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An'm '_ 's.‘ 33.2333 ”3‘.
THESIS ,- s.
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This is to certify that the
dissertation entitled
EVALUATION OF CHEMICALS FOR FLORAL INDUCTION AND
STALK ELONGATION IN SUGARBEET (BETA VULGARIS L.)
presented by
Martin D. Mahoney
has been accepted towards fulfillment
of the requirements for
Ph.D. d . Crop & Soil Sciences
egree in
< T 3
:_\_'{,"'2-14véNf/L L__d _
Major professor “2
Date June 20, 1982

 

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

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EVALUATION OF CHEMICALS FOR FLORAL INDUCTION AND

STALK ELONGATION IN SUGARBEET (BETA VULGARIS L.)

 

By

Martin D. Mahoney

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Crop and Soil Science

1982

ABSTRACT

EVALUATION OF CHEMICALS FOR FLORAL INDUCTION AND STALK

ELONGATION IN SUGARBEET (Beta Vulgaris L.)

 

by
Martin D. Mahoney

Floral induction and stalk elongation of sugarbeet (@223
vulgaris L.) were evaluated after applications of gibberellic
acid (GA3) at various photoperiods. Combination of GA3 with
plant hormones or hormone-like chemicals ethephon.[(2-
chloroethyl)phosphonic acid], 2,H-D[2,U-dichlorophenoxy)-
acetic acid], NAA(a-napthaleneacetic acid), and kinetin
(6-furfurylaminopurine), and herbicides reported to alter
plant lipid metabolism were also evaluated. GA3 applications
in combination with photoperiods of 18/6, 24/0 hr (day/night)
or lA/lO hr plus a 2-hr nightbreak substantially increased
flowering over the untreated controls. Growth chamber,
greenhouse or field application of GA3 in combination with
ethephon, 2,A—D, kinetin, members of thiocarbamate,

acetanilide, and benzoic acid herbicide classes, naptalam

(N-l-naphthylphtahalamic acid), TCA (trichloracetic

 

Martin D. Mahoney

acid), ethofumesate (2-ethoxy-2,3-dihydro—3,3-dimethyl-Sg
benzofuranyl methanesulfonate), dalapon (2,2-dichloro-
propionic acid), and glyphosate [N-(phosphonomethyl) glycine]
resulted in a synergistic increase in stalk elongation,
but not floral induction. Uptake of ltic-GAG by sugarbeet
foliage was not increased by pretreatment with alachlor
(an acetanilide herbicide) and did not explain that inter-
action.

GA3 may substitute for the cold temperature when foliar
applications of GA3 induce biennials to flower. A shift
in the fatty acid composition of membranes may be involved.
Low temperature, alachlor, 6A3, and GA3 plus alachlor
decreased the saturated and increased the unsaturated fatty -
acid composition of both mitachondrial and plasmalemma
membrane fractions. The unsaturated fatty acid content
of plasmalemma membranes of annual and florally induced
biennial sugarbeets increased with time, whereas the
mitochondria fraction showed no change. In non-induced
plants, there was a shift toward greater fatty acid
unsaturation in mitochondria but not plasmalemma membranes.
Fall applications of alachlor and vernolate which produce
similar effects as low temperature on plant cell membranes
increased the survival rate of sugarbeets in one study,
which suggests that these materials may aid in the cold
hardening of plants. In a second study, sugarbeet
survival in the absence of chemical treatments was too high

to adequately assess the chemical affects.

To my wife Chris and my sons Michael and Ryan

11

ACKNOWLEDGEMENTS

I am extremely grateful to Dr. Donald Penner for
giving me this opportunity, for his excellent guidance,
and for the use of his laboratory facilities. I would
like to thank Dr. George Hogaboam for his guidance
and for providing the financial assistance for this
project. :1 would alSo like to thank Dr. Anton Lang,

Dr. Alan futnam and Dr. Joe Saunders for thier guidance,
suggestions, and constructive criticism of the experi-
ments.

The technical assistance of Cathy Arne, Carla
Billings, Anne Gardiner, Anne Kerlikowske, John
Prioretti and Beth Robertson is gratefully acknowledged.
I would also like to thank Jackie Schartzer for typing
this manuscript.

I am grateful to Abbott Laboratories for supplying

formulated and technical Gibberellins.

iii

TABLE OF CONTENTS

 

PAGE
INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1
CHAPTER 1 Evaluation of Hormonal and Environmental
Influence of Floral Induction and Stalk
Elongation in Sugarbeet (Beta vulgaris
L.) . . . . . . . . . . . . . . . . . .‘. 7
ABSTRACT . . . . . . . . . . . . . . . . . . . . . 7
INTRODUCTION . . . . . . . . . . . . . . . . . . . 8

MATERIALS AND METHODS. . . . . . . . . . . . . . . 12
RESULTS AND DISCUSSION . . . . . . . . . . . . . . 17
CONCLUSION . . . . . . . . . . . . . . . . . . . . 2l
LITERATURE CITED . . . . . . . . . . . . . . . . . 22

CHAPTER 2 Influence of Herbicides Which Alter Plant
Lipid Metabolism on the Action of GA in

 

sugarbeet (Beta vulgaris L.). . . . . . . 35
ABSTRACT . . . . . . . . . . . . . . . . . . . . . 35
INTRODUCTION . . . . . . . . . . . . . . . . . . . 36
MATERIALS AND METHODS. . . . . . . . . . . . . . . 38
RESULTS AND DISCUSSION . . . . . . . . . . . . . . UN
CONCLUSION . . . . . . . . . . . . . . . . . . . . A8
LITERATURE CITED . . . . . . . . . . . . . . . . . 50

iv

PAGE

CHAPTER 3 Influence of Low Temperature, GA3, and
Herbicide Combinations on Membrane Lipid

Composition in Sugarbeets . . . . . . . . 66
ABSTRACT. . . . . . . . . . . . . . . . . . . . . . 66
INTRODUCTION. . . . . . . . . . . . . . . . . . . . 66
MATERIALS AND METHODS . . . . . . . . . . . . . . . 69
RESULTS AND DISCUSSION. . . . . . . . . . . . . . . 73
CONCLUSION. . . . . . . . . . . . . . . . . . . . . 77

LITERATURE CITED. . . . . . . . . . . . . . . . . . 79

CHAPTER A Effect of“Herbicides ThatAlter Plant Lipid
Metabolism on Survival of Sugarbeet

Seedlings . . . . . . . . . . . . . . . . 96

ABSTRACT. . . . . . . . . . . . . . . . . . . . . . 96
INTRODUCTION. . . . . . . . . . . . . . . . . . . . 97

’ MATERIALS AND METHODS . . . . . . . . . . . . . . . 100

RESULTS AND DISCUSSION. . . . . . . . . . . . . . . lOl

CONCLUSION. . . . . . . . . . . . . . . . . . . . . 102
LITERATURE CITED. . . . . . . . . . . . . . . . . . 10A
CHAPTER 5 SUMMARY . . . . . . . . . . . . . . . . . 108

APPENDIX I Additional Data of GA/Plant Hormone
Combinations not Present in the
Dissertation text . . . . . . . . . . . . llO

BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . . 122
RESOURCE REFERENCES . . . . . . . . . . . . . . . . . l2“

TABLE

LIST OF TABLES

CHAPTER 1

1.

Effect of GA; and GAu+7 applied once or as
repeated app ications on stalk elongation in
3-week-Old 'US H20' sugarbeets . . . . . .

Effect of GA3 on percent of plants that
flowered when applied to 3-week-old 'FC70l/5'
sugarbeets grown under various photoperiods.

Effect of GA3 in combination with leaf removal
on stalk elongation in 'FC70l/5' or 'US H20'
sugarbeets grown in a growth chamber .

Effect of GA in combination with various
sequences of leaf removal on stalk elon-

gation in 'US H20' sugarbeets grown in the
greenhouse . . . . . . . . . . . . . . . .

Effect of single applications of GA3/ethephon
combinations on stalk elongation in 2 to 3-
week-Old 'US H20' sugarbeets grown in the
greenhouse . . . . . . . . . . . . . . .

Effect of repeated applications of GA3/
ethephon combinations on stalk elongation

in 2 to 3-week-old 'US H20' sugarbeets grown
in the greenhouse. . . . . . . . . . . . .

Effect of repeated applications of GA3/
ethephon combinations on stalk elongation
in 2 to 3—week-old 'FC70l/5' and 'US H20'
sugarbeets grown in a growth chamber .

Effect Of repeated applications Of GA3/
2,A-D combinations on stalk height in 3 to
U-week-old 'FC70l/5' or 'US H20' sugarbeets
grown in the greenhouse. . . . . . . .

vi

PAGE

2“

25

26

27

28

29

3O

31

TABLE

10.

11.

CHAPTER

1.

Effect of repeated applications of GA3 in
combination with sequentially applied
2,u-D on stalk elongation and flowering
in 3 to A-week-Old 'US H20' sugarbeets
grown in the growth chamber or greenhouse-

Effect of single applications of GA/Kinetin
combinations on stalk elongation in 3-week-
old 'US H20' and 'ELHA' sugarbeets in the
greenhouse . . . . . . . . . . . . . . . .

Effect of GA in combination with EPTC and
diethatyl on stalk elongation in 3-week-
Old 'US H20' or 'ELHA' sugarbeets grown
in the greenhouse- . - . - -‘- - . - . -

2

List of herbicides evaluated in combination

with GA on sugarbeets» - . - - - .

Effect of single applications of GA,
diethatyl combinations on stalk elongation
in 2-week-old 'US H20', 'ELAO' and 'ELAA'
sugarbeets grown in the greenhouse . o -

Effect of single applications of GA,
acetanilide herbicide combinations on stalk
elongation in 'US H20' and 'ELAA' sugar-
beets from two greenhouse experiments. .

Effect of repeated applications of GA3,
thiocarbamate herbicide combinations on
stalk elongation in 3-week-old'FC70l/5'

sugarbeet seedlings grown in the greenhouse.

Effect of EPTC and EPTC plus R-25788 in
combination with GA3 on stalk elongation
when applied to the foliage of 3-week-old
'US H20' sugarbeet seedlings grown in the
greenhouse................

Effect of repeated applications of GA3 in
combination with alachlor and EPTC on
stalk elongation in 2—week-Old 'FC70l/5',
'US H20' and 'ELAO' sugarbeet seedlings
grown in pots outdoors -- - .

vii

PAGE

. 32

. 33

. 3’-l

. 53

. 5A

. 55

. 56

. 57

. 58

TABLE

10.

ll.

12.

13.

CHAPTER
I.

II.

Effect of repeated applications Of GA3 in
combination with alachlor and EPTC on
flowering and stalk elongation in H-week-
old 'FC70l/5', 'US H20', 'ELAO', and
'ELAA' sugarbeets in the field. . .

Effect Of repeated applications from two
experiments of GA, glyphosate combinations
on stalk elongation of A-week-old 'FC70l/5'
sugarbeets grown in the greenhouse . . . .

Effect Of single applications of GA, naptalam
combinations on stalk elongation in 2-week-
Old 'US H20', 'ELAO' and 'ELAA' sugarbeets
grown in the greenhouse. . . . . . . . .

Effect of repeated applications of GA,
naptalam combinations on stalk elongation
in u-week-Old 'FC7OI/5' sugarbeets grown
in the greenhouse. . . . . . . . . . . .

Effect of single applications of GA3 in
combination with benzoic acid-type chemicals
on stalk elongation in 2-week-old 'ELAA' and
H-week-Old 'FC70l/5' sugarbeet seedlings
grown in the greenhouse. . . . . . . . . .

Effect of repeated applications of GA3 in
combination with TCA, dalapon, ethfumesate
and pyrazon on stalk elongation in 3-week-
Old 'FC70l/5' and 'US H20' sugarbeets grown
in the greenhouse. . . . . . . . . . . . . .

Effect of alachlor in combination with GA3
on the uptake of 0.2uCi lAC-GA3 (luuCi/
umole) in 3-week-old 'FC70l/5' sugarbeet
seedlings grown in the greenhouse.

3

Effect Of Cold Temperature, Alachlor, and
GA3 Combinations on Stalk Elongation in
Sugarbeet 30 Days Following Treatment. .

Effect of Cold Temperature, Alachlor, and
GA3 Combinations on the Palmitic Acid
(Cl6:0) Composition of Mitochondria and
Plasmalemma Membranes in Biennial Sugar-
beet . . . . . . . . . . . . .

viii

PAGE

59

. 6O

. 61

- 62

. 63

- 6A

.65

-82

.83

TABLE PAGE

III. Effect of Cold Temperature, Alachlor, and
GA3 Combinations on the Stearic Acid (18:0)
Composition Of Mitochondria and Plasmalemma
Membranes in Biennial Sugarbeet. . . . . . . . . 84

IV. Effect of Cold Temperature, Alachlor, and
GA3 Combinations on the Oleic Acid (Cl8:1)
Composition of Mitochondria and Plasmalemma
Membranes in Biennial Sugarbeet. . . . . . . . . 85

V. Effect Of Cold Temperature, Alachlor, and
GA3 Combinations on the Linoleic Acid (18:2)
Composition of Mitochondria and Plasmalemma
Membranes in Biennial Sugarbeet. . . . . .«. . . 86

VI. Effect of Cold Temperature, Alachlor, and GA3
Combinations on the Linolenic Acid (018:3)
Composition of Mitochondria and Plasmalemma
Membranes in Biennial Sugarbeet. - - - - - - - - 87

VII. Effect of Alachlor in Combination with Non-
inductive (IA/10 hr day/night incandescent)
and Inductive (2A/O hr day/night incandes-
cent) Photoperiod on the Palmitic Acid
(Cl6:O) Composition of Mitochondria and
Plasmalemma Membranes in Biennial Sugarbeet. - - 88

VIII. Effect of Alachlor in Combination with Non-
inductive (IN/O hr day/night incandescent)
and Inductive (2A/0 hr day/night incandescent)
Photoperiod on the Oleic Acid (Cl8:l) Com-
position of Mitochondria and Plasmalemma
Membranes in Biennial Sugarbeet. . . - . - - - . 89

IX. Effect of Alachlor in Combination with Non-
inductive (IA/O hr day/night incandescent)
and Inductive (2A/O hr day/night incandes-
cent) Photoperiod on the Linoleic Acid
(Cl8z2) Composition Of Mitochondria and
Plasmalemma Membranes in Biennial Sugarbeet. - - 90

X. Effect of Alachlor in Combination with Non-
inductive (IA/10 hr day/night incandescent)
and Inductive (2A/O hr day/night incandescent)
Photoperiod on the Linolenic Acid (C18z3)
Composition of Mitochondria and Plasmalemma
Membranes in Biennial Sugarbeet. - - - - - - - ~91

ix

TABLE

XI.

