Some endogenous factors affecting root formation on hardwood cuttings of two clones of apple (Malus sylvestris Mill.) rootstocks : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University

Copyright is owned by the Author of the thesis. Permission is given for

a copy to be downloaded by an individual for the purpose of research and

private study only. The thesis may not be reproduced elsewhere without

the permission of the Author.

SOME ENDO GENO US FACTORS AFFECTING ROOT FORMATI ON

ON HARDWOOD CUTTINGS OF TWO CLONES OF APPLE

(

MALUS SYLVESTRIS MILL.₎ ROOTSTOCKS

A The s i s Submi t ted in Partial Ful filment of the Requirements for the Degree of

Doctor of Phi l o s ophy at

Mas sey University

DONALD STUART TUSTIN

1976

ACKNOWLEDGEMENTS

The author woul d l ike t o extend h i s sincere grati tude and apprec ia­

tion to Mr Murray Richards and Dr David Woolley for their c on tinued encouragement and as s i s tance in thei r c apci ty as supervis ors of thi s thesi s.

Thanks are al s o extended to the following people and organi sati ons:

The N . Z . Fru i t growers Federation Nursery and the Levin

Horti cul tural Res earch Stati on for supplying apple roots t ocks.

The As ian Vege table Re search and Devel opment Centre for the gift of mung bean seed.

Me ssrs B. Wil cox , A . Watson and D . Anderson for techni cal assis tance.

Mrs Sara Grant for the typing o f the the s i s.

The Univers i ty Grant s Commit t ee , whose award of a Pos t graduate Scholarship made thi s s tudy pos s ible.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS

ABSTRACT

REVIEW OF LITERATURE 1

A . Introduc t i on. 1

B. Part I . Phys ical Fac tors Affect ing the Propagati on o f

Apple Roo t s tocks from Cut t ings. 2

( 1 ) Phys i o l ogi cal Age of Parent Plant

(2)

Choice of Material from Parent Plant

(3) Seasonal Effec ts and the Time of Taking Cuttings

Part II . Phys ical Treatment s Pri or to Planting.

( 1) Treatment wi th Synthe t i c Growth Substance s

( 2 ) Temperature Treatments Pri o r to Planting

(3) Wounding and Bud Removal as an Aid to Roo t

2

5 7

8 8 1 2

Ini ti ation 14

Part I I I . Phys i cal Treatment during Root Ini tiat i on . 1 6

( 1 ) High Temperature Storage o f Cutt ings 1 6

C . PLANT GROWTH REGULATORS AND THEIR EFFECT ON ADVENTI TIOUS ROOT INITIATION

( 1) Endogenous Auxins ( 2) Gibberellins (3) Cytokinins

(4)

Abs c i sic Acid

( 5 ) Rooting Cofactors

(6) Endogenous Roo ting Inhibi tors

(7)

^Ethylene

18 18 23

27 3 0 3 2 38 4 0

D . METABOLISM OF INDOLEACETI C ACID DURING ADVENTITI OUS ROOT I NITIATION

RATIONALE OF THE PRESENT WORK

MATERIALS AND METHODS A . PLANT MATERIALS

B . COLLECTI ON AND PREPARATI ON OF CUTTING MATERIALS C . TREATMENT AND PLANTING O F CUTTINGS

D . PRODUCTION OF MAIDEN TREES E . RECIPROCAL DONOR GRAFTING

F . SAMPLING OF MATERIAL FOR HORMONE EXTRACTION

G. EXTRACTI ON OF PLANT MATERIAL FOR HORMONE ANALYSIS ( 1 ) Ini tial Extraction

( 2 ) Procedure for Separati on of Auxins , Inhibi tors

and Cytokinins

(3) Extracti on and Separati on of Rooting Cofactors

H . CHRO MATOGRAPHY OF EXTRACTS FOR HORMONE ANALYSI S

( 1 ⁾ Colunm Chromatography of the Acidic Ether Fraction

( 2)

^Colunm Chromatography of the Neutral Ether Fraction (3) Colunm Chromatography of the Butanol-solubl e

Cytokinins

( 4 ) Paper Chromatography of Auxins and Inhib i tors ( 5 ) Paper Chromatography of Roo ting Cofac tors

( 6 ) Paper Chromatography of Butanol-soluble Cyt okinins

I. BIOASSAY PROCEDURES

( 1 ) Tri ticum Coleoptil e Bioas say (2) Avena Coleoptile Bi oassay

(3)

Mung Bean Roo t Initiation Bi oas say

(4)

Radish Cotyledon Expansi on Bioas say

41

44

46 46 46 46 47 48 49 49 49

50

5 1 5 3 5 5 5 6

57 5 8 59 59

59 59 60 62 6 6

J . LANOLIN PASTES

K . RADIOACTIVE CHEMICALS L . RADIOASSAY OF 1 4c

( 1 ) Sc int i llation Cocktail s ( 2 ) 1 4co2 Trapping Sys tem

(3) Preparation of External Medium for Counting ( 4 ) Preparation of Samples for Counting

