph, s tained by safranin-f ast green

without prior removal of paraffin. A: apical

meristem; LP: leaf primordium; double arrows :

provascular tissue; Pi : pith; single arrows indicate the basal edges of the epidermis. Scale

bar = 64 pn. Fig. 3.13 8. Isolation plus partial puncturing (continued)

Figure 3.14 Transverse section at 70 prn from the summit in

an isolation-treated apex with for 2 weeks with I2 allowed to develop, showing a distinct provascular ring. LM rnicrograph, stained by safranin- f as t green with prior removal of paraf f in. Pi: pith;

pvt: provascular ring; 1,: the second leaf

primordium. Arrow: a leaf trace. Scale bar = 71 m-

Figure 3.15 Transverse section at 90 pm from the summit in

an isolation-treated apex with for 2 weeks with 1, allowed to develop, showing a provascular ring and

leaf traces of 1,. LM micrograph, stained by safranin-fast green with prior removal of

paraffin. 1,: the base of the second leaf prirnordium; arrow head: leaf trace; single arrow:

phloem element; double arrows: xylem element; Pi:

pith. Scale bar = 71 m.

9. Isolation plus partial puncturing (continued)

Figure 3.16 Transverse section at the base of the pith plug

in the same apex as in Figure 3.16 showing distinct vascular bundles related to the

unpunctured 1,. LM micrograph, stained by safranin-

fast green with prior removal of paraffin. Pg: pith plug; m: cutting mark; single arrow: phloem;

double arrows: xylem. Scale bar = 71 pm.

Figure 3.17 Transverse section through the base of the pith

plug in the same shoot apex as in Figure 3.16,

showing regenerated xylem elements which wi11 establish connection with original vascular system. LM micrograph, stained by safranin-fast green with prior removal of paraf fin. Pi: pith; hollow arrow: original vascular tissue; solid arrow: regenerated tracheary element; m: cutting

mark. Scale bar = 35 lm. Fig. 3.16

Fig. 3.17 10. Isolation pl us complete puncturing

Figure 3.18 Median longitudinal section of a shoot apex with isolation plus completely puncturing treatrnent after 2 weeks of operations. Newiy £ormed provascular cylinder extended in the isolated tissue rnicrograph, stained toluidine blue O without prior removal of para£ f in, A: apical meristem; Pi: pith; pvt: provascular tissue; hollow arrow: epidermis; solid arrow: the site of a punctured leaf primordium.

Scale bar = 35 p.

Figure 3.19 Transverse section at 120 pm from the summit of

a shoot apex with the fully operated treatment af ter 2 weeks of operations , showing provascular ring in the new growth. LM micrograph, stained by safranin-fast green with prior removal of

paraffin. Pi: pith; pvt: provascular ring; hollow arrow: epidermis; solid arrow: the site of a

punctured leaf primordium. Scale bar = 71 pm. Fig. 3.1@

Fig. 3.19 11. Isolation plus complete puncturing (continued)

Figure 3.20 Transverse section at 200 from the summit of

a fully' operated shoot apex, showing provascular

ring in the pith plug. LM micrograph, stained by safranin-fast green with prior removal of

parafiin. Pi: pith; pvt: provascular tissue ring.

Scale bar = 71 pm.

Figure 3.21 Portion of a longitudinal section of a shoot

apex with isolation treatment after 2 weeks of operat ions, showing newly formed provascular

tissue extended in the isolated tissue plug. LM

micrograph, stained by toluidine blue O without

prior removal of paraffin. Pi: pith; E:

provascular tissue extension; vt : original

'vascular tissue; arrow indicates the basal edge of

the epidermis. Scale bar = 71 m. Fig. 3.20

Fig. 3.21 12. Esterase activity in içolated shoot apices:

Figure 3.22 Longitudinal section of an isolated shoot apex,

showing esterase activity in the provascular

tissue and its extension. A: apical meristem; pvt: provascular tissue; Pi: pith; E: provascular tissue extension; arrows indicate the basal edge

of the epidermis. Scale bar = 71 pm, Fig. 3.22 Chapter 4 AUXIN EFE'ECTS ON WCULAR DIFFERENTIATION

IN THE SHOOT APEX OF CARROT

4.1 Introduction

In the surgical experiments described in Chapter 3, removal of leaf primordia did not prevent provascular tissue

formation by the apical meristem; however, further diff erentiation of the vascular tissue was obviously delayed or prevented. These results indicated that vascular tissue differentiation is strongly affected by developing leaves in angiosperms. It has been argued that the effect from the leaves is mediated by auxin (McArthur and Steeves 1972). This proposa1 is supported by several lines of evidence. It has been demonstrated that developing leaves are a major source of auxin in plants (Jacobs and Morrow 1957, Wetmore and Rier 1963). However, the most supportive evidence is from the in vitro culture of isolated shoot apices which have demonstrated that leaf primordia can be replaced by auxin. In cultures of shoot apices of angiosperms, it is necessary either to keep leaf primordia on or to put auxin into the medium (Smith and Murashige 1970,

Murashige 1974, Bal1 1980). It is reasonable to suggest that

171 172 auxin, either from leaf primordia or by addition, is essential because it plays a role in inducing vascular differentiation, Auxin is reported widely to be related to vascular differentiation (Sachs 1981; Aloni 1987a, 1991; Roberts, Gahan and Aloni 1988) and is regarded as the most important factor promoting differentiation of both xylem and phloem in plants (Roberts 1988a, Shininger 1979) . Jacobs (1952) demonstrated the role of auxin in the redifferentiation of parenchyma to form a xylem bridge around a severed vascular bundle in Coleus. Numerous studies have shown the capacity of auxin to promote vascular differentiation in such diverse systerns as isolated stem segments, plugs of pith tissue, callus cultures and ce11 suspensions (Shininger 1979, Roberts 1988b) . In some studies which dealt with vascular tissue regeneration from parenchymatous cells the results could be explained by the hypothesis of auxin flow through the plant (Sachs 1981).

Experiments involving auxin are many but few experiments have been performed to detemine the ef fects of auxin on the apical meristem in relationship to early vascular differentiation. There have been a few experiments dealing with IAA in situ replacement of leaf primordia in seed plants but in ferns only one species has been reported. In experiments on

Geum, IAA alone promoted the provascular tissue to undergo 173 more distinct differentiation but provascular tissue was not

advànced to procambium (McArthur and Steeves 1972). When ïAA

and 2% sucrose were applied to the shoot apex of Geum, a

procambial cylinder was observed but IAA was not able to replace leaf primordia totally (McArthur and Steeves 1972).

In Coleus IAA was reported to replace a leaf primordium and restore al1 vascular bundles which were related to the removed leaf primordium (Bruck and Paolillo 1984) . In

Lupinus, IAA was able to prevent meristematic cells under a removed leaf primordium from differentiating into parenchy-

matous cells (Young 1954) . In the fern Matteuccia, IAA was demonstrated to have a different ability and caused potential vascular tissue under the removed leaf primordium to dif-

ferentiate into parenchymatous cells (Ma and Steeves 1992). The present work is a morphological analysis of the

effects of IAA in relation to growth of the shoot apex and vascular differentiation in carrot. The complex interactions between the apical meristem and the rest of the plant will be minimized by isolation and puncturing. IAA will be applied to surgically treated apices by two carriers. The amount of IAA and position of IAA application will Vary. Analysis of the treated shoot apices will contribute to an understanding of the role played by auxin in the control of vascular differentiation by the apical meristem and leaf primordia. 4 -2 Material and methods

4.2.1 Plant material and growth conditions The material used in auxin application work was the same as that used in surgical experiments. The shoot apices were obtained from carrot plants (Daucus carota L, var, Little

Finger) grown in the Biology Department garden from seed obtained from Speers Seeds, Saskatoon, Saskatchewan. Seeds were sown in May; plants were used in August. The plants after treatment were put into a growth chamber under fluorescent and incandescent lights at 215-305 p mol m-2 s'l and with a photoperiod of 8 hours day and 16 hours night. Temperature was set at 23°C day and lg°C night and relative humidity at

90-95 8,

4.2.2 Preparation of IAA carriers

Although several auxins, indole acetic acid (IAA), 4- chloro-indole-acetic acid (4-chloro IAA) (Hofinger and

Bottger 1979, Engvild l986), phenylacetic acid (PAA) (Wight- man and Lighty 1982, Noda et al. 1989, Leuba and LeTourneau

1990) and indole butyric acid (IBA) (Schneider et al. 1985,

Epstein et al. 1989), have been found in plants and are considered to be natural auxins, IAA has commonly been used in experiments on vascular tissue differentiation, In order to compare results with those of others, IAA was used in the 175 present work. ïAA is usually applied in water, agar, lanolin or resin beads. Lanolin and resin beads were used in the present work as IAA carriers.

4.2.2.1 Lanolin emulsion The disadvantage of lanolin emulsion is that it is difficult both to localize and to remove after a tîmed application. However, it has a large carrying capacity for

IAA in a relatively small volume and is still being used in experimental work (Sachs 1993) . The procedure of preparing an IAA lanolin emulsion was given in detail by Aloni et al.

(1990). IAA was dissolved in 100% ethanol and then mixed with warm lanolin. To evaporate the ethanol, the liquid mixture was kept hot but not boiling while being stirred with a magnetic stirrer for at least 30 minutes (Aloni et al.

1990). In order to compare results with those from Geum, IAA was applied in the form of a lanolin paste in 0.5% (w/w) concentration similar to that used by McArthur and Steeves

(1972). As a control, lanolin emulsion was prepared in the same manner except without IAA.

4.2.2.2 Resin beads

The method using resin beads was first applied to plant tissues by Gee and Greyson (1969) and followed by Davies et 176

al. (1976), Bruck and Paolillo (1984) and Ma and Steeves (1992). Because resin beads are solid carriers, they are easy to handle and stick readily to plant surfaces. They have the advantage of providing precisely localized appli-

cation of IAA. The disadvantage of resin beads as IAA carriers is that the carrying capacity per volume is srna11 (Sachs 1993) .

4.2-2.3 Concentrations

The preparation of ïAA loaded resin beads in the present work was based on the procedure of Bruck and Paolillo (1984) with some modifications. The concentrations of IAA used by earlier workers to soak resin beads have varied. Some have used a 0.5 mM IAA solution (e.g. Bruck and Paolillo 1984,

Davies et al. l976), while others used 0.1 mM (Ma 1994) or 1 mM (Ma and Steeves 1992) . In order to determine an optimal concentration, two series of concentrations of IAA, front 0.01 to 0.1 and 0.1 to ImM, were prepared, The absorbent optical densities at a single wave length (A=278 nm) of a series of concentrations of IAA water solutions were measured by an ultraviolet spectrophotometer (Bechan Mode1 Du-7). A standard curve was obtained by plotting the measurements against the series of concentrations 0.1 to 1 mM (Fig. 4.1).

The curve of 0.1 to 1 mM solutions reveals that O.D. in the range of 0.1 to 0 -5 rnM IAA has a linear relationship with concentration. Beyond 0.5 mM, the reduction of O.D. not have a significant linear relationship to the concentrations. The curve of the 0.01 - 0.1 mM series was also plotted (Fig. 4 -2). There was a linear relationship, but the solution of 0.1 mM contained so little IAA that each bead might not absorb sufficient IAA. Therefore, in the present work, 0.5 mM IAA concentration was used to soak resin beads.

4 -2.2.4 Resin beads

There are many different types of ion exchange resin beads available for the binding of most ionizable molecules and they are in the form of solid spheres of many sizes (Gee and Greyson 1969) . 'As in the Ma and Steeves study (1992), anion exchange beads (Dowex-1, chloride form strongly basic anion exchange resin beads, 8% cross-linked) were used in the present work. Beads of 100-200 dry mesh, which were approximately 100 pn in diameter, were used in experiments. Beads of 50-100 dry mesh with a diameter of about 330 - 400 p were also prepared. They are able to take up more IAA, but they are too large for a point source on the shoot apices of carrot. 4.2.2.5 Soaking time The soaking time reported previously was one hour (Gee and Greyson 1969, Davies et al. 1976, Bruck and Paolillo 1984, Ma and Steeves 1992, Ma 1994) . In preparing the beads, it was found that the O. D. was reduced more by extending soaking time. This indicated that more IAA was absorbed by beads . Tests of beads on radish hypocotyls (Ma 1994) confirmed that extension of soaking time could cause beads to take up more IAA.

4.2.2.6 Procedure

Fifty rnilligrams of beads were soaked in 100 ml of a 0.5 rnM (8.76rng/lOOml) solution of IAA. The concentrations of the solution were determined by the ultraviolet spectrophotometer beiore and after the beads were soaked. The reduction in optical density, comparing before and after soaking the beads, indicated the extent of absorption of IAA by the beads. In the present work, based on the standard curve (Fig. 4.1 obtained from plotting the absorbency measurements at a single wave length (A=278nm) for each concentration of IAA, reduction of the optical density(Abs O.D.) indicates the amount of IAA taken up by the 50 mg of beads. By means of variation of soaking time, two kinds of beads with different contents of IAA were obtained. Low 17 9 content IAA beads (LBs) which were soaked in IAA solution for one hour contained 0.003 pg per bead (1.14 x 10-'' mol IAA-

/bead) and high content beads (HBs) which were soaked in IAA solution for 3 hours contained 0.009 pg IAA per bead (3.42 x 10-Il mol/bead) . The amount of IAA contained in a low content bead may be the same as that used by Ma (1994), However, the beads used by Bruck and Paoli110 (1984) were large (AG21K ' 50-100 mesh' BioRad, Richmond, California) and contained more IAA (l.58~lO-~mol/bead) , 1400 times as much as LB or 460 times as much as HE! in the present study. Control beads were soaked oniy in distilled water. The beads were then removed from the solution without being washed and stored dry at room temperature or in the refrigerator. According to Gee and Greyson (1969), the IAA loaded resin beads can be autoclaved or stored up to 7 weeks at room temperature or in the refrigerator without loss of activity.