XII.

XIII.

XIV.

CHAPTER
1.

Effect of Alachlor on the Palmitic Acid
(016:0) Composition of Mitochondria and
Plasmalemma Membranes in Annual and
Biennial Sugarbeet. . . . . . . . .

Effect of Alachlor on the Oleic Acid
(018:1) Composition of Mitochondria and
Plasmalemma Membranes in Annual and
Biennial Sugarbeet. . . . . . . . . . . .

Effect of Alachlor on the Linoleic Acid
(018:2) Fraction of Mitochondria and
Plasmalemma Membranes in Annual and
Biennial Sugarbeet. . . . . . . . . .

Effect of Alachlor on the Linolenic Acid
(018:3) Fraction of Mitochondria and
Plasmalemma Membranes in Annual and
Biennial Sugarbeet. . . . . . . .

14

Effect of Foliar Applications of Alachlor
and Vernolate on the Survival of 'US H20'
Sugarbeets in the Field During the
Winter. . . . . . . . . . . .

Effect of Foliar Applications of Alachlor
and Vernolate on the Survival of Three
Sugarbeet Lines in the Field During the
Winter. . . . . . . . . . .

APPENDIX I.

1.

Comparison between soil and foliar applied
GA3 for stalk elongation in 'US H20'
sugarbeets grown in the greenhouse

Evaluation of GA3 as a seed treatment on
stalk elongation in 'ELAA' and ‘US H20'
sugarbeets grown in the greenhouse.-

Effect of single applications of GA3 on
stalk elongation in 'ELHA' sugarbeets

at various stages of growth in the green-
house. . . . . . . . . . . . . . .
Evaluation of single applications Of
formulated and unformulated GA3 on stalk

elongation in 3-week-Old sugarbeets
grown in the greenhouse- - - -

PAGE

92’

93

9A

95

106

107

111

112

113

ll“

TABLE

10.

11.

PAGE

Evaluation of single applications of GA3

on stalk elongation in 6-week—old 'ELAH

sugarbeets when applied foliarly using

three different techniques in the green-

house. . . . . . . . . . . . . . . . . . . . . 115

Effect of repeated applications of GA3 in
combination with leaf removal on stalk

elongation in 3-week-Old greenhouse grown

'US H20' sugarbeets. . . . . . . . . . . . . . 116

Effect of single applications of GA3 and

GAu+7 in combination with ethephon on

stalk elongation in 2-week-old 'US H20'

and 'ELAA' sugarbeet seedlings grown in

the greenhouse . . . . . . . . . . . . . . . . 117

Effect of GA3 applied once in combination

with ethephon on stalk elongation in

3-week-Old 'US H20' and 'ELAU' sugarbeets

grown in the greenhouse. . . . . . . . . . . . 118

Effect of single applications of GA3 in

combination with etehphon on stalk

elongation in A-week-Old 'US H20' sugar-

beets grown in greenhouse soil . . . . . . . . 119

Effect of GA3 in combination with

sequential applications of NAA and 2,h-D

on stalk elongation in 3-week-Old

'F0701/5' sugarbeets in the greenhouse . . . . 120

Effect of GA3 in combination with

sequentially applied 2,A-D on stalk

elongation and flowering in 3-week-Old

'FC701/5' and 'US H20' sugarbeets grown

under greenhouse and growth chamber

environment. . . . . . . . . . . . . . . . . . 121

xi

1/
INTRODUCTION—-

Considerable research has been directed toward
elucidation of the mechanisms involved in floral induction
of seed plants and has been summarized in several reviews
(6, 13, 1A, 2A, 25). Unfortunately, there have been no
major advances in recent years in determining the under-
lying biochemical and physiological mechanisms involved
in floral induction (2“).

Flower initiation in plants represents the transition
from vegetative growth to reproductive development. The
flowering process encompasses several steps. It often
starts with perception of an environmental stimulus
(temperature, daylength), followed by changes in the shoot
apex, and terminates in the appearance of flower or
inflorescence primordia (24).

Two environmental parameters of prime and specific
importance in floral initiation are daylength (photoperiod)
and low temperature (vernalization). Photoperiodic plants
require a specific daylength, (usually called inductive),
and they fall into two main response types, long-day plants
and short-day plants (7). The long-day types require .

exposure to photoperiods longer than a critical daylength

l

‘in order to undergo rapid floral induction whereas short-
day plants require a photoperiod shorter than a critical
daylength. There is generally an overlap in daylength
necessary for floral induction of long- and short-day
types of different species (1A). The number of days
necessary for photoinduction varies widely with species,
and long- and short-day plants can be facultative or
obligate with respect to their photoperiodic requirements
and generally have to reach a certain age requirement for
flowering (1h).

Two additional but apparently much less frequent
photoperiodic response types are characterized by a dual
daylength requirement; the long-short-day and the short-
long-day plants (18, 22). A fifth group or response type
to photoperiod are those plants which will flower irre-
sepctive of daylength and are called day-neutral plants.

The major site of photoperiodic perception has been
shOwn to be the leaves in both long- and short-day plants
(12). In several cases, if only a single leaf perceived
the inductive photoperiod flowering will be initiated.

The promotion of floral induction by low temperature
is a process known as vernalization and is generally
associated with plants in nature that have to pass through
a winter period before they are capable of flowering (1M).
Winter annuals and biennials are plants that have this

cold requirement, the latter being of a facultative nature

for winter annuals but obligate for biennials (23).
Plants with a cold requirement for flowering or
promotion of flowering have a subsequent requirement for
higher temperatures and in most cases, also for long-days

before flower primodia are formed. In such plants, the
cold period is of a strictly inductive character with
the actual initiation of floral parts occurring in warmer

temperatures and long-days (23). Winter annuals such as

 

fall or winter cultivars of Triticum aestivum L. and other
cereals have a facultative requirement for both cold
temperatures and long-day as they will flower in the
absence of these conditions although at a considerably
slower rate (1“). On the other hand, biennials such as

sugarbeets (Beta vulgaris L.) have an obligate requirement

 

(for both cold temperatures and long-days.

Other characteristics of the low temperature effect
include site of perception at the shoot tip, an optimum
temperature generally in the range from O to 10 0, depending
on species, and reversal of the inductive effect by high
(greater than 15 C) temperatures (23). However, after
a plant has been subjected to low temperatures for long
periods of time (several weeks), the vernalized state is
said to be stabilized and will not be reversed by high
temperatures..

Since perception of photoinduction occurs in the

leaf but the response occurs in the shoot apex, a

"communication" is clearly taking place. This fact has
given rise to the flower-hormone (florigen) concept as
proposed by Chailakhyan (2). This gained further support
from grafting experiments which showed that a receptor
plant, maintained under non-induCtive conditions would
flower when a florally induced donor was grafter to it
(9, 20). It has further been proposed that plants which
require vernalization produce an additional hormone-like
substance (vernalin) and that the presence of this substance
is necessary for the formation of florigen (1A), the
ultimate factor required for flowering in cold requiring
plants. Despite a considerable amount of research, these
substances unfortunately have never been isolated and the
biochemical processes involved have not been elucidated.

As mentioned above, most sugarbeet cultivars are
biennial plants with an obligate or qualitative require-
ment for cold temperatures for floral induction. The
effective range of low temperature is from 2.75 to 10 C, with
an optimum of 4.4 C and these temperatures have to be
applied for approximately 1 to 2 months, followed by a
requirement for warmer temperatures (21 to 27 C) and long-
days (1H to l6-hr photoperiod) (15, 19, 21). This property
of biennial sugarbeet poses a problem for the plant breeder
because of the length of time required to obtain seed
under natural conditions. Thus, if sugarbeets could be
induced to flower in one growing season, it would

facilitate sugarbeet improvement through accelerated

breeding programs (11).

It was recently observed that young biennial sugar—
beets were induced to flower within 30 days in a particular ’
growth chamber (11). This phenomenOn lasted for a period
of approximately 2 years but could not be reproduced
afterwards. Examination of reCords of photoperiod and
temperature indicated that climatic induction of these
plants was precluded. Although the cause of floral in-
duction in this chamber was not readily apparent, a chemical
induction may have been involved.

Gibberellic acid (GA3) can induce floral initiation
without any cold treatment in several biennials but not
.in beet (1). Sugarbeet was induced to flower by GA3, only
under partial photothermal induction (continuous illumin-
ation with temperatures of 7 to 8 C for A3 days)(8).

It is thus apparent that GA3 does not substitute completely
for the cold temperature requirement in sugarbeet.

Cold temperatures cause a shift in the fatty acid
content of plant cell membranes toward greater unsaturation
(10). The herbicides diethatyl [N-(chloroacetyl)-N-(2,6-
diethylphenyl)g1ycine] and vernolate (S-propyl dipropylthio-
carbamate) have been shown to have an effect similiar to
that of low temperature on plant cell membranes under
warm temperature (30 C), causing a shift to a higher per—
centage of unsaturated fatty acids (17). Since these
herbicides have been shown to substitute for the low

temperature effect on membranes, the possibility exists

that these chemicals plus GA might substitute completely

3
for the cold temperature induction necessary for flowering
in sugarbeets.

The herbicide EPTC(S-ethy1 dipropylthiocarbamate)
(a vernolate analog) interacts antagonistically with GA3
in corn (g3a_m§ys L.) (A). GA3 has also been shown to
interact with other plant hormones on various processes
including flowering in plants other than sugarbeet (3, 5,
16).

The Objectives of this investigation were to evaluate

the effect of GA3 and GAu+ in combination with photo—

7
period, other plant hormones, and herbicides that alter

lipid metabolism on floral induction and stalk elongation
in sugarbeet and to determine the basis for any observed

interaction.

- Chapter 1, 2, 3 and A will be submitted to Agronomy
Journal, Weed Science, Plant Physiology, and Journal of
the American Society of Sugar Beet Technologists,
respectively, for publication. Discrepencies between
chapters with respect to format and listing of units
occurs because of different requirements for each
journal.

CHAPTER I

Evaluation of Hormonal and Environmental Influence on
Floral Induction and Stalk Elongation in Sugarbeet

(Beta vulgaris L.)

 

ABSTRACT

If biennial sugarbeet (Beta vulgaris L.) could be

 

induced to flower in the first year, it would facilitate
sugarbeet improvement through acelerated breeding

programs. Gibberellic acid (GA3) applications to the
foliage of 2 to H-week-old 'ELAA', 'US H20' and 'FC70l/5'
sugarbeets receiving various photoperiods or in combination
with the chemicals ethephon (2-chloroethyl)phosphonic acid,
2,A-D(2,U—dichlorophenoxy)acetic acid), NAA (naphthalene-
acetic acid), kinetin (6-furfury1aminopurine), diethatyl
(N-chloroacetyl-N-(2,6-deithylphenyl)glycine) and EPTC
(S-ethyl-N,N-dipropylthiocarbamate) were evaluated. GA3 .
application in combination with photoperiods of 18/6, 2H/O
hr (day/night), or lA/IO hr plus a 2-hr nightbreak substan—
tially increased flowering over the untreated controls.

GA3 application in combination with ethephon, 2,A-D or
kinetin resulted in a significant increase in stalk
elongation over either chemical alone, indicating a

synergistic interaction.

 

8

An understanding of the control of the biochemical
and physiological mechansims involved in floral induction
of plants presents many opportunities. Among, these are
increasing yield, quality and/or harvestability of crops
and an increase in the number of generations of seed in a
relatively short period of time for the purpose of crop
inprovement. Crop improvement through accelerated breeding
programs would be particularly useful for biennial crop

species such as celery (Apium graveolens L.), carrots

 

(Daucus carota L.), beets (Beta vulgaris L.) and members

 

of the genus Brassica.

Control of flowering in biennial sugarbeet would be
beneficial in two ways. Inhibition of flowering in
sugarbeet would allow control of these plants where they
are growing as weeds in a sugarbeet crop grown for sugar
(Arnold, 1980). This problem is widespread throughout
the sugarbeet growing regions in Europe and is starting
to become a problem in certain sugarbeet growing regions
of the United States. A second benefit from the control
of flowering in sugarbeet would be to induce these plants
to behave as flowering annuals for the improvement of
the crop through accelerated breeding programs (Hogaboam,
1982).

Although sugarbeet is predominately a biennial
species, annual types do exist. These flower under long

days, but do not require a cold induction period; whereas,

9

the biennial types require a cold induction period
(2.75 C to 10 C with an optimum of 4.4 C for approximately
one to two months) followed by a requirement for warmer
temperatures (21 to 27 C) and long days (14 to l6-hr
photoperiod) (Pack, 1925; Shaw, 1917; Stout, 1946).
Biennial sugarbeet lines and cultivars exhibit a bolting
tendency which is dependent on temperature, day length
and length of the photothermal inductive periods as described
above. It was found that without any cold temperature
exposure, a certain strain of sugarbeet could be induced
to flower at 23 C by use of continuous high-intensity
illumination (Steinberg and Garner, 1936). Under 18-hr
photoperiods, flowering did not occur at 23 C but was
induced if the temperature was lowered to 16 or 18 C.
Other research has shown that biennial sugarbeet will
flower under 14-hr fluorescent plus continuous incandescent
illimination (Hogaboam, 1982), eliminating the need for
continuous high-intensity lighting. Combinations of cool
temperature and incandescent light have also been shown
to shorten the time to flower in sugarbeets. Thus, it
appears that the effects of light and temperature are comple-
mentary on sugarbeet floral induction.

Adjusting environmental parameters to induce flowering
in sugarbeets does not lend itself well to field production
of seed in one growing season which would be necessary

for accelerated improvement of this crop. However, recent

10

research indicated that it was possible to induce biennial
sugarbeet to flower within one growing season (Hogaboam,
1982). Sugarbeets grown in a particular growth chamber
flowered within 30 days following seeding and this
phenomenon occurred in several experiments over a period
of more than two years. This chamber was set at a non-
inductive 14/10 hr light/dark period with 22/14 0 day/night
temperature. Careful examination of records of photoperiod
and temperature conditions did not provide any indication
that the sugarbeets were climatically induced to flower in
this chamber. Additional experiments in this chamber
revealed that not all lines evaluated, flowered, while in
others, almost all of the plants were induced. Another
experiment also indicated that this induction occurred
within 2 months from the time of seeding. Duplicate
sets of experiments done in another chamber under the same
set of environmental conditions did not induce flowering.
Although it was not readily apparent what caused the
flowering response, this observation holds out hope that
a purely chemical induction might be possible. The
possibility of ozone or freon leaks within the chamber
suggests examination of these agents for their potential
for floral induction.

