THE RELATTONSHTP OF ’ENDOGENGUS HORMONES
T0 GROWTH CHARACTERISTICS AND T f
' 'DWARFTNG TN MALUS L 7

Thesis for the Degree of Ph. D,
MICHIGAN STATE UNIVERSITY
HENRY ARTHUR. ROBITAILLE '

1970

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thesis entitled

THE RELATIONSHIP OF ENGOGENOUS HORMONES T0

GROWTH CHARACTERISTICS AND DWARFING
presented by

HENRY ARTHUR ROBITAILLE

has been accepted towards fulfillment
of the requirements for

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ABSTRACT

THE RELATIONSHIP OF ENDOGENOUS HORMONES TO

GROWTH CHARACTERISTICS AND DWARFING IN MALUS L.

by Henry Arthur Rohitaille

This research was concerned with the role of certain plant hormones
in the dwarfing phenomenon, including a comparison of dwarf and vigorous
trees. The level of endogenous hormones in xylem sap and shoot extracts,
and effects of exogenous hormone applications were studied.

Top/root ratios were variable for two year old trees on different
rootstocks growing under a constant environment. There was no difference
in the percent moisture of stems, leaves and bark, even though the dwarfing
effects were already evident with certain rootstocks.

Terminal growth measurements on five year old trees, taken every
two to three days over the growing season, indicated that shoots grew at
equal rates, but that shoots on dwarf trees ceased growth earlier than shoots
on vigorous trees.

One year old trees on rootstocks of different vigor responded differ-
entially to injections of 10 ppm of gibberellic acid (6A3). No differential
response was shown to abscisic acid (ABA) injections. A complicating factor
was the summer dormancy in the greenhouse of very dwarf trees after mini-

mal growth.

 

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Henry Arthur Robitaille

ABA reduced the terminal growth of one year old Malling Merton (MM)
111 non—grafted trees in proportion to the concentration injected. One
hundred ppm of ABA was temporarily toxic to the MM 111 trees, but "ter-
minal buds" characteristic of summer dormancy were induced in 'Red Prince
Delicious' trees. GA3 overcame ABA-induced inhibition of terminal growth.

A gibberellin A4 and A7 mixture (GA4+7) was more effective in pro-
moting lateral shoot growth than GA3. GA4+7, unlike GA3,was toxic to the
terminal growing point and caused the growth of lateral buds. GA4+7 was
not toxic to lateral shoot growth.

6-benzy1aminopurine overcame apical dominance and caused axillary
buds to produce spurs and lateral branches but did not alter growth. Indole-
3-acetic acid promoted growth slightly at 1 and 5 ppm, but was very inhibi-
tory to growth when injected in the tree trunk at 10 ppm.

A new technique for collecting non—contaminated bleeding sap was de-
veloped. There were no differences in the level of GA-like substances in
bleeding sap from dwarf and intermediate size trees when using the lettuce
hypocotyl bioassay.

Sap was collected centrifugally from both dwarf and vigorous trees on
seven dates over the growing season. There was a significant difference in

the level of a 0A3 -like substance only on April 30, although the level of this

 

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llenry Arthur Robitaille

gibberellin was always slightly higher in dwarf trees. Levels of an ABA~

like inhibitor were similarly higher in dwarf trees on most sampling dates.
There were no differences in the levels of GA-like substances in

apple shoot extracts. Methanol vs aqueous extraction methods were com-

pared.

THE REI...A'I‘IONSl-IIP OI? ENDOCENOUS IIORMONES TO

GROWTH Cl'hXiiACTERlS'l'ICS AND DWARFINC IN MALUS L.

By

Henry Arthur Robitaille

A T HES IS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR O F PIII LOSOPHY

Department of Horticulture

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AC KNOW LEDGMENTS

The author wishes to express his appreciation and indebtedness
to Dr. Robert F. Carlson who provided the assistantship, advisement.
and encouragement which made this dissertation possible.
Special thanks are expressed to Dr. Frank G. Dennis whose
valuable advice played a major role in the development of the thesis.
Appreciation is also expressed to Drs. David R. Dilley, Derek T.
Lamport, H. Paul Rasmussen, and Harold M. Sell for serving on the
guidance committee. and for their critical review of the manuscript.
Most importantly, the author wishes to give special thanks to his

wife, Phyllis, for her enduring patience, confidence and encouragement.

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TABLE OF CONTENTS

Page
LIST OF TABLES.. .......... O... 0000000000 OOIOOOOOOOOIOOOOOOOOO V
LIST OF FIGURESOOOOOOOO. ooooooooooooooooooooooooooooo o ooooooo Vii
INTRODUCTIONQOIOOOOIO OOOOOOOOOOOOOOOOOO .0... 00000 000.000.... 1
REVIEW OF LITERATURE... ....... . ..... ..... 3
Scion/Rootstock Relationships in Apple . . ........... . ....... ii
General-00000000000000.coo-o ooooooo 000...... oooooooooo 00 3
Rootstock Effects on the Scion....... ..... 3
Scion Effects on the Rootstock................... ..... .. 7
Interstock Effects on the Rootstock and Scion ... ......... 9
Rootstock, Interstock, and Scion Interaction . . . . . . ........ 12
Relationship Between "Stock" Effects on Vegetative vs
ReprOduCtive GrOWt11 OOOOOOOOOOIOOOOOOOOOOOOOOOOOOOOOO l4
By-paSSing the Dwarfing Component in the Tree . . . . . . . . . . . l7
Localization of the "Stock Effect" . . . . . . . . . . . . . . . . . . . . . . . l7
Dwarf and Vigorous Tree Differences . . . . . . . . . . . . . . ...... 19
Explanations of "Stock Effects" ......... ...... 31
Gibberellins, Abscisic Acid and the Apple Tree.... ....... .. 40
MATERIAL AND METHOIE
Moisture Content of Plant Parts and Top/Root Ratios .. . . . 46
Growth Pattern Studies. . . . . . . ...... . . ....... . . . . ........... 46
Injection Experiments . .................................... 46
Experiment 1............ ........ ............. . 50
ExperimentzOOOOOOOOOOOOOOOIOOOOOOOOOOOOOOCOOOOOO0.0.0 5-1
Experimerlt3O'COOOOOOOOIOOOOOOOOOOCOOOOOOOOOOOOOOOO... 51
Experimellt40000000.00.000000000000000... ooooooo 000000 52
Apple Sap: Collection, Purification and Assay . . . . . . . . . . . . . . 53
Bleedingsap000.0000.oo-ooooooooooooooooooooooooooooooo 53

iii

Pa ge

Centrifugal Sap . ....... . ...... . . . . . ..................... 58

Shoot Extracts: Purification and Assay . . ....... . . . . ...... . . 04
Activity of Methanol and Aqueous Extracts. . ..... . . . . . . . . . ()4

GA Activity in Trees of Decreasing Vigor . . . ..... . ...... 68
RESULTS AND DISCUSSION ...... ....... . 70
Moisture Content of Plant Parts, and Top/Root Ratios. . . . . . . . 70
Growth Pattern StudieS...... ......... 72
Injection Experiments .. ...... ..... 79

Differential Response of Dwarf and Vigorous Trees to
GA3 and ABA (Experiment 1) 79
Interaction of ABA With the Apple Tree (Experiment 2

aIld 3.0.00OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO00.0.0000 85
Interaction of GA3 , GA4+7, BA, and [AA With the Apple
Tree (Experimerlt 4). O O O O 0 O I O O O O O O O O O O O O O O O O O O 0 O O O O O O O O 96

Activity of Gibberellin-and Abscisic Acid-Like Substances

m Apple sapooooooooooooooooococo-000000...oooooooooooooo 107
Activity of GA-Like Substances in Bleeding Sap... ..... .... 107
Gibberellin Activity in Centrifugal Sap.................... 110
Inhibitor Activity in Centrifugal Sap ............. ..... 117

Activity of Gibberellin-Like Substances in Apple Shoot Extracts 120

Activity of Methanol and Aqueous Extracts . . . . . . . . . . . . . . . 120

Activity in Trees of Different Vigor . . . . . . . ..... . . . . . . . . . . 124
SUMMARY ...................................................... 128
LITERATURE CITED ............................................ 131

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LIST OF TABLES

Table Page

1. Mean dry weights of roots, stems and leaves, and the
top/root ratios of five randomly selected trees from
each of three rootstock types, with one scion, 'Red

Prince Delicious '. . ................ . . . . . . . . . . . ........ 70
:2. Mean percent moisture of roots, stems and leaves from

five randomly selected trees of 'Red Prince Delicious'

on three rootstock typeS........... ..... ...... .. 71

3. Uptake (ml) of 10 ppm of GA3 and 100 ppm of ABA solu-
tions by one year old 'Red Prince Delicious ' trees on
EM IX, VII and MM 111. Each number equals the mean
of four replications.................................. 82

4. Growth response of one year old 'Red Queen Delicious'/
MM 111 apple trees to ABA in the presence of increasing
concentrations of GA3................ ...... 95

5. Means for total uptake, terminal extension growth and
lateral growth, calculated for the various treatments
mjeCtedOOCCOOOOO....OIOOOOOOOCOOOOOOOOOOO.0.00.00... 97

6. GA3 equivalents (pg) in three fractions extracted from
125 ml of bleeding sap from 'Golden Delicious' trees
on EM IX and on EM VII. Means, calculated from
the total activity on each chromatogram, represent
two replicate trees on each rootstock. . . . . . . . . . . . . . . . . 108

7. A comparison of the Rfs of 6A3, GA4+7, and one of
the centrifugal sap gibberellins in three solvent systems. 111

. The stage of shoot development and levels of a GA3-like
substance in centrifugal sap from 1. 5 g of stem samples
taken from dwarf and vigorous trees on each sampling
datein l969............... ...... 112

 

Table Page

9. The stage of shoot development and the level of an ABA-
like inhibitor in centrifugal sap from 3 g stem sam-
ples, taken from dwarf and vigorous trees on each
sampling date.......... ........... .............. .118

vi

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LIST ()I" I*‘I(}UIIICS

Injection Experiment 2, a typical injection experiment.
MM 111 non-grafted trees were injected with one con-
contration of Alar and seven concentrations of ABA,
using 20 ml disposable syringes separated from the
needles by 10 inch sections of tygon tubing....... . .. .. ..

The procedure for collection of bleeding sap in the
orchard. Sap was frozen from the time it left the
tree to the time of extraction in the laboratory........ ..

Flow sheet illustrating the procedure for extraction of
bleedmg SaPOOOOIO0.0.000...0....OOOOOOOOOOOOOOIOOOOOOO

Flow sheet illustrating the procedure for extraction of
Centrifugal SaPCICIOOOOOOO.O..00...OOOCOOOCCOOOOOCIOOOO

Flow sheet illustrating the procedure for methanol ex-
traction of apple shoots................................

Flow chart illustrating the procedure for aqueous ex-
traCtion Of apple ShOOtSOOOOOOOOOOOOOOOOOOOOOOOOOOOOO...

Mean shoot growth of vigorous and dwarf trees measured
every two to four days during the 1969 season. Ten
shoots on four trees of each type were measured....... .

Mean shoot growth on vigorous trees minus that of dwarf
trees between each two measuring dates. Ten shoots
on four trees of each type were measured..... ..

The percent non-growing (summer dormant) shoots on
four dwarf and four vigorous trees. Ten shoots on
each tree were measured every two to four days during
the 1969 season.......................................

vii

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10. The response in terminal shoot growth, of one year old
'Red Prince Delicious' scions on three rootstocks to
injections of 10 ppm of GA3 (G) and 100 ppm of ABA
(A)ooooooo ooooooo o ooooooo o ooooooooo 000000000000 oooooo II()

11. The response in lateral growth (shoot diameter), of one
year old 'Red Prince Delicious' scions on three root-
stocks to injections of 10 ppm of GA3 (G) and 100 ppm
ofABA (A) .......... 80

12. A "terminal bud," characteristic of summer dormancy,
induced on a one year old 'Red Prince Delicious'/
EM IX tree by injections of 100 ppm of ABA ..... 83

13., Response, in terminal shoot growth, of one year old
MM 111 non-grafted trees to injections of increasing
concentrations of ABA ...... ....... 86

14. Terminal shoot growth curves for MM 111 non-grafted
trees injected with 4 ppm of ethanol (controls), 100
ppm of ABA, and 10 ppm of Alar. Injection dates are
indicated by small arrows............................ 88

15. Response obtained from five injections of a 100 ppm
solution of ABA (approximately 3 mg of ABA) on one
year old MMlll non-grafted trees. showing toxicity
symptoms at the growing point” ........... ..... .. 90

.16. A comparison of one year old MM 111 non-grafted trees
injected with 100 ppm of ABA (left), 4 ppm of ethanol
(controls) and 10 ppm of Alar (right). The ABA injected
trees had stopped growing, and new leaves were chlorotic
and twisted, while Alar injected trees had shortened
internodes, telescoping foliage and dark green coloration. 9O

17. Total uptake (ml) vs concentration of the ABA solution
injected. Solution uptake was inversely proportional .
to ABA concentration................................. 93

viii

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Figure

18.

19.

20.

21.

22.

23.

Total increase in lateral growth (shoot diameter) of
four one year old 'Red Queen Delicious' / EM IX
trees to three concentrations each of GA3, GA4+7,
IAA, and BA applied in five injections four days
apart ......... ......

Total terminal shoot growth of four one year old 'Red
Queen Delicious'/EM IX trees to three concentra-
tions each of GA3 and GA4+7 applied in five injec—
tions four days apart... .....

Total terminal shoot growth of four one year old 'Red
Queen Delicious'/EM IX trees to three concentrations
of IAA applied in five injections four days apart

'Red Queen Delicious'/EM IX trees injected with 5 ppm
of GA4+7 9left) and 10 ppm of GA3 (right). Note
the small, malformed leaves and side shoots on the
GA4+7 treated tree. Trees had resumed normal
growth at the time photograph was taken . ..... . .

'Red Queen Delicious '/ EM IX trees injected with 5 ppm
of BA. A11 main shoot foliage was removed to show
spur and side shoot development typical of cytokinin
effects on apple ....

Histograms illustrating the activity of GA-like substances
in aqueous and methanol extracts of shoots from one
year old 'Red Prince Delicious'/EM VII trees. The
standard deviation (3.7 mm) for the assay was sub-
tracted from the value for each column fraction.
Fractions showing no activity prevented seed germina-
tion, or hypocotyl growth was equal to or less than
the standard deviation. The histograms are A, MeOH
bound fraction; B, MeOH free fraction; C, aqueous
free fraction; D, aqueous bound fraction; E, NH4SO4

fraCtion .00...00.000.00.000.0.0.0.0000...0.0.0...O.

ix

Page

98

100

102

105

105

121

Figure Page

.24. Histograms showing the activity in 25 g fresh weight
of shoots collected from five year old 'Red Prince
Delicious' trees in August. Because there were no
differences between trees of increasing vigor, the
average of the data obtained from the four types of
trees is represented. Shaded portions of the histo-

grams represent activity abovc the standard deviation. .. 120

 

 

 

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INTRODUCTION

Dwarf apple trees are of commercial importance in most fruit areas
of the world. These smaller, precocious trees increase yields and simplify

cultural practices, thus lowering production costs and allowing a more

rapid turnover of varieties to meet changing market requirements. According

to the 1968 Michigan Fruit Tree Survey, dwarf and semi-dwarf trees account

for 31 percent of the apple trees in Michigan.

Dwarf apple trees have been extensively studied, and although much is
known about their behavioral characteristics, the mechanism by which dwarfing
interstocks and rootstocks can superimpose their growth and fruiting charac-

teristics on the scion variety is unknown.
The purpose of this study was to determine the role of gibberellins and

abscisic acid in the dwarfing phenomena. That they may be important is a

feasible hypothesis in light of what is known about dwarf trees and how exo-

genously applied hormones affect apple tree growth. Other plant hormones

may be involved, and indeed, it is likely that hormonal interaction is an
important factor. Gibberellins and abscisic acid were studied because:
1- gibberellins have been shown to increase terminal growth and inhibit flowering
in the apple tree; 2. the effect of abscisic acid on the growth and flowering

of the apple tree is unknown; and 3. the ratio of gibberellins to abscisic acid

has been proposed as the mechanism which controls growth and flowering in

 

 

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woody perennials.

This research was conducted to further the understanding of the
mechanisms controlling growth and flowering in woody perennials. The
use of component trees is an excellent technique for studying the roles of
various organs in the growth and development of the whole tree. Certain
of the dwarfing rootstocks are not well adapted to Michigan and other
production areas (Simons 1966). A better knowledge of the mechanisms in-
volved in scion/rootstock interaction will facilitate breeding and selection
programs designed to produce trees better suited to specific environmental

conditions .