4 -2.3 IAA application experiments

Surgical manipulations of shoot apices for the hormonal experirnent were performed by isolation of the promeristem followed by continuous puncturing of al1 leaf primordia up to and including the incipient leaf primordium position as described in Chapter 3, while lanolin or beads were applied. 180 The pot of experimental plants was covered with two sheets of Kleenex tissue or Scott towels (Scott Paper Company, Canada) and one sheet of clear plastic tightened with a rubber band after each operation, The apices were covered not only to keep rnoisture between beads and the surface of the apex, but also to protect against light-induced IAA breakdown. Based on the two carriers, two groups of IAA application experiments were planned.

4-2.3.1 Experiments with lanolin

Shoot apices were prepared as described in Chapter 3. After one day recovery, isolation operation was applied and

IAA was applied three days later, In experiments where IAA was mixed with lanolin, it was applied as a cap on the isolated apex; the hormone-containing lanolin paste was weighed three times. The average weight of paste applied was about 0.5 mg. Because the concentration of IAA in the lanolin is 0.5%, each cap contained approxirnately 2.5 pg IAA. As controls, lanolin emulsion, which was prepared in the same mannes except without hormone, was applied. Al1 experiments laçted two weeks and were repeated three times during 1991, 1992, 1994, with 12 plants in each treatment. The lanolin cap was replaced every day and new leaf 181 primordia, if any, were punctured. These manipulations were carried out under a dissecting microscope with cool light.

4.2.3.2 Experiments with resin beads The preparation of shoot apices was the same as des-

cribed in Chapter 3. In order to increase the survival rate, the recovery time was extended to three days. For applications of beads there were several treatments: 1) Only one high content IAA-containing bead was applied on the site of

the punctured 1, position, abbreviated as IHB (Fig. 4.3); 2)

one low content IAA-containing bead (LB) was applied sequen- tially on each site of a punctured leaf primordial position

from 1, to I, (Fig. 4 .4 ) , abbreviated as 3LB; 3) more than 3

LBs were applied to the shoot apex, abbreviated as LBs; 4)

as a control, beads containing no IAA were applied to similar

locations on the apex as in treatments 1, 2 and 3, abbreviated as BC. Each bead, either IAA-containing or without IAA, was

placed in a fixed position on the apex, for example, the

position of 1, for the one bead application. An attempt was made not to change the position of the beads when they were replaced about every 24 hours. For each apex, diagrams recorded changes at each operation and this information 182 served as a reference for the next operation and as the developmental history of the apex for future analysis.

Experimental treatments with resin beads were done in

1992 and 1995. Each treatment including control initiaily comprised 12 plants and experiments were repeated at least twice. In the experiments with resin beads as carrier, treatment 1 yielded 18 apices; treatment 2, 18 apices; treatments 3, 18 apices; treatment 4, 24 apices. Experiments were planned to last a maximum of three weeks. Some apices were fixed during this time in order to observe developmental processes. 183

4 .3 Results

The following account will describe the effects of IAA application on vascular differentiation. When IAA was applied to the shoot apices whether by lanolin or resin beads, mature vascular tissue was observed in al1 treated apices. The development of the vascular cylinder as affected by IAA will be described based on shoot apices in 1) experiments with IAA lanolin applied to the whole apical dome and 2) experiments with IAA beads applied on the sites of punctured leaf primordia. In the following description, the term "new growth", as used by Ma (19941, describes the tissue formed during the experiment by the shoot apex after the treatment. The term

"pith plug", following McArthur (McAirthur l967), describes the isolated pith under the isolated shoot apex. The pith plug did not consist of pith tissue only. As observed immediately after isolation, this was the case, but as the isolated shoot apex developed, especially under IAA treatments, vascular tissue developed in the pith plug. Two other terms will be used in the following description: provascular tissue and provascular tissue extension. Provascular tissue as defined in Chapter 2 is a tissue produced by the apical meristem which is the first stage of differentiation of the vascular system in the shoot. Used in isolated shoot apices, 184 the term refers to the provascular tissue produced by the isolated apical meristem and located in the new growth. Provascular tissue extension will be specifically used to

describe the provascular tissue which occurred in the isolated pith plug.

4-3.1 IAA lanolin application experiments

4.3.1.1 Control shoot apices

Previous workers have reported that lanolin used as a

carrier for IAA or other hormones has no effect on the experimental material (McArthur and Steeves 1972, Varnell and

Vasil 1978, Aloni et al. 1990) , Lanolin control shoot apices

had many similarities to the completely operated shoot apices described in Chapter 3, The new growth of the isolated shoot apices was very limited. The height from the lowest level of

epidermis or the site of severed P, to the tip of the apex was about 100 - 160 m. The apical meristem showed 3 - 5 layers of isodiametric, densely stained cells at the tip of

the apex but a zonation organization was not evident. Cortex

differentiation was earlier than in normal development at the

level of 50-60 p; this is similar to a completely operated apex. Pith differentiation was also affected. In normal plants, pith differentiation could start at the level of 40-

50 pm from the tip, while in the control shoot apex, pith 185 differentiation was identified at the level of 70-80 pm as in a completely operated apex. As in the cornpletely operated shoot apex, the provascular tissue extended into the apical meristem and basipetally into the pith plug (Fig. 4.5) . In longitudinal view, provascular cells in new growth were nearly rectangular in shape but not longer than the surrounding cells in the new growth. The L/W (L: length, W: width) ratio of provascular cells was 2.5: 1. This ratio is similar to the ratio of provascular tissue in n~rmaldevelopment. Vascular differentiation in control plants did not advance beyond the provascular stage above the severed P, in the new growth. No mature phloem or xylem were observed in the new growth of the controls. In transverse view, an uninterrupted provascular ring could be observed clearly in the new growth al the level of

100 pxrt from the tip of the shoot apex as a result of cortex and pith development. At the level of the severed Pl (about 150 from the tip of the shoot apex), the provascular ring was more distinct (Fig. 4.6) and the diameter of the ring was 125 - 165 p. The cells of provascular tissue were nearly isodiametric and about 10 in diameter in transverse view.

Cells were smaller than parenchymatous cells and darker stained with large nuclei. The area of the provascular ring in cross section view can be calculated by measuring the 186 diameters of provascular ring and of pith. The area of the

provascular ring at the level of the severed P, was 1.1 x IO4 w2- In the pith plug, peripheral cells oriented their division plane parallel to the surface and produced a protective sheath covering the cut surface. The provascular tissue extension in the pith plug originated from regeneration of the parenchymatous pith cells . The provascular extension was not connected to the original vascular system and was suspended in the pith plug. The cells in the provascular extension were rectangular in shape and the L/W ratio

was in the range of 2:l to 3:l. Longitudinal division had occurred in these provascular cells but elongation was

limited. They were no longer than surrounding parenchyma cells. No mature phloem or xylem were observed in the pith plug of the controls.

4.3.1.2 IAA in lanolin The most evident change in IAA-lanolin treated apices was the vertical growth of the isolated apex. In the controls, the height of the new growth was 100-160 pm, but with IAA treatment, it reached 210 pm. The apical meristem which was observed in the new growth had not been seriously affected by IAA treatment during the experimental period. A 187 zonation organization was not identified but 3 - 4 layers of densely stained meristematic cells were present in the tip of the new growth of the isolated shoot apex (Fig. 4.7) . At the level of 50-60 pm, cortex differentiation was evident. Pith differentiation occurred later at the level of 100 p. Cortex differentiation was similar to the controls but pith differentiation was delayed. Similarly to controls, provascular tissue extended to the apical meristem and downward into the pith plug. In the new growth, the provascular tissue was more densely stained than the surrounding parenchyrna cells but appeared to be similar to the apical meristem. In longitudinal view, provascular cells were not remarkably elongate, The elongate shape was the result of longitudinal division. The L/W ratio was 3.5:l and this rnight indicate they were entering the procambial stage in spite of limited elongation. The provascular tissue appeared as an uninterrupted ring in transverse section in new growth and no distinct axial bundles or procambial strands were observed in IAA treated shoot apices. The ring was clearly blocked out at the 100-

110 pm level as cortex and pith were becoming well developed (Fig. 4.8). The size of the provascular tissue ring increased slowly downwards. At the severed P, level (about

210 pm from the tip of the shoot apex), the provascular ring in cross section was 190 pm in diameter. Pith diameter did 188 not increase very much and at the same level, its diameter

was 100 p. The axea of provascular tissue was 2x104 pz, double that of controls. In the pith plug, the surface was covered by layers of protective sheath. Provascular tissue extension cells were not very elongated in the pith plug in longitudinal view. The L/W ratio reached 4:l. In transverse view of the pith plug, the provascular extension tissue appeared as a continuous ring but no distinct bundles could be identified. In the lower part of the pith plug, a few parenchyrna rays could be identified in the ring. In contrast to controls, a few mature vascular elements were observed in this ring in the new growth of IAA treated shoot apices (Fig. 4.9) and the pith plug. Although these mature vascular elements were sparse, they occurred in relation to the provascular ring. Phloem was at the outside of the ring and xylem was at the inside of the ring. Therefore, these phloem and xylem elements were differentiated from provascular tissue by promotion of IAA. Phloem elements could be identified in the new growth at the level of 140 - 150 m. The xylem elements were found at the 150 - 170 pm level from the smit of the apex in the new growth and both phloem and xylern continued downward into the pith plug. Compared to normal development where the phloem was found at

the level of 150 - 250 pm and xylem elements were found at 189 the level of 210 - 380 prn, the maturation of vascular tissue, especially xylem elements, in IAA treated shoot apices was earlier than normal. Following the pith plug downward, the vascular system was found to be suspended in the pith plug.

4.3-2 IAA bead application experiments In IAA lanolin experirnents, provascular tissue became very distinct but only a few mature vascular elements were observed. In the bead experiments, initially, the IAA beads were applied in the same way as the IAA lanolin, that is, as a cap covering the whole apical dome including the sites of punctured leaf primordia (Treatment 3, LBs) . It was found that IAA beads applied in this way had so strong an effect that the apical meristem and shoot apex were swollen (Fig.

4-10)but a large amount of xylem and phloem was formed under the influence of IAA in the new growth (Fig. 4.11) and the pith plug- From this treatment, beads appeared to be more efficient than lanolin in releasing IRA and a high 1A.A dosage caused extensive vascular differentiation. Since so much xylem and phloem were formed, it was difficult to correlate the mature vascular tissue precisely with the applied IAA source

In the 1HB and 3LB treatments, IAA beads were used as point sources applied to the sites of the punctured leaf 190 primordia. The IAA effect could then be limited to the part of the apical meristem where a new leaf primordium would be initiated and mature vascular tissue could be traced to the IAA point source.

4.3.2.1 Three low content IAA beads applied 4.3.2.1.1 New growth

The new growth in controls was about 200 pm while in IAA treated shoot apices it was in the range of 300 to 500 W.

The effect on vertical growth of the ïAA bead application was more evident than in IAA lanolin application, In addition to new growth, the IAA treated shoot apices had a higher survival rate and some could last up to three weeks, so that five to six leai primordia could be punctured in the three week experimental period. In the controls, almost no shoot apex could last so long and only three or four leaf primordia were punctured. The average length of the plastochron in IAA treated shoot apices was longer than that in the controls and close to the normal average length.

4.3.2.1.2 Apical meristem

IAA application had little effect on the apical meristem when three IAA beads were applied to the sites of the punctured leaf primordia. In the shoot apices of control 191 plants, the apical meristem was densely stained and four - f ive layers could be identified in longitudinal view (Fig. 4.12) . Similarly, a group of meristematic cells, about five layers, could be recognized in the tip of the new growth in longitudinal sections of IAA treated plants (Fig. 4.13).

4.3.2.1.3 Provascular tissue

In controls, as in fully operated shoot apices, the provascular tissue extended into the peripheral zone of the apical meristem. The provascular tissue zone was about 3-4 ce11 wide in longitudinal view and densely stained (Fig.

4 -12). Under the IAA beads, the provascular tissue zone was 4-5 cells wide and was more densely stained than in controls

(Fig. 4.13). The provascular cells under IAA influence became elongate. The ce11 size in IAA treated provascular tissue was about 25 prn x 5 prn in longitudinal view and the L/W ratio was 5: 1. The provascular cells in controls were

12.5 pm x 5 pm in size and the ratio was 2.5:l. Therefore, based on the ratio, procambium was formed in the new growth in the IAA treated shoot apices. In the transverse view, the provascular tissue showed an almost continuous ring but occasionally was interrupted by parenchma cells (Fig. 4,141. Cells in the provascular ring were more densely stained and were smaller than surrounding cells. 4.3.2.1.4 Mature vascular tissue In the controls, the provascular tissue and its extension did not show morphological evidence of further differentiation. In contrast to the controls, mature vascular tissue could be observed in the new growth of the IAA treated shoot apices and the pith plug. Xylem was found at the level of

240 p, phloem at the level of 210 pm, indicating that phloem differentiated earlier than xylem in the IAA treated shoot apex. Compared to normal development, the vascular maturation in IAA treated shoot apices was earlier. In transverse sections, xylem elements appeared to be correlated with the three IAA beads and formed an almost complete or continuous circle (Fig. 4.15). The phloem formed a ring of the outside of the vascular tissue (Fig. 4.15). Between the phloem and xylem, the tissue appeared to be procambium or cambium like (Fig. 4.15). The newly formed vascular system finally connected to the original vascular system.

4.3.2.2 One high content IAA bead applied

The effect of IAA in the one IAA bead treated shoot apices was similar to the three IAA beads treated in rnost aspects. A difference was noted in the apical meristem and provascular tissue. After 3 weeks, the shoot apex became 193 smaller in diameter but a group of densely stained rneris- tematic cells was still present in the tip of the new growth.