After it had been demonstrated that gibberellic acid
(GA3) acted as a growth promoting substance, it was found

that it can cause seed stalk development in plants

11

including sugarbeets (Brian et a1., 1954; Marth et a1.,
1956) and hasten the reproductive development of sugarbeet
seedlings (Gaskill, 1957). These results offer a potential
for field production of sugarbeet seed under non-inductive
climatic conditions. However, GA fulfilled only part

of the requirements for flowering as it was effective

only when applied under continuous incandescent light and

a temperature of approximately 8 C. Other research
substantiated these results in that a non-bolting sugarbeet
line would flower under continuous light plus GA3 (Stout,
1959) and a biennial variety of intermediate bolting
tendency flowered under an 18 but not a 9 hr photoperiod

in combination with gibberellin treatment (Snyder and
Wittwer, 1959). However, the minimal day length needed

for flower induction by GA was not determined, and GA3

was the only gibberellin tested. In addition, the

optimum stage of GA application to sugarbeet was not
established in these studies.

In recent literature, it has been shown that GA
interacts with other chemicals in plants. Gibberellins
and N6-benzyladenine were shown to have synergistic effect
on flowering in Chrysanthemum morifolium cv. Pink
Champagne (Pharis, 1972). Gibberellin also interacted
synergistically with kinetin in purple nutsedge (Chetram
and Bendixen, 1974) and with ethylene to reverse induced
dormancy in lettuce seed (Dunlap and Morgan, 1977).
Antagonistic interactions of GA with herbicides in barley

endosperm (Devlin and Cunningham, 1970), Avena seedlings

l2

(Chang et al., 1975) and corn (Harvey et al., 1975; Donald,
1977) have been reported.

In this study, the objectives were to determine
relative effectiveness of two gibberellins, critical day
length plus GA, freon, ozone, GA in combination with other
hormones and selected herbicides on flowering. The
influence of leaf removal on GA action in sugarbeet was

also of interest.

MATERIALS AND METHODS

Plant Material, Growth Conditions, Experimental

 

Design. The following procedures were used unless specified
otherwise.

Five to ten sugarbeet seeds, selected from lines 'EL44'
(intermediate bolting tendency) and 'FC701/5' (high bolting
tendency) and cultivar"US H20'(intermediate bolting
tendency) were placed 2.5 cm deep in a commercial potting
mixture (Metro-Mix 300) in 948 ml styrofoam cups. Prior
to chemical treatment, the seedlings were thinned to one
plant per pot of a uniform size. All formulated chemicals
(GA3 formulation was Pro Gibb) were diluted in tap water
on a mg/l basis and applied to the foliage until runoff
using a model No. 152 DeVilbiss atomizer with 0.35 to
0.70 kg/cm2 pressure. During the growth period, all pots

received 100 m1 of a 5000 mg/l 20:20:20 NPK fertilizer

13

solution once every 2 weeks. After cessation of stalk
elongation (4—6 weeks after the last chemical application)
stalk height was measured and flowering data recorded.

All experiments were done in the growth chamber, which

had fluorescent and incandescent lamps or in the greenhouse‘
under natural or supplementary (16/8 hr, day/night)

lighting from fluorescent or sodium vapor lamps. The

growth chambers were set at a 14/10 hr, 22/14 0 light-
temperature‘ regime with a photosynthetic photon flux

density (PPFD) that ranged from 300 to 400 umol - m'2 -

sec-l. Greenhouse temperatures ranged from 16 to 29 0

from late fall through early spring and 20 to 35 C from late
spring through early fall. The PPFD under fluorescent and
sodium vapor lamps in the greenhouse was 150 and 300

umol ' m"2 - sec'1 and that under natural lighting was

750 umol . m-2 - sec-1

during the summer months and 300
umol - m-2 - sec.l during the winter months. All pots

were placed in their respective growth environments (green—
house or growth chamber) under conditions listed above
immediately after seeding.

The experimental design was a randomized complete
block with three to five replications. The data were
analyzed using a two-way factorial analysis of variance
when two treatment factors were used and means were
compared by Duncan's multiple range test. All experiments

were repeated to confirm results.

Comparison of GA3 and GAu+7. GA3 and GA!4+7 (Pro Gibb

 

47 from Abbott Laboratories) were applied to 3-week-old

14
'US H20' sugarbeet seedlings at 0, 1, 20, 100, 500 and 2500
mg/l either once or eight times over 3 weeks. The experi-
ment was done in the greenhouse under natural lighting
from July 25 through.October 18, 1980.
3. GA3
was applied to the foliage of 'FC701/5' sugarbeets in

Critical Photoperiod in Combination with GA

growth chambers at 0, 200, 250, 500 and 1000 mg/l under
a 14/10 hr photoperiod; 0 and 250 mg/l under a 16/8 hr
photoperiod; 0, 500 and 1000 mg/l under 18/6 and 14/10
(fluorescent) plus 24/0 (incandescent) hr photoperiods.

GA was also applied at 0, 500, 1000, and 5000 mg/l to

3
plants grown under a 14/10 hr photoperiod with a 2 hr or 20
minute night break with incandescent lighting in the middle
of the dark phase.

Effect of Freon and Ozone on Sugarbeets. Ozone was
generated by passing a stream of air (20 cc/min) along an
ultraviolet light within a sealed container. The generated
ozone was forCed into a sealed growth chamber containing
'US H20' or 'FC701/5' sugarbeets. The plants were grown

under sodium vapor lamps with a PPFD of 450 umol ° m-2 -

sec-1.

In another experiment, freon (freon 12 by Dupont) was
applied to sugarbeets grown in this chamber. This material
was forced into the chamber as a gas at 3 cc/min from a
13.6 kg cylinder used for recharging refrigeration units.
Plants were examined for flowering and/or stalk height 8

to 10 weeks after seeding.

Leaf Removal in Combination with GA3. GA3 was applied

15
to the foliage of 2 to 3-week-old.’US H20'and 'F0701/5'

sugarbeet seedlings at 0, 50, 200, 500, 1000 and 2500 mg/l
in the growth chamber or greenhouse. In the growth
chamber experiment, the Chemical was applied in 7 weekly
applications. Prior to the fourth application, all except
the four youngest visible leaves were removed from the stem.
The photoperiod was 16/8 hr (day/night) with the temper-
atures at the usual settings. In the greenhouse experiment
(under sodium vapor lamps from September 7 through December
21, 1981) GA3 was applied in six weekly applications with
various combinations of leaf removal. In one set of plants,
all but the youngest four leaves were removed throughout
the duration of the experiment. In three other sets, leaves
were removed as follows: 3 i A
Set 2 - All leaves except the four youngest leaves
once prior to the fourth application.
Set 3 - As for set 2 except leaves were removed once
prior to the first application.
Set 4 - The four youngest leaves were removed once
prior to the fourth chemical application.

Combinations of GA3 with Ethephon [(2-Chloroethyl)-

 

phosphonic acid]. Combinations [GA3 and ethephon solutions

 

mixed together (tank-mixed)] of GA at 0, 50, 250, 500,

3
1000, 3300 and 5000 mg/l with ethephon (Ethrel) at 0, l, 2,
5, 10, 20 and 100 mg/l were applied to the foliage of 2 to
3-week-old 'FC701/5' or 'US H20'sugarbeet seedlings grown

in greenhouse or growth chamber. In the greenhouse

16

experiments, the chemicals were applied once or the
applications were repeated four times at weekly intervels.
The plants were grown under natural lighting from June 17
through September 4, 1980. In the growth chamber, the
chemical combinations were applied as repeated applications
three times per week for 2 weeks to 'FC701/5' sugarbeets
and in four weekly applications to 'US H20'sugarbeets.

Combinations of GA with Auxin-Type Chemicals.

3
Combinations (tank-mixed or sequential) of GA3 at 0, 10, 50,
200, 500, 1000 and 2500 mg/l with 2,4-D (2,4-(dichlorophenoxy)_
acetic acid) at 0, 0.125, 0.25, 0.50, 1.0, 2.5, 5 and 25

mg/l were applied to the foliage of 3 to 4-week—old 'FC701/5'
or 'US H20'sugarbeet seedlings. The tank-mixed combinations
were applied in three weekly applications to plants grown

in the greenhouse from November 15, 1980 through February

4, 1981. For the sequential applications, GA3 was applied

at four weekly intervals. Two weeks following the last

GA3 application, 2,4-D was applied in three weekly
applications. One of the Sequentially applied chemical
experiments was done in the greenhouse under sodium vapor

lamps and included NAA (a-naphthaleneacetic acid)/GA3
combinations. The other experiment was done in the growth

chamber.

Combination of GA with Kinetin (6-furfurylaminopurine).

 

GA3 and GA4+7(2% solutionfkmmiAbbott Laboratories) were

applied at 0, 1000 and 5000 mg/l in combination (tank-mixed)

with kinetin at 0, 0.01 and 1.0 mg/l on 3-week-old

17

'US H20'and 'EL44' sugarbeets grown in the greenhouse
under natural lighting from October 30, 1979 through
January 4, 1980. The form of kinetin used was Cytex
which is a seaweed extract containing 100 ppm kinetin.
Combinations of GA with Diethatyl [N—Chloroacetyl-N-
(2,6-diethylphenyl) glycine ethyl ester]and EPTC (S-

 

 

ethyl-N,N-dipropyl thiocarbamate). GA3 and GA“+7

(2% solution) were applied at 0, 1000 and 5000 mg/l in
combination (tank-mixed) with the herbicides diethatyl
(Antor 4E) and EPTC (Eptam 7E) at 0, 10 and 100 mg/l

on 3-week-o1di'USlH201and 'EL44' sugarbeet seedlings grown
under natural lighting in the greenhouse from October 30,

1979 through January 4, 1980.

RESULTS AND DISCUSSION

Comparison of GA and Critical Photoperiod in Combin-

 

ation with GA3. GA was found to be most effective for

 

inducing stalk elongation in sugarbeets when given as
repeated applications to the foliage of young sugarbeet
plants (Table 1). GA induced stalk elongation but not
flowering under non-inductive environments with no
differences between GA3 and GA4+7’ Biennial sugarbeets

have been induced to flower under continuous (24 hr)
incandescent light (Gaskill, 1952). -By increasing the length
of the photoperiod in increments from 14/10 to 24/0 day/

night with light from incandescent plus supplemental

l8

fluorescent lamps flowering occurred only at the 24/0 day/
night photoperiod in the absence of GA3 (Table 2). When

GA3 was applied to plants growing under various photoperiods,
flowering was enhanced with a minimum day length of 16 hr

for the response to occur. Flowering was also enhanced

with GA3 applications when sugarbeets received a 2 hr
nightbreak in the middle of the dark period of the l4/10

hr, day/night, cycle. Flowering was not enhanced when the
plants were given a 20 minute nightbreak.

Leaf Removal in Combination with GA Non-induced

3.
leaves have been reported to produce an inhibitory effect

on flowering (Lang, 1965). GA3 applications to sugarbeet
seedlings which had mature or young leaves removed, resulted
in no flower initiationznni a generally negative effect on
stalk elongation (Tables 3 and 4). Only one treatment

(GA at 200 mg/l on 'FC701/5') produced a positive effect

3

when leaves were removed (Table 3).

Combinations of GA with Ethephon. Ethylene has been

3
reported to induce flowering in members of the Bromeliaceae
(Zeevaart, 1978) and to interact synergistically with GA3
on lettuce seed germination (Dunlap and Morgan, 1977).
Ethephon alone or in combination with GA3 did not induce
flowering in sugarbeets under various environmental con-
ditions (Tables 5, 6, 7). However, a synergistic inter-

action was observed as GA3/ethephon combinations resulted

in greater stalk height than either chemical alone.

19

Combinations of GA3with Auxin-Type Chemicals. Auxin-

type materials are not generally associated with floral
induction (Zeevaart, 1978) but have been shown to interact

with GA for increased hypocotyl elongation in cucumber-

3
(Cucumis sativus L. cv. National Pickling) (Kazama and

 

Kasuni, 1974), the strongest effect being obtained when
GA3 was given to the plant material as a pretreatment.

Combinations of 2,4-D with GA at a given combination of

3
' rates (tank-mixed or sequential) resulted in a synergistic
interaction in the form of a significant increase in stalk
height over that of either chemical alone (Tables 8 and 9).
In these and similar experiments, 2,4-D did not induce

flowering alone or in combination with GA , except for one

3
experiment (Table 9). However, it Should be noted that
sugarbeets growing in this chamber were subjected to 2
weeks of 3 C daytime temperatures due to a malfunction of
the heating system. The cool temperature in combination
with the chemical treatments may have been responsible for

the induction. NAA did nOt appear to enhance the GA effect

on stalk elongation nor did it have any effect on flowering.

 

Combinations of GA with Kinetin. Cytokinins have been
shown to interact with GA to induce flowering in Chrysanthe-
mum and other plant responses (Pharis, 1972, Chetram and

Bendixen, 1974). Kinetin in combination with GA3 or GAA+7

did not induce flowering after single applications (Table 10).

However, kinetin interacted synergistically with GAu+7 for

increased stalk enlongation. Combinations with GA3 resulted

in an antagonistic interaction, indicating that the rates

20

of kinetin may be too high.

Combinations of GA with Diethatyl and EPTC. Combin-
ations of GA3 and several herbicides have produced varied
interactions in plants as described previously (DeVlin
and Cunningham, 1970; Chang et al., 1975; Donald, 1977).
The interactions were of an antagonistic nature and the
work was done on monocotyledons. Single applications of
GA in combination with diethatyl or EPTC did not induce
flowering or produce a significant interaction for
increased stalk elongation (Table 11). However, rates of
EPTC may have been too high as decreasing stalk heights were
obtained with high rates of GA for "US H20'and all GA rates
in 'EL44' sugarbeets. .This indicates that lower EPTC
rates might produce a synergistic interaction. Diethatyl/
GA combinations resulted in a trend toward increased stalk
height with increasing rates of the herbicide in 'US H20'.
The same effect was observed in 'EL44' with low rates of
GA plus the herbicide, but diethatyl plus high GA rates
resulted in a trend toward decreasing stalk height.

Ozone and freon gas applied to sugarbeets did not
induce flowering or stalk elongation. Ozone appeared to

injure the plants slightly.i

CONCLUSION

Repeated applications of GA3 induced greater stalk
elongation than single applications, were found to produce
a response equal to GAu+7, and neither chemical induced
flowering under non-inductive photoperiod. GA3 induced
or enhanced flowering as day length increased, but
only induced stalk elongation in plants with certain leaves
removed. GA3 in combination with other hormones resulted

in a significant increase in stalk height but did not

induce flowering.

21

11.

12.

LITERATURE CITED

Arnold, M. 1979. Weed Beet; whose problem: the
farmer's or the seed producer's? British Sugar Beet
Review. 47(1):5—7.