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REVIEW OF LITERATURE

Scion/Rootstock Relationships in Apple

General

Rootstocks and interstocks have been shown to affect the scion with
respect to many horticultural parameters. Tubbs (1951), in a resume of
scion/rootstock relations, listed 29 scion characters found to be influenced
by the rootstock. The most striking effects related to rootstock control
of growth and flowering. The problem was complex because the scion also
affected the rootstock. Furthermore, interstocks were capable of exerting
effects upon the scion similar to those of the rootstock. The literature in
this area is voluminous as shown in reviews by a number of workers (Hatton
1930, 1935, 1938; Swarbrick 1930; Tubbs 1951, 1967; Preston 1956; Rogers
and Beakbane 1957; Tukey 1964). This review cannot be complete, but will

attempt to give some understanding of our present knowledge of scion/stock

relations in apple.

Rootstock Effects on the Scion

Beakbane (1941) showed that the rootstock influence on fruit bud formation
Was evident in both stem and root grafted trees two years after grafting.
There was no apparent difference between one year old apple scions on dwarf

and vigorous rootstocks (Tubbs 1967). The first major difference between I

 

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rootstock effects upon the scion clone was in the growth of the lateral
buds. On dwarf rootstocks, many short laterals were formed, often

rosette-like rather than spur laterals. Vigorous rootstocks produced fewer

but longer lateral shoots.

The characteristics of many of the East Malling (EM) clonal rootstocks

were described by Hatton (1927). He found that trees on EM XII (very

vigorous) averaged five times more wood growth than the same scion variety

on EM IX (very dwarfing). The other EM clonal stocks made up a continuous

gradation of vigor between these two extremes. He found good correlation

between vigor expressed as height and spread and as total shoot growth.

Generally, productivity was correlated with vigor. Notable exceptions were

EM VII (semi-vigorous) and EM IV (vigorous). Interesting comparisons were

made between yields and cropping area. EM VII and EM I were similar in

Size, but EM VII produced considerably more fruit than EM 1. In most cases.

however, the more vigorous trees eventually equaled the yields of the dwarf

trees due to their much larger bearing surface. In a later paper, Hatton

(193 5) re-emphasized the precocity of certain rootstocks, and showed that

after 14 years trees on EM IX had produced seven times their own weight in

fruit while trees on EM XII had equaled their weight in fruit. Preston (1954)
fouhd that trees on 3426, the most dwarfing rootstock, EM IX and EM VII,

respectively, produced 20, 10, and 5 times their tree weight in fruit.

 

 

 

 

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EM IX and 3431, two dwarfing rootstocks of approximately equal
vigor, significantly shortened the juvenile phase of apple seedlings (Tydeman
.and Alston 1965). Nine years after germination, 88 percent of the seedlings

budded on these dwarfing rootstocks had flowered compared to 49 percent

of the seedlings on their own roots.
The evidence for an invigorating effect of rootstocks upon the scion is

weaker, perhaps because most scion varieties are vigorous. Barker (1928)

concluded that rootstocks produced no real increase in the growth of any

scion. Grubb (1939), however, found that five foot lengths cf three vigorous

apple varieties used as stem builders enhanced the vigor of the second scion

in four year old trees. Stembuilders, unlike interstems, usually form the

Only scion on a rootstock for one or two years, and are allowed to bear
foliage (Rogers and Beakbane 1957). Roberts (1929) found an invigorating

effect of rootstocks, but only on the slow growing variety, Jonathan.

"The same varieties growing on different rootstocks had distinct growth

Characteristics indicating rootstock effects upon the seasonal growth of the

trees;'(Swarbrick 1928). Elongation (shoot) growth of trees on EM IX showed

a more rapid and often earlier fall in the growth rate of leader shoots. The

amount of extension growth was nOt determined as much by differences in the

time at which trees began growth in the spring as by marked differences in

 

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the rate at which growth fell off during the latter part of the season. Rao

and Berry (1940) and Colby (1935) verified these results.

Rootstocks exhibited an effect on internode length (Swarbrick and Naik
1932). Shoots of trees on EM IV were characterized by an early falling

off in internode elongation. The mean internode length of current season

growth from EM IX was short, with the longest intemodes produced late in

the season. Mean internode lengths were the same on the rootstocks EM I

(vigorous), EM IV, EM X (very vigorous), EM X11, and Bristol 5 (vigorous).

The vigor of a rootstock was characterized by the shape of the resultant

tree (Hatton 1935). Trees on EM IX were easily recognizable by their stunted

habit of growth (Rao and Berry 1940). Trees on low -yielding rootstocks were

taller with a more upright habit of growth than trees on heavily cropping

rootstocks.

Rootstocks also modified leaf size (Swarbrick and Naik 1932). EM I

induced the largest leaf size, but EM IX trees had the largest amount of leaf
area per unit length of shoot. EM VII and Bristol 5, the most vigorous root-

Stocks tested, produced the least leaf area per unit length of shoot. There

Was a direct correlation between the ratio of mean leaf area to internode

leng‘th and fruitfulness. Vyvyan and Evans (1932) found the mean stem length.

the mean internode length and the mean and total leaf areas to be greater

on EM V (vigorous) than on EM IX, but the leaf area per total length of stem

 

 

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was greater on EM V. This was due to a larger total number of shoots
on EM V, with a smaller proportion of small-leafed bearing spurs on trees
on this rootstock. Also, mean leaf size was greater on all but secondary

shoots of trees on EM V. Colby (1935) reported finding fewer leaves per

spur with trees on EM IX than on EM XII.

In testing leaves of various apple combinations for photosynthetic
capacity, Friedrich (1953) found the highest rate of photosynthesis occurred

in leaves of spindle bushes on the rootstock EM IX.

Scion Effects on the Rootstock

Tukey and Brase (1933), working with 14 varieties grafted to seedling
piece roots, noted that the scion had an influence upon the growth and
character of the rootstock. This was a direct effect in that vigorous scions
induced vigorous root systems and less vigorous scions produced roots with
Sharp angles to the vertical. Swarbrick and Roberts (1927) and Roberts
(1929 and 1931) showed that the scion affected the growth and habit of the
rootstock when grafted directly on pieces of seedling root. Scions did not
Show the same dominance over seedling root systems when budded on the
Stem portion of the rootstock.

Rogers, 5;; a1. (1939) found that root systems of EM IX were invigorated

in trees with five foot lengths of vigorous stem builders. Amos, gt a.

 

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of] ..
I)« )(I |_K .

 

(1930) concluded that the scion effect on the total size of the root system
could be marked, although these differences were small when compared to
differences between unworked root systems. They also found that scions
altered the percentage of fibrous roots indirectly by altering the size of
the root system. Beakbane and deWet (1935) concurred, but found that
scion varieties did not alter distinctive rootstock characteristics. Working
with EM I, II, and IX rootstocks, they found no visible difference between
the root systems using two varieties on these rootstocks. Vyvyan (1930)
also reported that two year old root systems of EM II and IV retained
their distinctive morphological features irrespective of the variety used as
the scion. He went further, however, and showed that seedling root systems
did not take on distinctive morphological characters according to the scion
variety used, whether or not the tree was root or stem grafted.

Workers, therefore, took opposing stands on the nature of scion in-
fluence on seedling roots. Roberts (1932) obtained and rooted several English
varieties. From grafting studies, he agreed that English varieties did not
Show a scion influence when grafted on seedling roots, but that those varieties
used produced similar root systems. Therefore, if scion influence existed,

it could not. be detected by a comparison between these varieties.

-_5

v -,. a

’F' Y‘ n
\, ll

_...8 s

L ILLDH
.“ dIl
“lets: it“

Interstock Effects on the Rootstock and Scion

Many investigators have reported that interstocks influenced the
size and the fruiting characteristics of apple trees (Blair 1938; Rogers
and Beakbane 1957; Carlson 1965). Stem pieces of rootstock clones,
therefore, did not require their root systems to induce "stock" effects
in the scion.

Grubbs (1923 and 1939) and Hatton (1930) observed that interstock
effects on fruit bud formation in the scion were proportional to the length
of the interstock. Hatton (1930), Grubbs (1939), and Bueller and Borck
(1953) described similar effects of interstock length on scion growth.
Roberts and Blaney (1967) found no effect of interstem length on growth,
but flowering was proportional to the length of the interstem. They also
found an invigorating effect of long EM XVI interstocks over short EM XVI
interstocks on the scion. That interstocks could invigorate scions was
verified by Parry and Rogers (1968). Carlson and Robitaille (1970) recently
reported an effect of interstock length on both vigor and flowering. Inter-
stocks of EM VIII, a dwarfing rootstock, induced size control and precocity
in 4, 8 and 12 inch lengths when compared to non-interstock standard trees.
Interstock effects were proportional to their length, particularly with the

vigorous scion 'Red Delicious '.

 

the same 1

{:1

.3, posztior.
Dakar
dwarf I'iHJT
had a spr:
branches.
of the ma:
origin. int

COmDIIIaIIO

A “w-.
Uts'tl mmu
5' '

10

Interstock position in the tree may be important (Roberts and
Blaney 1967). When placed low in the trunk the amount of rootstock
stem was reduced and vigor was curtailed more effectively than when
the same interstock was placed higher in the trunk. There was, however,
no positional effect on flowering.

Dwarf interstock trees exhibited the characteristic growth habits of
dwarf rootstock trees (Dana, e_t a_l_. 1961). Trees with dwarfing interstocks
had a spreading habit of growth with little upward curvature of the scaffold
branches. Trees with own-stem interstocks showed sharp upward bending
of the main scaffolds. Lower branches on dwarf trees had a wider angle of
origin, indicating a significant interaction between crotch position and stem
combination.

The root system as well as the scion could be dwarfed with dwarfing
interstocks (Parry and Rogers 1968). Swarbrick, e; a], (1946) found that
the effect of intermediate stem pieces could be more pronounced on root
development than on top growth. Root systems of trees with EM IX inter-
stocks resembled, in some ways, the EM IX root system.

The question of interstock effectiveness in inducing "stock" effects
has been the subject of much study. Vyvyan (1938) found that while inter-
mediate stem pieces of dwarfing and vigorous rootstock clones had a mea-

surable effect, it was small when compared to the influence of either type

3f IOOE

EM IX 1

sini‘nr :
Blaney II

Of the 11».

ll

of root system. Tukey and Brase (1944) agreed that the influence of an
EM IX interstock was less than that of an EM IX rootstock. Swarbrick,
g _al_. (1946), however, showed that trees with EM IX interstocks were
similar to trees grafted on EM IX in the normal manner. Roberts and
Blaney (1967) indicated that the interstock influence was subordinate to that
of the rootstock. Trees on EM IX rootstocks were dwarf and those on
EM XVI rootstocks were vigorous regardless of opposing interstock influence.
Their data further indicated that EM IX interstocks compared to EM IX root-
stocks reduced growth by only 16 percent as opposed to 67 percent, respec-
tively. EM IX as a rootstock increased fruit set over EM IX as an inter-
stock by approximately 10 percent. Parry and Rogers (1968) studied trees
with combinations of two scion varieties and two rootstocks with and without
interstocks of varying degrees of vigor. All interstocks but EM IX produced
trees intermediate in vigor between that of trees on the two stock components.
In other words, 'Cox'/EM 26/MM 104 was intermediate in size between 'Cox'/
EM 26 and 'Cox'/MM 104. EM IX was as dwarfing when used as an interstock
as when used as a rootstock.

Dwarfing interstocks are often associated with overgrowth and swelling,
either over the length of the interstock or specifically at the graft unions
(Vyvyan 1938; Swarbrick, _e_t_ a}, 1946; Dana, g_t_ 9;. 1961; White 1962; Parry

and Rogers 1968).

A’ « _l a
relltttOch:
" v‘,"~ .
N. kil'ul : J
.IHH fi?’ «0
‘ c “I.Lr§~

RODCJ

v' _ .
IIIU 5i]

T‘.‘ .
1 k

 

12

Rootstock, InterstockJ and Scion Interaction

The resultant apple tree is the product of an interaction between
its two or three component parts. White (1962) demonstrated this inter-
action by grafting, in all combinations, 4 scions, 5 interstocks, and 10
rootstocks-”600 trees in a five year study. Height was most affected by
the rootstock in the first year, by the scion in the second year, and by
the interstock in the last three years. The scion/interstock interaction
showed up in the third year and increased thereafter. 'Golden Delicious'
and 'Jonathan' exhibited precocity in all combinations with EM IX and EM
VIII.

Roberts (1929) and Colby (1935) noted that all scion varieties were
not aflected in the same way by the rootstocks EM IX and EM XII. Al-
though in general EM IX produced dwarf trees and EM XII produced vigorous
trees, he found that some varieties on EM IX made taller trees than other
varieties on EM XII.

Ramirez and T abeunca (1964) reported that EM IX scions significantly
increased the bark percentage of EM XVI rootstocks, while EM XVI scions
had no effect on the bark percentage of EM IX rootstocks. EM XVI root-
stocks, however, significantly reduced the bark percentage on EM IX scions
while EM IX rootstocks had no effect on the bark percentage of EM XVI

scions .

“'tdicated tb.
‘9 51:;
on Its.

. V
. . _ I.
The below

13

Vyvyan (1955) considered the question of which component, stock
or scion, was the dominant partner in the grafted tree by studying
trees composed of EM IX, IV, and X11 in different combinations. Results
indicated that the rootstock component had a greater effect than the scion
on tree size, with composite trees usually resembling the stock variety.
The scion variety had some effect on tree size when the vigorous EM XII
was the scion. Tubbs (1967) found that the scion influenced rootstock weight;
however, the rootstock was the dominant partner.

The relative growth rate of a composite tree was mainly the resultant
of the growth rates of the component parts (Knight 1925; Vyvyan 1934, 1955;
Tubbs 1967; Parry and Rogers 1968). Knight (1925) wrote: "The scion makes
an intermediate amount of growth when double-worked on a vigorous stock
grafted on a dwarfing rootstock than when grafted on each of the stocks. "
Forty years later, Parry and Rogers (1968) reached the same conclusion
when they found that "The performance of various interstocks could have been
generally predicted with the assumption that the scion is subject to a blending
of the influence of the two other components." Tukey and Cline (1962) disa-
greed, however, and felt that there were interactions between clones, especially
between interstock and scion, that could result in individuality in the behavior
of each combination.

The ratio of top to root weights was constant for apple trees on

14

clonal rootstocks (Rogers and Vyvyan 1934). This constant existed
for a given variety on a given soil, independent of rootstock and
irrespective of the size of the tree and the crop it had carried. This
ratio was shown to be constant even after heavy top pruning (Knight 1934).
This indicated that there was no real competition between stock and scion
(T ubbs 1967), and that some mutual mechanism regulated the respective
growth rates of both roots and shoots (Vyvyan and Maggs 1954).
In addition, a direct adaptation by the scion to the limited root develop-
ment of dwarfing rootstocks may be indicated (Rogers and Vyvyan 1934).
Humphries (1958) found that removal of up to 50 percent of the roots of
rye and barley plants had no effect on the growth rate of the roots, but,
instead, the growth rate of shoots decreased as the roots were removed.
Colby (1935), disagreeing somewhat with other workers, noted that the
varieties 'Whitney' and 'Snow'/EM IX had slightly greater top/root ratios

than on EM XII.

Relationship Between "Stock" Effects on Vegetative vs Reproductive Growth
There is much disagreement on the relationship between growth and
precocity effects as induced by rootstocks and interstocks. With exceptions,
productivity is inversely correlated with vigor. Much evidence exists indicating
that flowering and vegetative growth are antagonistic processes. McLean (1940)

demonstrated that the checking of growth by looping stems would dwarf plants

15

and induce flowering. Wareing and Nasr (1958) checked the growth
rate and promoted flowering in apple and larch by pulling down branches.
Swarbrick (1929) made a survey of conditions most conducive to flower bud
formation in the apple, and found that they were those which tended to
prevent excessive amounts of elongation growth and induce cessation of
growth early in the season. He pointed out, together with other evidence,
that ringing, droughty weather and a sod cover were all conditions which
retarded growth and induced flower bud formation. In addition, spur buds,
often the first buds to initiate flowers, were also the first buds to stop
growth during the season. Swarbrick and Naik (1932) observed that EM IV,
although vigorous, was characterized by "an early falling off in internode
length. " Swarbrick (1929) admitted, however, "that under conditions of
acute deficiency of one or more substances essential to growth, a tree
may show these growth characteristics, yet there may be little or no flower
bud formation. " Roberts (1929) felt that rootstock influence upon flower bud
formation was associated with the diameter and not the length of terminal
growth.

Although growth characteristics may influence flower bud formation, the
reverse situation may also be true. Barlow (1963) found that cropping re-

duced growth only after reaching certain threshold values. Maggs (1963)

 

In t ”1.1;:
\~"
.\ r‘

r:Iatcd.

has a q;
Knight ft”?
reduced ;
“is naff
in pairs

EM [X \N.‘
Eli I, at;
“WI, \lc
Rubens .
fiOWGring

”C1011 VI
:4

 

“Ill-1e EM

i

DUI “(I p.

 

 

 

16

observed considerable reductions in the root growth of trees when fruits
were allowed to set. He also showed that deblossoming was followed,
in young trees, by an increased rate of sh00t growth (Maggs 1955).