Provascular tissue below the site of 1, had differentiated into mature vascular tissue. Because only one IAA bead was applied to the sit2 of a punctured leaf primordium (1,) , when the apical meristem of the shoot apex had grown beyond the site of the IAA bead, there was no direct influence on the provascular tissue which was now above the 1, level. The provascular tissue was not very distinct, In transverse view, the provascular ring above the 1, was almost continuous but became smaller in diameter (Fig, 4.16). The major difference was in the pattern of the vascular system in the new growth and the pith plug. The xylem in the newly formed vascular system was related to the position of the IAA bead applied, When only one IAA bead was applied for three weeks as a point source of IAA on the site of a punctured leaf primordium, xylem elements were differentiated in the vascular cylinder only on the side of that bead (Fig.

4.17). However, the phloem was not related to the location of IAA beads and it formed a ring in the transverse view

(Fig. 4.17) . No distinct leaf traces or widely separated axial vascular bundles could be identified. This pattern is different from the shoot apices in which leaf traces and their related axial vascular bundles are distinct when one leaf primordium is allowed to develop (see Chapter 3). The 194 newly formed vascular system finally connected to the original vascular system below the base of the pith plug. 4.4 Discussion

In Chapter 3, experiments demonstrated that the apical meristern could give rise to a provascular cylinder without development of leaf primordia. Without leaf primordia, this provascular tissue did not undergo further morphological differentiation, but esterase activity indicated that differentiation had begun in it. Because leaves are a most probable source of auxin, it is reasonable to suggest that

IAA could promote the further morphogenetic response. The

present IAA application work showed that this substance has strong effects on both initial vascular differentiation and the later stages of maturation in angiosperms.

4.4.1 IAA involvement in the early stages

The most evident response of provascular tissue to IAA was that it became more distinct in the IAA-lanolin or IAA- bead treated shoot apex. There was no apparent difference between the effect of IAA applied by lanolin or resin beads in terms of distinctiveness. In general histological staining, the provascular tissue in IAA treated shoot apices was more densely stained than in controls. This dense staining reflects some cellular changes occurring in these provascular cells, Although the present work did not include a cytochemical study of IAA effects, cytochemical analyses in 196 the lupin shoot apex revealed significant increases in concentrations of RNA, protein, and unsaturated lipids in the peripheral zone of the apical meristem in response to IAA application (Varnell and Vasil 1978). An increase of RNA and protein is necessary for ce11 division and tissue differentiation and could cause the increased density of staining. It is reasonable to conclude that active differentiation of provascular tissue is promoted in response to IAA application. The volume of provascular tissue was also increased in response to IAA application. In IAA lanolin treated shoot apices, the area of the provascular region in transverse sections was almost double that in controls. The provascular tissue ring in IAA treated shoot apices was not broken into segments as in normal plants. The increase in area in transverse sections was an increase of provascular tissue and was not caused by production of parenchyma cells as in the fern Matteuccia (Ma and Steeves 1992) . A possible parallel effect has been reported in lupin by Young (1954). When P, was removed, parenchyma was formed under the site of the rernoved P,. When the site of the removed P2 was treated by IAA - lanolin, the meristernatic condition of the cells was maintained. Since the meristematic cells were presumably 197 provascular tissue, this appears to have been IAA effect on this stage of vascular differentiation. Exogenous IAA enhances provascular tissue differentiation but clearly is not necessary. This raises the question whether auxin is necessary at al1 for provascular formation. If provascular tissue formation needs IAA, it is reasonable to presume that it is produced by the apical meristem. There is evidence to suggest that the apical

meristem can produce auxin. It has been estimated by mass

spectrometry that IAA levels in Phaseolus shoot tips were in

the order of 0.1 to 0.7 ng per shoot tip (White et al. 1975). If the apical meristern does produce auxin, why can the provascular tissue not be differentiated to the mature stage when leaf development is suppressed? The possible answer is that the ability to produce auxin is limited. Procambium formation may be another evident response to

IAA application. Procambium is not easily distinguished from provascular tissue because the provascular tissue is gradual-

ly and continuously differentiated to procambium. Procambium is not well defined and has been considered the same as the provascular tissue in seed plants (Esau 1960). In the study of Matteuccia, "the cells which have an elongate shape as a reçu1t of longitudinal. division, i . e. they are longer than they are wide, are considered procambial cells" (Ma 1994, 198

page 38) , In the study of normal development in carrot, the L/W ratio was introduced to distinguish the provascular tissue from the procambium. This L/W ratio may be a good standard for the study of normal development where, although

vertical elongation is limited, lateral expansion is extensive. In surgically treated shoot apices, both the vertical elongation and lateral expansion were limited. The L/W ratio rnay not reflect the real status of cells. Therefore, both the elongate shape and L/W ratio should be considered in

surgically treated shoot apices. Although in IAA lanolin treated shoot apices, the L/W ratio was not increased very much, the elongate shape indicates that the provascular

tissue was entering the procambium stage. In addition, in IAA bead treated shoot apices, the L/W ratio of provascular tissue was increased. Under the influence of IAA, provascular tissue thus appears to have differentiated to the procambial stage. This conclusion is supported by other studies. In Geum, IAA in lanolin with sugar caused the provascular tissue to becorne procambium (McArthur and Steeves 1972). 4 -4-2 IAA effects on maturation of vascular tissue In an experiment with IAA application via lanolin,

McArthur and Steeves (1972) pointed out that the emulsion formed a cap over the isolated shoot apex and could be expected to result in an even hormonal distribution across the isolate. They (1972) ernphasized that if a ring, rather than scattered elements of mature vascular tissue, occurred within the provascular cylinder, it would indicate that the provascular cylinder does in fact represent a blocking out of the vascular system, under the influence of the apical meristem. However, further hormonal influence from the leaves is required in order to continue its differentiation as procambium. In the present work, a ring pattern was indeed observed when the IAA was applied to the shoot apex as a cap. This result was very similar to that in the Geum study except that no vascular differentiation beyond procambium occurred in Geum (McArthur and Steeves 1972).

In the present work, application of IAA in lanolin to the shoot apical meristem produced a limited response of xylem dif ierentiation as well as phloem. With IAA beads as a point source application, by contrast, a large arnount of xylem was formed so that the relationship between IAA and vascular differentiation was dernonstrated more clearly. The difference in the amount of xylem between lanolin and bead 200 applications may reflect that the beads are more efficient in

releasing IAA, Xylem and phloem diff erentiation provided strong evidence that IAA regulates vascular differentiation and prornotes the maturation of vascular tissue. However, the basic pattern of the vascular system is established at the provascular stage by the apical meristem,

In Coleus, the quantity of induced vascular tissue following IAA application varied with the amount of IAA applied (Bruck and Paolillo 1984) . In the present study, the amount of xylem was not quantitatively analyzed in relation

to the amount of IAA applied, but the correlation between the

IAA and the xylem was very clear. With three IAA beads applied to the sites of the punctured leaf primordia, xylem elements formed a ring'in transverse sections. With one high

content IAA bead applied to the shoot apex, only half of the ring below the IAA bead had distinct xylem elements but phloem occurred around the ring. This finding agrees with reports that the amount of auxin required for phloem diffe-

rentiation is less than that for xylem (Aloni 1987a).

4 -4-3 IAA and leaf primordia

Sachs (1981) proposed that the flow of auxin through cells plays an important role in the pattern of the vascular system. The initial local differences in response to the 201 flow of auxin through cells lead to the establishment of preferred channels of auxin transport. Then these channels become progressively improved pathways of auxin movement and drain the surrounding regions at the same time that their cells are induced to undergo differentiation as vascular elements. Connections are established with preexisting channels that are highly preferred pathways but whose auxin supply has been depleted. In lupin, removal of one leaf primordium caused a parenchyma gap under the site of the removed leaf primordium, but removal of al1 leaf prirnordia caused a meristematic or provascular ring to persist (Young 1954). Moreover, the parenchyma gap could be prevented by application of UA. When one leaf primordium was rernoved, the even distribution of auxin was disturbed. Auxin was probably drained by other leaf bundles, causing parenchyma cells to differentiate between bundles. Exogenous IAA replaced the auxin from the site of a removed leaf prirnordium so that the parenchyma cells did not form. With xemoval of al1 leaf primordia, distribution of the preswned auxin from the apical meristem was evenly balanced, and the meristematic ring persisted.

In the present work (see Chapter 31, in the experiments in which one leaf primordium was allowed to develop, there were axial vascular bundles related to the leaf primordium while provascular tissue extension was not observed. Pos- 202 sibly, the vascular bundles related to the unpunctured leaf primordium affected the formation of the provascular tissue by drawing away auxin from the apical meristem. In the one

IAA bead application (the present chapter), the result was different from allowing one leaf primordium to develop. No bundles formed but provascular tissue and its extension developed in response to the exogenous IAA. This suggests that while IAA accounts for some of the influence of leaf primordia, it cannot entirely replace them.

In Coleus, it was reported the IN4 could replace the leaf primordia and restore the vascular bundles which were lost when a leaf primordium was removed (Bruck and Paolillo 1984) . This conclusion in Coleus was qpestioned by Ma because the provascular tissue existed befare the IAA -was ------applied (Ma 1994) . The present work further supports Ma's opinion. In carrot, it was found that before a leaf primordium reached 10 pm in height, the initial of the main axial vascular bundle which is related to it has been formed (see Chapter 2). Although there could be variation in different species, the vascular bundle initials must have been formed under the removed leaf primordium in Coleus. Therefore, IAA application promoted a pre-existing bundle initial to develop and mature instead of creating a vascular systern. Although the conclusion is misleading in the light of the present 203

work, the IAA replacement experiment in Col eus did demonstrate that IAA promotes vascular differentiation,

4.4.4 Other substances in vascular differentiation

Although the present work emphasized the effect of IAA alone, it is not intended to overlook the possible importance of other substances. There are recent indications that

cytokinins rnay be necessary at the early stage of vascular dif ferentiation. Cytokinins stimulate ce11 division in tissue cultures (Skoog and Miller 1957) and are involved in the control of differentiation of both tracheary and secondary xylem fibres in tissue culture (Aloni 1987b, Roberts et

al. 1988).

Direct evidence that cytokinins are involved in the control of vesse1 regeneration in intact plants was recently reported in wounded Coleus internode tissue (Baum et al. 1991) . These authors proposed that cytokinin may increase the tissue sensitivity to auxin. When kinetin ( 6-furfuryl- anirnopurine) and other hormones such as gibberellin (GA3) were applied to the apical meristem, no effect on initial vascular differentiation was reported (Varne11 and Vasil 1978) . The cytokinin apparently is effective in early vascular differentiation only in the presence of IAA (Aloni et al. 1990, Aloni 1993) . 204 In addition to cytokinins, many other hormones and substances can affect vascular differentiation (Shininger 1979, Aloni 1992, Roberts 1988a, Northcote 1995) . In corn- bination with IAA, sucrose (McArthur and Steeves 1972) and gibberellins (Wareing, Haney and Digby 1964, Reberts 1976,

Aloni 1979) have been show to play a role in vascular differentiation in many species. If these substances are needed for vascular difieren- tiation in carrot they must be present endogenously in sufficient quantity in the experimental plants. In the absence of leaf primordia in carrot, procambium and even matured vascular tissue could form under IAA influence alone. In the earlier experiments with Geum (McArthur and Steeves

1972), the rhizome was cultured without roots and procambium was observed only if IAA was combined with sucrose, No mature vascular tissue was produced. In the present study, the experimental plants retained a well developed root which may have been an endogenous source of cytokinins as well as carbohydrate and other nutrients. Figure 4.1 Standard cuwe obtalneâ by plofting the absorbency measurements at 278 nm of each concentration of IAA against the known IAA concentrations (simM)

IAA concentrations (mM)

205 Flgure 4.2 Standard curve obtalned by plottlng the absotbency masurements at 278 nm of each concentration of IAA agalnst the known IAA concentratfons (S.1mM)

O F CU C3 * V) CD P- m Q, f 9 9 9 9 9 9 9 9 9 O O O O O O O O O O

IAA concentrations (mM)

206 1. Top view of shoot apices

Figure 4.3 Top view of an exposed shoot apex (indicated by an arrow head) showing al1 leaf primordia removed

and 1 bead applied at the site of 12. This is a demonstration in which the vertical cuts were not made and the resin bead was stained with

erythrosin. Scale bar = 450 pm.

Figure 4.4 Top view of an exposed shoot apex (indicated by

an arrow) showing al1 leaf primordia removed and 3 beads applied. In order to show the shoot apex, one of beads at the right hand was a little away from the site of a punctured leaf primordium. This is a demonstration in which the vertical cuts were not made and the resin beads were stained

with erythrosin. Scale bar = 250 pm. Fie. 4.3

Fig. 4.4 2. Lanolin controls

Figure 4.5 A median longitudinal section of a shoot apex

with lanolin without IAA applied for 2 weeks, showing provascular tissue and its extension in the pith plug (below the two arrows). Section was stained in safranin-tamic acid-iron alum after paraffin was removed (Sharman 1943). LM

micrograph. A: apical meristem; pvt: provascular

tissue; Pi: pith; Cx: cortex; E: provascular

extension; arrows : the basal edge of epidermis ; arrow heads: the sites of punctured leaf

primordia. Scale bar = 103 p.m.

Figure 4.6 A transverse section at 150 p.xn from the summit

of a shoot apex with lanolin without IAA applied

'for 2 weeks showing provascular ring. Section was stained in safranin-tannic acid-iron alum after paraffin was rmoved (Sharman 1943). LM micrograph. Pi: pith; pvt: provascular tissue.

Scale bar = 53 m. Fig. 4.5

Fig. 4.6 3. IAA - lanolin application: Figure 4.7 A median longitudinal section of an isolated

shoot apex treated with IAA lanolin for two weeks, showing distinct provascular tissue and its

extension in the pith plug (below the two arrows). Section was stained in safranin-tannic acid-iron alun af ter paraffin was removed (Sharrnan 1943) . LM micrograph. A: apical meristem; pvt: provascular tissue; Pi: pith; Cx: cortex; E: provascular extension; arrows: the basal edge of epidermis; arrow head: the site of a punctured

leaf prirnordium. Scale bar = 99 W.