Brian, P. W., G. W. Elson, H. C. Henawing and M. Radley.
1954. The plant growth promoting properties of
gibberellic acid a metabolite of the fungus Gibberella
fijikori, J. Sci. of Food and Agr., p 602.
Bukovac, M. J. and S. H. Wittwer. 1957. Gibberellin
and higher plants, II: Induction of flowering in
biennials. Quar. Bull. Mich. Agr. Exptl. Sta. 39:
Chang, T. 0., H. V. Marsh, Jr. and P. H. Jennings.

1975. Effect of alachlor on Avena seedlings:

Inhibition of growth and interaction with gibberellic
acid and indoleacetic acid. Pest. Biochem. and Physiol.
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Chetram, R. S. and L. E. Bendixen. 1974. Gibberellic
acid plus cyotokinins induced basal bulbs of purple
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Devlin, R. M. and R. P. Cunningham. 1970. The
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Donald, W. W. 1977. The role of gibberellins in EPTC
(S-ethyl dipropylthiocarbamate) injury to corn (£33
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Dunlap, J. R. and P. W. Morgan. 1977. Reversal of
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gibberellic acid. Plant Physiol. 60:222-224.

Gaskill, J. O. 1952. A new sugar-beet breeding tool
two seed gfineration in one year. Agron. J. 44:338.
Gaskill, J. O. 1957. A preliminary report on the use
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in sugar beet seedlings. J. Am. SOC. Sugar Beet
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Harvey, B. M. R., F. Y. Chang and R. A. Fletcher. 1975.
Relationship between S-ethyl dipropylthiocarbamate
injury and perioxidase activity in corn seedlings.

Cana. J. Bot. 53(2):225-230.

Hogaboam, G. J. 1982. Early induction of flowering

in sugarbeets. Agron. J. 74:151-152.

 

22

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23

Kazama, H. and M. Katsumi. 1974. Auxin-gibberellin
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pretreatment on IAA-induced elongation. Plant and
Cell Physiol. 15:307-314.

Lang, A. 1965. Physiology of Flower initiation.
Encyclopedia of Plant Physiol. 15/2:1380-1536.

Marth, P. 0., W. V. Audia and J. W. Mitchell. 1956.
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118(2):106-lll.

Pack, D. A. 1930. The seed production of sugar beets.

Facts About Sugar. 25:37-39, 48.

Pharis, R. P. 1972. Flowering of Chrysanthemum under

non-inductive long days by gibberellins and Nb-

benzyladenine. Planta 105:205-212.

Shaw, H. B. 1917. Climatic control of the morphology

and physiology of beets. Sugar 19:387-381, 431-434,
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under controlled conditions. J. Agr. Res. 52:
943-960.

Stout, M. 1946. Relation of temperature to repro-

duction in sugar beets. J. Agr. Res. 72(2):49-68. .
Stout, M. 1959. Some effects of gibberellic acid on

the physiology of sugar beets. J. Am. Soc. Sugar Beet

Technologists. 10:305-310.

Snyder, F. W. and S. H. Wittwer. 1959. Some effects
of gibberellin on stem elongation and flowering in
sugar beets. J. Am. Soc. Sugar Beet Technologists.
10:553-561.

Zeevaart, J. A. D. 1978. Phytohormones and flower
formation. Phytohormones and related compounds-A
comprehensive treatise, Vol. 11. 291-323. Elsevier/

North Holland Biomedical Press.

 

24

Table 1. Effect of GA3 and GA4+7 applied once or as
repeated applications on stalk elongation in 3-week—
old 'US H20' sugarbeets .

 

 

 

 

 

 

Single Application Repeated Application
Treatment
Rate GA3 GAu+7 GA3 GA4+7
(ms/l) (mm)
0 4 A* - 22 A -
1 11 A 5 A 22 A 29 A
20 12 A 6 A 47 A 50 A
100 24 A 18 A 110 B 143 B
500 44 AB 33 A 241 C 287 C
2500 48 A. 47 A. . 279 C 246 C

 

*Treatment means followed by the same letter are not
significantly different at the 5% level according to
Duncan's multiple range test.

25

.COHAOQ xnmo on» no OHCCHE on» CH xoopnpzwfic mpscfie om m oo>fiooop COHLOQODO£m

 

 

 

 

A

.UOASOQ xnmo mo canoes on» Ca xwopnpnwfic poo: m w oo>fiooon oofimeOpocm+

I I I as as I ooom man
I I me I I I comm mae
ooa ow I om om .o coca mac
ooa so I I em a com mas
I I I I I o omm m<o
I . I I I I 0 com m<w
o: o o om o o o m<w

; . _ 1 A5 3E5

o\:m m\wa m\ma . oa\:a +oa\za oa\=H opmm psoEpmone

A

Ampsom cfi.p£wHZ\zmQV.UOHAOQODOQm

 

.mUOHpooouoso wooeno> noon: czonm mpoonpowSm .m\Ho~om.
CHOIxoozIm o» condemn 20:3 oopozoam pan» mucmaa mo ucoonoq :o m<o mo poommm .m canoe

26

.oo>oEop mo>moH u + moo>oEmp no: mo>moH u +

\

.pmop mwcmn oaofipase m.cmocsm on wcfionooom Ho>oa am can no
peopOMMHo zapcmOHMchHm no: one amppoa beam on» an oozoaaom mtaumamm> Cannes memozx

 

 

 

 

mm o.wwH m O.HHN mm N.:m: DO w.mwm OOOH m<w

an m.mea a a.aam man o.aaa a a.mmm com mag

00 m.ama Do 3.0NH o o.owm m N.ONN oom m<o

ma e.ea em N.EA a a.ae ma a.ma om mam

m< N.mH < w.m < N.mm a< N.mH o m<w
255 :R5

+ I + I boom psoEpmonB

 

 

Hm>osom Moog Hm>oEom Moog

+
ON: m: m\.mo>o.m

 

.ponsmco npzopw m CH czonw mpoonpmwzm ommmflr so .m\ao~om.
:H soapnwcoao xawpw so Hm>oEop mama so“: cofiuocHnEoo ca m<u mo poommm .m manna

27

mo>ooa nzom now
u :IEoupon mCOHDBOHHQom panda noumm coco oo>oEon mo>oOH Loom do» poooxo Ham

.pmmp owcon bandpass m.coo::o on wcfionooom
Ho>OH Rm on» an DCOAOMMAC hauzmoHMHQme no: who sonata been on» an oozoaaom mammz*

.COHDBOHHQQm cannon noumo coco oo>oEon
do» neonpwowaoam condom mount mono oo>oEon mo>moH msom don uqooxo Haw

HIEODDOB

 

 

 

 

mucosfinoaxo pzonmzonnp oo>oEop mo>ooa 930m do» poooxo Ham u QIEoupon moo>OEop mo>moa o: u +
a a.amm a m.eoa was a.eaa m m.mem e a.mam comm mac
can a.oea a a.emm am e.ema was m.mm= ea e.mem oooa mas
Q0 0.:ma QI< :.omH mom m.mmH mom w.mmH Q m.mam oom m<m
QI< m.w~ om< o.H: om< m.mm m< m.mm 0mg m.mm oom m<o
< o.w < 0.: < o.o < 0.0 *< o.m o m<w
lees . 1A\aev
doe :IEoupom .,HIEouuom o Eouuom I ovum, HmOHEono

 

+Ho>oEom mood

 

.omsoncoopw on» :H 239% mpoonpmwSm 5% m? 5 coaumwcoao xaoum
co Hw>oEms mama mo moocosvom m30finm> can; coapmcfinEoo :a mac mo poommm .: manna

.pmop omens bandpass m.:mo::o Op wcfionooom
HC>OH em on» we uconohmfio >HpCM0Hmacwfim no: 0pm sonata beam on» an oozoaaom memoze

 

28

 

 

 

 

I I mum m.mHH a o OOH cocamcpm

one m.ooa muo o.mm . a 0.5m a o om cocameem
mo 0.:NH mom m.mm a :.mm a :.m OH nonethem
mum :.moa cue 0.0m ma :.om a o m sesamepm
m m.o:a om m.:o ma a.mm a o H scenteem
moo m.me muo m.mm ma m.mm ea 0 o sesamepm

Assv Afistv
ooom comm oooa 0 them phosphate
Ad\wsv mac

.omsoccoonw Ono EH szonw mummnpmwsm .ommmfl: CHOIxoozIm on m 2H coapmwcoao
xampm so mCOHpmcfinEoo conoozuo\m<o.mo macauMOHHoQo oawcfim mo poommm .m manna

29

.nwon ow:m: oaoflnflss m.:wo:so on w:fionooom

Ho>OH mm o:n nm n:oLOMMHo zan:mOHMH:me no: one :onnoa meow o:n an oozoaaom m:moz*

 

 

 

 

 

I om m.=mH m m.mma a m.mm can eosamhnm
cam a.mmm eIm a.amm no a.mmm a o.mm om eoeamhem
cam 3.0mm oIa m.mom om m.mam a m.em o.m coeaeenm

ca a.nam mao a.mom moo o.nmm a 0.0m o.m cocaeenm
oIa m.emm ea o.wmm mac a.wmm a m.om o.H eoeamenm
a m.emm Ema a.nmm om «.mnm e< m.ma o sesamehm
Assn An\wev
coon com cm 0 them phosphate
An\wsv mac

 

.omzo::oo:w o:n :fi :3o:w m monnmwsmfimm ms. CHOIxoosIm on m :H :oanmw:oao

xamnm :o m:OHno:anEoo :ondonno\ <9 no m:0antoaaqam oonmoom: no noommm

.o manna

3O

.nmon ow:en oHQHane m.:eo:sa on w:Honoooe Ho>oH am o:n ne n:ononnHo
mHn:e0HnH:me no: one nonnoH oEem o:n an oozoaHon ne>HnHzo no o:HH noonnewsm :nnnnz m:eoz*

 

 

 

 

 

 

 

 

 

a m.mmH n m.HmH mo m.mHH a m.e I om conaosnm
m m.an me m.m:H mo m.e0H a o.m I OH eoeaocnm
me m.mHH no m.m0H o m.me < 0.: m m.mmm a o m eoeaoenm
I I I I m m.Hmm a o H connoenm
mm m.OMH ma o.mmH om m.ee a m.m m o.mmm ea 0 o connoEnm
Asev AH\wev
com omm om o omm o onem unmaneone
HH\meV m<c HH\weV mac
om: mo m\HoSn
.nonee:o

znzonw e :H :3onw onoonnewSo.QwHma.o:e.m\ao>om. GHOIxoozlm on m :H :onnew:oao

xHenm :o o:OHne:HnEoo :o::o:no\m<u no m:oHneonHoae ooneooon no noonnm

.w oHneE

31

.nwon ow:en oHQHnHSE m.:eo:em on m:Honoooe Ho>oH Rm o:n ne n:ononnHo
an:e0HnH:me no: one nonnoH osem o:n an ooonHon ne>HnHSo no o:HH noonnewSm :n:nn3.m:eoz*

 

 

 

 

 

 

 

I me 0.0m om< 0.5H A w.>om Ax w.wma m< 3.0H m.m alzam
Hmc m.mHH mmm m.m© m< w.OH ax o.mmH Dom o.mm m< m.m o.H leam
Hm w.mHH on o.w: m< z.m mlm m.NOH ma N.w> m< m.OH om.o lenm
mum w.om mma :.m> m< m.m xIH N.mmH Dom o.mm m< ©.m mm.o leam
H o.oma me :.wm m< m.HH xlm 3.:mH Do o.wm m< o.m mmH.o lenm
mmo 0.50 on N.N: om< :.>H mmm m.wm mmm :.>w mom¢ N.NH 0 lenm
AEEV AH\mEv
comm com o comm com o onem n:oEneonB
AH\mev m<u AH\wEV m<o
0mm m: m\HoN.om

.omso::oonw o:n :a :30nm mnoonnemzm.omm m:;_no.m\flowom. UHOI xoo3I: on m :H ncwfio:

xHenm :o m:oHne:finEoo QI : m\m<c.fio o:oHne0HHooe Coneodon no noonnm

.m oHnee

32

.noon ow:en oHQHnHSE m.:eo:=n on m:ficnoooe Ho>oH Rm o:n ne n:ononnH©
an:eOHnH:wHo no: one nonnoH oEem o:n an oosoHHon :OHnnc:oo :nzonw :Hnnfiz m:eoz*

.UonoZOHn mnoonnew5m o>Hn no nso neon.
H

.vonozoan mnoonnewso o>Hn no nso o:o

 

 

 

 

 

+
no o.mHH ma :.mm ma e.mH a m a o oasoeeoono mm eIe.m
no e.ooH ma o.mH ma =.w < o < o onsoeeoono m eIa.m
o H.ANH m e.oe e 0 me m.n a o omaoeeoonu H eIe.m
o e.Hm ma m.nm a o < m.m < 0.: omaoncoonu o eIe.m
no o.mmH ma m.mH ma a.mH a :.m a o nonseeo mm aIe.m
n epzono
ma m.nm ma o.mm < e.» a o a o nonseeo m eIz.m
enzono
em a.mn ma e.om a :.e a o a o nonseeo H oIe.m
enzono
em me me m.mH e o a 0 ea 0 noneeeo o mIa.m
. enzone
AEEV AH\wEv
OOOH com om OH o . whennHeeoo onem unmaneonn
enzonu
HH\eev mac

 

.omso::oonw no nonseno :nzonw o:n :H :zonw mnoonnewsm

.omm.w:;.cHOIxoo3I= on m :« w:mnoonn o:e :OHnew:oao xHenm :o QI:.N cofiaaae
nHHeHn:osoom :na: :oane:HnEoo :H <0 no o:oaneOHHQQe coneoaon no noonnm .m oHnee

33

. .nmon owcen oHaaanE e.:eo:=o on m:Hunoooe Ho>oH
um onn ne ncononnno anceoHnficwnm no: one nonnoH osem o:n an eoonHon ne>nano no oan noonnewsm :ngnnz mceoza

 

oI< n.em one m.zm no n.mm ma o.eH a m.m I I I I I o.H ennoenx
m o.mm ma o.om om< n.nm com n.mm m< e.m a o.om oma m.oH aI< m.m~ .om< n.0H oma m.m H.o chocHx

I I I I I one n.mm one e.mH he n.3m one n.eH one n.3H nc.o ereene

 

 

 

 

 

 

 

 

 

QI< ~.mm m< o.mH a m.mo m< 0.x < m.m QI< o.mm om< m.mH .oo m.mm oI< m.wm e< o.H o :npocnx
Assn . ,-. AH\mev
ooom coca ooom coca o ooom oooa ooom oooa o onem n:oEneone
Hwaev n<=<o HH\wev mac Hmeev eases Aerev mac
aaqm ONE ”3

 

.omzoscoonm osn :« mnoonnewze.:=am. ece
.ommmfl: UHOIxoozIm :n :OnnemcoHo xHenm :o m:onne:nnsoo :Hnocnx\<o no ecoHneOHHoae oncnm no noonnm .CH oHnee

l Jittn'lltlr.ua I4

 

 

CE. 092nm Eu“?— CCanCNQECO Cfi. <C.KO OOOInInnm .HH THEME.