Not all workers considered growth and precocity to be intimately
related. Gregory (1957) pointed out that Knight discriminated between
these two aspects of scion-stock relations when he suggested that vigor
was a quantitative rootstock effect and precocity was a qualitative effect.
Knight found that the amount of growth on EM IX and EM I was greatly
reduced by budding several scions on the same stock, whereas precocity
was unaffected. When these two rootstocks were inarched and combined
in pairs with a single scion, the vigor of the scion on the pair EM I and
EM IX was intermediate to the vigor of the scion on the pairs EM I and
EM I, and EM IX and EM IX. Scions on the pair EM I and EM IX, how-
ever, were as late in flowering as scions on the pair EM I and EM I.
Roberts and Blaney (1967) also considered stock effects on growth and
flowering to be independent, because EM IX induced cropping before reducing
scion vigor; EM XVI invigorated the scion without affecting its precocity

while EM I both invigorated the scion and induced earlier flowering; and

 

trees of 'Starlcing Delicious'/M: sikkimensis were small and weak-growing

but not precocious. Cline's (1960) work showed that the expression of

 

h-“ _

growing

dE'lay {3d .

17

dwarfing by a rootstock was influenced by both the quantity and availability
of nutrients. Nutrient concentration influenced the growth rate of trees
but had little effect on precociousness. From this he concluded that growth

and fruiting were not directly related.

Bypassing the Dwarfing Component in the Tree

 

When nonvigorous trees on 'Northern Spy' roots were inarched with
young EM XVI trees, Hearman, gt; a1. (1937) saw a significant increase in
the vigor of the trees after three years. When EM XVI was inarched into
a single branch. that branch increased in vigor. In a follow-up study on the
same trees, Thompson, _e_t_ a_._l_. (1954) reached similar conclusions. Hatton
and Bagenal (1934) reported that when the Bramley's Seedling apple variety,
growing on EM IX rootstock, scion rooted. it grew away very rapidly with
delayed fruiting.

Dana, e; 41, (1961) concluded that 'Golden Delicious' bridge scions
nullified the effect of Clark Dwarf (very dwarfing) interstocks as a factor

in growth control of 'Golden Delicious' trees on Virginia Crab seedlings.

Localization of the "Stock Effect"

 

One of the most interesting controversies in the literature on rootstock,
interstock and scion interaction concerned the localization of the stock effect.
Roberts (1929) concluded that rootstock influence resided in the stem portion
of the rootstock. He found a marked influence of the scion upon the rootstock

when the scion was grafted directly on piece roots of apple seedlings. The

 

 

m

\«z

18

scions, however, did not show the same dominance over seedling roots

when grafted on the stern portion of the rootstock. Work reported earlier

in this review showed that stem portions (interstocks) induced the rootstock
effect in a scion variety. Hatton (1931) showed that rootstock influence

was also apparent in trees worked directly on the roots of clonal stocks.
Other workers (Knight 1925; Beakbane and Rogers 1956) determined that the
root portion of a rootstock was primarily responsible for the rootstock effect,
but they agreed that the stem portion contributed to the influence on the scion.

In a study of rootstock influence on winter hardiness, Kalasnikova and
Krajuskina (1964) grafted four apple varieties in the branches and on the root
trunk of five seedling rootstocks. Results showed that rootstock type had a
greater influence on the winter hardiness of the resulting tree than the
position of grafting. Roberts and Blaney (1967) agreed, and stated that root-
stock influence was found in certain genetic materials whether used as scion,
rootstock or interstock in the composite tree.

Messer and Lavee (1969) studied metabolism relating to apple root-
stock vigor at the cellular level. Cultures were isolated from one year old
branches of EM XIII and EM IX. Subcultures used for study were taken
during the second year, "and grown on a modified White's medium (Miller
and Skoog 1963) adapted for tree callus culture by Lavee (1963)." Their

Work indicated that the vigor and dwarfism characteristics of those rootstocks

 

_-—_—¢‘ —

 

19

was present at the cellular level. They found differences in growth rates
between EM IX and EM XIII cultures using optimum concentrations of
naphthalene acetic acid (NAA) and kinetin. The optimum concentrations of

both growth regulators were the same for both rootstock cultures.

Dwarf and Vigorous Tree Differences

Many variations have been found between dwarf and vigorous trees.
Anatomical, biochemical, physiological and metabolic differences have been
reported. Stems and roots of dwarfing rootstocks contained a more highly
parenchymatous xylem and phloem and a higher ratio of bark to wood than
those of vigorous rootstocks (Colby 1935; Beakbane and Renwick 1936;
Beakbane and Thompson 1939; Dana, .e_t_ §l_. 1961; and McKenzie 1961). The
percent areas of fibers, vessels, rays and, to a lesser extent of parenchyma,
showed a definite connection with the vigor induced in the scion (Beakbane
and Thompson 1939). The number of vessels and vessel cross-sectional
area were negatively correlated with tree size and with the dwarfing ability
of stems when used as interstocks (Scholz 1957). Vessels in roots of self-
budded EM IX, II and VII showed a positive correlation with size and tree
Vigor, but the vessels were smaller than those in the roots of EM IV

(McKenzie 1961). EM IV had large amounts of bark and wood ray tissue

'1 i 1'“!

 

upon the)
iksse 1V7
[fat “13::
ejects, I

Tag"-
rootstot' k
stock Hg
from dihdl
m those 1'

“EH: not Q:

centtatior

 

20

together with very large vessels. These dwarf and vigorous characteristics
in the same rootstock were reflected in the scion in field studies.

Anatomical rootstock characteristics were transmitted to scions grafted
upon them, and scions modified those particular characteristics (Colby 1935;
Mosse 1951; Thompson 1952; McKenzie 1961). McKenzie (1961) suggested
that anatomical differences were the result and not the cause of rootstock
effects, because flowering increased bark and wood ray tissue in roots.

Taper and Ling (1961) studied electrical resistance in twigs of 13 apple
rootstocks ranging from very vigorous to very dwarfing as a measure of root-
stock vigor. They felt that the greater electrical resistance found in twigs
from dwarfing rootstocks was a measure of the greater amount of living tissue
in those rootstocks. Significant differences in morphological characteristics
were noted in tissue cultures of EM IX and XIII rootstocks at optimal con-
centrations of NAA and kinetin (Messer and Lavee 1969). EM XIII cultures
were friable and contained a greater proportion of larger cells which had a
higher water content and more diverse cellular forms.

The respiratory rate of root tissues was higher in vigorous than in
dwarfing apple rootstocks (Hasson 1953). Miller, et a}. (1961) rated apple
rootstock selections as to the vigor they produced in a scion variety on the

basis of the percent bark present in cross-sectional areas of adventitious

 

‘v‘Jere f0 LL11

SEIGCUORS

 

21

roots. Respiration was calculated in leaf disks and root tips using mano-
metric techniques. Of 18 selections studied, leaf disks from seven selec-
tions had a significantly lower rate of 02 absorption than leaf disks from

the vigorous Malus baccata 5. Of these seven selections, five were con-

 

sidered dwarfing and one borderline on the percent bark index. A highly
significant regression was obtained when the percent bark in the roots was
related to the respiration rate of the leaf disks. A highly significant re-
gression coefficient was found between the amount of 02 absorbed by root
tips and the percent bark on the roots. No significant differences, however,
were found between the respiration rates of root tips from the various
selections.

Messer and Lavee (1969) found similar RQ values in tissue cultures of
EM IX and XIII. The EM IX cultures, however, had higher respiration rates
with a Q02 1.4 times greater on a dry weight basis.

The net assimilation rates (NAR) of all rootstocks grown under a wide
range of cultural conditions varied with the vigor of the stocks, as
greater vigor always accompanied a higher assimilation rate (Ruck and Bolas
1956). Nitrogen deficiency markedly reduced the NAR of both dwarfing and
Vigorous rootstocks. Gregory (1957) found that 'Cox's Orange Pippin' had

a higher NAR on EM XVI than on EM IX. There was no difference in NAR

22

between the two rootstocks early in the season, but in August and
September the NAR of EM IX trees fell 52 percent while that of EM XVI
trees rose 12 percent. Similar results were obtained with unworked
rootstocks. Using 10 rootstocks, a good correlation was found between
NAR data and vigor. Gregory (1957) suggested that the late season drop
in NAR might be due to more rapid leaf senescence on dwarfing rootstocks.
The possible relationship between the late season decrease in NAR and the
early cessation of shoot elongation in dwarf trees was not discussed.
Working with detached stems from apple rootstocks of different vigor,
Knight (1926) found no significant differences in water translocation under
pressure. Graft unions of trees on EM IX, however, offered greater re-
sistance to water flow than did unions of trees on EM XII (Warne and Raby
1939). The water content of scions on EM IX was appreciably less than
for scions on EM XII (Rao and Berry 1940). Friedrich (1953), however,
showed that the water balance of trees on EM IX was particularly efficient.
Although the dwarfing and vigorous rootstocks themselves had different trans-
piration rates, Knight (1925) observed that a single scion worked on those
different rootstocks showed no difference in transpiration rate. Upward
movement of food reserves from the roots was limited in dwarf trees, how-

ever, as a result of early suberization of young roots thereby limiting the

 

 

 

 

 

23

water supply to the tops of those trees (Colby 1935). In contrast, up-
ward movement of water occurred freely in vigorous trees. Dana, gt a_1.
(1962) found that interstocks did not impair water transport.

The bromine ion was used to measure root absorption in BM IX,
II and XIII rootstocks (Berry 1939). Concentrations of bromine absorbed
increased in the same order as the vigor of the rootstocks, with significant
differences in absorption between EM XIII and EM IX but not between EM
XIII and EM II, and EM IX and EM II. Pearse (1940) determined that
although trees on EM XII absorbed more water per unit leaf area, trees
on EM IX absorbed more water per unit increase in fresh weight. Trees
on EM XII absorbed considerably more phosphorus and potassium than
did trees on EM IX, but the ratio of total growth to the amounts of each
of these nutrients absorbed did not differ significantly for trees on the two
rootstocks. Twenty-one elements were shown to be absorbed by roots and
transported to the scion in trees on EM IX and X11 (Roach 1931). Lead
was absorbed but remained restricted to the roots of both rootstocks.
Molybdenum was found only in the EM IX rootstock.

Using sand culture techniques, Cline (1960) found little difference

32

. . 86 .
between rootstocks in the translocation of Rb and P, or in the accumu-

lation of those isotopes in the leaves of the scion. Bukovac, gt 11, (1958),

 

SC

1a

the

‘lk a

Ea

din

mat
of 1

lCO]

[—2,
’1
L—q

[01,11

24

however, were able to relate the transport rates of 32F and 45Ca from
solution cultures to the field observed vigor of 'McIntosh' scions on
EM IX, VII and XVI. Similar results were demonstrated with rooted
layers of the same rootstock clones. Crab C was 73 percent more
vigorous than EM IX when grown in solutions of 5 ppm nitrogen (N),
but: only 12 percent more vigorous when grown in solutions of 200 ppm
N (Ruck and Bolas 1956). The authors suggested that translocation in
the vigorous Crab C rootstock was more efficient at the lower than at
the higher N level.

Dana, g a1_. (1962) found that seedling-grafted trees, whose N level
was lower at the start of the test, accumulated leaf N more rapidly than
dwarf interstock trees. Self interstock trees also accumulated more N
than dwarf interstock trees, ruling out the extra graft effect. Under con-
ditions of limited N supply, however, dwarf trees accumulated more N
than vigorous trees. They concluded that this was because dwarf trees
made less growth than vigorous trees. Spurs and two year old branches
0f fruitful dwarf trees accumulated both starch and nitrogen as the season
advanced, Unfruitful vigorous spurs and branches showed a low N level
(Colby 1935). In an analysis of bark samples from single-shoot trees of
EM IX and XVI, Martin and Williams (1967) found higher levels of N and

total amino acids in the EM IX bark. Seasonal cycles of nitrogenous

,7
-- ,-
"1

~o~

m
'1".

 

 

 

(F3
F

on
m 1
mo;

ear]

min;
and
of [h

Wink

materials were examined by Kench (1937), including total N, total non-
protein N, ammonia, acid amide, humin, basic, imide, monoamino and
proteose N in the wood, bark and leaves of terminal shoot portions of
'Lanes Prince Albert' on EM II, V, VII, IX and Malling Borne (vigorous).
No significant differences were observed between the composition of shoots
grown on EM II, V, VII and Malling Borne. Shoots grown on EM IX had
significantly higher levels of total N and acid amide in the wood. Wame
and Wallace (1935) studied total N, dry matter, total ash and the ash con-
stituents calcium, magnesium, potassium and phosphorus in shoots, and re-
ducing sugars, sucrose, total sugars and titratable acidity in fruits, in re-
lation to dwarfing and precocity, They could not explain rootstock effects
on the basis of those chemical characters. Vaidya (1938) found differences
in levels of calcium, phosphorus and magnesium in trees on EM IX, VII and
more vigorous rootstocks. Seasonal cycles of ash and phosphorus were
earlier in trees on EM IX.

Several workers noted rootstock, interstock and scion effects on the
mineral composition of apple foliage (Cline 1960; Tukey, _e_t_ al_. 1962; Titus
and Gosheh 1963). Tukey, g a_l_. (1962) analyzed leaves from adjacent trees
0f the same scion variety on different stock combinations for five major

mltrients. There were significant differences not only between different

26

stock combinations but also between trees on standard seedling rootstocks.
When combinations of scions, interstocks, and rootstocks were analyzed,
all components were found to be capable of influencing the nutritional
status of the foliage. Scions had the greatest effect and rootstocks the
least. Cline (1960) found that the leaf composition of scions on BM 11
and VII differed significantly from that of the scions on standard seedling
rootstocks but not from each other. When those clones were used as
interstocks, significant differences in major nutrient levels in the foliage
were noted, apparently related to the scion and interstock but not to the
rootstock variety.

The scion and rootstock had an effect on the level of nitrogen in
apple tracheal sap, but had little effect on the proportions of nitrogenous
compounds present (Bollard 1957).

Differences in levels of alcohol soluble and alcohol insoluble nitrogen
were found between EM IX and EM XIII cultures. EM IX cultures contained

Significantly more protein nitrogen, while EM XIII cultures contained more
soluble nitrogen on a dry weight basis (Messer and Lavee 1969).

Shoots of very fruitful dwarf trees showed an early accumulation of
starch when compared to shoots of unfruitful varieties on EM XII (Colby
1935; Rao and Berry 1940; Martin and Williams 1967). Smyth (1938) found

great uniformity in the seasonal cycles of total alcohol soluble matter,

 

 

 

 

 

d1

27

reducing sugars, sucrose, total sugars, starch, hemicellulose, cellulose
and lignin, in leaves, wood and bark of terminal shoots of 'Lanes Prince
Albert' on BM 11, V, VII, IX and Malling Borne. The only exceptions
were that starch and hemicellulose tended to accumulate from the end of
June onward in trees on EM IX, leading to a higher winter starch content
in trees on that rootstock. Rao and Berry (1940) observed that starch
accumulation preceded the cessation of extension growth in trees on both
rootstocks.
Leaves of azions on EM VIII interstocks and on EM IX rootstocks had
higher reducing sugars than did leaves on more vigorous trees (Dana, £3; 11.
1962; Leonard 1938). Similar results were obtained in studies of bark and
wood from EM IX trees (Rao and Berry 1940; Martin and Williams 1967).
The ability of dwarf trees to store larger quantities of starch and
soluble carbohydrates than more vigorous trees was associated with the
greater number of living cells in the dwarf trees by a number of workers
(Rao and Berry 1940; Rogers and Beakbane 1956; Priestley 1963).

The early growth stages of EM IX tissue cultures had poorly defined
iOdj-ne staining areas as opposed to well defined plastids, few in number,
in BM XIII cultures. Older EM IX cultures contained fewer iodine staining

areas and more distinct plastids (Messer and Lavee 1969).

 

 

92:2
and
net
3114
Run:

.v\

and

[Ween
in [h{
0)iidas
plddec

28

Beakbane (1941) noted that roots and stems of EM IX and II

contained more oxidases and peroxidases than similar tissues of EM X11

and standard seedling rootstocks. This finding, coupled with our know-

ledge of the role played by those enzymes in auxin destruction, may
lead to a better understanding of the dwarfing phenomena.

Scholz (1957) studied auxin inhibitors in ether extracts of bark from
EM IX, VIII, VII, II and Clark Dwarf rootstocks. EM VIII and Clark
Dwarf extracts were most inhibitory; EM IX extracts were intermediate;
and EM VII and II extracts were least inhibitory. Inhibition of indole-3-
acetic acid (1AA) activity was directly correlated with rootstock vigor.
Dilute sodium hydroxide extracts of leaves from EM 11, VII, DC, Mags
Robusta 5 and six Malus baccata selections were tested on the germination
and growth of stratified apple seeds and wheat seeds (Miller 1965). The
Percent bark in 3 mm root sections was used as the index of rootstock

dwarfing ability. There was a strong correlation between germination and

Percentage bark in the sections. Bioassay of extracts in the watercress

germination and growth test (Wheeler 1962) showed an inhibitory zone be-
tween Rfs 0.4 and O. 5, with small differences in the amount of inhibition
in the order of decreasing vigor of the rootstocks. A study of the IAA-
OXidaSe activity of extracts using crude enzyme preparations from black-
POdded bean plant roots showed that rate of IAA breakdown in the inhibitory

zone was greater in dwarfing than in vigorous rootstocks. The differences

29

were again small.