( 5 ) Treatment of Tissue Re s idue ( 6 ) Counting of Radioactive Samples ( 7 ) �uench Correction

EXPERI MENTAL

SECTION I . SEASONAL CrUlliGES IN ROOT INITIATION , BUD DORMANCY AND HORMONAL STATUS IN MM 1 06 AND EM XII APPLE ROOTSTOCK HARDWOOD CUTTINGS

A . SEAS ONAL CHANGES I N ROOT INITIATION O F HARDWOOD CUTTINGS

Introduct i on and Methods Re sul t s

B. THE RELATI ONSHIP BETWEEN BUD DORMANCY AND ROOT INITIATION

Intro duction and Methods Resul t s

C . SEASONAL CHANGES OF ENDOGENOUS GROWTH REGULATORS OF MM 1 06 AND EM XII APPLE ROOTSTOCKS

Methods Re su l t s

(1 ) Auxins

( 2) Abscisic Acid

Page 6 7 6 9 69 6 9 7 0 7 0 7 1 7 1 7 1 7 2

7 5

7 5 7 5 76

7 9 7 9 80

80 80 81 81 85

(3) Ro o ting Cofac tors

( 4 ) Butanol-s oluble Cyt okinins

Page 86 89

DISCUSS ION 9 5

SECTION I I . DEMONSTRATION AND LOCATION OF A TRANSMI S SIBLE ROOT PROMOTER IN APPLE ROOTSTOCK HARDWOOD CUTTINGS

A . ROOT INITIATION TRIALS OF RECIPROCAL DONOR GRAFTED ROOTSTOCKS

Introduct i on and Methods Re sul ts

B . EXAMINATION OF ENDOGENOUS GROWTH REGULATORS FROM RECIPROCAL DONOR GRAFTED

MM

1 06 and EM XI I APPLE ROOTSTOCKS

Methods Resul ts

( 1 ) Auxins

( 2 ) Abs cisic Acid

(3) Roo ting Cofac t ors

( 4 ) Butanol -soluble Cytokinins

1 07

1 07 1 07 1 07

1 09 1 09 1 09 1 09 1 1 1 1 1 1 1 1 1

DI SCUS SION

116

SECTION I I I . EXAMINATION OF

THE

ROLES OF IAA AND IBA IN ROOT FORMATION OF DIFFICULT AND EASY-TO-ROOT CUTTINGS OF

APPLE

ROOTSTOCKS 1 20

Introduc tion 1 20

A . THE EFFECT OF IAA AND IBA ON ROOT FORMATION WHEN

APPLIED

ALONE OR IN COMBINATION , I .

Methods Resul t s

1 20 1 2 0 1 2 1

B. THE EFFECT OF IAA Al1"D IBA ON ROOT FORMATION WHEN APPLIED ALONE OR IN CO�ffiiNATION, I I .

Introduction Me thods

( 1 ) EM XII Hardwood Cut t ings Res.J.l ts

( 2 ) _MM 1 06 Hardwood Cutt ings Introduction

Me thods Re sul ts

C. THE EFFECT OF VARYING CONC��TRATIONS OF IBA ON ROOT FO ill1ATI ON OF

MM

1 06 AND EM XI I HARDWOOD CUTTINGS

D . THE METABOLISM O F 1 4C-IAA IN HARDWOOD C UTTINGS AND EM XI I APPLE ROOTSTOCKS

Methods Resul t s

( 1 ) ( 2 )

(3)

( 4)

1 4co Evolution 2

External Solut i on s

Alcohol Extrac ts of Cut tings Alcohol Ins olubl e Compounds

DI SCUSSION

SUMMARY AND GENERAL DI SCUSSION

APPENDIX I : Buffer solutions

APPENDIX I I : Summary of s tat i s ti cal analys es

BIBLIOGRAPHY

OF MM 1 06

1 2 5 1 2 5

1 2 5 1 2 5 1 2 5 1 27 1 27 1 2 8 1 28

1 34

1 36 1 36 1 37 1 37 1 39 1 4 3 1 45 1 47

1 57

TABLE OF FI GURES

Fi gure 1

2

Summary o f Extrac tion Pro c edure

Summary of Revi sed Mung Bean Extraction Procedure

3 Standard curve for wheat c o leoptile response to abscisic acid. Standard errors shown.

4 Standard curve for oat col eoptile re s ponse to i ndole-acetic aci d

5 Standard curve for oat col eoptile response to indole­

ac e toni trile

6 Standard curve for radish cotyl edon response to kinetin. Standard errors shown.

7 Co l our quench correcti on curve

8 Seasonal changes in root ini t i ation of hardwood cuttings o f apple rootstocks

9 Seasonal change s of an aci di c gro wth promo ter , the same as or simi l ar to IAA , de termined by the oat coleopti l e bioas say of the acid ether frac tions o f s tem tissue srunples o f apple roots tocks