 

 

 

 

34

.nmon ow:en oHQHnHze e.:eo::o on m:«cnoooe Ho>oH um onn ne n:ononnHo
nnneeonnucwnn no: one nonnoH osem on» an eoonHon oenonnnon noeo non ne>Hnaso no ocHH noeo :Hnnnz mceoza

 

one m.Om ma m.mm one O.Hm ea n.em m.m om m.m= one n.mm o n.OO om n.HO < n.O OOH Hnoeeoono

m.m om< o.mm om< m.mH om m.mm om< m.mm < O.H o Hanennofin

a
Oom n.nm ma m.a~ Oo e.om ma m.ON < O.m om< m.nm oea.0.0m om m.HO m< n.HH om<.O.nH OH Hnoeeoono
OI< n.mm ma 0.0H O m.~o ea 0.0 a

<

 

 

 

 

 

 

I a O.Hm me O.mm < n.OH m.m ea n.mm me n.O~ ma n.O~ m< O.m~ a m.a OOH onmm
ma O.Nm a n.mm m O.HO a n.m~ a O.n m< m.Hm me m.mm ma 0.0~ m< O.HH a 0.0 OH oeam
ma e.mm a 0.0H m m.mo < 0.0 < m.m ma 0.0m me m.OH e m.om m< m.om aa O.H O oeam

Assn AH\wev
OOOO OOOH OOOm OOOH O OOOm , OOOH OOom OOOH . O them Octaneonn
oneeO Naaao nH\on mao HoneO eazao HmeeO m<o
:Onm OQnma

 

.oezo::oonw o:n :« :zonm enoonnemsm
.zaqm. no 6% mPoHonoozIm :H :onnew:o.Ho xHenm :0 Hanennono 23 09mm :33 :onnegnsoo 5 5.. no noonnm .HH oHnen.

CHAPTER 2

Influence of Herbicides Which Alter Plant Lipid
Metabolism on the Action of GA in sugarbeet

(Beta vulgaris L.)

 

ABSTRACT

Application of GA in combination with herbicides
reported to alter plant lipid metabolism were evaluated
for their effect on stalk elongation and floral initiation

in sugarbeets (Beta vulgaris L.). Combinations of GA with

 

members of thiocarbamate, acetanilide, and benzoic acid

classes of herbicides, naptalam (N-l-naphthylphthalamic acid),
TCA (trichloroacetic acid), ethofumesate (2-ethoxy-2,3-dihydro-
3,3-dimethy1-S-benzofuranyl methanesulfonate), dalapon (2,2-
dichloropropionic acid) and glyphosate [N-(phosphonomethyl)-
glycine] resulted in a synergistic increase in stalk elon-
gation . Flowering was induced by GA3 alone and in combination
with alachlor [2-chloro-2',6'-diethy1-N-(methoxymethyl)-
acetanilide] and EPTC (S-ethyl dipropylthiocarbamate) only,

in one field experiment, in 5-15% of ‘FC701/5' sugarbeets.

14

Uptake of C-GA by sugarbeet foliage was not increased by

3
pretreatment with alachlor, and thus was not the basis for

the observed interaction.

35

INTRODUCTION

Floral induction of biennial sugarbeet in one growing
season would facilitate sugarbeet improvement through accel-
erated breeding programs (8). Biennial sugarbeets require a
cold induction period (2.75 to 10 C with an optimum of 4.4 C
for approximately 1 to 2 months) followed by a requirement for
warmer temperatures (21 to 27 C) and long days (14 to 16 hr
photoperiod) (12,14,15).

Recently, it was observed that young biennial sugarbeets
were induced to flower within 30 days from seeding in a
particular growth chamber (8). Examination of records of
photoperiod and temperature conditions precluded environ-
mental induction of sugarbeet flowering in this chamber.
'This phenomenon occurred in several subsequent experiments
over a period of approximately 2 years. Although the cause
of the floral induction was not readily apparent, a chemical
induction may have been involved.

Gibberellic acid (GA3) can induce floral initiation

without the cold treatment in the biennials Hyoscyamus niger

 

(10), carrot (Daucas carota L.), celery (Apium graveolens L.)

 

 

but not beet (2). However, sugarbeet was induced to flower
if GA3 was applied under a partial photothermal induction
(continuous illumination and temperature of 7 to 8 C for

43 days) (6). GA3 did not cause floral initiation without
36

#4

Av

.au

any

nu.

AC

~\~

 

a...
Au

37

the cold treatment in sugarbeets. Wheatly and Johnson
(15) were unable to induce flowering with GA3 applications
to April planted sugarbeets.

Cold temperatures cause a shift in the fatty acid
content of plant cell membranes toward greater unsaturation
(7). The herbicides diethatyl [N-(chloroacetyl)-N-(2,6-
diethylphenyl)glycine] and vernolate (S-propyl diphropylthio-
carbamate) act similiarly as the low temperature effect on
plant cell membranes under warm temperature (30 C) causing
a shift to more unsaturated fatty acids (13).

EPTC (S-ethyl dipropylthiocarbamate) and BASF 13-338
[4-chloro-S(dimethylamino)-2-pheny173(2H) pyridazinone]
induce a shift to more saturated fatty acids in soybean

(Glycine max (L.) merr.) and wheat (Triticum;aestevum L.)

 

 

respectively (17, 18). EPTC, TCA, and ethofumesate have
been shown to reduce epicuticular wax depositiOn in cabbage

(Brassica oleracea L.) (6, 9, 11).

 

Since diethatyl and vernolate have been shown to sub-
stitute for the low temperature effect on membranes, there
exists the possibility that these herbicides could also
mimic the low temperature effect on other plant processes.
Thus it might be possible to substitute completely for the
cold temperature induction necessary for flowering in sugar-
beets with combinations of these herbicides and GA.

The objectives of this study were to evaluate the effect
of combinations of GA with diethatyl and vernolate on floral

induction and stalk elongation in sugarbeet. Since EPTC

38

(a vernolate analog) and other herbiCides also affected other
aspects of plant lipid metabolism, they were also evaluated
in combination with GA for their effect on floral induction .

and stalk elongation in sugarbeet.
MATERIALS AND METHODS

Plant material, growth conditions, experimental design.

 

Five to ten sugarbeet seeds, selected from lines 'EL40'

(low bolting tendency), 'EL44' (intermediate bolting tendency)
and 'FC701/5' (high bolting tendency) and cultivar 'US H20'
(intermediate bolting tendency), were placed 2.5 cm deep in

a commercial potting mixture (Metro-Mix 300) in 948 ml
styrofoam cups. Prior to Chemical treatment, the seedlings
were thinned to one plant of a uniform size per pot. All
formulated chemicals [GA3(Pro Gibb formulation) and herbicides
(Table 1)] were diluted in tap water on a mg/L basis and
applied in combination [solutions of herbicide and GA3 mixed
together] to the foliage until runoff using a model No. 152
DeVilbiss automizer with 0.35 to 0.70 kg/cm2 pressure. During
the growth period all pots received 100 m1 of a 5000 mg/L
20:20:20 N-P-K fertilizer solution once every 2 weeks. After
cessation of stalk elongation (4-6 weeks after the last
chemical application) stalk height was measured and flowering
data recorded. The experiments were done in a growth chamber,
which had fluorescent and incandescent lamps; in a greenhouse

under natural or natural plus supplemental (16/8 hr day/night)

39

lighting from fluorescent or sodium vapor lamps; or in pots
grown outdoors. The growth chambers were set at a 14/10 hr,
22/14 0 day/night light-temperature regime with a photo-
synthetic photon flux density (PPFD) that ranged from 300

to 400 umole - m—2 . sec-l. Greenhouse temperatures ranged
from 16 to 29 C from late fall through early spring and

20 to 35 C from late spring through early fall. The PPFD

under fluorescent and sodium vapor lamps in the greenhouse

was 150 and 300 umol - m-2 - sec-l, respectively, and
that under natural lighting was 750 umol - m"2 - sec-l
during the summer months and 300 umol - m“2 - sec.l during

the winter months. All pots were placed in their respec-
tive growth environments (greenhouse, growth chamber, or
outdoors) under the conditions listed above immediately
after seeding.

The experimental design was a randomized complete block
with three to five replications. *The data were analyzed
using a two or three-way factorial analysis of variance
for two or three treatment factors, respectively, and
means were compared by Duncan's multiple range test. All
experiments were repeated to confirm results.

Chloroacetanilides applied in combination with GA. In

 

the first greenhouse experiment (from January 3 through
February 20, 1980, under supplementary fluorescent lighting)
diethatyl solutions (made from Antor 4E) were applied once
at 0, 5, 100, and 200 mg/L in combination with GA3 and
GA”+7 (2% solution from Abbott Laboratories) at 0, 500,

1500 and 5000 mg/L to the foliage of 2-week-Old 'US H20',

-=\

4O

'EL40' and 'EL44' sugarbeet seedlings. In two subsequent
greenhouse experiments (from January 31 through March 15,
1980 and April 8 through June 12, 1980, under natural
lighting), other members of the Chloroacetanilides
[alachlor (Lasso 4E), metolachlor (Dual 8E) and acetochlor
(5E formulation)] and diethatyl were applied once at rates

ranging from 0.5 to 500 mg/L in combination with GA and

3
GA“+7 at 0 and 5000 mg/L to the foliage of 2-week-old
'US H20' and 'EL44' sugarbeets.

Thiocarbamate herbicides applied in combination with

 

GA3. Applications of GA3 in combination with commercial

formulations of four thiocarbamate herbicides [EPTC,
vernolate, butylate + R-25788 (N,N-diallyldichloroacetamide)
and cycloate] were made seven times over 3 weeks to the
foliage of 3-week-old 'FC701/5' sugarbeet seedlings. The
plants received GA3 at 0 and 200 mg/L in combination with
the thiocarbamates at 0, 0.25, 0.5 and 1.0 mg/L for the
first six applications and ten times their respective
rates for the last application. The experiment was done
in the greenhouse under natural lighting from July 7
through October 6, 1980.

For the third experiment, combinations of EPTC and EPTC
plus the antidote R-25788 (Eradicane 6.7E) at 0, 25 and 100
mg/L and 0+0, 25+2.1, and 100+8.3 mg/L, respectively, with
GA3 at 0 and 2000 mg/L were applied twice to the foliage
of 3-week-old 'US H20' sugarbeet seedlings grown in the

greenhouse under sodium vapor lamps.

41

Thiocarbamate and acetanilide herbicide combinations

with GA3 in outdoor experiments. GA3 was applied at 0,

 

500, and 2500 mg/L in combination with EPTC at 0, 10 and
50 and alachlor at 0, 50, and 250 mg/L to the foliage of
2-week-old 'FC701/5', 'US H20'and 'EL44' sugarbeet seed-
lings. The plants were grown in a greenhouse potting‘
mixture (sand:peat:clay, 1:1:1 by volume) and placed in
the greenhouse under sodium vapor lamps. At the time of
Chemical treatment the plants were moved outdoors and
received five weekly applications. The experiment was
done from April 13 through July 14, 1981 and all treat-
ments were replicated six times.

On May 1, 1981, a field experiment was initiated on
a sandy loam soil. Prior to plowing, a 12-12-12 (N-P-K)
fertilizer was applied at 448 kg/ha broadcast, followed
by a 7-28-18 fertilizer with 2% Mn and 0.25% B applied at
140 kg/ha in the band at planting. 'EL40', 'EL44',
'FC70l/5' and 'US H20' sugarbeets were seeded 5 cm apart
in rows 0.7 m apart by 9.1 m in length. One row of each
sugarbeet cultivar or line was randomized and planted
within each plot. The plants were thinned to 15 cm 8
weeks after seeding and weeded throughout the growing
period as necessary. GA3 (Pro Gibb Plus from Abbott
Laboratories) was applied at 0, 300, and 1200 mg/L in
combination with EPTC at 0, 25, and 100 mg/L and alachlor

at 0, 50, and 250 mg/L to the foliage of 4-week-old sugar-

beet seedlings at the two to four leaf stage in seven

42

weekly applications. The chemicals were applied in water
at 34 L/ha plus 0.25 (v/v) X—77 surfactant with a tractor-
mounted plot sprayer for the first time. The other six
applications were made in 73 L/ha plus 0.25% v/v X-77
surfactant with a knapsack sprayer, with pressure supplied
from a cartridge of compressed 002. All treatments were
replicated three times and flowering and stalk height

data were collected 16 weeks after the first application.

Evaluation of glyphosate for flowering and stalk

 

elongation. Glyphosate was applied to the foliage of

 

clones from a selection of early bolting 'FC701/5' sugar-
beets developed through tissue culture. The herbicide
was applied once at 0, 1, 10, 100, and 1000 mg/L to the
foliage of the sugarbeets in the six to eight leaf stage.
The plants were grown in a chamber under 14 hr light from
fluorescent plus continuous light from incandescent lamps.
In two subsequent experiments, glyphosate was applied
from 0 to 100 mg/L in combination with GA3 from 0 to 2000

mg/L and GA (Pro Gibb 47) from 0 to 500 mg/L to the

4+7
foliage of 4-week-Old 'FC701/5' seedlings. In the first
experiment, the chemicals were applied twice, 11 days
apart, to plants grown under sodium vapor lamps in the
greenhouse. In the second experiment, the chemicals were
applied six times over 5 weeks to plants grown in the
greenhouse under natural lighting from July 22 through
October 19, 1981.

GA combinations with naptalam. GA and GA“+7 (2%

q

 

43

formulation) were applied from 0 to 5000 mg/L in combin-
ation with naptalam (Alanap 2E) from 0 to 20 mg/L to the
foliage of 2-week-old 'US H20', 'EL40' and 'EL44'
(experiment 1) and 4-week-old 'FC701/5' (experiment 2)
seedlings. The treatment was made once in experiment 1
and in 3 weekly applications in the second experiment.
Both experiments were done in the greenhouse under
supplementary light from fluorescent lamps.

GA3 combinations with benzoic acid—type chemicals.