Martin and Stahly (1967) studied growth promoters and inhibitors in
bark samples of EM IX and XVI rootstocks collected at five stages of
development. The level of promoters was greater in EM XVI extracts than
in BM IX extracts at all stages of growth. Sections coinciding with the Rf
of standard 1AA were more promotive in EM XVI than in EM IX extracts.
Growth inhibition was greater in EM IX extracts from samples collected
during the initial flush of growth and later in the fall. There was twice
the promotion in coleoptile growth in EM XVI leaf extracts, at an Rf beyond
that of IAA. There was less promotion in the zone adjacent to that for
standard 1AA in EM XVI than in BM IX extracts due to large quantities of
phloridzin smeared from the origin of Rf 0.4. The authors considered their
leaf extract work to be less significant since leaves are not required for
rootstock effects.

Using root and stem sections, Gur and Samish (1968) found a significant
negative correlation between IAA destruction and rootstock vigor. The rate
0f IAA-destruction between different rootstocks was greater when root bark
rather than whole root disks was utilized. Two chromatographic zones were
correlated with vigor. Those zones acted as inhibitors of coleoptile growth
When EXtracted from all but two rootstocks. The occurrence of promotion
in thOSe two cases indicated the presence of both a promoter and inhibitor

in the same area. The promoter was identified as IAN.

30

Different levels of endogenous growth substances in dwarf and vigorous
trees were also reported by Lavee (1960).

There is little evidence to support a role for gibberellins in the
rootstock effect. Beakbane (1965), studying the effects of gibberellic acid
(GA 3) on the anatomy of apple leaves, found that anatomical alterations in
structure induced by commercial GA3 were similar to those characteristics
of dwarfing apple rootstocks. The similarity between leaves of very dwarfing
apple rootstocks and the commercial GA3 treated leaves of EM VII was in

the wide separation of palisade cells by large air spaces, in palisade cell
Shape, and in the distribution of spongy mesophyll cells.

Although the optimal concentrations of NAA and kinetin were the same
for both EM IX and XIII cultures, they responded differently to varied con-
centrations of those hormmes (Messer and Lavee 1969). Increasing kinetin
levels decreased the growth of EM IX cultures, but increased the growth of

EM XIII cultures. An increase in NAA concentration inhibited the growth
0f EM XIII cultures but had no effect on EM IX cultures.

Inhibition of growth due to phenolic compounds may be an important
factor contributing to the growth differences due to rootstocks (Mendel and
Cohen 1962). Phloridzin, the principal phenol in _M_§_11_i§_, was present in
higher levels in EM xv1 bark than in EM 1x bark throughout the growing

Season (Martin and Stahly 1967; Martin and Williams 1967). Total phenols

31

were higher in the bark of single shoot trees of EM XVI than in the bark
of similar EM IX trees. They also reported that chlorogenic acid levels
were higher in EM XVI bark during initial bud swell, but were higher in
the EM IX bark at the stage where 10 percent of the trees had set terminal
buds- EM IX contained higher levels of p-coumaric acid at initial bud swell,
initial flush growth, and when 10 percent of the trees had set terminal buds.
During the fall coloration stage, p-coumaric acid levels were higher in the
EM XVI bark. No differences were found in levels of protocatechuic, caffeic,
ferulic and p-hydr0xybenzoic acids between the two rootstock clones, but
there were many differences in alkaline hydrolysis products.

Tissue cultures of EM IX contained higher levels of all the phenolic
constituents present in the cultures of both rootstocks. However, EM IX

cultures contained at least one additional phenolic compound (Messer and

Lavee 1969).

ESPl-anations of "Stock Effects"

NIany of the differences between dwarf and vigorous rootstocks and
interstocks have been tentatively postulated as being causal factors in growth
and f10Wering control. The fact that top/ root ratios remained constant for
trees on dwarfing rootstodts (Rogers and Vyvyan 1934; Vyvyan and M3883
1954) Was evidence that their root systems were not limiting (Martin and

Stahly 1967). Some workers felt the presence of two graft unions in interstock

32

trees was partly responsible for interstock effects (Tukey and Brase 1933;
Beakbane and Rogers 1956). Other workers disagreed with this theory
(Blair 1938; Dana, 3t 31. 1962). The introduction of virus diseases, a
particular hazard when double-working trees, was also suggested as an
explanation for interstock effects (Beakbane and Rogers 1956). Virus
diseases in apple cause a reduction in growth (Prentice 1950; Posnette and
Cropley 1956), but Rogers (1957) showed that all clones in the original
East Malling rootstock trials were not infected with rubbery wood or mosaic
virus.

There is also evidence that phloridzin can retard growth (Bomer 1957;
Hancock, pg _a;l_. 1961). 111 apple trees, this phenol was found in highest
concentrations in vigorously growing tissues (Harvey 1925; Martin and Stahly
1967; Martin and Williams 1967). Hutchinson (1959), however, found no
correlation between phloridzin and vigor, while Pieniazek (1965) showed that
phloridzin did not inhibit the growth of _Ma_ly_§. Martin and Stahly (1967)
concluded that differences in phloridzin levels did not account directly for
the contrasting growth habits of EM IX and XVI. They suggested, instead,
that differences in growth were related to the metabolism of phloridzin in
the two plant systems. Growth control could be influenced by the equilibrium
between phloridzin and its breakdown product, phloretic acid (Sarapuu 1965;

(Jrochowska 1966). Higher levels of p-coumaric acid and inhibitory compounds

33

as well as lower levels of phloridzin, chlorogenic acid and growth pro-
moting compounds were related to the growth characteristics of EM IX
as compared to EM XVI (Martin and Stahly 1967).

Beakbane (1956) postulated five theories as possible mechanisms of
rootstock effect. They were concerned with competition for food; transport
of water and metabolites; active transport of ions; the capacity of each
component to make characteristic elaborated compounds; and living tissue
to plant surface ratio.

Most of the explanations suggested as the mode of action of rootstocks
and interstocks can be classified in three catagories: l. translocation, 2.
nitrogen and carbohydrate relations, and 3. growth regulators.

Translocation: Translocation has been implicated in the dwarfing

 

phenomena. Thomas Knight, as early as 1822, concluded that the effect
of dwarfing rootstocks on growth and precocity was due to an obstruction
of the descending sap. Beakbane and Rogers (1956) suggested that the speed
of transport of water and metabolites might be controlled by the number
and size of the elements of the conducting tissue. Passage of mineral salts
through the small vessels in the wood of dwarfing stocks could be slower
than through the large vessels of vigorous stocks, thus, restricting shoot
growth (Beakbane and Thompson 1939). Storage in phloem and xylem has

also been considered a dwarfing mechanism (Beakbane 1956). Scholz (1957)

34

theorized that dwarfing was based on reduced water conduction through
dwarfing interstock xylem resulting in reduced shoot growth and earlier
flowering. Fruiting would accentuate the dwarfing phenomena. Bukovac,

gt a_l_. (1958) obtained similar experimental results, but arrived at a

slightly different conclusion. They suggested that "a limited supply of

an essential nutrient or metabolite from the rootstock, or a number of such
during early summer when the growth of the scion is at a maximum, may
be partially responsible for the dwarfing response observed in the scion. "
Interstocks might alter transport or affect uptake by "altering the physiology
of the tree. " Colby (1935) disagreed that xylem transport rate was a de-
termining factor in tree vigor. He found trees on EM IX had smooth unions
which, he assumed, presented no serious translocation problem. There was,
however, a low water supply in EM IX trees moving from the root to the
scion 111 early summer due to early suberization of young roots. He felt
this might interfere with the upward movement of organic reserves from roots
to shoots.

Colby (1935) found that EM IX interstocks also resulted in extreme
retention of organic reserves in the roots below the interstock. EM XII
interstocks allowed free upward movement. It was proposed by Gregory
(1957) that a difference in transport rate of five percent would account for

the extreme differences in the growth of these trees. A bark ring graft only

35

7/8 inches wide was sufficient to temporarily induce stock effects in a
scion variety (Roberts 1934). He contended that starch accumulation in
the tissues above the ring was related to the amount of new shoot growth.
There is evidence that phloem transport may play a role in dwarfing
(Sax 1954). Phloem blocks were created by inverting bark rings, and by
exposing stem segments to ionizing radiation. Treated trees made little
growth and were extremely precocious. By adjusting the width of the in-
verted rings, and the length of a normally oriented central segment of bark
between them, any desired degree of dwarfing was obtained. Sax attributed
these results to the inability of nutrients and auxins to move freely through
the inverted phloem tissue. With X-ray induced blocks, he suggested that
nuclear injury, which occurred without killing the tissue, prevented cell
division and sieve tube formation. Dickson and Samuels (1956) fed 32?
through petioles and followed its downward movement and accumulation in
phloem tissues. Trees with inverted bark rings showed an accumulation of
radioactivity for a considerable distance above the bark inversion. Five
year old trees of 'Mclntosh'/EM IX/M. sikkimensis showed a high level
of radioactivity in the interstem when compared to the trunk above and below
the interstem. Three year old 'Starking'/Clark Dwarf/Virginia Crab trees

also showed a high accumulation of radioactivity in the interstem, with a

36

greater accumulation above than below the interstem. Experiments with
a four year old ’McIntosh' tree with a % sargenti interstock tied into a
knot showed much less striking localization of radioactivity. It was noted
earlier that dwarfing interstocks and rootstocks were often associated with
over-growth at the graft union. Peach trees dwarfed on the rootstock

Prunus tomentosa showed no retardation of isotope and no characteristic

 

over-growth. Dickson and Samuels (1956) concluded that dwarfing was asso-
ciated with retardation of phloem transport, but that it was also caused by
other factors.

Dana, 39 a_l_. (1962) found that translocation of sugars was impeded by
Clark Dwarf interstocks. Bridging these interstocks with scions of the top
variety decreased the accumulation of sugars by providing an alternate route
past the dwarfing interstock. When the varieties 'Baldwin' and 'Stayman'
were grafted on rootstock No. 33340, they made little growth (Sax 1953).

When No. 33340 was used as an interstock on M: sikkimensis roots, how-

 

ever, those varieties made good growth. Restricted growth on 33340 root-
stocks, therefore, could not have been due to poor graft unions or inter-
ference of conduction between stock and scion.

In experiments transferring EM IX cultures to the medium upon which
EM XIII cultures had been growing, Messer and Lavee (1969) obtained no

evidence that a secreted substance was responsible for the differences between

37

cultures. Grafting experiments between cultures were also unsuccessful.

Nitrogen and carbohydrates: Finch (1935) indicated that differences

 

in the formation and utilization of carbohydrates and N compounds might
account for the differences in growth and fruiting habits of apple clones.
Thomas (1932) felt the accumulation of carbohydrates in apple trees was
the direct cause for cessation of terminal growth. Preston (personal com-
munication) felt that because dwarf trees had a higher crop/wood ratio,
carbohydrates were being diverted into fruit rather than wood production.
Trees grafted on dwarf rootstocks had more reserve carbohydrates available
for fruit bud formation (Beakbane and Thompson 1939). Rao and Berry
(1940) suggested that due to early suberization of young roots, the dwarf
root system was incapable of maintaining an adequate water supply. The
reduced water content of the scion decreased growth resulting in a rapid
accumulation of starch in the scion wood. Martin and Williams (1967) con-
cluded that reducing sugars, sorbitol, starch, N and total amino acids did
not have a direct bearing on the reduced growth rate of EM IX.

Stock effects are not due to starvation of the root system (Rogers and
Beakbane 1957). According to Priestley(l969), differences in the relative
proportions of leafy shoot and root growth may result from the metabolism
of surplus carbohydrates in the roots.

Messer and Lavee (1969) suggested that starch accumulation in cells

38

of EM IX cultures represented a block in the utilization of sugar in that
rootstock. This was also indicated by the lack of a positive growth
response by EM IX cultures to increasing sucrose concentrations in the
medium. They theorized that individual cells of dwarfing rootstocks may
be predisposed to store greater amounts of carbohydrates than individual

cells of more vigorous rootstocks.

Growth regulators: It was reported that synthetic auxins increased

 

the width of crotch angles on the scaffold branches of fruit trees (Verner
1939; Preston and Barlow 1951). Other reports showed that 2, 3, 5-tri-
iodobenzoic acid (TIBA)- an antiauxin - induced several dwarfing responses
in vigorous apple trees, including growth reduction, increased‘branch spread,
and precocity (Scholz 1957). If IAA was responsible for shoot extension in
the apple tree, then the greater rate of IAA oxidation that occurred in
dwarfing rootstocks might be an important factor in the dwarfing mechanism
(Miller 1956). Pieniazek (1968), however, found that NAA was slightly in-
hibitory to the growth in \_I_i_t_r_g of isolated apple shoot tips.

Sax (personal communication) suggested that excessively high auxin
levels in the tops of dwarf trees, perhaps due to slowed downward translo-
cation, were responsible for checking the growth of those trees.

Gur and Samish (1968) indicated that the auxin supply to the roots in

39

dwarf trees was reduced by destruction in both stem and root bark, thus,
affecting the growth of the tree. Although they did not identify IAA in
their extracts, they gave evidence that IAN could be converted to IAA by
apple root tissue. They proposed that the IAN - IAA equilibrium was
directly influenced by IAA oxidases. Inhibitors found in bark tissue were
acting as IAA oxidase cofactors.

Other proponents for a role of growth regulators in the dwarfing
mechanism were McKenzie (1956), Mendel and Cohen (1962) and Martin
and Stahly (1967). Messer and Lavee (1969) disagreed, due to the failure
of NAA to stimulate or inhibit growth of EM IX cultures and because mor-
phological differences between EM IX and EM XIII cultures were maintained
in all treatment combinations of NAA and kinetin supporting growth. They
suggested that the compact cultures of EM IX might have resulted from a
high cytokinin/auxin ratio, and that likewise, the soft cultures of EM XIII
might have resulted from a high ratio of auxins to cytoldnins. They ad-
mitted, however, that their results did not support a simple well-defined
conclusion.

Work on gibberellin transport from roots to shoots prompted Carr,
et a1. (1964) and Jones and Lacey (1968) to suggest that the differential

dwarfing effect of apple rootstocks might be partially explained on the basis

40

of gibberellins originating in the roots.

Tubbs (1967) speculated on "the stock influence arising from the
production, in relative proportions characteristic of the clone, from stem
tissue but more freely from root tissue, of substances, respectively, in-

creasing and decreasing the activity of apical and lateral meristems. "

Gibberellins, Abscisic Acid and the Apple Tree

 

Gibberellins were reported to induce parthenocarpy in apple (Luckwill
1959; Davison 1960; Bukovac 1963; Dennis and Edgerton 1966). Gibberellins
A4 and A7 were more active in inducing parthenocarpy than was GA3 (Dennis
and Edgerton 1966; Bukovac and Nakagawa 1967).

Fulford, gt §_l. (1967) reported that commercial GA3 was effective in
delaying bud scale formation in the apple. Aqueous sprays of 200 ppm of
GA3 applied four times at weekly intervals to shoots of EM XVI delayed the
formation of resting buds for several nodes, leaves developing instead of
bud scales. Walker and Donoho (1959) found that sprays of 1000 and 4000
ppm of GA3 were ineffective in breaking the rest period of apple trees.
Terminal buds began growth soon after the GA3 treatment, but only small
tufts of leaves developed.

Hull and Lewis (1959) reported no effect on the flowering and vegeta-

tive growth of mature bearing apple trees from a single application of a

41

100 ppm spray of GA3. Karnatz (1963) made eight weekly applications

of 10, 100 and 200 ppm solutions of GA3, beginning in early May, to apple
seedlings. He measured trunk diameter at the ground line as an index of
growth, and found that GA3 promoted growth one year and reduced it the
next. Five years after treatment, two of 15 trees treated with 200 ppm of
GA3 and one of 15 control trees had bloomed. He concluded that GA3 had
no effect on flowering or vegetative development of apple trees.