Figure 4.8 A transverse section at 150 prn from the summit

in an isolation-treated apex with IAA-lanolin applied for 2 weeks showing distinct provascular

ring. LM micrograph; the section was stained by

safranin-fast green without prior removal of paraf f in. Pi: pith; pvt: provascular tissue.

Scale bar = 50 m. Fig. 4.7

Fig. 4.8 4. ïm lanolin application (continued)

Figure 4.9 A transverse section at 280 pxn from the summit

in an isolation-treated apex with IAA-lanolin applied for 2 weeks showing a vascular tissue ring. LM micrograph; the section was stained by safranin-fast green without prior removal of

paraf fin. Arrows : phloem; double arrows : xylem. Scale bar = 30 m. Fig. a.9 5. Several low content IAA beads applied as a cap (LBs):

Figure 4.10 Top view of an isolated shoot apex which was

treated with low content IAA beads for 3 weeks.

Hollow arrow: centre of the apical dome. Scale

bar = 400 pm,

Figure 4.11 A transverse section of a shoot apex with low

content IAA-containing beads applied as a cap for three weeks, showing a cylinder of mature vascular tissue extended into the pith plug. LM

micrograph; the section was stained by safranin-

fast green with prior removal of paraf f in. xy:

wlern; double arrows: phloem; single arrows: oil

ducts. Scale bar = 57 W. Fia. 4.10

Fig. 4.11 6. Three bead application

Figure 4-12 A rnedian longitudinal section of a shoot apex

with 3 resin beads without IAA applied after two weeks , showing provascular tissue and its

extension in the pith plug. LM micrograph; the section was stained by safranin-fast green with

prior removal of paraffin. A: apical meristem;

double arrows : provascular tissue; single arrow: the site of a punctured leaf primordium. Scale

bar = 39 W.

Figure 4.13 A median longitudinal section of a shoot apex

with 3 resin beads with IAA applied for two weeks, showing provascular tissue and its extension. LM

micrograph; the section was stained by saf ranin-

fast green with prior removal of paraffin. A: apical meristem; double arrows: provascular tissue; arrow head: procambium; single arrows: the

sites of puictured leaf primordia. Scale bar = 35 w- Fig. 4.12

Fig. 4.13 7. Three bead application (continued)

Figure 4.14 A transverse section at 100 pxn from the surmnit

in a shoot apex with 3 IAA-containing beads

applied for 3 weeks, showing provascular tissue.

micrograph; the section was stained safranin-fast green with prior remova1 of

paraffin. pvt: provascular ring; Pi: pith; Cx:

cortex. Scale bar = 67 m.

Figure 4.15 A transverse section 340 pm below the summit of

the shoot apex showing mature vascular tissue was formed under the influence of three IAA beads after 3 weeks. LM micrograph with polarized

light; the section was stained by safranin-fast

green with prior removal of paraffin. Pi: pith;

Cx: cortex; single arrows: phloem; double arrows:

xylem. Scale bar = 106 p. Fig. 4.15 8. One IAA bead application :

Figure 4.16 A transverse section at 80 pm from the summit

in a shoot apex with 1 IAA-containing bead applied

for 3 weeks, showing provascular tissue. LM micrograph; the section was stained by safranin- fast green with priot removal of paraffin. pvt:

provascular ring. Scale bar = 53 W.

Figure 4.17 A transverse section below the site of I2 in a

shoot apex with one high content IAA-containing

bead applied after 3 weeks showing distinct phloem and xylem elements formed. LM micrograph with polarized light; the section was stained by safranin-fas t green with prior removal of

paraffin. ph: phloem; xy: xylem elements; Cx:

cortex. Scale bar = 53 p. Fig. 4.16

Fig. 4.17 Chapter 5 EVIDENCE ERûM OTHER HERsACEOUS

DICOTYLEDONS

5.1 Introduction In the previous Chapters, the study of the shoot of carrot revealed that the process of vascular differentiation is complex. The differentiation of provascular tissue in the shoot apex was shown to be independent of the leaf primordia. A provascular ring could be observed above the divergence point of the youngest leaf trace. The surgical experiments further confirmed this independence. When leaf development was suppressed at an early stage, the provascular tissue still could be observed in general histological staining and in esterase tests. However, differentiation in the treated shoot apices never advanced to later stages; no provascular tissue became mature vascular tissue. Auxin application experiments demonstrated that auxin is one of the factors that prompt further maturation of vascular tissue. When an exogenous auxin was applied to the shoot apices on which the leaf primordia were suppressed, the provascular differentiation was promoted and provascular tissue could even advance to the mature stage. 216 Based on the above facts, vascular differentiation can be proposed to occur in two stages. In the first stage, the provascular stage, cells in the peripheral zone of the apical meristern are committed to provascular development. In this stage, the provascular tissue is independent of the leaves similarly to the initial stage of ferns (Ma 1994) . In the second stage, the provascular tissue advances to procambium and mature vascular tissue, This stage is dependent on the leaves in dicots. This is quite different from the development of vascular tissue in ferns where, whether initial or late stage, leaf participation is not necessary and leaves do not play an essential role in the maturation of vascular tissue (Ma and Steeves 199Sb).

The present study in carrot and the study in Geum

(McArthur and Steeves 1972) have thus contributed to an understanding of vascular diiferentiation in the shoot apex of dicotyledons, However, dicotyledons are a large group of flowering plants with great diversity, Carrots and Geum are short stemmed plants and may represent special cases in this group. For this reason it was felt that a wider survey of developmental patterns should be made and two other species in this group have been selected for study. Potato (Solarium tuberosum L. CV. Pontiac) represents a shoot apex of small size and lupin (Lupinus albus L.) has a much larger shoot 217 apex; both have elongate shoots. Initial vascular differentiation has been investigated by earlier workers. In potato, Sussex (1955a) has described a cylinder of meristematic tissue as "residual meristem which is vascular tissue in its initial stage". In lupin, a similar meristematic cylinder, which was blocked out in relation to the differentiation of pith and cortex, was termed residual meristem in order to distinguish it from eumeristematic tissue in the peripheral region of the apical rneristem (O'Neill 1961). Although different terms were used to describe this tissue by Sussex and O'Neill, what they described is comparable to the provascular tissue. In this chapter, the term provascular tissue is used following McArthur and Steeves (1972). Because normal development in these species has been studied by other workers (Sussex 1955a, Snow and Snow 1947, Bal1

1949, O'Neill 1961), the present work will emphasize surgical experiments. 5.2 Material and methods

5.2.1 Material and growth conditions

5 .2.1.1 Potato (Solanum tuberosum L. CV. Pontiac) Rapidly-growing juvenile shoots of potato were obtained by a method described by Sussex (1955a). The tubers of potato were washed and sterilized for 10 minutes in a 5%

Javex solution. Using a 15 mm diameter cork borer, the tuber was cut into small plugs each with an axillary bud. The plugs of tuber tissue were then trimmed basally to a length of 10-20 mm, Cylindrical plugs each bearing a bud were placed on sterilized filter paper moistened with sterilized distilled water in covered glass dishes (Fig. 5.1) in an incubator at 20°C, under fluorescent lights at 25 p mol. m-2 s-1 with a photoperiod of 8 hours day and 16 hours night. Buds after germination for 10 days were 5-10 mm in length. The shoot apices of juvenile buds from potato tubers were used in both the normal developmental study and the experiments .

5 .2.1.2 Lupin (Lupinus albus L. )

Lupin is an annual plant. Plants were grown in a growth chamber from seeds. Seeds were sterilized with 95% ethanol for 1 min followed by a 50% Javex solution for 5 min (a few drops of a surfactant, Tween 80 or Triton X-100 were added to 219 the bleach solution) followed by 24 hours soaking in sterile water. The seedlings were grown in a growth chamber under fluorescent and incandescent lights at 215 - 305 p mol. m-2 s'l with a photoperiod of 8 hours day and 16 hours night.

Temperature was set at 23'~day and 19'~night with relative humidity at 90-95 %. Seedlings 15 days old after gernination were used in both the normal developmental study and the experiments (Fig. 5.2).

5.2.2 Methods

5.2.2.1 Light and scanning electron microscopy Shoot apices of juvenile buds from potato tubers were fixed in FAA and ernbedded in paraffin. Sections were cut at

10 pm thickness and stained with safranin and fast green without removal of paraffin (Ma et al. 1993) . In some cases, sections were stained after removal of paraffin by the safranin-tannic acid-iron alun method (Sharman 1943). Shoot apices of 15 days old lupin plants were fixed in

FAA, embedded in paraffin, cut at 10 prn thickness and stained with safranin and fast green (Ma et al. 1993) or toluidine blue O (Sakai 1973) without rernoval of paraffin. The staining rnethod using safranin-tannic acid-iron alum after removal of paraffin (Sharman 1943) also was used in some cases. Some of the shoot apices were fixed in glutaraldehyde 220

and osmium tetroxide and ernbedded in plastic resin (Berlyn

and Miksche 1976), cut at 1 p thickness with a glass knife on a MT2 microtome (Reichert-Jung), affixed to glass microscope slides and stained with 1% toluidine blue O in 1% borax solution (Trump et al. 1961) for observation by light microscopy . For scanning electron rnicroscopy (SEM), the samples were fixed in glutaraldehyde, dehydrated in an acetone series to

100%, critical point dried, coated with gold and observed under SEM. Photographs were taken on polaroid type 665 positive/negative black and white instant pack film. Al1 printing used Kodak polycontrast RC paper or Polycontrast III

RC paper for enlarging photographs,

5.2.2.2 Histochemistry The esterase test has been described in detail in

Chapter 2. IU1 material described in esterase tests in this chapter was fresh frozen sectioned. The results were interpreted based on the conclusion from inhibitor tests which were carried out in Chapter 2.

5.2.2.3 Surgical operations

The surgical methods used in Geum (McArthur and Steeves

1972) and carrot were followed. The manipulations including isolation of the shoot apex and puncturing of leaf primordia were carried out under a dissecting microscope (Wild M5 stereo), illuminated by a cool light lamp (Fibre optic illuminator, Cambridge instruments).

5.2.2.3.1 Experiments in potato The operations in the present experiment are different from that used by Sussex, but there is much in his general method that can be used for reference (Sussex 1955b). Buds

of potato after germination for 10 days, about 5 mm in

length, were selected. The shoot apex was exposed by rernoving outer scaly leaves until the last two or three

youngest leaf primordia were clearly visible on the apical dome (Sussex 1955b). During this process, some fluid escapes

from the wounded leaf bases. In order to minirnize the

wounding effect on the shoot apex, it was usual to allow a recovery of one day before further surgical operation such as isolation or puncturing was carried out. The method of isolation was similar to that described by

Sussex (1955b). The isolation operation was carried out by fine scalpels made from fragments of stainless steel razor blades. Three or four vertical cuts, about 200 pn deep into the pith, made a triangle or square enclosing the apical dome. Vertical cutting was just inside the bases of the 222 youngest existing leaf primordia. Puncturing of leaf primordia was carried out after plants had recovered from either the removal of leaf primordia or the isolation. Under the dissecting microscope, a fine needle was used to pierce the site on the apical meristem where the next primordim was expected to arise or the buttress was arising. After the operation, the shoot was covered with a water- rnoistened filter paper cap to maintain a high humidity around the apices and placed on filter paper in a covered glass dish (Fig. 5.1). Dishes containing experimental shoots were first left in a cool part of the laboratory for a time (Sussex 1955b), then returned to the incribator and maintained in the conditions previously described.

Two experiments were carried out in potato. In one experirnent, the shoot apices were isolated and newly emerged leaf primordia were punctured. The apices were fixed in FAA at the following intervals after isolation: 1) immediately;

2) 1 hour, 3) 2 hours; 4) 1 day; 5) 2 days; 6 4 days; 7) 21 days. In the other experiment, new leaf primordia were punctured without any prior isolation- The shoot apices were fixed after 3 weeks. Surgical experiments, especially isolation were difficult. In order to obtain enough samples, experiments were repeated with one hundred and sixteen plants, of which sixty seven shoot apices survived and were fixed. For general 223 histological study the same methods were used as described above.

5.2.2.3.2 Experiments in lupin Preparation of the shoot apex of lupin is different from that of potato because lupin has a long shoot with larger leaves. In 15 day old seedlings, older leaves were first folded down. The shoot apex was exposed by removal of leaf primordia until the last two or three youngest ones were visible. After one day of recovery, the surgical operations began. The rnethod of surgical operation has been described in detail in Chapter 3 and above. Three or four vertical cuts were made about 200 ysn to 500 p deep downwards into the pith to separate the large apical dome from the youngest leaf primordia (Bal1 1952). Puncturing was carried out in combination with prior isolation (McArthur and Steeves 1972) or without (Young 1954). Under a dissecting microscope using a fine needle, successive leaf prirnordia could be punctured as or just before they appeared.

After surgical operation, the shoot apex was covered by a water-moistened cotton wool cap (Fig. 5.2) . The folded leaves were then pushed back and in some cases their petioles were tied together to hold the cotton cover. The experimen- 224 ta1 plants were exposed to air and could not be covered. After operation, they were returned to the growth chamber with the same conditions. Both isolation plus puncturing and puncturing only experiments lasted from one to three weeks and were repeated at least twice. The treated shoot apices were fixed in FAA and examined under a light microscope. In experiments with lupin, seventy eight plants have been treated and sixty three surviving shoot apices were obtained from the experiments. For general histological and histochemical studies the same methods were used as described above. 5.3 Results

5-3.1 Potato (Solarium tuberosum L. CV- Pontiac)

5.3.1.1 Normal developniant in the shoot apex

The swollen potato tuber is a subterranean shoot, the

leaves of which are reduced to srna11 scales. When ger-

minated, several vegetative buds emerge from the axil of each leaf scale. The leaves on a tuber bud shoot are small,

spirally arranged with a short petiole, Each leaf has three leaf traces connected to the stem vascular system. The shoot

apices of juvenile buds from potato tubers have been extensively studied (Sussex 195Sa) . In the present study, no attempt was made to describe every detail of the shoot apex, but emphasis was be put on establishing if there is provascular tissue and if so, where it is located as a basis for further experiments.