GA3 was applied once at 0 and 5000 mg/L in combination with
commercial formulations of benzoic acid-type chemicals
[chloramben, dicamba, and TIBA (technical grade)] 'from

0 to 250 mg/L to the foliage of 2-week-old 'EL44' and
4-week old 'FC701/5' sugarbeet seedlings. These experi-
ments were done in the greenhouse under natural lighting
from January 31 through March 31, 1980, and August 13
through October 15, 1980, respectively.

GA3 combinations with TCA, dalapon, ethofumesate, and

 

pyrazon. GA3 was applied at 0 and 1000 mg/L in combination
with TCA (83% active ingredient) at 0, 100, 500, and 1000
mg/L; dalapon (DOWpon M) at 0, 10, 50, and 100 mg/L;
ethofumesate (Nortron 1.5E) at 0, 5, 10, and 20 mg/L; and
pyrazon (Pyramin 65 W) at 0, 10, 25, and 50 mg/L to the
foliage of 3-week-old 'FC701/5' and 'US H20' sugarbeets.
The treatments were made in foUr weekly applications, and
the plants were grown in the greenhouse under sodium vapor

lamps.

44

ltic-GAB uptake study. This experiment was done in

 

the greenhouse under sodium vapor lamps with three
replications per treatment. GA3 was applied at 1000 mg/L
in combination with alachlor at 0 and 50 mg/L to the
foliage of 3-week-old 'FC701/5' seedlings. After these
solutions were allowed to dry on the leaf, 0.2 uCi [1,

7, l2, l8-lu0] GA (14 uCi/umole) in 5 ul (10% isopropyl

3
alcohol in water) was spotted on the center of the first

true leaf of the sugarbeet seedling. Three and seven

days after treatment, the plants were harvested. In one

set of plants, the tip and base of the l”Cu-CA3 treated

leaf (tip harvested 0.5 cm above and base harvested 0.5

14

cm below C—GA3 spot) and the youngest visible leaf

were oxidized and analyzed for 1’40 by liquid scintillation
radioassay.

RESULTS AND DISCUSSION

Chloroacetanilides applied in combination with GA.

 

Combinations of GA with diethatyl, alachlor, acetochlor,

3
and metolachlor resulted in a Significant increase in

stalk height over either chemical alone (Tables 2 and 3).
This was found in all lines or cultivar tested except for
GA3 plus acetochlor on 'US H20' (Table 3). A significant

increase in stalk height with GA,4+ was found only when

7
applied in combination with diethatyl to 'EL40' and 'US H20'
sugarbeets. These results indicate a synergistic inter-

action occurred between the herbicides and GA. The

45

interaction was dependent on chemical, chemical rate, and
sugarbeet type used. Flowering was not induced by any
of the treatments.

Thiocarbamate herbicides applied in combination with

GA3. Combinations of thiocarbamate herbicides (EPTC,

vernolate, butylate plus R—25788, and cycloate) with GA3
resulted in a significant increase in stalk height over
either chemical alone (Table 4). Butylate plus the antidote
R-25788 at the highest rate in combination with GA reduced
the interaction to a non-significant level. Since R-25788
protects corn from butylate and EPTC (antagonistic response)
injury (3), this may explain why the interaction between
butylate and GA3 was reduced. A second experiment lends
support to this idea as R-25788 also reduced the inter-

action between EPTC and GA at the low but not the high

3
rates (Table 5). The inability of R-25788 to reverse

the interaction between GA3 and EPTC at 100 mg/L may be
due to the high herbicide rate. None of the treatments

induced flowering.

Thiocarbamate and acetanilide herbicides in combination

 

with GA in outdoor experiments. Various combinations of

3

GA with EPTC and alachlor resulted in a significant

3
synergistic interaction for increased stalk height on plants
grown in pots outdoors or in the field (Tables 6 and 7).
Alachlor with GA3 produced a significant increase in all

sugarbeet types grown in pots and on 'FC701/5' and 'El44'

46

in the field. EPTC with GA produced a significant

3
increase in stalk height in 'FC701/5' and 'US H20' in pots

and 'EL44' in the field. Flowering was Observed on a few
plants of 'FC70l/5', in various treatments in the field
experiment (Table 7).

Evaluation of glyphosate for flowering and stalk

elongation. Glyphosate has been reported to inhibit

 

meristems in several plant species (1). However, when
applied to the foliage of a photoperiodically (24/0 day/
night incandescent plus 14/10 day/night fluorescent)

induced selection of 'FC701/5' sugarbeet clones, to deter-
mine if flowering could be inhibited, l and 10 mg/L enhanced
flowering over the untreated control. Therefore, subsequent
experiments were initiated to determine the effect of
glyphosate in combination with GA3 and GA“+7 on flowering
and stalk elongation in sugarbeet. Combinations of
glyphosate and GA3 significantly increased stalk elongation
(Table 8), but no flowering was induced by any of the
treatments. Glyphosate plus 100 mg/L GAu+7 increased

stalk length, but these results were not significantly

different than those obtained with’GAu+7 alone.

GA combinations with naptalam. Naptalam was evaluated

 

with GA because of its classification as an amide, similar
to the Chloroacetanilides (1). Naptalam interacted syner-

gistically with GA!4+ on all sugarbeet types tested

7

(Tables 9 and 10). Combinations of GA with increasing

3
rates of naptalam increased stalk height, but these results

47
were not significantly different than those obtained with
GA3 alone. None of the treatments induced flowering.

GA3 combinations with benzoic acid-type chemicals.

 

GA3 combinations with dicamba and chloramben resulted in a
synergistic interaction as stalk height was significantly
increased over that induced by either chemical alone in
'FC701/5' sugarbeets (Table 11). The chloramben rates
used appeared to be too high for 'EL44' sugarbeets as a
trend toward decreasing stalk height resulted. TIBA
interacted with GA3 for increased stalk height in. 'EL44'
but not in 'FC701/5' sugarbeets.

GA3 combinations with TCA, dalapon. ethofumesate, and

 

pyrazon. GA3 also interacted synergistically with dalapon,
TCA, ethofumesate, and pyrazon. Combinations with the first
three herbicides resulted in significantly longer stalks
than with GA3 alone (Table 12). Although the differences
were not statistically significant, GA3 combinations with
pyrazon resulted in increased stalk elongation over that
obtained from GA3 alone. The high degree of variability
may be responsible for lack of significance between GA3

and this combination. In a second experiment, GA3/pyrazon
combinations resulted in a significant increase in stalk
height over GA3 alone.

14

C-GA3 uptake study. As mentioned above, several

 

of the herbicides evaluated in these experiments have been
reported to alter epicuticular wax deposition in plants.

This might explain the synergistic interaction observed

48

with the GA, herbicide combinations. luC-GA was applied

3
to sugarbeet plants treated with alachlor and alachlor plus
GA3 to determine if the herbicide had any effect on GA3

uptake. The results indicate that alachlor or alachlor
plus GA3 did not significantly alter 1LiC-GA3 uptake
(Table 13). In fact it appears that alachlor produced a

14

trend toward decreased uptake of C-GA into the treated

3
leaf for both harvest times. Based on these results, it
appears that the interaction between GA and the herbicides,

may be taking place inside the plant.
CONCLUSION

GA exhibited a synergistic interaction in combination
with several herbicides, which were reported to alter lipid
metabolism, in the form of a significant increase in stalk
height over that obtained with either chemical applied.
alone. The interaction was dependent on chemical, chemical
rate, type of GA and sugarbeet line or cultivar.

Two models commonly used in reference to chemical
interactions are additive and multiplicative types. Use
of the F-test (as in this report) to determine if any
interaction between two chemicals is significant only
tests for additive responses, i.e. both chemicals
competing with each other for a common site of action. A
multiplicative model (Colby's (4) test for an interaction

between chemicals) determines if the chemicals are

49

affecting different sites of action. Colby's test was
used where the F-test showed a significant increase in
stalk height over either chemical alone in this study.
Results Showed the measured value to be greater than the
expected value in all cases, which indicates the syner-
gistic interaction arises from chemicals affecting
different sites of action.

None of the treatments induced the plants to flower
except in the field experiment where some of the treat-
ments (low rates of CA3 plus alachlor and EPTC) induced
several 'FC70l/5' plants to flower.

luC-GA showed that alachlor did not

3
result in increased movement of luC-GA

Treatments with
3 into the plant,
a finding which indicates that the interaction between

herbicides and GA may be taking place inside the plant.

LITERATURE CITED

Ashton, F. M. and A. S. Crafts. 1981. Mode of Action,

 

of Herbicides, Second Edition. Wiley Interscience

 

Publ. New York. 525 p.

Bukovac, M. J. and S. H. Wittwer. 1958. The effect of
gibberellin on economic crops. Economic Botany 12:213-
255.

Chang, F. Y., J. D. Bandeen, and G. R. Stephenson. 1972.
A selective antidote for prevention of EPTC injury in
corn. Can J. Plant Sci. 52:704-714.

Colby, S. R. 1967. Calculating synergistic and
antagonistic responses of herbicide combinations. Weeds
15:20-22.

Gaskill, J. O. 1957. A preliminary report on the use
of gibberellic acid to hasten reporductive development
in sugar beet seedlings. J. Am. Soc. of Sugar Beet
Technologists. 9(6):521-528.

Center, W. A. 1966. The influence of EPTC on external
foliage wax deposition. Weeds. 14:27-31.

Harris, P. and A. T. James. 1969. The effect of low
temperatures on fatty acid biosynthesis in plants.

Biochem. J. 112:325—330.

50

10.

ll.

12.

13.

14.

15.

16.

51
Hogaboam, G. J. 1982. Early induction of flowering

in sugarbeets. Agron. J. 74(1):151—152.
Kolattukudy, P. E. 1965. Biosynthesis of wax in

Brassica oleracea. Biochem. 4:1844-1855.

 

Lang, A. 1956. Induction of flower formation in

biennial Hyoscyamus by treatment with gibberellins.

 

Die Naturwissenschaften 43:284-285.

Leavitt, J. R. 0., D. N. Duncan, D. Penner and W. F.
Meggitt. 1978. Inhibition of epicuticular wax
deposition on cabbage by ethofumesate. Plant Physiol.
61:1034—1036.

Pack, D. A. 1930. The seed production of sugar beets.
Facts About Sugar 25:37-39, 48.

Rivera, C. M. 1977. Effect of temperature and various
agricultural chemicals on phospholipid fatty acid

composition of soybean (Glycine map (L.) Merr.). PhD

 

Dissertation, Michigan State Univ., p. 80-99.

Shaw, H. B. 1917-1918. Climatic control of the
morphology and physiology of beets. Sugar. 19:387-391,
431-434, 479-486; 20:23-27, 68-70, 109-112, 150-154.
Stout, M. 1946. Relation of temperature to repro-
duction in sugar beets. J. Agric. Res. 72(2):49-68.
Wheatley, G. W. and R. J. Johnson. 1959. Studies

on the use of gibberellic acid to induce flowering

in sugar beets. J. Am. Soc. of Sugar Beet Technolo-

gists. lO(4):335-343.

17.

18.

52

Willemot, 0. 1977. Simultaneous inhibition of
linolenic acid synthesis in winter wheat roots and
frost hardening by BASF 13-338, a derviative of
pyridazinone. Plant Physiol. 60:1-4.

Wilkinson, R. E., A. E. Smith and B. Michel. 1977.
Alteration of soybean complex lipid biosynthesis by
S-ethyl dipropylthiocarbamate. Plant Physiol. 60:
86-88.

53

Table 1. List of herbicides evaluated in combination with

GA on sugarbeets.

 

Common Name

Chemical Name

 

acetochlor

alachlor

butylate
chloramben
cycloate
dalapon
dicamba

diethatyl

EPTC

ethofumesate

glyphosate

metolachlor

naptalam
pyrazon
TCA

TIBA

vernolate

2-chloro-N(ethoxymethyl)-6'-ethy1-g-
acetotoluidide

2-chloro-2',6'-diethy1-N-(methoxymethyl)
acetanilide

S-ethyl diisobutylthiocarbamate
3-amino-2,5-dichlorobenzoic acid
S-ethyl N-ethylthiocyclohexanecarbamate
2,2-dichloropropionic acid
3,6-dichloro-g-anisic acid

N-(Chloroacetyl)-N-(2,6-diethylphenyl)
glycine

S-ethyl dipropylthiocarbamate

(i)-2-ethoxy-2,3-dihydrO-3,3-dimethyl
-5-benzofuranyl methanesulfonate

N-(phosphonomethyl)glycine

2-chloro-N-(2-ethyl-6-methylphenyl)-N-
(2-methoxy-l-methylethyl)acetamide

N-l-naphthylphthalamic acid
5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone
trichloroacetic acid

triiodobenzoic acid

S-propyl dipropylthiocarbamate

 

54

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56

Table 4. Effect of repeated applications of GA3, thiocar-
bamate herbicide combinations on stalk elongation in
3-week-old 'FC701/5' sugarbeet seedlings grown in the
greenhouse.a

 

 

 

 

Rateb GA3 (ms/L)
Treatment (mg/L) 0 200
mm
Check 0 8 A 226 BC
EPTC 0.25 30 A 348 D
EPTC 0.5 18 A 531 F
EPTC 1.0 5 A 456 EF
Vernolate 0.25 4 A 457 EF
Vernolate '0.5 16 A 393 DE
Vernolate 1.0 15 A 366 DE
Butylate + R-25788 0.25 + 0.01 5 A 136 B
Butylate + R-25788 0.5 + 0.02 12 A .333 D
Butylate + R-25788 1.0 + 0.04 11 A 311 CD
Cycloate 0.25 20 A 158 B
Cycloate 0.5 6 A 156 B
Cycloate 1.0 9 A 373 DE

 

aMeans followed by the same letter are not significantly

different at the 5% level according to Duncan's multiple

range test.
b

above for the last application.

Plants received 10 times the corresponding rate listed

57

Table 5. Effect of EPTC and EPTC plus R-25788 in combination
with GA on stalk elongation when applied to the foliage
of 3-week-old WHSIfifll'sugarbeet seedlings grown in the

 

 

 

 

greenhouse.a
GA3 (mg/L)
Rate
Treatment (mg/L) 0 2000
mm
Check 0 O A 58 B
EPTC 25 O A 95 C
EPTC 100 0 A >105 C
EPTC + R-25788 25 + 2.1 0 A 41 B
EPTC + R-23788 100 + 8.3 0 A 100 C

 

aMeans followed by the same letter are not significantly
different at the 5% level according to Duncan's multiple
range test.