Powell, _et pl. (1959) measured several parameters in relation to GA3
applications at 1000 ppm sprayed weekly or twice weekly. Sprayed seedlings
had more linear growth per tree, more growing points per tree, a greater
number of leaves per tree, smaller leaves, and a smaller root/top ratio.
There was no significant change in the dry weights of sprayed trees. Kato
and Ito (1962) found that gibberellin foliar applications promoted the growth
of elongating apple shoots, but failed to stimulate growth in shoots which
had ceased elongation. Apple trees treated with lto 2 ml of 300 ppm solu-
tion of GA3 twice per week throughout the growing season were larger and
formed twice as many lateral shoots as control trees (Sironval, 53; gal. 1962).
Localized application of gibberellins produced a limited region of internode
extension higher up the stem, the extent and site of response varying with
the gibberellin used and the method of application (Fulford 1967). On BM 26,

GA3 had a small effect on internode growth, GA7 a rather great one while

42

GA4 was strongly promotive and affected internodes for some distance
up the stem. Of the gibberellins A1, A3, A4, A5, A7 and A9, Barlow
(1967) found that 10 apple clones only reacted strongly to GA4 and less
markedly to GA7. GA3, when supplied in water solutions to decapitated
stems and petiole stumps of apple trees, stimulated the elongation of buds
previously forced into growth by wounding or 6-benzylaminopurine (Pieniazek
and Saniewski 1968). Naphthalene acetic acid and GA3 synergistically in-
creased the rate of cambial divisions. Wood produced was abnormal, com-
posed mainly of short vessels, parenchyma cells and very few fiber-tra-
cheids. Gibberellic acid induced no changes in the growth, ill 3129, of
isolated apple shoot tips from young seedlings (Pieniazek 1968). Leaves
on GA3 treated plants, however, were abnormally narrow and long.
Greenhalgh and Edgerton (1967) found that gibberellin treatments ex-
tended the period of apical meristem activity, increased shoot growth, and
strongly inhibited flower bud formation. Guttridge (1962) first reported an
inhibition of fruit bud formation in apple with GA3. He applied 10 and 50
ppm of GA3 to leaves and stems at weekly intervals from May 31 to
August 29. The percentage of spurs bearing blossom clusters was reduced
from 40 percent on unsprayed branches to 14. 7 percent on sprayed branches.
Marcelle and Sironval (1963) treated Golden Delicious' trees with 100 and

300 ppm solutions of GA3 according to the method of Sironval, 9: 21.0962).

43

At the end of the growing season, 300 ppm trees were 15 cm higher
than control trees, and had 9 to 11 lateral shoots as compared to

4 to 5 lateral shoots on control trees. One-hundred ppm trees were
intermediate. Gibberellic acid treatments increased the total number of
flower buds, but decreased the percent flower buds. The authors con-
cluded that enhancement of shoot growth by GA3 corresponded to a de-
crease in the capacity of the buds to form flowers. Dennis and Edgerton
(1966) suggested that the inhibitory effect of GA3 on flower bud formation
might be dissipated when growth was prolonged. Gibberellic acid inhibited
flower bud formation in 'McIntosh' which flowers only on spurs, but did
not inhibit flower bud formation in 'Rome Beauty' which flowers on long
shoots. Grochowska (1968) replaced seeds in apple fruitlets with growth
regulators, and studied effects on flowering. Naphthalene acetic acid had
a stimulatory effect on flower bud formation, but GA3, at higher concen-
trations, suppressed flowering by 8 percent.

Nitsch (1958) demonstrated the presence of gibberellin-like substances
in apple seeds. These substances were later characterized as gibberellins
A4 and A7 by Dennis and Nitsch (1966). Luckwill, _e_t _al_. (1969) confirmed
the presence of GA4 and GA7 in apple seeds using thin-layer and gas-liquid
chromatography methods. Gibberellin activity first appeared in the seeds

five weeks after full bloom, increased to a maximum concentration nine weeks

44

after full bloom, and subsequently decreased. Hayashi, _et _a_l. (1968)
identified GA3 in parthenocarpic apple fruit by biological response, paper
and thin-layer chromatography and gas --liquid chromatography methods.
An additional component was present having a retention time similar to
that of standard GA4+7. 1

Little information is available on the presence of gibberellins in
vegetative apple tissues. The extraction of GA-like substances from apple
shoots was reported by Kato and Ito (1962). Other reports involved
gibberellins in apple xylem sap. Using the dwarf pea epicotyl bioassay,
Jones and Lacey (1968) found appreciable gibberellin activity in the acid
fraction of both bleeding and stem sap. With the oat leaf base section
bioassay additional active peaks were found, and levels corresponding to
0. 77 pg of GA3 equivalents per liter of bleeding sap were reported. No
gibberellins were identified. Luckwill and Whyte (1968) found gibberellin
activity in stem sap collected in early spring. They reported no activity
in bleeding sap, and low levels of 0.01 to 0.02 pg of GA3 equivalents per
liter of stem sap.

Abscisic acid (ABA) has often been reported ineffective in decreasing
the growth of perennial plants (Weaver and Pool 1969; Meyer, g; _a_l. 1969).
HOlubowicz and Boe (1969) found tint ABA increased the hardiness and

decreased the photosynthetic rate of 'Rome Beauty' apple seedlings. ABA

45

also inhibited the germination of apple seeds, the inhibiting effect ending
when the seeds were rinsed (Pieniazek and Rudnicki 1967).

Luckwill (1957) identified "Malus Inhibitor 2," and differentiated
between this inhibitor and certain phenolic acids. Pieniazek and Grochowska
(1967) later suggested that "Malus Inhibitor 2" was ABA. Davison (1963)
identified zones similar to the "Inhibitor B" zone (Kefford 1955) in xylem
sap from cape honeysuckle, willow and apple. Davison (1965) later reported
similar results using the acid fraction of stem sap assayed with the wheat
coleoptile test. In willow, maximum inhibition occurred in sap collected
at leaf fall and minimum inhibition in sap collected one month before bud-
break. Chromatography indicated the presence of a single substance in this
"B-inhibitor" zone. Milborrow (1967) identified ABA as the main compo-
nent of "inhibitor B. "

Pieniazek and Rudnicki (1967) and Pieniazek and Grochowska (1967)
confirmed the presence of ABA in apple leaves, seeds, fruit juice and xylem
sap using optical roto ry dispersion techniques. Rudnicki (1969) found ABA
to be present in non-stratified apple seeds. His evidence was based on
chromatographic behavior, fluorescence, UV - spectroscopy and growth

inhibition in the wheat coleoptile bioassay.

MATERIALS AND METHODS

Moisture Content of Plant Parts and Top/Root Ratios

Fifteen two year old 'Red Prince Delicious' trees on EM II, on
EM VII and on EM IX were removed and placed in storage. Five trees
on each rootstock were randomly selected and were divided into three
parts: leaves, stems, and roots. Fresh and dry weights, percent moisture,
and top-root ratios were calculated for each. Data were analyzed by analy-

sis of variance and the lsd procedure.

Growth Pattern Studies

 

Ten uniform branches on four trees each of 'Jonathan'/8 inch EM Vll/
Alnarp-Z and 'Jonathan'/seedling were selected and tagged before bud break.
These trees were five years old and their vigor characteristics were very
evident. Current season's terminal growth was measured one week following
full bloom, and subsequeme every two to three days for the remainder of
the growing season. Stem diameter was measured with a vernier caliper

one inch above the 1969 growth.

Injection Experiments

 

A typical injection of exogenous hormones is shown in Figure 1.

One year old trees were potted in a soil-sand-peat mix and grown in the

46

Figure l.

47

Injection Experiment 2, a typical injection experiment.
MM 111 non-grafted trees were injected with one con-
centration of Alar and seven concentrations of ABA,
using 20 m1 disposable syringes separated from the

needles by 10 inch sections of tygon tubing.

_ _ vs.- h-“ -—.#-— _a— —_._—

 

49

greenhouse under 16 hour photoperiods. Supplemental lighting was
provided by two rows of fluorescent lamps (cool-white) between every
two rows of trees, approximately 12 inches above the trees. Lamp
fixtures were adjustable according to the height of the trees.

Only one shoot was allowed to grow per tree.

Solutions were injected using a "Plastipac" disposable 20 ml syringe
with a 10 inch section of tygon tubing between the syringe and the needle
(Yale No. 18GL) (Figure 1). The needle was inserted into the trunk of
the tree at a higher point in the trunk with each injection. Atmospheric
conditions, the availability of water to the plant roots and particularly depth
of insertion were important factors in determining uptake.

Although no preliminary experiments were conducted, uptake and trans-
location probably occurred in the xylem as solution temperature did not
affect uptake, and conditions conducive to high transpiration rates gave maxi-
mum uptake.

Treatments were begun after trees had made approximately 8 to 12
inches of growth. Injections were repeated every three or four days and
records of the amount of solution taken up by each tree were maintained.
The response parameter was usually plotted against the actual number of mg
injected per treatment.

Hormones injected were GA3 (Gibrel, 94.6 percent GA3 - Merck

Chemical Division, Rahway, N. 1.), GA4+7 mixture (Plant Protection, Ltd. ,

50

Surrey, England), ABA (50 percent cis,trans - 50 percent trans, trans -
Research Division, Reynolds Tobacco Company), 6-benzylaminopurine
(Nutritional Biochemical Company) and IAA (Nutritional Biochemical Company).
Solutions were stored ill plastic bottles at ~200C, with the required amount

thawed at each injection date.

Emeriment l

 

The response of both dwarf and vigorous trees to GA3 and ABA was
studied using trees of 'Red Prince Delicious' on EM IX, VII and MM 111
Trees were potted in two gallon plastic containers and arranged in a com-
pletely randomized design. There were four replications per rootstock of
the following three treatments: control (4 ppm of ethanol), 10 ppm of GA3,
and 100 ppm of ABA. Six injections were made between October 2 and
October 21 and the data were recorded October 28. The increase in ter-
minal growth and the diameter of the new shoots one inch from their base
were measured.

Analysis of variance and Duncan's multiple range test were used to
test differences between treatment means. Subsequently, the trees were
placed in storage at 400C for 90 days and then forced in the greenhouse to

determine any effects on flowering.

51

Experiment 2

The effect of ABA on the growth of apple trees was studied using
MMlll non-grafted trees. Trees were growing vigorously in eight inch
pots at the start of treatments. Forty-five uniform trees were selected
and blocked according to size prior to injections. Each tree received
0.003 moles of NH4 nitrogen equivalents and 0.0037 moles of N03 nitrogen
equivalents in 500 ml of water two weeks prior to the initial injection.
Five injections each of 100, 80, 60, 40, 20, 10, 1 and 0 mg/l of ABA
and 10 mg/l of Alar (succinic acid 2,2-dimethyl hydrazide) were made.
Terminal growth measurements were taken 13 times between January 8
and April 9.

Regression analysis was used to determine the relationship between

terminal growth and the concentration of the injected solution.

Experiment 3

One year old trees of 'Red Queen Delicious'/MM 111 were used to
determine the effect of GA3 on ABA inhibition of terminal growth in the
apple tree. Trees were potted in two gallon plastic containers, and were
growing vigorously at the time of treatment. Slight symptoms of iron de-
ficiency were observed, which could not be corrected during the short course
0f the experiment, despite inclusion of a low concentration of chelated iron

in the watering solution. Five injections were made, beginning June 6.

52

Terminal growth was measured at the initial injection, and one week
after final injection. The solutions injected were 0, l, 5, 10, or

15 mg/l of GA3, each concentration plus or minus 50 mg/l of ABA.
The design was a randomized block with four replications per treatment.

Analysis of variance was used to determine treatment effects.

Experiment 4

 

Trees of 'Red Queen Delicious'/EM IX were potted in two gallon
plastic containers in May and grown in the greenhouse for approximately
two weeks, at which time they ceased growth and set "terminal buds"
characteristic of summer dormancy. This experiment tested the effec-
tiveness of certain growth regulators in inducing regrowth in these trees.
Gibberellin A3, GA4+7, IAA and 6-benzylaminopurine (BA) were injected,
each at concentrations of 1, 5, and 10 mg/l. Trees were arranged in
a randomized block design, with four replications per treatment. A total
of five injections were made beginning June 13. Terminal growth was
measured at the first injection and seven days after the final injection.
Stem diameter (lateral growth) was measured seven days after the final
injection. Analysis of variance, orthogonal components and Tukey's pro-

cedure were used to test significance between treatments.

53

Apple Sap: Collection, Purification and Assay

 

Bleeding Sap

 

Bleeding sap was collected from eight year old 'Golden Delicious'/
EM IX and 'Golden Delicious'/EM VII trees. The technique used to
collect sap samples for analysis is illustrated in Figure 2. Trees were
decapitated approximately 12 inches above the ground, the cut being made
V-shaped at a 450 angle. A metal spout was placed immediately below
the "V", and led to a collection bottle immersed in an acetone-dry ice
bath. The frozen sap was collected and the dry ice replenished twice daily.
The sap was stored at ~200C until processed. Sap from two trees of each
combination (two replications) collected from June 6 to 12, 1969, was
used for study.

Dibasic potassium phosphate was added to the sap after thawing to give
a concentration of 0.33 M, and the pH was adjusted to 8 with 5 N potassium
hydroxide. The buffered sap was partitioned three times with petroleum
ether and twice with ethyl acetate. The neutral-alkaline ethyl acetate frac-
tion (Figure 3, Fraction 1) was saved for later assay. All solvents used
in extraction work were glass distilled. The sap was then partitioned three
times with ethyl acetate at pH 2. 5, yielding the acidic ethyl acetate fraction

(Figure 3, Fraction 2) and the remaining aqueous phase. The aqueous phase

54

Figure 2. The procedure for collection of bleeding sap in the
orchard. Sap was frozen from the time it left the

tree to the time of extraction in the laboratory.

 

 

 

56

Bleeding Sap

KHZPO4 added to a concentration of
O. 33 M, pH adjusted to 8.0

Partitioned 3x w1th Petroleum Ether

Petroleum Ether Phase Buffer Phase
(Discarded)

Partitioned 2x with Ethyl Acetate

Buffer Phase Ethyl Acetate Phase
(Fraction 1)

pH adjusted to 2.5
Partitioned 3x with Ethyl Acetate

Buffer Phise Ethyl cetate Phase

(Fraction 2)

Acid Hydrolysis
Partitioned 3x with Ethyl Acetate

\

Buffer Phase Ethyl Acetate Phase
(Discarded) (Fraction 3)

Figure 3. Flow sheet illustrating the procedure for

extraction of bleeding sap.

57

was adjusted to 0.4 N hydrochloric acid for hydrolysis and held in a
water bath at 600C for one hour. It was then adjusted to pH 2. 5 with
5 N potassium hydroxide and partitioned three times with ethyl acetate
yielding the "bound" ethyl acetate fraction (Figure 3, Fraction 3). The
three ethyl acetate fractions were reduced to dryness _ln_ w, redissolved
in 2 ml of methanol, and chromatographed by descending paper chroma-
tography (Whatman No. 3) in isopropanol: ammonium hydroxide: water
(10:1:1 v/v). Chromatograms were divided into 11 zones, including a
control zone below the origin, eluted three times with ethanol, and assayed
with a modified lettuce hypocotyl test (Frankland and Wareing 1960), as
outlined below.

Residues remaining after vacuum evaporation were redissolved in
0.3 ml of ethanol and transferred to 1. 5x6 cm vials containing filter paper
disks. The ethanol was evaporated _i_n_ yggup and five drops of distilled
water were added to each vial and evaporated under vacuum. Approximately

10 lettuce seeds (Lactuca sativa cv. Parris Island) were placed ill each vial

 

following the addition of 0.3 ml of distilled water to each. Seeds, germi-
nated under low light intensity for 24 hours at 22°C, were moved to high
light intensity at the same temperature for four days. Seedlings were dipped
in crystal violet, and the hypocotyls measured to the nearest 0. 5 mm under a

dissecting microscope fitted with an ocular micrometer.

lioassay of GA3 standards enabled a quantitative analysis of the
unknowns. Chromatogram sections below the origin were used as con-
trols, Results were analyzed by analysis of variance and Tukey's

procedure.

(’Ientrifugal Sap

 

The method of Goldschmidt and Monselise (1968) was used to obtain
centrifugal sap. Stem samples of previous season's growth (61) g), of
llHlfUl‘Ill diameter, were collected from five year old "Red Prince DeliciousV’
12 inch EM VIII/’Alnarp-LZ and 'Red Prince Delicious' /seedling trees at in-
tervals during the grmving season. Collection dates in 1969 were April 14
(silver-tip), April 30 (tight cluster), May 13 (full bloom), May 27 (rapid
elongation growth), June 10 (rapid elongation growth), july 5 (many dwarf
shoots summer dormant) and September 30 (onset of winter dormancy). Ill
1970, samples were collected on April 22 (silver-tip), May 6 (tight cluster)
and May 18 (full bloom).

Samples were immediately cut into 5 cm lengths, arranged right—side
up, fully immersed in 20 percent methanol, and centrifuged for 45 minutes
at approximately 1,500 x g in a clinical centrifuge. Since only 30 g of
stems could be centrifuged at one time, remaining stems were stored intact

under refrigeration. All centril‘ugation had been completed within two hours

after sample collection.

5‘.)

In 1969, dwarf and vigorous samples were collected between 8 and
9 a. m. on successive clear days, but in .1970 samples were collected
on the same day, one set between 8 and 9 11.111. and one between 9 and
1-0 a.m. The primary advantage of the centrifugation technique was that
centrifugates were obtained which were relatively pure and highly active
(Goldschmidt and Monselise 1968). Presumably, by centrifugation, the
sap present in the conducting elements and the compounds in the bark
soluble in 20 percent methanol were obtained. Most toxic compounds
resulting from cell rupture were excluded.