5.3.1.1.1 Organization of the shoot apex

The shoot apex of potato is smaller than that of carrot, the diameter of the apical dome is in the range of 50 to 100 pm at the level of the axil of the youngest leaf prirnordium. A layered pattern of the apical meristem is distinct in longitudinal sectional view at both the early (Fig. 5.3) and late plastochron (Fig. 5.4) . The tunica in the potato variety Kerr's Pink was considered to consist of two layers 226 (Sussex 195Sa) . In the present variety, there are two layers of cells which show a higher frequency of anticlinal divisions and the cells in the surface layer are not very different from those of the second layer (Figs. 5.3, 5.4) . The corpus comprises a group of relatively smaller cells with

no predominate division plane (Figs. 5 .3, 5 .4) . The zonation organization of the apical meristem is not distinct in the early plastochron (Fig. 5.3) but can be recognized in the late plastochron (Fig. 5.4) . The cells of the central zone are slightly less densely stained than the those in the peripheral zone and are approximately isodiametric in shape (Fig. 5.4) . The cells in the peripheral zone are more or less rectangular but more darkly stained (Fig.

5.4). Rib meristem is located under the central zone and its cells are horizontally rectangular in shape because the transverse divisions predominate (Fig. 5.4).

5.3.1.1.2 Development in the shoot apex The sequence of development in the shoot apex was traced in serial transverse sections of 10 p thickness. As in carrot, the uppermost section showing the tip of the apex will be used as a reference point in a vertical scale measured from the tip of the shoot dome downward toward the root. Examination of serial transverse sections of the shoot 227 apex shows that the distance from the axil of the youngest leaf primordium to the summit of the apical dome is in the range of 10 to 50 p depending on the plastochron stage. The following description is based on a representative shoot apex on which the youngest leaf primordium was less than 10 pm in height. The first section shows that the top portion of the apical dome is enclosed by three older leaf primordia (Fig, 5. 5) - The buttress of the youngest leaf primordium P, has formed and will be shown to be attached to the dome in the next section, facing the adaxial side of the P,. From the summit of the apical dome to the level of 50 pm, the cells in the peripheral zone of the apical meristem are generally strongly stained while the central zone cells show lighter staining .

At the level of 60 p, the bases of P, and P, have attached to the apex (Fig, 5.6) . Because pith and cortex have begun to differentiate at this level, a ring of rneris- tematic tissue is blocked out in the centre of the transverse section (Fig. 5.6). This provascular ring is about 100 pm in diameter. Because at this stage P, is only a buttress of less than 10 pm in height, the axial vascular bundle which is positionally related to the P, could not be easily identified in the provascular tissue. Therefore, the whole provascular ring is continuous (Fig. 5.6). 228 At the level of 70 p, the provascular ring becomes more distinct because of development of pith and cortex (Fig.

5-7). Because the leaf traces of P, and P, were observed

outside of the ring (Fig. 5-71, the provascular ring is above the divergence points of leaf traces of P, and P, The provascular tissue at the location facing the leaf trace of

P, has been reduced because the leaf trace is diverging from

the provascular ring (Fig. 5.7) . At the level of 80 p, the provascular ring was interrupted by the divergence points of

the leaf traces of P, and P,. At the 90 level, the provas-

cular ring includes the two axial vascular bundles which are connected to the leaf traces of P, or P, (Fig. 5.8) . At this level the ring becomes larger and consists of provascular tissue and procambium.

pppppppp----

5.3.1.1.3 Esterase activity in the shoot apex

In most respects, the pattern of esterase distribution

in the shoot apices of potato is similar to that in the

apices of carrot. No enzyme reaction was shown if unfixed frozen sections were incubated in the absence of naphthol

AS-D acetate. In full medium incubation, heavy esterase activity could be identified in most of the vascular tissue including provascular tissue. As in carrot,in longitudinal sections, a heavy reaction was observed in the central part 229 of the stem which includes provascular tissue and developing pith (Fig. 5.9). Unlike carrot, in some longitudinal sections, esterase positive cells were found to extend from the provascular tissue through the cells of the peripheral zone of the apex to the surface layer (Fig. 5.9). These cells were not different from other cells in the peripheral zone in size or shape. The blue colour was not so dark as in the procambium, but it was very distinct from that in the surrounding cells. The stained tissue was also continuous with provascular tissue underneath, The continuity with provascular tissue suggests that these cells would form the further provascular tissue as the shoot apex grows. A similar observation was reported in two other species, Pisum satiwm and Vicia faba, by Gahan and Bellani (1984).

5.3.1.2 Surgical oporation experiments

5.3.1.2.1 Isolation and puncturing Initial observation showed that experimental apices fixed at different intervals following the operation could be described in three groups based on sirnilar results, Group 1 included those fixed immediately after isolation and one and two hours after isolation. Group II included those fixed one 230 day, two days and four days after isolation. Group III included treatments of twenty one days.

5.3.1.2.1.1 Group 1

The shoot apices in Group 1 showed that the isolated

portion, either triangular or square, had a diameter of 60 to

100 p. The cuts were about 140 pm deep and leaf traces were separated from the shoot apex. Examination of the isolated

shoot apices in Group 1, however, showed that there was some provascular tissue left in the isolated shoot apex because provascular tissue is close to the apical meristem.

5.3.1.2.1.2 Group II

Shoot apices of Group II showed the early reaction of shoot apices to the isolation. One and two days after isolation, there was no significant change in the isolated shoot apex. The isolated apical meristem seemed not to be active in growth. From the tip to the level of 70 - 100 pm, pith and cortex differentiation were not evident. In the normal shoot apex, the first appearance of a provascular ring was at the level of 60 pm below the summit of the shoot apex. In isolation treated shoot apices, the provascular ring appeared at the level of 80 to 110 pm. The pith plug consisted of large pith cells and no vascular tissue was 231 observed in it in the one to two day treated shoot. In the four day treated apices, no vertical growth of the isolated shoot apex was evident but the appearance of the first new

leaf primordium (1,) indicated that the apical meristem was active. The apical meristem became irregular in shape because of the puncture of leaf primordia. An evident change in the pith plug was the occurrence of periclinal ce11 divisions which parallelled the cut surface and formed a protective sheath. Provascular tissue dif ferentiation had extended downward into the pith plug. The extension was suspended in the pith plug and no connection between the provascular tissue and the original mature vascular system was observed.

5.3.1.2.1.3 Group III

In Group III, there was initiation of new leaf primordia on the isolated shoot apices during the first and second weeks . With continuous puncturing, however, only two or rarely three leaf primordia were punctured and after two weeks the isolated apex became smaller. After 21 days of experimental treatrnent, pith dif ferentiation in the shoot apex was still delayed. Because the pith was not well developed, the distalmost provascular tissue and rib meristem were not distinguishable and they had the form of a disk in 232 transverse section. This solid core could be traced down to

the 110 pm level (Fig, 5.10). Then pith formation delimited the provascular tissue as a ring in transverse section. At

the 120 to 130 pm level, the provascular tissue was observed in a ring-like configuration under the isolated shoot apex (Fig. 5.11) . In the normal shoot apex, at this level protophloem has been formed and even protoxylem could be observed. In contrast to the normal development, no mature vascular tissue was found in this ring.

5.3.1.2.2 Puncturing wi thout isolation

Because the shoot apex is small, isolation is very difficult and the rate of survival is low. Therefore, in another experiment, after the initial removal of leaf primordia, the treatment consisted only of continuous puncturing of leaf primordia without isolation. The basic pattern of vascular tissue differentiation was not different from that in the isolation and puncturing experiments. The shoot dome became smaller after three weeks of puncturing. Pith differentiation was noticeably affected.

From the 60 pm level to the level of 100 pm, a group of cells in the central area were strongly stained by safranin (Fig. 5.12). This central disk represented the provascular tissue and rib meristem. At the level of 110 pm, because of the 233 appearance of pith, the disk became ring like (Fig. 5-13) This may be compared with the normal shoot apex where the provascular ring appears at the level of 60 m. Because there was no isolation, the cortex was evident and expanded laterally, However, pith expansion was not evident. In the

normal shoot apex, a continuous provascular ring was only observed in one to two transverse sections at the level of 50

- 60 prn. In contrast to the normal, the continuous provascular ring in experimental plants could be traced dom to the

level of 160 pm where a mature vascular system has formed in normal plants, The problem with the puncturing only treatment is that it is not certain which part of the tissue was produced prior the treatment, However, compared to the normal shoot apex in which at the 110 p level mature vascular tissue was observed, the results of surgical experiments clearly demonstrated that provascular differentiation did not advance to the maturation stage when leaf influence was suppressed, 5.3.2 Lupin (Lupinus albus L. )

5.3.2.1 Normal development in the shoot apex Lupin is an annual plant with a height of 0.5 - 1 m at maturity. The shoot apex is surrounded by helically arranged young leaves and leaf primordia at the tip of the stem (Fig. 5.14) . Three leaf traces diverge from the stem and enter each leaf, The normal development of the shoot apex of lupin has been studied by several workers (Snow and Snow 1947, Ball 1949, Young 1954, O'Neill 1961). The present study will provide only essential details with emphasis on the early development of vascular tissue.

5.3.2.1.1 Organization of the shoot apex

The vegetative shoot apex of lupin is larger than that of carrot. Its apical dome has a convex shape and is ca 220 pn in diameter (Fig. S. 15) . The general organization of the shoot apex of lupin has been described as consisting of tunica and corpus (Ball 1949). The present work based on longitudinal paraffin sections confirmed Ball's observation

(Fig. 5.16). In median longitudinal plastic section through the shoot apex, the outermost layers of the apical dome are characterized by a predominantly anticlinal division plane and only a few periclinal divisions were observed in the second layer of the tunica (Fig. 5.17) . Below the two tunica 235 layers, cells of the corpus are not regularly arranged because ce11 divisions occur in al1 planes (Fig. 5.17). Corpus and tunica together constitute the apical meristem which gives rise to al1 tissues of the stem and haves. The epidermis is derived from the first or outer tunica layer (Fig. 5.16). Other tissues are derived from the second layer of tunica and cells of the corpus (Fig. 5.16) . Under the terminal meristem, the rib meristem region was characterized by cells arranged in files contributing cells to the pith below (Fig. 5.16) . The tunica-corpus and zonation organizations are not mutually exclusive in the shoot apices of carrot (see Chapter

2) .- However, cytohistologicaI zonation is diff icult to recognize to the shoot apex of lupin. Cells in the terminal meristem appear uniform histologically and zonation was not observed when sections were stained by toluidine blue O, safranin-tannic acid-iron alun or safranin-fast green (Fig.

5.16). Based the ratio of L/B (greatest dimension / least dimension of a cell) , Young (1954) concluded that there is only one ce11 type in the apical meristem tissue from the summit to the level of 40-50 pm and Bal1 (1949) also found no evidence of structural differentiation. 5.3.2.1.2 Development in the shoot apex The process of development in the shoot apex related to leaf primordial occurrence was traced in serial transverse sections of 10 prn thickness. As in carrot, the uppermost section showing the tip of the apex will be used as a reference point. Examination of serial transverse sections of the shoot apex showed that a new leaf primordium was initiated at a position close to the summit of the apical dome. With growth of the shoot apex, the riew leaf primordium is displaced downward from the summit to the peripheral region. The minimum vertical distance from the axil of the youngest leaf primordium to the extreme summit of the shoot apex was

20 - 30 pm. The maximum vertical distance was 40 pm below the summit of the shoot apex. Above this level the next leaf primordium had not formed or was not large enough to be recognized. The following description is based on a representative shoot apex on which the youngest leaf primordium was at the level of 40 p. The first three sections through the tip of the apex showed a portion of the apical dome and the surrounding young leaf primordia. No leaf primordium was observed to be attached to the dome. The axil of P, was observed at the 40 pm level. Here the youngest visible leaf prirnordium, Pl, was large and easily identified but no leaf trace could 237 be observed in it (Fig. 5.18). From the summit of the apical dome to the 80 p level, the cells in the apical dome were strongly stained by safranin - fast green and no evidence of histological or morphological differentiation could be found

(e.g. Figs- 5-19, 5.20).

The earliest differentiation was found at the 90 pm level where pith differentiation was indicated by vacuolation in the cells of the centre of the shoot apex (Fig. 5.21) Since the centre was occupied by the developing pith cells, a meristematic ring could be observed in transverse sections. However, the ring at this level was the precursor of both cortex and provascular tissue (Fig. 5.21).

The axil of P, was below the 60 p level and the leaf trace of P, had just started to develop (Fig. 5.19). The axil of P, was at the level of 120 pn (Fig. 5.22). The leaf trace of P, was not observed above this level, but could be identified as a group of darkly stained cells surrounded by young cortex cells at the base of the P, in this section (Fig. 5.22) . Meanwhile, pith and cortex became more evident and a provascular ring could be identified above the divergence points of the leaf traces of P, and P, (Fig. 5-22). The differentiation of the cortex was more evident at the 140 ~;imlevel, The divergence point of the leaf trace of

P, was at this level too because in the next section it would 238 be replaced by an axial vascular bundle. A ring of provascular tissue could be recognized clearly at the 150 pm level

(Fig. 5.23). This provascular ring was about 220 pm in diameter and the pith was about 125 pm in diameter. At this level, the axial vascular bundle which serves Pl had not become distinctly procambial. Therefore, the whole provascular ring was continuous.

5.3.2.1.3 Esterases in the shoot apex A simple cytochemical study was carried out on this species in the 19SOfs (Ball 1949, Young 1954) . This method measures oxygen release from hydrogen peroxide to determine the catalase activity of a tissue. Young found that the tissues surrounding the provascular tissue could cause the hydrogen peroxide to evolve oxygen but neither provascular tissue nor apical meristem could- Ball (1949) had obtained a similar result. These results suggest that the provascular tissue and apical meristem have similar features. Esterase activity in the shoot apex of this species has been not studied before. In the present work, no enzyme reaction was shown in any tissue if the incubation medium lacked naphthol AS-D acetate or fresh sections were treated by boiling water. In contrast to this, when unheated fresh 239 sections were put in a full incubation medium, the es terase reaction could be seen in a few minutes.