58

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25 SEE
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63

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65

Table 13. Effect of alachlor in combination with GA3 on
the uptake of 0.2u01 l4c-GA3 (14uCi/umole) in 3-week—old
'FC70l/5' sugarbeet seedlings grown in the greenhouse.a

 

 

 

l4C
Harvest
Treatment Rate Time Treated leafb Newest leaf
(mg/L) (days) ———————(DPM/gm F. w.)
GA3 1000 3 9312 A 1066 A
Alachlor + GA3 50 + 1000 3 2359 A 132 A
GA3 1000 7 23,791 A 499 A
Alachlor + GA3 50 + 1000 7 4678 A 1034 A

 

aMeans within columns followed by the same letter are not
significantly different at the 5% level according to Duncan's
multiple range test.

bResults from tipznuibase of treated leaf were combined.

CHAPTER 3

Influence of Low Temperature, GA3, and Herbicide

Combinations on Membrane Lipid Composition in Sugarbeets
ABSTRACT

Fatty acid composition of mitochondria and plasmalemma
membranes of sugarbeet (Beta vulgaris L.) foliage was analyzed
in annual, florally-induced biennial, and non-induced biennial
plants which received treatment combinations of low temper-
ature, gibberellic acid (GAg), and the herbicide alachlor
(2echloro-2',6'-diethyl-N-(methoxymethyl)acetanilide). Low
temperature decreased the saturated (palmitic and stearic
acids) and increased the unsaturated (linoleic and linolenic
acids) fatty acid composition of both membrane fractions in

sugarbeet. Alachlor GA and alachlor plus GA3 induced an

3.
effect on membrane lipids similar to that of low temperature.
Plasmalemma membranes of annual and florally induced biennial
sugarbeets also exhibited a shift toward a higher percentage
of unsaturated fatty acids over time whereas the mitochondria
fraction showed no change. In non-induced biannial sugarbeet,

the unsaturated fatty acids increased over time in the

mitochondria membranes but not the plasmalemma.

 

Plant responses associated with low temperature include

vernalization (low temperature promotion of flowering),

66

67

breaking of seed dormancy, breaking of winter dormancy in
bulb of perennial woody plants, underground induction of
storage organs such as tubers, and effects on vegetative
form and growth of certain plants (14). Vernalization is
important for the induction of flowering in winter annuals
such as cereals, and biennials such as sugarbeets (Beta
vulgaris L.). Gnerally, the cold requirement in cereals
is quantitative, whereas in biennials it is considered
qualitative or absolute (l4, 16).

The cold requirement in sugarbeets impedes improvement
of this crop through breeding programs due to the length of
time needed between generations of seed (7). If this plant
could be induced to flower in one growing season it would
accelerate breeding programs.

It has been demonstrated that low temperatures have a
distinctive effect on plant cell membranes (3, 6, 12). The
saturated fatty acid [palmitic acid (16:0)] composition of
membranes from plants growing under low or a shift from high
to low temperatures decreased and the unsaturated fatty acids
[linoleic (18:2) and linolenic (18:3)] increased. This was
shown to occur in membranes of individual organelles such as
mitochondria and plasmalemma and the type of shift appeared
to be dependent on species and orgenelle examined (8, l2).

Giberellic acid (GA3) has been shown to induce flowering

in biennials such as Hyoscyamus niger (9), carrot (Daucus

 

m...

.mo

Cy

a:

fit.

0%

 

K

 

68

carota L.), and species and cultivars of Brassica but not

beet (2). It has been suggested GA substituted for the

3
cold requirement where flowering was induced but did not
substitute for the obligate photoperiodic requirement in
sugarbeets to induce flowering (l6). GA induced flowering

in sugarbeet only under partial photothermal induction
conditions (continuous illimination and temperature of 7

to 8 C for 43 days) (5).

Recent research has shown that certain herbicides
could substitute for the cold effect on plant cell membranes
by increasing the percentage of unsaturated fatty acids in
mitochondria and plasmalemma membranes of soybean (Glycine
ma§_(L.) Merr.) (13). Based on these results, herbicides
that influence lipid metabolism were applied in combination
with GA to the foliage of sugarbeet seedlings to determine
if the obligate cold requirement could be replaced (10).

The results indicated that several classes of herbicides
interacted synergistically with GA to induce stalk elongation.
However, none of the chemicals, alone or in combination with
GA induced flowering in plants grown under non-inductive
conditions.

The low temperature effectcmn sugarbeet plant cell mem-
branes has not been documented. In addition, it has not been
established that GA or herbicides mimic the low temperature
effect on cell membranes of this plant. Therefore, the
objective of this investigation was to determine the effect

of low temperature, GA and herbicides on cell membranes of

3

69
sugarbeet seedlings, and to compare these results to the
fatty acid composition of membrane lipids in annual and
florally induced biennial sugarbeet to determine whether

induced changes were similiar to naturally occurring Shifts.

MATERIALS AND METHODS

Experimental Conditions for Chemical and Temperature

Treatment in Biennial Sugarbeet. Two sets of 948 ml styro-

 

foam cups containing five to ten 'US H20' (intermediate
bolting tendency) sugarbeet seeds planted 2.5 cm deep in a
commercial potting mixture (Metro-Mix 300) were placed in
two growth chambers, respectively. Environmental conditions
within the chambers were 14/10-hr photoperiod with a day/
night temperature of 22/14 C. The lamps were cool white
fluorescent with supplemental incandescent and provided a
photosynthetic photon flux density (PPFD) of 300 umol

m-2 - sec'l. Seventeen days after seeding, all plants were
thinned to one plant per cup of a uniform size and the
temperature in one chamber was reduced to 15/5 C (day/night)
(Low temperature). Chemical treatments consisting of GA3
(Pro Gibb) at O and 2500 mg/L in combination (solutions
mixed together) with alachlor [2-chloro-2',6'-diethyl-N-
(methoxymethyl)acetanilide] (Lasso 4E) at O and 250 mg/L
were mede twice over 3 days to the foliage of l7-day-old
seedlings. One week after the initial chemical application,

the chamber with the low temperature was changed back to

22/14 C (day/night). The plants were watered as needed and

70

fertilized once every 2 weeks with a 5000 mg/L solution
containing a 20:20:20 (NPK) fertilizer. All treatments
were replicated three or four times and the experiment was
repeated to confirm results.

Experimental Conditions for Chemical Treatment of

 

Biennial, Annual and Florally Induced Biennial Sugarbeets.
An annual line and a biennial cultivar ('US H20') of sugar-
beet were seeded in pots and maintained as described above
with the exception of the following changes. Immediately
after seeding, both sugarbeet types were placed in one
chamber with a l6/8-hr photoperiod and a temperature of
22/14 C (day/night). In a second experiment, 2 sets of
'US H20' biennial sugarbeets were seeded and maintained as
described above. One set was placed in a chamber with 14/10-
hr photoperiod and 22/14 C, day/night temperature. The second
set was placed in a chamber under inductive conditions for
flowering (14 hr of light daily from fluorescent lamps plus
24 hr from incandescent lamps) (6). The temperature was
22 C for the 14 hr of illumination with fluorescent lighting
and 14 C for the remaining 10 hr.

Seventeen days after seeding, two alachlor applications
of O and 250 ml/L were made 3 days apart, to the foliage
of the sugarbeets described above. Extra pots of sugarbeets
for both experiments were seeded and remained in the growth
chamber until the onset of flowering.

Membrane Separation. Plants were harvested at O, 7

 

and 14 days following chemical treatment for fatty acid

71

analysis according to a slightly modified procedure
described by Rivera and Penner (l2). Plasmalemma and
mitochondria membranes were obtained by homogenizing fresh
foliage in a Virtis grinder for 90 sec in 30 ml of an ice-
cold medium consisting of 0.26 M sucrose, 3 mM EDTA, 50 mM
Tricine (N-Tris(hydroxy-methyl) methyl glycine), and 1%
(w/v) BSA (Bovine Serum Albumin) (fatty acid free) (pH 7.8).
The homogenate was strained through 4 layers of chessecloth
and centrifuged at 13,000 g for 15 min at 2 C. The 13,000
g pellet containing mitochondria was resuspended in the
homogenizing medium and centrifuged at 2500 g for 10 min to
remove cell walls and other large cellular fragments. The
resulting supernatant was then pelleted at 13,000 g for

15 min, the peliet rinsed and suspended in deionized H20,
and repelleted at 13,000 g for an additional 15 min. The
resulting mitochondria were then held at —15 C for further
analysis. The supernatant containing plasmalemma from the
original 13,000 g centrifugation was further centrifuged

at 80,000 g for 30 min. The resulting pellet was then
resuspended in 2 ml 20% (w/w) sucrose containing 1 mM MgSOu
and 1 mM Tris-Mes (2—N-morpholinoethane sulphonic acid),

pH 7.8. The suspension was layered onto a discontinuous
sucrose gradient consisting of 28 ml of 45% (w/w) sucrose
and 8 m1 of 34% (w/w) sucrose. The sucrose solutions each
contained 1 mM MgSOu and 1 mM Tris-Mes, pH 7.8. The
gradient tubes were centrifuged for 2 hr at 95,000 g in a

swinging bucket rotor (Beckman SW27 rotor). The plasmalemma

72

were obtained from the 34% to 45% interface, diluted in

deionized H 0, and pelleted at 80,000 g for 10 min. The

2
plasmalemma samples were held at -15 C for further analysis.‘

Extraction and Analysis of Phospholipids. The frozen

 

membrane samples were lyophilized and the lipids extracted
according to a modified procedure of Folch et a1. (4). Ten

ml of CHC13-Me0H (2:1) (C/M) was added to the lyophilized

tissue and the samples were shaken in a water bath at 33 C
for 30 min. The extract was filtered (Whatman No. 4) into
a second tube, and the residue re-extracted once with 5 m1

C/M by shaking in a H 0 bath for 15 min. The filtered

2
extracts were obtained and washed with 0.2 vols of 0.9%

NaCl solution in a tube stirrer for 30 sec. After the
mixture settled, the upper phase was discarded and the

lower phase washed twice with 0.2 vols of CHCl -Me0H-H 0

3 2
(3:48:37 by volume) containing 0.9% NaCl. The lower phase
containing the lipids was taken to dryness under N2 at 33 C

and the residue dissolved in 50 ul CHCl The lipids were

3.
.then applied to TLC plates (20 X 20 cm) precoated with
0.25 mm silica gel 60 F254. The phospholipids were sepa-

rated from the remaining lipids in Me2C0—MeCOOH-H 0 (100:

2
2:1 by volume) and the phospholipid band selected on the
basis of published Rf values (15) was scraped from the
plates, extracted with 2 m1 C/M followed by 1 ml MeOH and
the resultant solution taken to dryness under N2 at 33 C.

Fatty acid methyl esters were prepared according to a

modified procedure of Metcalfe et a1. (11). 0.5 N methanolic

73
KOH (1 ml) was added to the dried sample followed by boiling

for 5 min. After the tubes had cooled, 1 m1 of 14% BF3—MeOH
was added and the samples boiled for an additional 2 min.
One drop of saturated NaCl solution was then added and the
methyl esters extracted 3 times with 1 m1 hexane each.

The extracts were combined, dried under N2, and the residue
was taken up in 50 ul acetone for GLC analysis. Fatty

acid composition was determined by FID using a 1.83 m by

2 mm glass column packed with 12% stabilized DEGS on anakrom
ABS and operated at 165 C with N2 as the carrier gas. Peak
identification and quantification was performed by compar-
ison with authentic fatty acid methyl ester standards.

Data from the experiment with chemical and temperature
treatment, comparison between annual and biennial sugarbeet,
and comparison between induced and non-induced biennial
sugarbeets were analyzed as a three-way factorial with

a split plot, a two-way factorial with a split plot and a

split split plot, respectively.
RESULTS AND DISCUSSION

Effects of Low Temperature, Alachlor and GA3 in Biennial

 

Sugarbeet. The five major fatty acids found in membrane
fractions of sugarbeet foliage were palmitic (16:0), stearic
(18:0), oleic (18:1), linoleic (18 2) and linolenic (18:3).
Trace amounts of myristic (14:0) and palmitoleic (16:1) acids

were occasionally observed.

74

Low temperature resulted in measureable changes in the
fatty acid composition of both the mitochondria and
plasmalemma membranes (Tables II through VI). The per-
centage of palmitic, stearic and oleic acids of the mitochon-
dria membrane decreased with an increase in linoleic and
linolenic acid composition after exposure to low temperature
for 7 days. However, in the plasmalemma fraction, the
palmitic and oleic acid composition decreased with an increase
only in the percent of linoleic acid. This trend was observed
as treatment temperature was increased. This relationship
between temperature and fatty acid composition supports
previous investigations concerning other plant species (1, 3,
8, 12, 15, 17).

Treatments of alachlor, GA3, and GA plus alachlor

3
induced various shifts in the percent composition of palmitic,
linoleic, and linolenic acid in the mitochondria and
plasmalemma membranes (Tables II, V, and VI). Under low
temperature GA3 decreased the percent linolenic acid com—
position and GA3 plus alachlor decreased the linolenic and
increased the percent composition of palmitic acid in
mitochondria membranes. Under warm temperature linoleic

acid increased in the mitochondria fraction from all three
chemical treatments. In the plasmalemma membrane fraction,
only GA3 plus alachlor increased the percent linolenic acid
composition under low temperature. However, under warm

temperature, alachlor and alachlor plus GA3 increased the

percent linolenic but decreased the percent composition of

75

palmitic acid. These results support those of Rivera and
Penner demonstrating that membrane composition shifted
toward a higher percentage of unsaturated fatty acids
following herbicide treatment (13). Data from stalk height
measurements 4 weeks after treatment taken on other plants
that received these chemical and temperature treatments
showed that alachlor GA combinations produced a significant

3
increase in stalk height (Table I), indicating that the GA

3
herbicide interaction took place in this experiment. However,
it does not appear that results from the fatty acid analysis
offer an explanation of this interaction in warm temperature
treated plants. The chemical combination did not signifi-
cantly increase the unsaturated fatty acid content compared

to the increase induced by GA3 or alachlor alone.

Fatty Acid Analysis of Annual, Biennial and Induced

 

Biennial Sugarbeet. In these experiments, only trace amounts

 

of stearic acid were obtained and will not be discussed.
Percent fatty acid composition of mitochondria and plasma-
lemma membranes of biennial sugarbeets under non—inductive
and inductive conditions for flowering was compared. In
the mitochondria membranes of untreated, non-induced
biennial, the percent palmitic and oleic acid composition
decreased and the percent composition of linoleic acid
increased with time (Tables VII through X). In untreated
florally induced sugarbeet, only the percent composition

of oleic acid decreased. In the plasmalemma fraction the

76
palmitic acid composition decreased in both non-induced
and induced plants and only the percent linolenic acid
composition of induced sugarbeet increased after 2 weeks
(Tables VII and X). It should be noted that the per;
cent linolenic acid composition of the non-induced plants
increased after 7 days but decreased by 14 days, whereas
the increase in the induced sugarbeet was consistent through-
out the 2 week period (Table X). Thus, plants about to
flower did not undergo an extensive shift toward unsaturation
compared to plants exposed to low temperature.