The procedure adapted for extraction, purification and bioassay of
centrifugal sap is outlined in Figure 4. Centrifugates were evaporated to
dryness 1.1.1. vacuo and resuspended in 0. 33 M phosphate buffer at: pH 8.
This buffered phase was partitioned twice with both petroleum ether and
ethyl acetate, adjusted to pH 2.5 with concentrated hydrochloric acid, and
partitioned four times with ethyl acetate. The acidic ethyl acetate phase
was evaporated to dryness _il_1 vacuo. Residues were redissolved in 2 ml
of methanol and stored under refrigeration in paraffin-sealed vials.

The gibberellins were purified by thin layer chromatography (TLC)

on Eastman Chromatogram Sheets 1' (No. 6060, silica gel) in isopropyl ether:

 

Distillation Products Industries, Rochester, New York 14603

60

Uniform Stems

Cut to 5 cm Lengths
Immersed in 20% Solution of Methanol

Centrifuged 45 Minutes, 1,500 x g

Stems 20% Methanol Solution
(Discarded)
Evaporate to Dryness in vacuo

l

Resuspended in O. 33 M Phosphate Buffer Solution, pH 8.0

Partitioned 2x with Petroleum Ether

Petroleum Ether Phase Buffer Phase
(Discarded)
Partitioned 2x with Ethyl Acetate

Buffer Phase Ethyl Acetate Phase
(Discarded)

pH Adjusted to 2.5

Partitioned 4x wi h Ethyl Acetate

/

Ethyl Acetate Phase Buffer Phase
(Fraction 1)

Acid Hydrolysis (1970 Samples Only)

Partitioned 4x with Ethyl Acetate

Ethyl Acetate Phase Buffer Phase
(Fraction 2) (Discarded)

Figure 4. Flow sheet illustrating the procedure for extraction of
centrifugal sap.

61

acetic acid (95:5 v/v). Plates were divided into 11 equal sections in-
cluding a control section below the origin, eluted with methanol, and
tested for CA activity in the dwarf pea epicotyl bioassay (Phillips and
Jones 1964). The assay was modified by placing 2 ml of test solution in
each vial and by waiting four days at 220C before measuring. Quantitative
determination of GA activity was made from standard curves of GA3 (Gi-
brel, 94.6 percent GA3, Merck Chemical Co. ). All bioassays were re-
plicated three times, and analysis of variance and lsd were used to test
for differences in GA activity between dwarf and vigorous trees on a given
sampling date.

Attempts to study ABA levels in centrifugates after purification by
TLC failed, as inhibition was noted from Rf 0.2 to 1.0 in all solvent
systems used. This inhibition was not eliminated by successive chroma-
tography in different solvent systems, or by treatment with polyvinylpolypyr-
rolidon (PVP). * In all PVP work, conditions optimal for phenol binding to
PVP as determined by Anderson and Sowers (1968) were followed. The
failure of the PVP treatment to remove the interfering inhibition suggested
that these inhibitors were non-phenolic in nature. No further identification

of these inhibitors was attempted.

_—

Polyclar AT., G.A. F. Corporation, N°Y°

62

Because TLC methods failed to purify ABA in centrifugal sap
samples, silica gel colurrm chromatography (Powell 1964) was utilized.
Glass columns of 17 mm diameter were packed with 8 g of silicic
acid (Mallinckrodt, 100 mesh), previously hydrated with 4. 5 ml of 0.5 M
formic acid, and slurried with hexane. Silicic acid had previously been
washed with distilled water to remove smaller particles which reduced
the column flow rate. Samples were introduced on the column by drying
them on glass wool. Gradient elution with increasing concentrations of
ethyl acetate in hexane was accomplished using two glass cylinders con-
taining equal weights of hexane and ethyl acetate, attached in series and
connected to a Buchler* No. 2-6000 micro-pump to provide hydrostatic
pressure. All solvents were saturated with 0.5 M formic acid. Five ml
fractions were collected on an automatic fraction collector. Preliminary
experiments indicated that ABA was eluted between fractions 23 and 28;
therefore, only fractions 15 through 35 were collected. Fractions were
halved to allow duplicate bioassays for each column and evaporated ‘_11_1_ 113.259.
Five drops of water were added to the residue and rennved under vacuum.
A wheat coleoptile straight-growth assay, adapted from different sources,

was used to test for ABA activity as outlined below.

 

"' Buchler Instruments, Inc., Fort Lee, N. J-

63

Wheat seeds (cv. York Star.) were germinated in the dark for 72 hours.
Coleoptiles approximately 2. 5 cm long and of uniform diameter were selected,
and 4 mm sections were cut 3 mm below the apex with a specially designed
guillotine. Five sub-apical sections were incubated in 0.3 ml phosphate-
citrate containing two percent sucrose (Nitsch and Nitsch 1956) in tubes con-
taining residues from the column elutates. All operations were conducted
under green light. The tubes were placed on a rotating clinostat (1 rpm)
in the dark (Hancock, _e_t_: 11. 1953), and sections were measured after 22
hours. Quantitative determination of ABA concentration was determined from
standard curves of ABA (50 percent cis,trans - 50 trans, trans, Reynolds
Tobacco Co.). All bioassays were replicated twice, and analysis of variance
and lsd were used to test for differences in GA activity bwtween dwarf and
vigorous trees on any given sampling date.

Gas-liquid chromatography (GLC) was done on a Packard 7300 series
gas chromatograph with a six foot glass column packed with two percent
QF-l (fluorinated silicone polymer) on Chromasorb W at 180°C. The eluate
was monitored with a flame ionization detector. Centrifugates and standard
ABA samples were prepared for GLC by methylation with diazomethane
(Arndt 1943; Schlenk and Gellerman 1960).

Optical rotatory dispersion was carried out on a Jasco Model ORD/

UV—5 recording spectropolarimeter. Samples were either used directly off

64

the silica gel column, or after further purification by TLC in benzene:
acetone: formic acid (70:30:1 v/v/v) followed by elution and TLC in
isopropanol: ammonium hydroxide: water (10:1:1 v/v/v).

The location of apple ABA on TLC plates was monitored by co-
chromatographing standard ABA on all plates. Three 1 cm sections
adjacent to and below the standard ABA zone were eluted with ethanol,
dried, resuspended in water and and extracted at pH 2. 5 with ethyl

acetate.

Shoot Extracts: Purification and Assay

 

Activity of Methanol and Aqueous Extracts

 

GA-like substances were extracted from apple shoots using both
methanol (Aung, gt _al. 1967) and aqueous buffer (Jones 1968). Extraction
with aqueous buffers had the advantage of reducing pigment contamination
and preventing the loss of protein-bound gibberellins.

Shoots from 'Red Prince Delicious' trees budded on EM VII in 1968
were sampled on June 26, 1969, and stored at ~200C until extraction.

Methanol extracts (Figure 51: Shoots (15g) were frozen in liquid

 

nitrogen and homogenized in absolute methanol in a Virtis homogenizer.
Following filtration through cheesecloth, the filtrate was centrifuged at

1,500 x g for 30 minutes. The supernatant solution was evaporated

65

Stem Samples
Frozen in Liquid Nitrogen
Homogenized in Absolute Methanol

Filtered Through Cheesecloth

Debfis/ Methanol Solution

(Discarded)
Centrifuged; 30 Minutes, 1,500 x g

Methanol Precipitate
l (Discarded)
Evapora ed i_n_ vacuo

Resuspended in equal volumes of Petroleum
Ether and 0. 33 M KH2P04 Buffer Solution

Buffer Phase
Partitioned 3x with Petroleum Ether
Petroleum Ether Phase Buffer Phase
Partitioned 3x with Buffer

Petroleum Ether Phase Buffer Phase
(Discarded) 1
pH Adjusted to 2. 5

Partitioned 3x with Ethyl Acetate

/

Ethyl Acetate Phase Buffer Phase
(Fraction 1)
Ac1d Hydrolysis

Partitioned 3x with Ethyl Acetate

Ethyl Acetate Phase her Phase
(Fraction 2) (Discarded)

Figure 5. Flow sheet illustrating the procedure for methanol extraction
of apple shoots.

66

311 1332112, and the residue suspended in equal parts (v/v) petroleum ether

and 0. 33 M phosphate buffer at pH 8. 0. The buffer phase was partitioned
three times with petroleum ether; likewise, the petroleum ether phase was
partitioned three times with buffer. The petroleum ether was discarded,

and the aqueous phases combined, and partitioned three times with ethyl
acetate. The pH of the aqueous phase was adjusted to 2.5 with concentrated
hydrochloric acid and partitioned three times with ethyl acetate (Fraction 1).
Acid hydrolysis of the remaining aqueous phase followed by readjustment to
pH 2.5 and partitioning three times with ethyl acetate yielded the bound form
(Fraction 2) of the extract.

Buffer extracts (Figure 6): Shoots (15g) were frozen in liquid nitrogen
and homogenized with "Tris" buffer at pH 7.2 in a Virtis homogenizer. Fol-
lowing filtration through cheesecloth, the filtrate was centrifuged at 15,000 x g
for 30 minutes. The pellet was discarded and the supernatant saturated with
ammonium sulfate and re-centrifuged at 15,000 x g for 30 minutes. The
supernatant was decanted, and the precipitate hydrolyzed in 0. 4 N hydrochloric
acid in a water bath at 600C for one hour. This solution was partitioned three
times with ethyl acetate (Fraction 3). The ammonium sulfate supernatant was
adjusted to pH 2.5 and partitioned three times with ethyl acetate (Fraction 1).
Acid hydrolysis of the resulting aqueous phase followed by partitioning three

times with ethyl acetate yielded Fraction 2. All fractions were reduced to

67

Stem Samples

1

Frozen in Liquid Nitrogen

Homogenized in "Tris" Buffer pH 7. 2

l

Filtered Through Cheesecloth

\

Debris Buffer Solution
(Discarded)
Centrifuged; 30 minutes, 15,000 x g

Buffer Sdfion/ P\re?:ipitate

(Discarded)
Saturated with (NH4)2804

Centrifuged; 30 minutes, 15,000 x g

Precipitate Supernatant
Acid Hydrolysis pH Adjusted to 2. 5
pH Adjusted to 2. 5 Partitioned 3x with Ethyl Acetate

(Fraction 1)
Partitioned 3x with Ethyl Acetate

/ } Acid Hydrolysis
Ethyl Acetate Phase B er Phase
(Fraction 3) (Discarded) Partitioned 3x with Ethyl Acetate

./ \

Ethyl Acetate Phase Buffer Phase
(Fraction 2) (Discarded)

Figure 6. Flow chart illustrating the procedure for aqueous
extraction of apple shoots.

68

dryness 2 vacuo. Chromatography was by gradient elution on silicic acid

columns as outlined earlier. The activity of GA-like substances was

measured in the lettuce hypocotyl bioassay.

GA Activity in Trees of Decreasig Vigor

Current season shoots from trees of 'Red Prince Delicious '/A1narp-2
containing 4, 8, and 12 inch interstocks of EM VIII, and from trees of
'Red Prince Delicious'/seedlings were collected in early August, 1968. These
trees were five years old and exhibited dwarfing characteristics proportional
to the length of the interstock. Leaves were removed, and the shoots
immediately frozen in liquid nitrogen and extracted in methanol. The ex—
traction procedure was identical with that outlined in Figure 6, except tlat
samples were extracted for 49 hours on a shaker after homogenization.
Also, filtration through celite was utilized in place of cheesecloth filtration
and centrifugation because of the large volumes of the extracts.

Phloridzin was removed prior to chromatography by reducing the
sample volume to 0.5 ml of methanol, adding 25 ml of water at 900C, and
refrigerating overnight. GA-like substances were recovered after filtration
by acidifying the solution and partitioning three times with ethyl acetate.
Chromatography was by serial elution from silicic acid columns (Powell

1960). Glass colunms 2.7 cm in diameter were packed with 20 g of silicic

69

acid which was acidified with 12. 5 ml of 0. 5 M formic acid and slurried
in hexane saturated with 0. 5 M formic acid. Extracts from 25 g of fresh
weight of plant material were introduced on the column by drying on glass
wool. Columns were eluted serially with 10 ml each 01’ 0. 5, 5, 10, 20,
30, 40, 50, 60, 70, and 100 percent ethyl acetate in hexane. All solvents
were saturated with 0. 5 M formic acid. Fractions 1 through 9 were com-
bined and re-chromatographed to sharpen the peaks. The fractions re-
covered from the second colurrm were dried i_n__ 29933 and bioassayed in the

dwarf pea epicotyl test.

RESULTS AND DISCUSSION

Moisture Content of Plant Parts, and T gp/Root Ratios
Component weight and top/root ratio data are presented in Table 1.

Table 1. Mean dry weights of roots, stems and leaves, and the top/root
ratios of five randomly selected trees from each of three root-
stocks types, with one scion, 'Red Prince Delicious'.

 

 

 

 

Dry weights (my
Combinations Roots Stems Leaves Top/ root ratios
RPD/EM IX 119a 116a 41a 0. 97
RPD/ EM VII 206b 148a 58a 0. 71
RPD/EM II 255b 155a 52a 0. 62

 

1/ Means in each column followed by unlike letters significantly different
at the .05 level.

Although the only significant difierence occurred between root weights
of the EM IX trees and the EM VII and EM 11 trees, dry weights of roots
and stems increased in order of increasing vigor of the rootstocks. The
data agreed with the known vigor of the three rootstocks in that there was
a larger difference in the dry weights of roots and stems between EM IX
and EM VII than between EM VII and BM 11 trees. The difference between

EM IX and the other trees may have existed because only the EM IX trees

70

71

flowered the second season.

There were no differences in percent moisture between components
from the different trees (Table 2). This was unexpected since correlations
are known to exist between tree vigor and the ratio of living/non-living
cells in the plant tissues. These anatomical differences, however, may be

too small to be detected by gross weight measurements.

Table 2. Mean percent moisture of roots, stems and leaves from five
randomly selected trees of 'Red Prince Delicious' on three
rootstock types.

 

 

 

 

 

Percent moisture-y
Combinations Roots Stems Leaves Average
RPD/EM IV 48 53 35 45.3
RPD/EM VII 48 47 35 43.3
RPD/EM II 51 52 33 45.3
1/

- No significant difference between rootstock types.

Variation was obtained in top/ root (t/r) ratios (Table 1). The large
root systems of the EM ll trees were reflected in the small t/r ratios of
those trees. Results were not in agreement with the hypothesis that t/r

ratios remain constant for a given scion clone on different rootstocks under

72

uniform conditions (Rogers and Vyvan 1934). The data were collected
by sampling very young trees, which may indicate that a limiting root
system may be responsible for initiating "stock effects." As growth

continues, an adjustment takes place between the stock and scion to a

constant t/r ratio.

Growth Pattern Studies

 

Growth rates, calculated from means of terminal growth measure-
ments of dwarf and vigorous trees, are illustrated in Figure 7. These
data are plotted in Figure 8 as the mean difference in the growth of dwarf
and vigorous trees for each measuring date. Individual shoots from dwarf
trees grew at the same rate as the fastest growing shoots from vigorous
trees. A significantly greater number of shoots from dwarf trees, however,
stopped growth early in the season (Figure 9). Approximately 1/3 of the
dwarf shoots became summer dormant in June, but broke "terminal buds"
and resumed growth later in the season. This is seen in Figure 8, where
in mid-July the difference in shoot growth between dwarf and vigorous trees
began to decrease. These data agreed with that of other workers who found
that dwarf trees ceased terminal elongation earlier in the season than vigor-
ous trees. There were no significant differences between mean stem dia-

meters of dwarf and vigorous trees.

73

Figure 7. Mean shoot growth of vigorous and dwarf trees measured
every two to four days during the 1969 season. Ten

shoots on four trees of each type were measured.

vmonous

DWARF

74

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75

Figure 8. Mean shoot growth of vigorous trees minus that of dwarf
trees between each two measuring dates. Ten shoots on

four trees of each type were measured.

76

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Figure 9.

77

The percent non-growing (summer dormant) shoots on four
dwarf and four vigorous trees. Ten shoots on each tree
were measured every two to four days during the 1969

season.

 

78

3.53 oz...n=z<m
$2.582— 52. _ ~22. .22

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79

Although yield data were obtained in 1968 and 1969, correlations
between yields and the growth of dwarf and vigorous trees were meaning-
less since dwarf trees bore their crop on a smaller surface area than
vigorous trees. The significant factor was yields per linear branch foot,
since vigorous trees had a much higher ratio of leaves to fruit. Vigorous
trees produced more shoot elongation than dwarf trees while bearing an

equal or greater weight of fruit.

Injection Experiments

 

Differential Resppnse of Dwarf and Vigorous Trees to GAlmd ABA (Ex-

periment 1)

 

Trees of 'Red Prince Delicious' on EM IX, VII and MM 111 responded
differentially to injections to 10 ppm of GA3. Although uptake varied
greatly from tree to tree at each injection date, there was no significant
difference in uptake between trees over the six injection dates (Table 3).
When terminal growth was used as the measure of response, EM IX trees
were slightly more responsive to GA3 than EM VII trees (Figure 10). Trees
on the vigorous MM 111 rootstock did not respond to 10 ppm of GA3. Re-
sponse, therefore, increased in order of decreasing vigor of the rootstocks.
Differences in response were less marked when lateral growth (stem diameter)

was measured (Figure 11). MM 111 trees were slightly less responsive to

10 ppm of GA3 than were EM IX and VII trees.