As in carrot, in longitudinal sections of the shoot apex of lupin, a heavy esterase reaction was easily observed in the vascular tissue. The esterase reaction occurred in axial vascular bundles and acropetally along a leaf trace extending to a leaf primordium (Fig. 5.24). Unlike carrot in which esterase activity was present in the provascular ring and in the developing pith, in lupin it was in the provascular tissue of leaf traces and axial vascular bundles but no evident esterase reaction was observed in provascular ring and the rib meristem region. As reported in the general histological description above the level of 90 pm, the apical dorne of lupin has not differentiated. The esterase reaction supported this observation. No reaction was observed in the apical meristem. In this aspect, it is similar to carrot. However, the most distinct difference is observed below the dome. Unlike carrot in which the provascular ring shows high reaction, the ring in lupin does not. A section through the axif of P,, which is at or above the divergence of the trace of P, showed several discrete sites with high esterase reaction but no clear ring with high esterase reaction (Fig. 5.25). Two of these heavy reaction sites were identified as leaf traces of 240 P, and P,. One is an axial vascular bundle positionally related to the P,.

5.3.2.2 Surgical operation experiments A number of surgical studies on the shoot apex of this species have been done by earlier workers (e.g. Snow and Snow 1947; Bal1 1950, 1952, 1955; Young 1954) . These include isolation or splitting of the shoot apex and removal of leaf primordia without the isolation of the shoot apex. The present study included the isolation plus puncturing and puncturing leaf primordia only. In addition to general histology, esterase activity was tested in surgically treated shoot apices.

5.3.2.2.1 Isolation plus Puncturing

Generally, the result obtained in isolation plus puncturing treated shoot apices was similar to that in carrot. After the isolation treatment, the apical dome was seated on the tip of an isolated shoot apex (Fig. 5.26). The apical dome was bright green in colour while the pith plug was scarred. As the experiment continued, the apical dome also became scarred because of puncturing and desiccation. Although in the shoot of lupin elongation in normal development is very evident, the vertical growth was seriously 241 reduced by surgical treatment - During the experimental period, only three or four leaf primordia were punctured. After that, the unscarred part of the isolated apical dome became very small and no further puncturing could be done without damage to the shoot apex. No mature vascular tissue could be identified in either the isolated shoot apex or the pith plug after three weeks of treatment. In transverse sections of 3 week treated shoot apices, the apical meristem still maintained meristematic status above the level of the last punctured site (12 or IJ . A provascular ring in transverse section was observed at the level of the first punctured site (1,) (Fig. 5.27). In sections of the pith plug, a provascular ring extension also was observed. The cells in the provascular ring or provascular extension were densely stained and less vacuolated than surrounding cells. At first, the provascular extension was suspended in the pith plug, because in sections closer to the base of the pith plug, no provascular tissue could be found. After three weeks or more, the connection between the extension and the original vascular system could be established. 5.3 - 2 2.2 Esterases in treated shoot apices In the normal shoot apex of lupin, as described above, esterases were predominantly present in axial vascular bundles and leaf traces. However, no esterase activity in a provascular tissue ring in the axis was clearly demonstrated. This indicates that the distribution of esterases in the normal shoot apex of lupin is different from that in carrot. However, when esterase activity was tested in surgically treated shoot apices, the results were similar to surgically treated shoot apices of carrot, The tests showed that after treatment, the provascular ring in lupin demonstrated a high esterase activity.

In isolation plus purxturing treated shoot apices, al1 presumed provascular tissue whether in the new growth of the isolated shoot apex or in the pith plug showed a positive reaction in the esterase test- In 3-week treated plants, a near rnedian longitudinal section of a treated shoot apex shows that esterase activity occurred in the provascular region and the reaction became weaker as provascular tissue extended acropetally in the apical meristem of the isolated shoot apex (Fig. 5.28) . Although the cells in the provascular tissue region had not advanced structurally beyond the provascular stage, the active esterase reaction indicates that vascular differentiation was progressing. In transverse 243 view, a provascular tissue ring in the isolated shoot apex could be demonstrated by the esterase reaction (Fig. 5-29), In contrast, no positive esterase reaction was observed in the surxounding tissue, either pith or cortex,

5.3.2.2 .3 Puncturing without isolation Young (1954) found that after three weeks of removal of leaf primordia in lupin, a meristematic ring persisted dom to a level at which phloem and xylem elements could be observed in the controls. In the present work, after 3 weeks of treatment, provascular tissue cells were distinct from surrounding cells because pith and cortex cells became vacuolate. The provascular tissue was distinct because of dense cytoplasm and lack of central vacuoles (Fig. 5.30) . The ring could be traced down to the 500 pm level (Fig.

5.31)- Because there was no isolation of the shoot apex from surrounding tissues, the procambium which was previously present was continuous with newly formed provascular tissue.

Below the level of 600 p the ring was interrupted by some gaps and some procambial bundles were well developed, These bundles were formed before the experiment. However, their developrnent was arrested when the leaf primordia were removed. At the same level, normal plants have developed discrete vascular bundles separated by parenchyma. 244 A similar esterase test result was obtained from puncturing only treated shoot apices of lupin. Esterase tests showed a provascular ring with positive reaction in shoot apices with leaf primordia suppressed. This suggests that esterase activity in provascular tissue of defoliated shoot apices became stronger than in normal plants upon suppression of the leaf primordia. 5.4 Discussion

5.4.1 Development of provascular tissue

General conclusions are often drawn from experimental s tudies of a particularly favourable species but, wherever possible, t is desirable to confirm the results in other species. Potato, lupin and carrot represent herbaceous species in three diiferent orders of dicotyledons. There are differences and similarities in morphology and development among them. Unlike the carrot which has a very short stem, the shoots of potato and lupin elongate during development. The shoot apex of potato is smaller than that of carrot, while that of lupin is larger. The organization of the shoot apex of carrot showed both a layered pattern and zonation.

In potato, the layered pattern is distinct (Sussex 1955a) and a zonation organization can also be recognized in the late stage of a plastochron. Lupin shoot apices showed a layered pattern (Bal1 1949) but no cytohistological zonation organization could be identified (Young 1954) . In spite of differences in the organization of the apical dome, development in the shoot apices is very similar in terrns of the initiation of leaf primordia and the differentiation of provascular tissue. As in carrot, the initiation of leaf primordia near the summit of the apical dome and their displacement to the periphery (Sussex 1955a, Young 1954) 246 results in a periodic change in the form of the apical dome during each plastochron. In the early stage of vascular differentiation, both potato and lupin show a pattern similar to that in carrot. As demonstrated in the previous chapters, provascular tissue is clearly present in the shoot apices of carrot. The present work affinned the existence of a provascular stage of differentiation in the shoot apices of the potato and lupin. This provascular tissue is comparable to the residual meristem described in the shoot apices of potato and lupin by Sussex (1955a) and O'Neill (1961) respectively. This initial vascular differentiation occurs very close to the peripheral zone or region of the apical meristem. Because the peripheral zone or region is the precursor of the provascular tissue and cortex, it cannot be considered to be provascular tissue. Since there is periodic change in the development of the shoot apex, the position of the first appearance of the provascular ring varies during the plastochron. The presence of initial vascular tissue in lupin was questioned by Bal1 (1949), because he did not find a ring- like tissue region above the youngest leaf primordium. There appear to be only a few angiosperms in which this initial phase of differentiation (usually designated residual meristem) cm be observed above the level of the youngest primordium (Clowes 1961). However, in the present study it 247 has been found that a ring of provascular tissue can be observed constantly above the divergence point of the youngest leaf trace. If, as seems reasonable, the influence of a leaf primordium on vascular differentiation is associated with its differentiating trace, the presence of provascular tissue above the level at which that trace joins the stele is significant. It supports the interpretation that initial vascular differentiation does not depend on the leaf influence. The fact that a provascular ring cannot be observed above the axil of the first leaf primordium in angiosperrns may reflect a significant dif ference from the ferns in which the provascular tissue is directly under the promeristem and above the level of the youngest visible leaf primordium (Wardlaw 1944a, 1949b; Ma and Steeves 1994, 1995a).

5.4 .2 Histochemical evidence

At the present tirne, knowledge of esterase activity is based largely on animal tissues and only limited studies have been carried out with plants material. Nevertheless the use of carboxylesterase as a marker for vascular tissue has becorne well established (Gahan and Bellani 1984, Ma and Steeves 199Sa). Studies in carrot used this enzyme to delimit the initial stage of vascular differentiation and 248 showed that provascular tissue could be identified in the shoot apex above the divergence of the trace of the youngest leaf. Comparable but more limited examination of potato and lupin, while showing a close association of esterase activity with vascular tissue, gave variable results, in relation to provascular tissue. Histochemical tests for esterase activity in the shoot apex of potato showed (Gahan and Bellani 1984, Mueller 1995) that, as in carrot, activity was not limited to provascular tissue, but also appeared in other actively differentiating tissues including rib meristem and developing pith. In some shoot apices, an extension of esterase activity was observed in the peripheral zone of the shoot apical dome where leaf primordia might be initiating. Some cells in the peripheral zone which appeared to represent future leaf traces may have advanced to the provascular stage. A similar result was not found in carrot or lupin, but has been reported in other angiosperms by Gahan and Bellani (1984). They showed that carboxylesterase activity was present in a presumed leaf trace in the shoot apex of Pisum sativum and Vicia faba before any leaf buttress had emerged. This leaf trace probably was at the provascular stage because no cytological difference from other apical meristem cells was observed. 249 Esterase activity in lupin showed a different pattern from those in carrot and potato. Although the esterase reaction was present in the provascular tissue of leaf traces in lupin, the rib meristem and developing pith did not show esterase activity as in carrot and potato. The difference in the ~rovascular tissue ring in the axis was even more striking. Unlike the result in carrot and potato, the provascular tissue ring in the axis of lupin did not show a positive reaction although areas of higher activity representing potential axial bundles were found. This diff erence may have a taxonornic basis because a similar result was reported in two other species of Fabaceae (Gahan and Bellani

1984) . In another species in this family, Trifolium pratense, only discrete bundles in the stem gave a positive reaction (Mueller 1995). These results, including the present work, may reflect the presence of variation in angiospems in the timing of cornmitment ta vascular differentiation.

5 .4 .3 Surgical operations The surgical operations on carrot represent a technique for obtaining considerable information about the development of the shoot apex, including the initiation and development of vascular tissue. The surgical experiments demonstrated 250 conclusively the ability of the apical meristem in carrot to initiate provascular tissue without the participation of developing leaves or the mature vascular system. However the further dif ferentiation and development of vascular tissue needs some factors firom the developing leaves- The present experimental work on potato and lupin further confirms this conclusion based on the carrot study. The suppression of leaf primordia seems not to affect the basic functioning of the shoot apical meristem itself.

Growth of the shoot apex, in spite of limits, was indicated

by the formation of provascular tissue and new leaf pwimor- dia. The control of provascular differentiation by the apical meristem without the intervention of leaves has received strong confirmation from these studies. The response in lupin is of particular interest because the provascular ring in the axis did not give a positive esterase reaction. With suppression of leaf primordia, not only did the provascular ring become clearer as in other species, but in addition it produced a positive reaction for esterase. In the normal shoot apex this reaction was lacking in the ring- This raises interesting questions about the possible role of the leaves in suppressing esterase activity in the stem provascular system and might hold the key to understanding the variation in angiosperm apices, 251 The effect of suppression of leaf primordia on further maturation of vascular tissue was evident, Whether or not there was isolation of the shoot apex, provascular tissue did not undergo further differentiation into mature vascular tissue. Although there are differences in the normal organization of the shoot apices of carrot, potato and lupin, the results after suppression of leaf primordia are sme. One is tempted to suggest that, as in carrot, the influence of the leaves is largely mediated by auxin but this should be tested before final conclusions are drawn.

5.4.4 General conclusion

In spite of differences in the normal shoot apices, the histological, histochemical and surgical studies of potato and lupin confirm the conclusions derived from the carrot. 1. Demonstration of plants and basic apparatus used in

experimen tS.

Figure 5.1 The basic apparatus used in the experiments on

potato. Two tuber plugs with a juvenile shoot are in the covered glass culture dish (left) and two tuber plugs sit in the open glass culture dish on

filter paper (centre) . A needle used in surgery is shown at the right. Arrow: a filter paper cap used to prevent desiccation of the exposed shoot

apex. Scale bar = 19 mm.

Figure 5.2 Seedlings of lupin used in experiments. The

leaf primordia have been removed from shoot apices of the middle two plants and a water moistened

cotton (arrows) was placed on the apices and held

by the petioles of leaves to prevent the loss of

water from the exposed shoot apex. Control plants

are on the far right and left. Scale bar = 42 mm. Fie. 5.1

Fig. 5.2 2. Median longitudinal sections of the shoot apex of potato,

showing the organization of the shoot apex. LM mi crographs . Sections were s tained in safranin-tannic

acid-iron alum after paraffin was removed (Shannan 1943) .

Figure 5.3 The shoot apex at the early stage of plastochron; showing a layered organization. T:

tunica; C: corpus. Scale bar = 26 pm.

Figure 5.4 The shoot apex at the late stage of plastochron;

showing zonation organization. Cz: the central

zone, slightly less stained; Pz : peripheral zone;

RM: rib meristem. Scale bar = 26 W. Fig. 5.3 3. Transverse sections of the shoot apex of potato. LM

micrographs, Sections were stained in safranin-tamic

acid-iron al um af ter paraffin was removed (Shaman

1943).

Figure 5.5 The first section through the sdtof a shoot

apex; A: a part of apical meristem; 1-5: Pl to P5.

Scale bar = 60 W.