1 0 Seas onal change s of a neutral gTowth promo ter , the same as o r simil ar to ^{IAN ,} determined by the oat coleopti l e b ioassay o f neutral ether fracti ons of stem t i s sue samples of apple roots tocks

1 1 Seas onal change s of an aci dic growth inhibitor, the same as or s imil ar to ABA , determined by the wheat c ol e optile bi oas s ay o f ac idi c ether frac t i ons of stem ti ssue samples o f appl e roots tocks

1 2 Seas onal changes in an acidi c root i ni ti ation promoter from

MM 1 06 and H1 XII appl e roots tocks , determined by the mung bean b i oas say

52 54

6 1

6 3

6 4

6 8 7 3

77

82

84

87

88

Figure

1 3 H i s tograms of an aqueous promoter from s tem ti ssue samples of MM 1 06 and fl1 XII apple ro ots tocks, determined by the mung bean bioas say.

1 4 Histograms of an aqueous promoter from stem t i s sue sampl es of

MM 1 06 and EM XI I apple roo t s to cks , de termined by the mung bean

b i oas say, minus 1 ppm IAA

1 5 Seas onal changes in butanol-s oluble cytokinins from MM 1 06 and EM XI I appl e rootstocks

1 6 His tograms of IAA-like aci di c growth promoters from stem tissue sampl e s of rec ipro cal donor-grafted apple roots tocks, de termined by the oat coleoptile bioas say

1 7 Histograms of IAN-l ike neutral growth promoters f1om s t em t i s sue sampl es of reciproc al donor-grafted apple rootsto cks, determined by the oat coleoptile bi oas say

1 8 H i s t ograms of ABA-l ike aci dic growth i nhibi tors from stem t i s sue sampl e s of rec iprocal donor-grafted apple rootst o cks, determined by the wheat col eoptile bi oas s ay

1 9 Histograms of an aqueous promoter from stem tissue sampl e s of reciprocal donor-graft e d appl e roo t s tocks, determined by the

mung bean bioas say

2 0 H i s tograms o f a n aque ous promo ter from stem tissu e and centri fu ate of MM 1 06 appl e rootstock

2 1 His tograms o f IAA-like growth promoters from stem tissue and

22

centri fugate of MM 1 06 apple roots tock

Rate curves of 1 -1 4co 2 evolution ^by MM 1 06 and EM XII hard- wood cut tings

90

9 1

93

1 1 0

1 1 2

1 1 3

1 1 4

1 32

1 33

1 38 2 3 Rate curves o f 2-1 4co2 evolution by MM 1 06 and EM XII hard­

wood cuttings 1 40

24 H i s tograms of chromatogram segments o f the 1 -1 4C-IAA external s o luti on

2 5 Hi s to grams o f chromatogram segments o f the 2- 1 4c -IAA exterr-al s oluti on

1 4 1

1 42

Figure 2

6

27

H i stograms of chromatogram segments of the alcoholic extract s o f MM

106

^{and EM}

XII

hardwood cuttings treated with

1-14c-IAA

Histograms of chromatogram segments o f the al coholic extrac t s o f MM

106

^{and EM}

XII

hardwood cut tings treated with 2-14c- IAA

144

146

LIST OF TABLES

Table

1 Mean number of cut tings rooted on successive harve st dates 2 Time in days, to 5o% bud burs t of cut tings of MM 1 06 and

EM XII apple roots tocks

3 Results of roo t ini tiat i on trial s of stock-scion combinati ons of Mrvi 1 06 and ElvJ: XII apple ro otstocks

4 The number of Mrvi 1 06 cut t ings rooted, and the average number o f roo t s per cut ting when treated ei ther sing�y or in

c ombination with IAA or IBA

5 The number of EM XII cut tings rooted, and the average number of roots per cut t ing , when treated e i ther singly or in c omb ination with IAA or IBA

6 Th� number of EM XII cut t ings roo ted and the average number o f roots per cutting, when treated e i ther singly or in c ombination wi th IAA (1.CP/o) and IBA

7 The number of c entrifuged MM 1 06 cut ting·s rooted and the average number of ro ots per cut ting, when treate d ei ther singly or in combinat ion with IAA

(1.CP/o)

^{and IBA}

8 The effect of IBA conc entrati on ^on root initiati on of EM XII apple roots tock hardwood cutt ings

9 The e ffect o f I BA concentration on root ini tiation of

MM

^{1 06} app l e roo tstock hardwoo d cuttings

Page 78

eo

108

1 2 1

1 22

1 26

1 29

134

13 5

LI ST OF PLATES

Plate Page

1 A pho tographi c repre sentation of the effects of IBA and 0.1% IAA in lanolin , applied ei ther separat ely or in

c orr.binat ion , on root formati on of apple roots tock hardwood cuttings

2 A phot ographic repre s entati on of the effects of IBA and 1.0%

IAA in lanolin , applied ei ther separately or in combina­

tion , on root formati on of � 1 06 apple roots tock hardwood cut tings after centrifugati on and base removal

1 2 3

1 3 1