80

Figure 10. The response in terminal shoot growth, of one year old
'Red Prince Delicious' scions on three rootstocks to

injections of 10 ppm of GA3 (G) and 100 ppm of ABA (A).

Figure 11. The response in shoot diameter of one year old 'Red
Prince Delicious' scions on three rootstocks to injections

of 10 ppm of GA3 (G) and 100 ppm of ABA (A).

Significantly different from controls (C) at the -01 level.

‘ Significantly different from controls (C) at the .05 level.

Mean Terminal Growth (mm)

81

400
300
200
100 I I
A C G A C G. A C 6
EM IX EMVII MMIII
A6
E
E ,
c 5 i
’ ,
a.
0
E4
.9.
O
0 t
"" r
U! l a
2
A c e A c o A c 6

EM IX EM VII MMIII

82

Table 3. Uptake (ml) of 10 ppm of GA3 and 100 ppm of ABA solutions
by one year old 'Red Prince Delicious' trees on EM IX, VII
and MM Ill. Each number equals the mean of four replications.

 

 

 

 

Rootstocks}!
Treatments IX VII 111
Ethanol (4 ppm) 97 105 131
GA3 (10 ppm) 116 115 128
ABA (100 ppm) 82 87 87

 

1/

— No significant differences in uptake between rootstocks and treatments.

These results may indicate differences in the endogenous levels of
gibberellins available to the scion. They may also indicate a differential
supply of GA by the rootstocks to the scion.

There was no differential response to ABA, as trees injected with
100 ppm of ABA ceased growth and set "terminal buds" characteristic of
summer dormancy (Figure 12). This was of interest because of the
greater tendency of dwarf trees to become summer dormant in the orchard
(Figure 9). This complicated attempts to study differential effects of ABA
because young 'Red Prince Delicious '/EM IX trees grown in the greenhouse
showed a much greater tendency toward summer dormancy than young
'Red Prince Delicious' trees on more vigorous rootstocks. It should be

noted that young trees, which had never flowered, showed the same tendencies

. «“- .

 

 

83

Figure 12. A "Terminal Bud," characteristic of summer dormancy,
induced on a one year old 'Red Prince Delicious'/EM IX

tree by injections of 100 ppm of ABA.

 

84

 

85

towards summer dormancy as older bearing trees in the orchard.
There were no treatment effects on flower bud formation in this

experiment.

Interaction of ABA With the Apple Tree (Experiment 2 and 3)

 

One year old MM 111 non-grafted trees were responsive to injected
solutions of ABA. A concentration of 1 ppm of ABA was inhibitory to
terminal growth, while increasing concentrations of ABA decreased terminal
growth in a linear manner (Figure 13). Terminal shoot extension was in-
hibited almost immediately upon injection; similarly, trees recovered
quickly after injections were terminated (Figure 14). ABA injected trees
quickly resumed growth at the same rate as the non-treated trees after the
last injection.

Unlike 'Red Prince Delicious' scions, MM 111 trees did not set "ter-
minal buds" at high ABA concentrations. Instead, toxicity symptoms
appeared when a concentration of 100 ppm was injected (Figure 15). The
new leaves produced were small, chlorotic, twisted and brittle, and growth
ceased. This condition was reversed and growth resumed when injections
were terminated.

Alar injected at 10 ppm also stopped growth, but the toxicity symptoms
induced by ABA were not evident. Instead, typical responses of apple trees

to Alar including shortened internodes, telescoping foliage and dark green

86

Figure 13. Response, in terminal shoot growth, of one year old
MM 111 non-grafted trees to injections of increasing

concentrations of ABA.

 

87

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.95 1030.:- cotuszogou <n<
r...— o.— n6

 

00

00..

0!.

On—

can

(mun) mucus [nuguuog uoow

88

Figure 14. Terminal shoot growth curves for MM 111 non-grafted
trees injected with 4 ppm of ethanol (controls), 100
ppm of ABA, and 10 ppm of Alar. Injection dates are

indicated by small arrows.

89

800

CONTROL

AaA (Iooppm)

600

O

o
A.

.EE. .2326

.05-Ea...

200

ALAR (10.3me

60 80 100
Days aflar Initial Inlactlan

40

2O

Figure 15.

Figure 16.

90

Response obtained from five injections of a 100 ppm
solution of ABA (approximately 3 mg of ABA) on one
year old MM 111 non-grafted trees, showing toxicity

symptoms at the growing point.

A comparison of one year old MM 111 non-grafted trees
injected with 100 ppm of ABA (left), 4 ppm of ethanol
(controls) and 10 ppm of Alar (right). The ABA injected
trees had stopped growing, and new leaves were chlorotic
and twisted, while Alar injected trees had shortened inter-

nodes, telescoping foliage and dark green coloration.

 

92

coloration were evident (Figure 16). Although Alar was injected at a 10-
fold lower concentration than the highest level of ABA, trees required a
longer period of time to overcome Alar-induced growth inhibition than
ABA-induced growth inhibition (Figure 14). ABA, unlike Alar, is an
endogenous plant hormone which may explain why trees overcame its
effects more readily.

If the uptake of injection Solutions depends on transpiration, a
measure of this rate of uptake may be an indication of transpiration rate,
other factors, i.e., depth of needle insertion, plugging, and atmospheric
and soil conditions being constant. Total uptake from five injections was
plotted against concentrations of the injected solutions in Figure 17. The
scatter was possibly due to plugging of the needle either by insertion or
increased cell division around the injury. Nevertheless, there was a de-
finite trend toward decreased uptake at higher ABA concentrations. This
trend was significant, since it was equally evident on the first as well as
on the last injection date. This would not be true if differences were
caused by decreased growth and vigor of trees receiving the higher con-
centrations of ABA. Size differences between trees receiving different
treatments were not great enough to account for differences in uptake.
Also, only those trees receiving the highest concentration of ABA (100 ppm)

showed any visible toxicity symptoms. Little and Eidt (1968) previously

Figure 17.

93

Total uptake (ml) vs concentration of the ABA solution
injected. Solution uptake was inversely proportional

to ABA concentration.

*- i _ _.l,=< l“

Uptake (ml)

Total

340

320

300

280

260

240

220

94

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ABA Concentration

60 80
lnioctod (mg)

 

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95

reported an effect of ABA on transpiration in two softwood and two hard-
wood species.

GA3 overcame ABA-induced inhibition of apple shoot growth (Table 4).
These data support the theory that GA3 and ABA may interact to control
certain aspects of plant growth and development (Wareing 1968; Thomas
1965; DeMaggio and Freeberg 1969). A further discussion on the signifi-
cance of the ABA/GA ratio and its role in dwarfing will be given later.

Table 4. Growth response of one year old 'Red Queen Delicious '/MM 111
apple trees to ABA in the presence of increasing concentrations

 

 

 

of GA3.
Terminal growth Percent

Treatments (ppm) Net increase (mm)l/ of controls
Control (4 ppm EtOH) 206

GA3 (15) 228 111
ABA (50) 85 41
ABA (50) + GA3 (l) 87 42
ABA (50) + GA3 (5) 118 57
ABA (50) + GA3 (10) 186 90
ABA (50) + GA3 (15) 194 94

 

1/ Means of four replications.

96

Interaction of GA3, GA4+7, BA, and IAA With the Apple Tree (Experiment 4)

No information was obtained on the ability of the various treatments
to induce shoot growth from summer dormant buds because all trees re-
sumed growth, regardless of treatment received, during the course of the
experiment. Interesting qualitative information and data on terminal and
lateral growth were obtained. Means for total uptake and lateral and ter-
minal growth are given in Table 5. No significant differences were found
in total uptake between treatments. In T ukey's test, treatment means for
terminal growth were significantly different only between the highest con-
centration of IAA, which was strongly inhibitory, and the highest concentra-
tion of GA4,+.7, which was promotive. Gibberellins A4+7 promoted lateral
growth, and the amount of growth induced at 5 and 10 ppm was significantly
greater than the amount induced by all other treatments except the highest
concentration of GA3 (Figure 18). Indole-3-acetic acid exhibited increasing
inhibition of lateral growth with increasing concentration.

Meaningful orthogonal comparisons included GA3 vs GA4+7 and controls
vs the highest concentration of IAA. Gibberellins A4+7 were not significantly
more effective than GA3 in promoting terminal growth (Figure 19). T er-
minal growth of trees injected with 10 ppm of IAA was significantly less

than terminal growth of untreated trees at the .01 level (Figure 20).

97

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98

Total increase in lateral growth (shoot diameter) of
four one year old 'Red Queen Delicious'/EM IX trees
to three concentrations each of GA3, GA4+7, IAA, and

BA applied in five injections four days apart.

 

99

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100

Figure 19. Total terminal shoot growth of four one year old
'Red Queen Delicious'/EM IX trees to three concen-
trations each of GA3 and GA4+7 applied in five

injections four days apart.

 

 

 

101

(mm)

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Growth

Terminal
Treatments

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Figure 20.

Total terminal shoot growth of four one year old
'Red Queen Delicious'/EM IX trees to three concen-

trations of IAA applied in five injections four days apart.

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The effects of 5 and 10 ppm of GA4+7 are depicted in Figure 21.
Unlike GA3, GA4+7 at those concentrations inhibited the activity of the
apical meristem of the main shoot while simultaneously promoting lateral
bud break and rapid growth of one or more side shoots. New leaves
formed on the main shoot of GA4+7 injected trees were small and mal-
formed. No adverse effects of GA4+7 injections were noted on side
shoots. Main shoots resumed normal growth quickly when injections were
discontinued. Assuming the effects, although not typical, were induced by
GA4+7, interesting speculations are possible. Stimulation of lateral bud
break and side shoot growth at the expense of the main shoot may indicate
a role in apical dominance for either GA4, GA7 or both gibberellins.
Further experimentation using more highly purified material should be
carried out in this area.

A cytokinin, 6-benzylaminopurine, had no effect on either extension
or lateral growth. Injections of 5 and 10 ppm of BA, however, promoted
axillary bud break over the entire length of the shoot with buds developing
into spurs and lateral branches (Figure 22). Multiple spray applications of
BA were reported to promote bud break on apple and pear shoots (Poll 1968).
Benes, gt_ a. ([965), Pieniazek and Jankiewicz (1966), and Williams and Stahly

(1968) used cytokinins to break dormancy and rest in apple buds. Williams

Figure 2 1.

Figure 22.

105

'Red Queen Delicious'/EM IX trees injected with 5 ppm
of GA4+7 (left) and 10 ppm of GA3 (right). Note the

small, malformed leaves and side shoots on the GA4+7
treated tree. Trees had resumed normal growth at the

time photograph was taken.

'Red Queen Delicious'/EM IX trees injected with 5 ppm
of BA. All main shoot foliage was removed to show spur

and side shoot development typical of cytokinin effects on

apple.

 

 

 

107

and Stahly (1968) used two synthetic cytokinins -- N(purin-6-yl)phenylglycine
and 6-benzylamino-9-(tetrahydropyran-Z~yl)-9H-purine - to overcome apical
dominance and promote axillary bud growth on actively growing apple shoots.
Our work corroborated these findings.

Dwarf and vigorous trees differ primarily with regard to precocity
and habit of growth. Although no information was obtained relating to
flower bud initiation, many growth habit characteristics of dwarf trees are
not unlike those induced in this experiment with BA and GA4+7. Gibberellin
A3 promoted terminal growth; GA4+7 promoted terminal and lateral growth
and differentially favored lateral shoot growth to terminal shoot grOWth;

BA broke apical dominance producing spurs and lateral branches; and IAA
promoted growth at lower concentrations and inhibited growth at the higher
concentration.

Activity of Gibberellin and Abscisic
Acid-Like Substances in Apple Sap

 

Activity of GA-Like Substances in Bleeding Sap
There were 0.068 g of GA3 equivalents in 125 ml of bleeding sap
from 'Golden Delicious '/ EM IX trees, and 0.079 13 of GA3 equivalents

in 125 ml of bleeding sap from 'Golden Delicious'/EM VII trees. In Table 6,

108

 

 

 

 

Table 6. GA3 equivalents (ug) in three fractions extracted from
125 ml of bleeding sap from 'Golden Delicious' trees
on EM IX and on EM VII. Means, calculated from
the total activity on each Chromatogram, represent
two replicate trees on each rootstock.

. . 1/

Rootstock Fraction GA3 equivalents (1g)-

EM IX 1 (neutral) 0.020

EM VII 1 (neutral) 0.036

EM IX 2 (acid) 0.029

EM VII 2 (acid) 0.027

EM IX 3 (hydrolyzed acid) 0.019

EM VII 3 (hydrolyzed acid) 0. 016

1/

- No significant differences between means.

109

total activity is sub-divided into the contributions of the three fractions
extracted. Statistical analysis indicated that dwarf and vigorous trees had
similar levels of activity in all fractions, and that the neutral and hydrolyzed
acid fractions were not significantly lower in activity than the acid fraction.
Gibberellin activity in the hydrolyzed acid fraction was presumably due to
"bound gibberellins, " chemically identified as glucosyl-gibberellins in higher
plants, mostly from immature seeds (Tamura, gt 31. 1968). Bound gib-
berellins have also been identified in the bleeding sap of other trees (Sembdner,
g3 a_l. 1968), and a role for storage has been assigned to gibberellins cir-
culating in the xylem (Bowen and Wareing 1969). According to Lang (1970),
"Our understanding of the physiological role of bound gibberellins is still
quite fragmentary, but evidence for two functions seems to be emerging,
namely, as a storage and transport form. "

The role of gibberellins in the alkaline-neutral fraction is obscure.
Gibberellin activity in the neutral fraction has been reported from such
diverse sources as potato peelings, grape berries and bean seeds by a
number of workers (Hayashi, at al. 1968; Weaver and Pool 1965; Hashimoto
and Rappaport 1966 a and b; Iwahori, §_t:_ a_1_. 1968).

Hayashi and Rappaport (1966) crystallized and partially identified

Potato Factor II, a stimulator of dwarf peas, from the neutral fraction of

110

potato extract. They determined Potato Factor II was a non-GA-like
compound, similar to certain of the fatty acid derivatives shown by
Stowe and Obreiter (1962) to stimulate growth of excised pea epicotyl
sections. Potato Factor 11 differed from these derivatives in that it
stimulated growth of intact pea plants. There is the possibility, there-
fore, that activity in the alkaline-neutral fraction was due to substances
unrelated to gibberellins.

There is evidence that interconversions occur between acidic and
bound gibberellins and these neutral compounds. Hayashi and Rappaport
(1965) reported that a chemically neutral substance, Potato Factor I was
converted in Egg to an acidic GA-like substance. Hashimoto and Rappa-
port (1966b) found that when mature green seeds of two bean cultivars were
incubated with GAl prior to extraction and bioassay, there was a striking
increase in the activity of the neutral fraction. This increased activity
was accompanied by decreased activity in certain areas of the acidic and
bound fractions. Hashimoto and Rappaport (1966a) suggested that neutral
substances in bean seeds may be a reserve form of gibberellins since they

were present in fairly large amounts in mature dry seeds.

Gibberellin Activity in Centrifugal Sap

 

A GA3-like substance was identified in centrifugal sap using the dwarf

111

pea epicotyl bioassay. This GA was characterized on the basis of

migration on TLC plates in three different solvent systems (Table 7).

Table 7. A comparison of the Rfs of GA3, GA4+7, and one
of the centrifugal sap gibberellins in three solvent

 

 

 

 

systems.
Solvent systemsl/
Compound 1 2 3
Centrifugate 0. 07-0. 13 0. 33-0. 40 0. 07-0. 13
GA3 0. 08 0. 37 0. l4
GA4,+7 0. 26 0. 73 0. 42

 

1/

— Solvents: l, di-isopropyl ether/ acetic acid (95:5); 2, benzene/
n-butanol/ acetic acid (75:20:5); 3, chloroform/ethyl
acetate/acetic acid (60:40:5).

Gibberellins A3, GA4, and GA7 were previously identified in at
least some part of the apple tree. No GA4 or GA7 activity was identified
in centrifugates in this work. The dwarf pea epicotyl bioassay, however,
proved to be 10 times less sensitive to GA4+7 than to GA3. The lettuce
hypocotyl test showed the presence of another GA in centrifugates at an Rf
similar to that for GA4+7 in isopropyl ether; acetic acid (95:5 v/v). No

further work was done with this GA4+7-Iike substance.

112

Levels of the GA3 -like substance extracted from the equivalent of
1.5 g of centrifuged dwarf and vigorous stems are illustrated in Table 8.
together with the stage of shoot development at each sampling date.
Table 8. The stage of shoot development and levels of a GA3-like sub-

stance in centrifugal sap from 1. 5 g of stem samples taken
from dwarf and vigorous trees on each sampling date in 1969.