Figure 5.6 A section at the level of 60 from the apical

summit showing the first appearance of the

provascular ring. pvt : provascular tissue; 2-5 : P2 to Pg; arrow: the initiating axial vascular bundle

related to the Pl. Scale bar = 60 m. Fig. 5.5

Fig. 5.6 4. Transverse sections of the growing shoot apex of potato;

LM micrographs , Sections were stained in safranin-

tannic acid-iron alum after paraffin was removed (continued).

Figure 5-7 A section at the level of 70 prn from the apical

summit showing the provascular ring. pvt : provascular tissue; 2-5: Pz to P5; single arrow:

the leaf trace of Pa; double arrows: the leaf trace of P,, Scale bar = 60 m.

Figure 5.8 A section at the level of 90 pm from the apical

s&t showing the leaf traces of Pz and P3 joined to the provascular ring. pvt: provascular tissue;

4 and 5: P, and P5; single arrow: the axial vascular bundle related to the Pz; double arrow:

the axial vascular bundle related to Pj. Scale bar

= 60 m. Fig. 5.7

Fig. 5.8 5. Esterase in the nonnal shoot apex of potato. An Un f ixed

frozen section reacted in full medium at '37OC for I h

for esterase activi ty using AS-D acetate as substrate

and fast blue BE as diazonium salt. The thickness of

the section is 20 m.

Figure 5.9 A median longitudinal frozen section showing the

distribution of esterase activity in the shoot apex. A: apical meristem; LT: leaf trace; LP: leaf primordium; Pi: pith; arrow head: provascular tissue; hollow arrows: positive reaction in the

peripheral zone. Scale bar = 26 p. -

6. Surgi cal experiments (3 weeks) -isolation pl us puncturing.

Sections were stained in safranin and fast green without

prior removal of the paraffin (Ma, Sawhney & Steeves

1993 ) . LM micrographs .

Figure 5.10 A paraf fin embedded transverse section of an

isolated shoot apex of potato, 110 pxn from the apical summit, showing provascular tissue in the new growth af ter suppression of leaf primordia . Note the pith has not been well differentiated, therefore, provascular tissue and rib meris tem fonn a disc in the centre of the shoot. pvt:

provascular tissue; Rm: undeveloped rib meristem;

Scale bar = 26 pxn.

Figure 5 11 A paraffin embedded transverse section of an

isolated shoot apex of potato, 130 pxn from the apical summit, showing a provascular tissue ring in the pith plug after suppression of leaf primordia. pvt: provascular tissue; Pi: pith.

Scale bar = 26 pxn. Fig. 5.10

Fig. 5.11 7. Surgical experiments in potato - puncturing only.

Sections were stained in safranin and fast green without

prior removal of the paraffin (Ma, Sawhney & Steeves

1993)- LMphotographs.

Figure 5-12 A paraffin embedded transverse section of a

shoot apex of potato, 60 from the apical

sununit, showing provascular tissue after

suppression of leaf primordia. Note the pith has

not been we11 diff erentiated, . theref ore,

provascular tissue and rib meristem form a disc in the centre of shoot. pvt: provascular tissue; Rm:

undeveloped rib meristem. Scale bar = 26 p.

Figure 5.13 A paraffin embedded transverse section of a

shoot apex of potato, 100 pm from the apical summit, showing provascular tissue ring after

suppression of leaf primordia. Pi: pith; pvt:

provascular tissue. Scale bar = 26 W. Fig. 5.12

Fin. 5.13 8. Scaming el ectron micrographs of a dissected vegetative

shoot apex of lupin.

Figure 5.14 The whole view of a dissected vegetative shoot

apex of lupin. A: the dome of the shoot apex; LI

to L3: the first to third leaf primordium; S:

stipule. Scale bar = 88 m.

Figure 5.15 A shoot apex split in a median plane. A:

Apical meristem; L: Leaf primordium. Scale bar =

68 pm. Fig. 5.14

Fig. 9. Organization of shoot apex of lupin.

Figure 5.16 A median longitudinal paraffin-section of the

shoot apex of a seedling of Lupinus albus L.,

stained by safranin-fast green without removal of

paraffin (Ma, Sawhney & Steeves 19931.. Note: The

central zone is slightly less stained. T: Tunica; C: Corpus; RM: Rib meristem; LP: Leaf primordium; Scale bar = 60 m.

Figure 5.17 A median longitudinal section of a vegetative

shoot apex of lupin, showing predominantly anticlinal division plane in the surface layer of tunica, periclinal divisions (hollow arrows) occurring in the second layer of tunica and no predominant division planes in the corpus.

Plastic thin sectioning. Scale bar = 12.5 W. Fig. 5.16

Fig. 5.17 10. Transverse-sections of the shoot apex of lupin; LM

micrographs; sections were stained in safranin and fast

green without prior removal of the paraffin (Ma, Sawhney

& Steeves 1993).

Figure 5.18 A transverse section at the level of 40 pm from

the apical sumit. A: portion of apical meristem;

1-5 : Pl to P5. Scale bar = 60 pm.

Figure 5.19 A transverse section at the level of 60 pm from

the apical summit. A: portion of apical meristem;

1-5: Pl to P5. Scale bar = 60 m. Fig. 5.18 11, Transverse-sections of the shoot apex of 1 upin (continued).

Figure 5.20 A transverse section at the level of 70 pn from

the apical surtunit. 1: the base of Pl which has

attached to the apical meristem; 2-5: Pz to P5.

Scale bar = 60 p.

Figure 5.21 A transverse section at the level of 90 pm from

the apical summit. 1: the base of Pl which has

attached to the apical meristem; 2-5: P2 to P5.

Scale bar = 60 p.m. Fig. 5.20

Fig. 5.21 Transverse-sections of the shoot apex of lupin (continued)

Figure 5.22 A transverse section at the level of 120 pm

frorn the apical summit showing the first appearance of the provascular ring, 2: the base of PZ which has attached to the stem; 3-5: Pj to P5;

Pi : pith; arrow: the leaf trace of Pl. Scale bar

= 60 p.

Figure 5.23 A transverse section at the level, of 150 pxn

from the apical summit showing the provascular

ring. 2 and 3: the bases of P, to P3 which have

attached to the stem; 4 and 5: Pc and P5; Pi: pith;

arrow head: the axial vascular bundle related to

Pl, Scale bar = 60 m. Fig. 5.22

Fig. 5.23 13. Esterase activity in the normal shoot apices of lupin;

Unfixed frozen sections reacted in full medium at 37 OC

for 1 h using AS-D acetate as substrate and fast blue BB

as diazonium salt. The thickness of sections is 20 pm.

Figure 5.24 A median longitudinal frozen section of the

shoot apex of a lupin seedling shows esterase activity in the leaf traces- A: apical meristem; double arrow: provascular tissue; single arrow:

procambium; LP: leaf primordium. Scale bar = 32

Figure 5.25 A transverse section of a frozen shoot apex of

a lupine seedling, 100 ym froa the apical summit, shows esterase activity in the leaf traces. No

evident reaction was observed in the rernaining region of presumed provascular tissue. 1. the

site of axial vascular bundle related to Pl; 2 and 3: the sites of leaf traces of P, and Pj; Pi: pith.

Scale bar = 35 W. Fig. 5.24

Fig. 5.25 14. Surgical experiments- isolation and puncturing.

Figure 5.26 A top view of an isolated shoot apex of which

leaf primordia were punctured for three weeks.

hollow arrow: apical dome. Scale bar = 1 mm.

Figure 5.27 A transverse plastic thin section of the

isolated shoot apex of a lupin seedling, at the

level of the 1, showing a provascular tissue ring (indicated by arrows) after suppression of leaf

primordia. Scale bar = 22.5 pm. Fig. 5-26

Fig. 5.27

I 15. Surgical experiments - isolation and puncturing (continued) - Unfixed frozen sections of the surgical treated shoot apex reacted in full medium at 37 OC for 1

h for esterase activi ty using AS-D acetate as substrate

and fast blue BB as diazonium salt. The thickness of sections is 20 m.

Figure 5.28 A median longitudinal frozen section of the isolated shoot apex of a lupin seedling of which the leaf primordia were suppressed, showing esterase activity in the provascular tissue and its extension as welI as in original vascular

tissue. A: apical meristem; hollow arrows: provascular tissue; E: provascular tissue extension; VT: original vascular tissue. Scale

bar = 106 pm.

Figure 5.29 A traverse frozen section of a surgically treated shoot apex of a lupin seedling, 100 p from the apical sununit, showing esterase activity in the region of provascular tissue and an axial vascular bundle- Hollow arrows indicate a provascular ring. A dark spot in the ring is possibly a developing axial vascular bundle

related to a delayed leaf puncture. Scale bar =

53 pl-

1 6. Surgical experiments-puncturing only. Sections were

stained in safranin and fast green without prior removal

of the paraffin (Ma, Sawhney & Steeves 1993). LM mi crographs .

Figure 5 JO A transverse paraf fin embedded se-ction of the

shoot apex of a lupin seedling, 300 pm from the apical sunanit, showing provascular tissue after

suppression of leaf primordia, Arrows : provascular tissue ring; Pi: pith; hollow arrow: the site of a punctured leaf primordium, Scale bar = 106 m.

Figure 5.31 A transverse paraffin embedded section of the

shoot apex of lupine seedling, 500 pm from the apical smit, showing a continuous provascular tissue ring af ter suppression of leif prirnordia . Arrows: a provascular tissue ring; Pi: pith,

Scale bar = 106 p. Fig. 5.30

Fig. 5131 Chapter 6 GENERAL DISCUSSION

The present investigation was initiated as an attempt to clarify: 1) the respective roles of the shoot apical meristem and developing leaves in controlling the pattern of initial

dif f erentiation of the vascular system and its subsequent maturation in angiosperms and 2) the participation of auxin

in these controlling processes. To this end a series of developmental and experimental studies have been carried out on selected species of dicotyledons. It was also proposed that the results of the investigation would be interpreted as

far as possible in the context of current concepts of stelar evolution. The following discussion will assess the extent to which these studies have clarified the two major question and will consider their evolutionary implications with an emphasis on the similarities and differences between ferns and seed plants.

6.1 Control of primary vascular differentiation

6.1.1 Initial vascular dif ferentiation

As reviewed in the Chapter 1, the generally accepted pattern of primary vascular differentiation based on the 269 morphological evidence is described as pxoceeding from procambium to mature primary vascular tissue (e.g. Esau 1943b, 1954; Steeves and Sussex 1989). An initial or provascular stage preceding procambium has not generally been recognized (Ma and Steeves 1994, 1995a) . This initial stage concept can be traced to the early recognition of incipient vascular tissue in ferns by Wardlaw (1944a) and later extended to seed plants by the same author (Wardlaw 1949a). In a study of vascular differentiation in Geum, this concept

was emphasized by recognition of provascular tissue prior to procambium in this dicotyledonous species (Mmthur and Steeves 1972). More recently, investigations of initial vascular differentiation have progressed in both ferns and angiosperms. In recent studies of shoot apices of the ferns

Mat teuccia and Osmunda, the provascular stage was systematically investigated and further confirmed by a series of histological, experimental and histochernical investigations (Ma and Steeves 1994, 1995a, 199Sb). In other investigations

of primary vascular dif f erentiation in angiosperms, the procambium--mature vascular tissue pattern has also been challenged by evidence from histochemical studies. Based on esterase distribution in the root apex, a multi-step program has been proposed in primary vascular differentiation in the 270 roots of angiosperms (Gahan 1981, Rana and Gahan 1982). According to this hypothesis, the first step in primary vascular differentiation is a general preparing of the cells of the future vascular systern from derivatives of the apical meristem (Rana and Gahan 1982) . This first step is equivalent to the concept of a provascular stage. In a parallel study of shoot apices of angiosperms, the multi-step program in primary vascular differentiation was further elaborated based on carboxylesterase activity (Gahan and Bellani 1984) . In the first step cells are committed to form elements of the stele. This commitment is not a morphological change but a biochemical one (Gahan and Bellani 1984). A coarse programming of the genes involved in providing the basic guide for differentiation into the elements of the vascular tissue may be induced in this stage (Gahan and Bellani 1984). A further program involves individual cells which differentiate in more localized conditions under the control of the cell's immediate environment (Gahan 1981) - The multi-step program hypothesis suggests that determination of the vascular path has occurred in advance of the determination of the leaf primordia (Gahan and Bellani 1984). The first step of this program is equivalent to the provascular stage. The present work strongly supports the presence of an initial stage of primary vascular differentiation in the 271 shoot apex from apical meristem to provascular tissue. In the initial stage, cells derived from the peripheral zone of the apical meristem are merely prepared for their roles in

the primary vascular system. In this stage, although morphological differentiation has not been observed, molecular or biochemical 1eve1 changes occur under the control of the

apical meristem (Gahan and Bellani 1984, Demura and Fukuda 1994) . This differentiation occurs in the axial provascular tissue, which is above the divergence point of the leaf traces and is not related to leaf development. The later stages are from procambium to mature vascular tissue. This further differentiation is obviously under the influence of leaf developrnent. The initial stage has been largely overlooked because noticeable morphological f eatures are absent. However, as described in Chapter 2, the present study has provided evidence of carboxylesterase activity to confirm that initial vascular differentiation takes place in provascular tissue.

Because the diff erentiation is gradual, the differences between the peripheral zone of the apical meristem and the provascular tissue are not very distinct in the normal developmental process. The multi-step programming system was strongly supported by surgical operations. As discussed in Chapter 3, after the 272 suppression of leaf primordia the provascular tissue is persistent in the shoot apex and, in isolation experiments, its presence Is more cleaxly demonstrated because the connection between newly forrned provascular tissue and the original procambium is separated by cutting. In Chapter 5, experiments two other dicotyledons revealed that, while the distinction of provascular tissue normal shoot apices may Vary from species to species, after suppression of leaf development, provascular tissue in al1 investigated species became distinct. The essential conclusion from this phase of the study is that the provascular stage of differentiation establishes the basic cylindrical fom of the vascular system and that this differentiation is under the control of the apical meristem. It is also clear that the apical meristem cannot promote further differentiation beyond the initial stage in the absence of influence from the developing leaves.

6.1.2 Differentiation of the eustelic primary vascular system

The primary vascular system of seed plants is eustelic and the close relationship between the bundle pattern in the stem and the phyllotaxy of the shoot has been noted by many authors (Esau 1943b, Slade 1971, Beck et al. 1982, Kirchoff 1984). The experimental investigation carried out in the present study provided a basis for understanding this 273 relationship in developmental terms. The basic pattern of the stele is blocked out in a cylinder of provascular tissue under the control of the apical meristem. The further differentiation that establishes the final pattern is under the control of the leaf primordia. While it is not possible to associate al1 of the influence of leaf primordia with the auxin that they produce, the important role played by this substance is evident. Tt is reasonable to suggest that the polar flow of auxin (Sachs 1981) transported from a leaf primordium induces the further differentiation of provascular tissue in its pathway. Thus the correlation between phyllotaxy and the eustelic stem vascular pattern is to be expected. Auxin has also been implicated in the mechanism that controls the placement of leaf primordia at the shoot apex in the context of the physiological field theory proposed by

Wardlaw (1949a). When IAA-lanolin was applied to the presum- ptive site of a leaf primordium prior to emergence in lupin, there was an increase in the variability of divergence angles (VarneII and Vasil 1978) . Similarly, triiodobenzoic acid (TIBA), a substance that affects auxin transport, could modify the arrangement of leaf primordia (Schwabe 1971, Liu et al. 1993) . In the present work no evidence of a modifi-

cation of phyllotaxy by the application of IAA to the shoot 274 apex was obtained but it was shown that IAA restored the normal plastochron length where leaf primordia removal had shortened it. A similar observation was mentioned in the fern Matteuccia by Ma (1994),

6.1.3 The hormone determination model

Stein (1993) has proposed a hormone determination model to describe primary vascular differentiation in an axis under the influence of the flux of a single hormone derived from axial and lateral sources. Stein's model is concerned with stelar architectures in major groups of fossil and living vascular plants, but it provides an instructive way to interpret the results of the present study assuming that the single hormone is auxin. The basic feature of this model will be outlined. The shoot apex contains multiple sources of hormone termed organizing centres (OCs), which include the shoot apical dome and any number of lateral branch apices in leafless plants or leaf primordia (Fig, 61, The centre of the apical meristem is the axial OC, The leaf primordia are defined as lateral OCs. Although the Ocs are initiated on the surface of the apical dome, the interaction plane is at a certain distance from the surface. This plane in the shoot apex in which tissue is receptive to the influence of the OCs 275 is the window of opportunity (WO). The WO receives the influence from the OCs and responds to the influence by differentiating primary vascular tissue. The WO may be represented by a short cylinder which starts where the tissue is receptive to the influence of OCs and ends where the tissue irreversibly differentiates according to the hormone signal received. The lateral OCs originate from the centre of the apex and are displaced to the periphery. Each OC exhibits a latent phase at first,

then an active phase of hormone production (Fig. 6.2). The vertical distance from the OCs to the WO is supposed to be constant since, as the shoot grows, the WO moves acropetally.

In transverse view, the imaged OCs are imposed on the WO in a pattern comparable to the phyllotaxy. Because the WO moves acropetally with each plastochron, there is a fixed number of OCs imposed on each WO. Each OC has a concentration gradient in the plane of the receptive tissues. The distance between the axis centre and the OCs is the radius of ontogenetic (phyllotactic) helix. Therefore, the position of each new OC could be determined by the interaction of the existing OCs. This mode1 is a simplified picture of vascular differentiation but it is supported by substantial information. If the window of opportunity is equivalent to provascular tissue 276 and if the central OC (apical meristem) is considered to produce too low a flux of auxin to promote maturation of vascular tissue, the present work supports the interpretation provided by this model.

6.2 Comparison of vascular differentiation in ferns and

angiosperms

It was pointed out in the introduction that the current interpretation of stelar evolution in the vascular plants rejects the long held view that the eustele of the seed plants is derived £rom the dictyostele of the fern type (Beck et al. 1982). The ferns and seed plants are now recognized as separate evolucionary lines and the eustele is believed to have evolved directly from an ancestral protostele. Never- theless the reticulate fern vascular system, like the eustele, closely reflects the phyllotactic pattern of the shoot, suggesting that similar morphogenetic events may occur in the shoot apices of both groups (White and Weidlich 1995). The developmental and experimental analysis of vascular differentiation in several angiosperms in the present study following a similar study in a representative dictyostelic fern (Ma 1994) makes it possible to test this hypothesis and 277 to consider how, in developmental terms, the two vascular sys t ems have evo lved .

6.2.1 Provascular tissue

As mentioned above, the existence of provascular tissue in ferns was proposed by Wardlaw (1344a) and confirmed by Ma and Steeves (1994). Provascular tissue in angiosperms was recognized in Primula by Wardlaw (1949a) and further studied in Geum by McArthur and Steeves (1972). The present study supports the existence of provascular tissue in angiosperms. These studies and the present work have demonstrated that in the initial step of primary vascular differentiation, there is similarity between angiosperms and ferns. Provas- cular tissue is produced by the apical meristem independently of leaves- In ferns, provascular tissue is demonstrated to be subjacent to a one layered promeristem and occurs above the youngest leaf primordia (Wardlaw 1944a, Ma and Steeves 1994). Because of this location, obviously no leaf influence is involved in the formation of the provascular tissue. In angiosperms, provascular tissue is less distinct in the normal shoot apex, but suppression of developing leaf primordia results in a more distinct provascular tissue (Young 1954, McArthur and Steeves 1972)- The present work has demonstrated that the provascular ring is present above 278 the divergence point of the leaf trace of the youngest leaf primordium, Since the leaf influence is probably transported by leaf traces, this observation supports the interpretation that the initiation of provascular tissue is independent from leaf influence (McArthur and Steeves 1972). Although in the initial blocking out of the vascular system the two groups are similar, there are differences in the provascular tissue stage, In ferns the provascular tissue is subjacent to a one layered promeristem (Wardlaw 1944a, Ma and Steeves 1994), while in angiosperms, the provascular tissue is below the peripheral zone of the multi- layered apical meristem above the divergence point of the leaf trace of the youngest leaf primordium, The differences between the two groups are more distinct in the esterase test. In the fern Osmunda, there is strong carboxylesterase activity in the provascular tissue (Ma and

Steeves 1995a), while arnong angiosperms, the activity of the esterases shows some variations. In carrots, there is a relatively strong reaction at the base of the provascular cylinder. In the Pisum and Vicia (Gahan and Bellani 1984) and potato, esterase activity could extend to the peripheral zone. In Trifolium (Mueller 1995) and lupin, this reaction could be demonstrated in the axial bundles and leaf traces but not in the provascular ring. In a hydrogen peroxide 279 test, neither the provascular tissue nor the apical meristem in lupin could cause evolution of oxygen while the adjacent parenchyma cells could (Bal1 1949, Young 1954) . Therefore, provascular tissue in ferns appears to be more advanced as a partly differentiated vascular meristem (Wardlaw 1950, 1952b; Wetmore and Wardlaw 1951; Ma and Steeves 1995a). In angiospems, the differentiation of this tissue shows a range of diversity.

6 -2.2 Control of vascular differentiation Although shoot apices in both ferns and angiosperm plants have provascular tissue, further differentiation is controlled differently and the different organization of vascular tissue in ferns and angiosperms reflects the different control mechanisms. The experimental analysis of

Matteuccia demonstrated the control of the promeristem over cornplete vascular differentiation in ferns (Ma and Steeves

1995b). Once the prestelar system in the form of a tubular provascular tissue cylinder is formed, the general pattern of the future stele has been detemined in ferns. If there are no secondary factors acting upon the prestelar system, such as young leaf primordia, then the entire provascular tissue will be expressed in the mature vascular system without 280 fundamental changes of the configuration (Ma and Steeves l99Sb). Surgical experiments proved that leaf primordia play a di£ ferent role in angiosperms. Without leaves there is no further differentiation and no mature vascular tissue is formed (Young 1954, McArthur and Steeves 1972, Bruck and Paoli110 1984)- The present work supports several previous studies. Although both the fern dictyostele and the an-

giosperm eustele are reticulate patterns that may be correlated with phyllotaxy (White and Weidlich 1995), the relationship to leaves is different. In angiosperms, the leaves control the bundle pathways; in ferns they determine the position of parenchymatous gaps in the vascular system (Ma and Steeves 199Sb). The fern dictyostele is leaf influenced; the angiosperm eustele is leaf dependent. Experiments in the present study in which exogenous auxin was supplied to defoliated apices have confirmed the central role played by this substance in vascular differentiation and have revealed further differences between ferns and angiosperms. There is, however, as was emphasized in Chapter 4, no basis for ruling out the participation in the shoot apex of other factors which have been show to be significant elsewhere- There was evidence that auxin enhanced the development of provascular tissue in carrot shoot apices with leaf development suppressed. This might suggest that 281 auxin £rom the apical meristem is involved in the normal development of provascular tissue but it is more probable that the applied auxin merely advanced the differentiation of this tissue. If the apical meristem is a source of auxin, the supply is clearly inadequate to promote differentiation beyond the provascular stage. Furthemore, the application of auxin in lanolin as a unifom cap over the apex did not change the basic provascular pattern as would be expected if auxin were the controlling factor. The role of auxin in promoting the maturation of vascular tissues in angiosperms is abundantly documented in the literature (Lyndon 1994, Sachs 1991, Fukuda 1992) and clearly, in the present work, the essential role of leaves in advancing differentiation beyond the provascular stage is shown to be largely auxin mediated. Nevertheless, applied awcin, even as a point source, has not resulted in the differentiation of vascular bundles with the characteristic histological pattern of the normal vascular system. This deficiency might be overcome by varying the concentration or the timing of the auxin treatment, but it is equally possible that other factors are involved. In striking contrast to the situation in angiospenns, the apical meristem in ferns appears to produce sufficient auxin to bring about the complete maturation of the provascular tissue that it initiates (Ma and Steeves 1995b) . As in 282 the angiosperms, the possible role of auxin in the initial differentiation of provascular tissue remains unclear. The role of leaves in modifying the differentiation pattern of

the vascular system is, as in angiosperms, largely mediated by auxin and may be replaced by an exogenous source. However, the auxin, either from leaves or exogenous, promotes expansion of the parenchymatous tissue of the pith and the formation of leaf gaps rather than vascular maturation (Ma and Steeves 1992 ) . It has been suggested (Ma and Steeves 1992) that this supplemental auxin supply may create a concentration above the optimum for vascular differentiation, resulting in a different response.

6.2.3 Hypotheeie of shifting auxin source In a study on stelar evolution in Devonian progymnosperms Scheckler (1978) proposed that observed trends could be interpreted as reflecting that the production of auxin in the axial promeristem had been reduced and the axial auxin influence replaced by the influence of lateral appendages. In a study of stelar architecture in a range of fossil plants, Stein (1993) developed a quantitative mode1 to interpret changing patterns in terms of a postulated shift in centres of auxin production in the shoot apex. Although Stein's (1993) study dealt with the evolutionary line that 283 1ed to the seed plants and did not specifically concern ferns, his proposa1 has considerable significance for the present cornparison. Stein postulated that in the original protostelic ancestral forms of both ferns and seed plants of the Devonian Period, the leafless shoot apex was the sole

auxin source. As more advanced forms appeared in the seed plant line with lateral appendage (ultimately leaves) , the main source of auxin shifted to those lateral sources. As a consequence, the protos tele became lobed and ultimately

dissected into strands under the hormonal control of the appendages. The leaf-dependent eustele is the final result of this trend. The demonstration in the present study that al1 differentiation beyond the provascular stage depends upon the leaves whose influence can, at least in part, be replaced by applied awcin, provides strong developmental support for this hypothesis based on an evolutionary analysis.

A parallel but different sequence may be postulated for the ferns. Beginning with the ancestral protostelic form, it may be assumed that the sole auxin source, adequate for full

differentiation, was in the apical meristem. As lateral appendages evolved, they became accessory auxin sources but the role of the central auxin source was not diminished. The hormone from the lateral sources, perhaps because they provided an excess, exerted a different effect, one of pazenchymatization and expansion rather than the maturation 284 of vascular tissue. This interpretation is supported by the experimental studies of Ma and Steeves (1992). Thus, the similar but separate evolution of ferns and seed plants has resulted in steles which, although they show similarities in their relationship to phyllotaxy (White and Weidlich 1995), are significantly different in the developmental process that produce them. Figure 6.1 Parameters of the hormone determination model in polar (transverse view). Hypothetical meristematic tissue is observed during its "window of opportunity" for receiving hormone signals from the shoot apex. A phyllotactic system involving five organizing centres (OCs) at the shoot apex is observed by projection ont0 the surface of the receptive tissue. Phyllotactic parameters include primary divergence angle, a, between successive elernents along the ontogenetic helix, and radius from axis centre for each element, rl, r2, etc. The ratio r2/rl is the plastochron ratio (Richards 1951) held constant in the model, The hormone signal from each OC composes a concentration gradient in the plane of the receptive tissues diagrammed here as a topoqraphic map consisting of a set of concentric hormone concentration contours. (After Stein 1993) .

Figure 6.2 Simplified diagram of the development of a shoot apical meristem, viewed £rom the side, showing terminology employed in the hormone detemination mode1 described in the text. Curxed lines represent surface of the apical dome at successive time intervals, tl - t4. Axis centre is marked by the central vertical line. Lateral branch and / or leaf primordia senring as hormone sources are termed "organizing centresn (OCs). Each OC, produced at the axis centre, becomes displaced toward the periphery of the axis oves successive time intervals and exhibits variable length "latentn,L, and "activen,A, phases of hormone production. Hormone is actively transported to developing tissues below the apex that are receptive to the hormone signal during a short time interval, termed "window of opportunityn at corresponding tirne intervals, Cl - t4. (After Stein 1993).

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