 

 

 

 

Sampling Stage of shoot Vigor GA3 1
date development type equivalents (pg)-/
4 / 14 silver-tip dwf 0. 000
vig 0. 000
. 2/
4/30 t1ght cluster dwf 0. 055'"
vig 0. 007
5/ 13 full bloom dwf 0. 005
vig 0. 005
5/ 27 rapid shoot elongation dwf 0. 003
vig 0. 001
6 / 10 summer dormancy dwf 0. 001
vig 0. 001
7/ 5 summer dormancy dwf 0. 000
vig 0. 000
9/ 30 winter dormancy dwf 0. 001
vig 0. 001
1/

- Means of three replications

-2-/ Difference significant at .01 level

113

These values represented the means of three replicate bioassays. The
only significant difference in sap activity occurred on April 30. On
that date activity in dwarf stems was 7.64 times greater than activity
in vigorous stems.
Levels of this GA rose early in spring and reached a peak at the tight
cluster stage of shoot development. Levels then decreased gradually over
the next two months and disappeared completely in early July. A secondary
rise in activity occurred in early fall prior to the onset of winter dormancy.
The function of this GA in centrifugal sap poses a complex question.
Activity is high, particularly on April 30 when levels up to 0.05 pg of GA3
equivalents were measured in sap from 1.5 g of stems. That GA3 could have
a role in shoot elongation was demonstrated in the injection experiments. Kato
and Ito (1962) suggested that gibberellins were involved in the shoot growth
process. They extracted larger amounts of GA-like substances from vigorous
shoots than from weak or non-growing shoots. Luckwill and Whyte (1968) ob-
served that the cessation of shoot growth by late July in 'Red Stoke' apple
seedlings and by September in the more vigorous 'Cheddar Cross' seedlings
corresponded in both cultivars to the time when Substance K (a cytokinin) and
Substance A (unidentified) disappeared from the xylem sap. If the GA3-like

substance, identified in centrifugates, was solely responsible for shoot growth,

114

we should have found higher levels in the vigorous trees. The possibility
exists that we were not examining metabolically important hormones, but
that hormones occur in xylem sap for storage and eventual breakdown.
Bowen and Wareing (1969) found that some breakdown of GA3 and considerable
degradation of kinetin occurred when these hormones were applied to the
xylem of rooted willow cuttings. There is good evidence that gibberellins
present in the xylem stream are produced in the roots (Carr, _e_t_il. 1964;
Phillips and Jones 1964; Jones and Phillips 1966; Sitton, gt ;_a._l_. 1967; Jones and
Lacey 1968; Reid and Burrows 1968; Reid, gt _a_1_. 1969). It is feasible, there-
fore, to propose that GA metabolism plays a role in dwarfing either through
differences in production by dwarfing and vigorous rootstocks, or by inacti-
vation of GA activity by dwarfing interstocks as gibberellins pass through them.
If, however, gibberellins responsible for promoting shoot growth are produced
by young leaves, as suggested by Kato and Ito (1962) when they inhibited shoot
growth by removing the young upper leaves, then GA relations could not be
solely responsible for the dwarfing effect of rootstocks and interstocks. Re-
moval of leaves, however, would alter many other parameters affecting growth.
The difference in activity between dwarf and vigorous trees occurred at
a stage when there was little morphological or developmental difference be-

tween the two types of trees. GA responses usually take place in the presence

115

of the hormone. Nevertheless, hypotheses can be proposed. Nanda and
Purohit (1965) showed that GA3 initiated the release of sugars from starch

in twigs, and Lee and Rosa (1969) showed that GA3 regulated starch and

sugar levels in tobacco leaves. Gibberellins, then, may affect growth by
influencing carbohydrate relations in the stem. The final answer will doubtless
be a complex one. Reid and Burrows (1968) suggested that "The activities of
cytokinins and gibberellins in bud burst are likely to be diverse since they are
involved in the regulation of phenomena ranging from the mobilization of
assimilates and inorganic substances to cell division and protein synthesis. "
The same statement could be repeated with growth regulators and the dwarfing
phenomenon.

One important hormonal interaction which may be important in dwarfing
is the growth promoter/inhibitor ratio, since there is good evidence that this
ratio may be an important control mechanism in the growth and reproductive
physiology of some plants. Working with the short day (SD) plant , black currant,
under long day (LD) conditions, Wareing (1968) was able to induce cessation of
growth and the formation of typical bud scales at the growing point with ABA
applications. Similar results were obtained with birch and sycamore seedlings.
However, if gibberellins were applied together with ABA the plants continued

to grow normally. The effect of ABA was overcome by GA. Endogenously

116

these hormones acted similarly; the level of ABA decreased and that of
GA increased as dormancy proceeded from January to March. The same
hormone balance seemed to regulate flowering. Inhibitor levels increased
while GA levels decreased prior to visible signs of flower initiation. ABA
when applied to black currant under LD conditions at 20 ppm daily for four
weeks caused cessation of terminal growth and the early stages of flower
initiation in axillary buds. Under SD conditions flowering was inhibited with
GA3 applications. ABA also induced flowering in strawberry, but had no
effect on many other SD plants. Because ABA reduced the endogenous GA
level in black currants, as did (2 -chloroethy1) trimethylammonium chloride
which also promoted flowering, Wareing suggested that the decrease in endo-
genous gibberellins, compounds inhibitory to flower bud initiation in the apple,
might be responsible for decreased growth and initiation of flower buds.
DeMaggio and Freeberg (1969) found that whole buds from dormant maple trees
were stimulated by GA3 under both LD and SD conditions. ABA inhibited this
GA-stimulated bud growth.

Gibberellins and ABA are endogenous hormones in the apple tree. Gib-
berellins promote growth and inhibit flowering, while ABA inhibits growth of
apple trees. Commercial gibberellic acid will overcome this inhibitory effect

of ABA. Although ABA did not induce flower bud initiation in young apple trees

117

under our experimental conditions, a study of ABA levels in centrifugal sap
samples was undertaken to determine the nature of the endogenous ABA/ GA

interaction.

Inhibitor Activity in Centrifugal Sap

 

A zone of strong inhibitory activity in the wheat coleoptile bioassay, eluted
from columns in the same fraction as standard ABA, was found in centrifugal
sap. Further evidence that the endogenous inhibitor was ABA was obtained by
co-chromatographing, using GLC, 2 pg of standard ABA with the equivalent
of 2 1g of the inhibitor from centrifugal sap. Only one peak was obtained
at the retention time for ABA, with a peak height equivalent to 4 ug of ABA.

No interfering peaks were present.

Negative results were obtained in attempts to characterize ABA using
ORD methods, probably because the concentration of ABA in centrifugal sap
samples was too low for detection.

Levels of the ABA-like inhibitor in centrifugal sap obtained from the
equivalent of 3 g of dwarf and vigorous stems, measured in the wheat coleoptile
bioassay, are illustrated in Table 9 together with the stage of shoot development
at each sampling date.

Dwarf shoots were higher in activity on all sampling dates excepting

July 7 and September 30 when differences were negligible. Differences in ABA

118

Table 9. The stage of shoot development and the level of an ABA -like
inhibitor in centrifugal sap from 3 g stem samples, taken
from dwarf and vigorous trees on each sampling date.

 

 

 

 

Sampling Stage of shoot Vigor ABAl/
Year date development type equivalents (pg)
1969 4/ 14 silver-tip dwf 0. 67
vig 0. 45
4 /30 tight cluster dwf none detectable
Vig I! H
5/13 full bloom dwf 0. 55
vig 0. 15
5/27 rapid shoot dwf 0. 33
elongation vig 0. 03
6 / 10 summer dormancy dwf 0. 47
vig 0. 14
7/ 5 summer dormancy dwf 0. 04
vig 0. 07
9/30 winter dormancy dwf 0. 06
vig 0. 14
19 70 4 / 22 silver -tip dwf none detectable
vig H M
5/6 tight cluster dwf " "
vig I I '3
5/18 full bloom dwf 0. 67
vig 0. 50
1/

- Means of two replications. There was no significant difference between
dwarf and vigorous trees on any sampling date.

119

levels between dwarf and vigorous trees began to increase at approximately
full bloom, reached a peak two weeks after full bloom, and finally de-
creased to a low in July. Highest ABA levels occurred before bud break,
and during the period of summer dormancy. On April 14, buds on both
dwarf and vigorous trees were at the same stage of development, and

ABA levels were approximately equal. Between May 27 and July 5, how-
ever, many more dwarf shoots than vigorous shoots became summer dor-
mant while ABA levels were higher in dwarf than in vigorous stems. The
high level of ABA in the sap of dwarf trees during mid-summer, therefore,
may be partly responsible for summer dormancy in those trees. The fact
that summer dormancy was induced in young trees of the same cultivar by
ABA injections into the xylem stream was further evidence for this.

It was interesting that ABA levels were high when GA levels were low
and vice versa. This was particularly noticeable on April 14 and 30. Since
good separation of the two substances was obtained by chromatography, they
could not have been interacting in the bioassays. Also, GA3-induced elonga-
tion of dwarf pea epicotyls was not inhibited by ABA except at very high
ABA concentrations (1 Hg), and GA3 was not effective in overcoming ABA-
induced inhibition of wheat coleoptile elongation.

Several different cultivars were utilized in the study. For example,

120

growth curves were taken from 'Jonathan' trees, while centrifugal sap
was collected from 'Red Delicious' trees. These trees, however, were
the same age and were located in the same orchard. Finally, only an
ABA-like inhibitor and a GA3-like GA were studied. Surely auxins and
cytokinins also play a role in growth and flowering processes, not to
mention the additional GA present, tentatively identified as a GA4+7-
like substance. For these reasons, caution must be exercised in inter-

preting results.

Activity of Gibberellin-Like

 

Substances in @ple Shoot Extracts

 

Activity of Methanol and Aqueous Extracts

 

The 32 fractions from each of the five columns in this experiment
were tested in the same bioassay, and the standard deviation for the
bioassay was subtracted from the value for each fraction. Only activity
greater than the standard deviation is illustrated in Figure 23.

The methanol (Histogram B) and aqueous (Histogram C) ethyl acetate
free fractions had several active areas in the lettuce hypocotyl assay. No
attempt was made to characterize active substances. The methanol extract
showed higher activity between fractions 13 and 18. Other active zones

showed less difference in activity between methanol and aqueous extracts.

Figure 23.

121

Histograms illustrating the activity of GA-like sub-
stances in aqueous and methanol extracts of shoots

from one year old 'Red Prince Delicious'/EM VII trees.
The standard deviation (3.7 mm) for the assay was
subtracted from the value for each column fraction.
Fractions showing no activity prevented seed germina-
tion, or hypocotyl growth was equal to or less than the
standarddeviation. The histograms are A, MeOH bound
fraction; B, MeOH free fraction; C, aqueous free fraction;

D, aqueous bound fraction; E, aqueous NH4SO4 fraction.

JquflN uouoou

”th" nouaou

122

Hypocotyl Hypoco'yl
Growth (mm) Growth (mm)

9! 3|

Hypocofyl
Growth (mm)

'3

 

123

Less activity occurred in the ethyl acetate bound fractions. Most
interesting was the aqueous ammonium sulfate precipitated fraction
(Histogram E), which showed the usual peak over colurrm fraction 18,
plus considerable activity in column fractions obtained earlier than 18.

Jones (1968) gave evidence to show that protein-GA complexes
were precipitated and lost when organic solvents were used for tissue
extraction. That activity differences in the acid fraction, between aqueous
and methanol extracts, were present only in one zone, was evidence that
the activity in the ammonium sulfate fraction was due to precipitated pro-
tein-GA complexes and not to contamination from the free or bound frac-
tions. Although protein bound gibberellins have been reported by a number
of workers (McComb 1961; Jones 1964; Hayashi and Rappaport 1962; Rein-
hard and Sacher 1967; Jones 1968), Lang (1970) recently pointed out the
lack of entirely convincing proof for their existence. He stated that "Pre-
cipitation of low-molecular compounds together with protein is a common,
non-specific phenomenon and can be observed with e. g. , gelatine solutions. "

If it is assumed that the activity in the ammonium sulfate precipitated
fraction belonged in the free fraction, then there was little difference in

activity obtained by extraction with either methanol or "Tris" buffer. If,

however, the activity in the ammonium sulfate precipitated fraction was due

124

to hydrolyzed protein-GA complexes, then a considerable portion of the
total GA activity was lost by extracting with methanol. Buffer extractions,
then were less efficient in obtaining free GA-like substances from the
acid fraction.

Activity in Trees of Different Vigor

 

No differences were found in the activity of GA-like substances, in
the dwarf pea epicotyl bioassay, between shoots collected in early August
from five year old trees representing four gradations of vigor. The dwarf
pea epicotyl bioassay was a good choice for this study because the pea
epicotyls measured activity in heavily contaminated column fractions. As
mentioned before, however, this assay was lO-fold less sensitive to GA4,+7
than to GA3.

Figure 24 represents the average of the data obtained from the four
'types of trees utilized in this study. These histograms represent the
activity in 25 g fresh weight of shoots. The presence of several distinct
GA-like substances was confirmed by TLC, but no gibberellins were charac-
terized. It should be noted that activity in the bound fractions was as high
or higher than activity in the free fractions. If one speculates on a correla-
tion between growth and GA activity, the decrease in growth occurring in

August may be related to the large percentage of the total gibberellins found

‘ l

 

125

in the bound form at that time.

No further work was done with extracts because it was felt that
the problems encountered in purification might make it impossible to
detect small differences between dwarf and vigorous samples. In this

respect, xylem sap was far superior to extracts.

Figure

[0

126

Histograms showing the activity in 25 g fresh weight

of shoots collected from five year old 'Red Prince
Delicious' trees in August. Because there were no
differences between trees of increasing vigor, the
average of the data obtained from the four types of
trees is represented. Shaded portions of the histograms

represent activity above the standard deviation.

127

 

Bound

.001 ug

mmmmmws

mwmmmmms

Gibberell

6
4
120

Houanoo co «use you

100

raction Number

F

SUMMARY

Differences in growth characteristics of dwarf and vigorous apple
trees are well understood, but the mechanism by which rootstocks and
interstocks dwarf scions is not known. Since characteristics associated
with dwarfing are related to growth and flowering, a role for plant
hormones in the dwarfing mechanism was hypothesized.

No significant quantitative or qualitative differences in gibberellins
were found in shoot extracts made in August from trees representing four
degrees of vigor. Experiments indicated little difference between extrac-
tion with methanol or buffer solutions. Bleeding sap collected in June
from trees of low and intermediate vigor showed no quantitative or quali-
tative differences in gibberellins. Centrifugal sap collected from dwarf
trees at the tight flower cluster stage of shoot development contained a
significantly higher level of a GA3-like substance than did centrifugal sap
collected from vigorous trees on the same date. Levels of an ABA-like
inhibitor were also higher in centrifugal sap collected from dwarf trees
than in centrifugal sap collected from vigorous trees in early June.

Terminal growth measurements showed that shoots grew at equal
rates, but that dwarf shoots ceased growth earlier than vigorous shoots.
Early growth cessation and high ABA levels occurred simultaneously in

dwarf trees.

128

129

One year old trees on rootstocks of different vigor responded
differentially when injected with 10 ppm of GA3. There was no
differential response to ABA injections due to summer dormancy in
dwarf trees.

ABA reduced the terminal growth of one year old trees proportional
to the concentration injected. One hundred ppm of ABA was temporarily

I!

toxic to MMlll trees, whereas it induced "terminal buds characteristic
of summer dormancy in 'Red Prince Delicious' trees. Gibberellin A3
overcame ABA-induced inhibition of terminal growth.

Gibberellins A4+7 were more effective than GA3 in promoting terminal
extension and lateral growth when injected in one year old trees. Gib-
berellins A4,+7 were toxic to the terminal growing point, and induced growth
of lateral buds. Although BA did not alter growth rate, it overcame apical
dominance and caused axillary buds to produce spurs and lateral branches.
Indole-3-acetic acid promoted growth at 1 and 5 ppm, but inhibited growth
when injected at 10 ppm.

Dry weights of stems, roots and leaves of two year old trees in-
creased in order of increasing vigor of the rootstocks. There were no
differences in the percent moisture of plant parts from the various trees.

T op/root ratios were not constant for one scion cultivar on different root-

stocks. This result did not agree with studies on older trees by other

workers.

130

Future research on the role of growth regulators in dwarfing should

include the following studies:

1.

Examination of the seasonal levels of the GA4+7~like substance

in centrifugal sap.

Extraction of gibberellins from buds at the tight cluster stage

of shoot development.

Extraction of ABA from summer dormant vs non-summer dormant
shoots.

Injections of GA3 above and below dwarfing interstocks, measuring
effects on terminal growth.

Examination of the levels of endogenous gibberellins above and
below a dwarfing interstock.

Examination of the levels of other plant hormones in the centri-
fugal sap of dwarf and vigorous trees.

Definite characterization of the plant hormones studied.

Control of tree Vigor and flowering by injections of factorial

combinations of plant hormones.

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