Alachlor increased the degree of saturation in mito-
chendria membranes of non-induced sugarbeet as percent
composition of linoleic acid increased and linolenic acid
decreased after 14 days (Tables IX and X). In the plasma-
lemma of noninduced beets, alachlor increased the percent
palmitic and linoleic but decreased the percent composition
of oleic and linolenic acid after 7 days. However, after
14 days, only the percent palmitic acid was significantly
different from the untreated control as the level of the
others decreased. Alachlor also decreased the degree of
unsaturation in induced sugarbeets as oleic acid increased
and linoleic acid decreased. These results are in contrast
to those reported earlier in this paper and by Rivera and
Penner (13).

There were no changes in percentage of fatty acids in
mitochondria membranes from untreated annual sugarbeet over

time (Tables XI through XIV). However, the degree of

77

unsaturation increased in the biennial type as the percen-
tage of palmitic acid decreased and linoleic acid increased
(Tables XI and XIII). In the plasmalemma there were no
changes in the fatty acid composition of biennial sugarbeet,
whereas the percent palmitic acid composition decreased
while the percent linolenic acid of the annual type increased
(Tables XI and XIV). Alachlor had no effect on the fatty
acid composition of either membrane fraction. These results
are similiar to those obtained in the experiment comparing
induced and non-induced biennial sugarbeet. The sugarbeets
capable of flowering (photoperiodically induced biennial

and the annual type) exhibited a shift toward a higher
percentage of unsaturated fatty acids in the plasmalemma,
but no shift was observed in the fatty acid composition of
mitochondria membranes. Both the annual and induced

biennial sugarbeets flowered 6 to 8 weeks after seeding.

CONCLUSION

Low temperature, alachlor and GA alachlor combinations

3
caused an increase in the percent of unsaturated fatty acids
in mitochondria and/or plasmalemma membranes in sugarbeet.
However, the alachlor and GA3 combinations did not signifi-
cantly increase the level of unsaturation over either

chemical alone.

Flowering sugarbeets (photoperiodically-induced

78
biennial and the annual type) exhibited a shift toward a
higher percentage of unsaturated fatty acids in the
plasmalemma but not the mitochondria membrane. A shift to
a greater percentage of unsaturated fatty acids in the
mitochondria but not the plasmalemma membrane fractions
occurred in non-induced biennial sugarbeets. Alachlor
generally caused, a shift toward a larger percentage of
saturated fatty acids.

Alachlor, GA3 or alachlor plus GA3 treatments did not
completely substitute for the low temperature effect on
membrane lipids, or produce effects on fatty acid composition
similiar to those found in flowering sugarbeet.

A positive correlation was not apparent between changes
in membrane fatty acid content in florally non-induced
biennial sugarbeets treated with low temperature and/or
chemical and changes in membrane fatty acid content of

florally induced sugarbeets.

LITERATURE CITED

Bartholomew L. and K. D. Mace. 1972. Isolation and
identification of phospholipids from root tip cell

of plasmalemma of Phaseolus limensis. Cytobios.

 

5:241-27.

 

Bukovac, M. J. and S. H. Wittwer. 1958. The effects
of gibberellin on economic crops. 'Economic Botany
12:213-255.

De la Roche, I. A., C. J. Andrews, M. K. Pomeroy, P.
weinberger, and M. Kates. 1972. Lipid changes in

winter wheat seedlings (Triticum aestivum) at temper-

 

atures inducing cold hardiness. Can. J. Bot. 50:2401-
2409.

Folch, J., M. Lees, and G. H. Sloane—Stanley. 1957.

A simple method for the isolation and purification of
total lipides from animal tissues. J. Biol. Chem.
226:497-509.

Gaskill, J. O. 1957. A preliminary report on the use

 

of gibberellic acid to hasten reporductive development
in sugar beet seedlings. J. Am. Soc. Sugar Beet
Technologists. 9(6):521-528.

Harris, P. and A. T. James. 1969. The effect of low
temperature on fatty acid biosynthesis in plants.

Biochem J. 112:325-330.
79

10.

11.

12.

13.

14.

80

Hogaboam, G. J. 1982. Early induction of flowering in
sugarbeets. Agron. J. 74(1):151-152.

Keenan, T. W., R. T. Leonard, and T. K. Hodges. 1973.
Lipid composition of plasma membranes from Avena
sativa roots. Cytobios. 7:103-112.

Lang, A. 1956. Induction of flower formation in

biennial Hyoscyamus by treatment with gibberellins.

 

Die Naturwissenschaften 43:284-285.

Mahoney, M. D., G. J. Hogaboam, and D. Penner. 1982.
Influence of herbicides which alter plant lipid
metabolism on the action of GA in sugarbeet (Beta
vulgaris L.). Weed Sci. Submitted.

Metcalfe, L. D., A. A. Schmitz and J. R. Pelka. 1966.
Rapid preparation of fatty acid esters from lipids

for gas chromatographic analysis. Analyt. Chem.
38:514-515.

Rivera, C. M. and D. Penner. 1978. Rapid changes in
soybean root membrane lipids with altered temperature.
Phytochem. 17:1269-1272.

Rivera, C. M. 1977. Effect of temperature and various
agricultural chemicals on phospholipid fatty acid

composition of soybean (Glycine max (L.) Merr.) pp.

 

80-90. PhD Dissertation, Michigan State University.
Salisbury, F. B. and C. W. Ross. 1978. Plant
responses to temperature and related phenomena. In:

Plant Physiology. Wadsworth Publishing Co., Inc.,

 

Belmont, CA. pp 317-331.

15.

16.

17.

81
Schwertner, H. A. and J. B. Biale. 1973. Lipid
composition of plant mitochondria and of chloroplasts.
J. Lipid Res. 14:235—242.
Snyder, F. W. and S. H. Wittwer. 1959. Some effects
of gibberellin on stem elongation and flowering in
sugarbeets. J. Am. Soc. Sugar Beet Technologists.
10:553-561.
Thomas, L. W. and S. Zalik. 1973. Lipids in rye seed-
lings in relation to vernalization. Plant Physiol.
52:268-273.

82

Table I. Effect of Cold Temperature.Alachlor,and GA3
Combinations on Stalk Elongation in Sugarbeet 30
Days Following Treatment.a

 

 

 

 

 

 

 

GA3 (mg/L)
Cold Warm
Treatment Rate 0 2500 0 2500
(mg/L) (mm)
Alachlor 0 0 A 21.8 B 0 A 22.4 B
Alachlor 250 0 A 35.6 C 0 A 44.8 C

 

aMeans followed by the same letter are not significantly
different at the 5% level according to Duncan's multiple
range test.

83

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85

 

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86

 

 

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CHAPTER 4

Effect of Herbicides That alter Plant Lipid Metabolism on

Survival of Sugarbeet Seedlings.
ABSTRACT

Alachlor [2-chloro-2',6' diethyl-N—(methoxymethyl)
acetanilide] and vernolate (S-propyl dipropylthiocarbamate)
were evaluated for their effect on winter survival of
sugarbeet seedlings in the field when applied in the fall
to 2-month-old plants. Alachlor at 100 mg/l and vernolate
at 50 mg/l increased in survival rate of 'US H20' in the
first experiment. In the second experiment, the sugarbeet
survival rate of the untreated controls was too high to
adequately assess chemical effects. .The high survival

rate may have been caused by an unusually heavy snow-cover.

96

INTRODUCTION

If increased winter survival of sugarbeets could be
acheived in the (HIMi temperate climates of the Northern
sugarbeet growing regions (such as Michigan), improvement
of this crop could be facilitated through more efficient
breeding programs producing seed in the north (4).
Currently, the sugarbeets are removed from the field in the
fall and the roots, with crown buds intact, are placed in a
cold room (4 C) with the buds exposed to continuous illumin-
ation from incandescent lamps for 2 to 3 months. The
following spring, the roots with the florally induced buds
are replanted in the soil for purposes of flowering, cross
pollination, and seed harvest. Although effective, this
process reduces the amount of sugarbeet breeding research
that can be accomplished.

Cold hardy temperate zone crop plants are able to with-
stand winter temperatures of -30 C or less, but in the
spring and summer months, they are susceptible to cold and
can be easily killed at temperatures near 0 C (12). Cold
hardiness of these species is dependent on their genetically
controlled acclimation to survive freezing temperatures

and their ability to express this trait.

97

98

Prevailing ambient temperature appears to be the most
important environmental parameter for imparting cold
hardiness to cereals (7, 8). Low, above-freezing temper-
atures impart cold hardiness in the fall as most of these
plants acclimate as temperatures gradually fall below
10 C (l), with optimal temperatures for cold acclimation
near 3 C for cereals (7).

Stage of plant growth is important to acclimation
and the maintainance of hardiness to cold temperature. It

was found that winter wheat (Triticum aestivum L.),

 

growing 11 weeks or more in the fall, prior to cessation
of growth, suffered more winter injury than younger plants
(10). The four to six leaf stage was the optimum stage
for aquiring winter hardiness in this species.

There is considerable controversy on the biochemical
and physiological processes involved in cold hardiness of
higher plants (12). Much of this research has dealt with
lipids in relation to the cold hardening phenomenon.
Examining the fatty acid composition of plant cell membranes
in cereals exposed to optimum temperatures for cold hardening
resulted in an increase in the linolenic acid portion of
lipids (2). Membrane lipids containing a higher percentage
of unsaturated fatty acids have been shown to be more fluid
at low temperatures, which would aid in maintaining
membrane integrity at lower temperatures (6). However,
additional research has shown that this shift toward greater

fatty acid unsaturation in cereals may only be a

99

low-temperature response and not involved in cold accli-
mation, per se (3).

Chemical effects on membrane lipids and their relation—
ship to cold hardening and chilling injury have also been
investigated. Willemot (13) found that BASF 13—338 [4-chloro-
5(dimethylamino)-2-phenyl-3(2H)-pyridazinone] inhibited
linolenic acid accumulation and frost resistance in l2-day-
old winter wheat plants. This chemical was alSo shown to

affect cotton (Gossypium hirsutum L.) seedlings similiarly,

 

ultimately leaving the plants more susceptible to chilling
injury (11). Other research demonstrated that the herbicides
diethatyl [N-(chloroacetyl)-N—(2,6 diethylphenyl)glycine]

and vernolate (S-propyldipropylthiocarbamate) increased cold

hardiness in soybean (Glycine max (L.) Merr.) and this

 

result corresponded to an increase in the unsaturated fatty
aicd content of plasmalemma membrane in the root (9).
Research by Mahoney et a1. (5) demonstrated that alachlor
treatments to young sugarbeet foliage increased the un-
saturated fatty acid content of mitochondria and plasmalemma
membranes similar to a cold temperature treatment.

The objective of this investigation was to evaluate
chemicals that increase the unsaturated fatty acid content
of plant cell membranes for their potential to increase

winter survival of sugarbeet seedlings in the field.

5‘?

.1 D-

In

MATERIALS AND METHODS

Field experiments were conducted during the winter
periods of 1980 - 1981 and 1981 — 1982, near East Lansing
and Haslett, Michigan, respectively.

The first experiment was initiated on August 25, 1980
in a sandy loam soil. 'US H20' sugarbeet seeds were planted
2.5 cm deep and 5 cm apart in rows 0.3 m apart in plots 1.2
m wide by 1.2 m long. On October 27, 1980, the sugarbeets
(in the 6 to 8 leaf stage) received foliar applications of
solutions of alachlor (Lasso 4E) at 100 and 200 mg/L and
vernolate (Vernam 7E) at 50 mg/L with 0.5% v/v Tween 20
surfactant. Applications were made with a hand-pump
sprayer to the foliage until solution runoff occurred. All
treatments were replicated three times. Two weeks following
treatments, all plots were covered with 30 cm of wheat straw.

The second experiment was initiated on August 21, 1981
in a loamy sand soil. All plots contained one row each of
three sugarbeet lines [G—O (Seed mixture produced at Sorenson
in 1980), J—O (81B1-1) and I-O (50% each 81B2-00 and 8185-00)
types] planted 2.5 cm deep and 5 cm apart in rows 0.6 m
apart and 4.3 m long. On October 28, 1981, the sugarbeets

(in the 6 to 8 leaf stage) received foliar applications of

100

101
alachlor at 100 and 200 mg/l and vernolate at 25, 50
and 100 mg/l in 73 1/ha H20 with 0.5% (v/v) X-77 surfactant.
Treatments were made with a knap-sack sprayer, under 2.1
kg/cm2 pressure supplied by a cartridge of compressed CO2.
Treatments were replicated four times and all sugarbeet
lines were randomized within each plot. There were two
sets of treatments for this experiment. One set received
a cover of 30 cm of wheat straw 3 weeks after application,
whereas the other received no cover.

Early the following spring (March 30 and April 16 for
experiments one and two, respectively) the straw was
removed from the plots, stand counts taken and flowering
observed. The data were analyzed by analysis of variance

and treatment means compared by Duncan's multiple range test.
RESULTS AND DISCUSSION

In the first experiment, alachlor at 100 mg/l and
vernolate at 50 mg/l applied to runoff, increased the
number of sugarbeets that survived during the winter
compared to the untreated control (Table 1). This
indicates that a relationship may exist between herbicides
that increase the fatty acid unsaturation and their ability
to impart cold tolerance to plants as reported by Rivera
(9). The percentage of plants that survived during the
winter was not calculated becauSe stand counts were not

taken in the fall. Of the surviving plants approximately

102

80 percent from every treatment flowered indicating that
the chemicals had no adverse affect on flowering.

In the second experiment, the chemical treatments
had no effect on winter survival of sugarbeets (Table 2).
It should be noted that approximately 100 percent of the
plants without any cover and 80 percent of the plants with
straw—cover survived the winter. Snowfall during the
winters of 1980-1981 and 1981-1982 was 98 and 143 cm
respectively. The larger amount of snow-cover during the
second winter might explain the increased sugarbeet
survival in this experiment. Because of the unexpectedly
high survival rate chemical effects on winter survival
could not be adequately assessed in this experiment. The
lower survival rate of plants under the straw-cover than
those with no cover was probably the result of rodent
damage. As in experiment one, over 80 percent of the

plants flowered in all treatments.
CONCLUSION

Treatments of alachlor at 100 mg/l and vernolate at
50 mg/l increased the number of 'US H20' sugarbeets that
survived in the field during the winter. In the second
experiment the survival rate of the untreated controls of
covered and uncovered sugarbeets was too high to assess
herbicide effects on survival. Approximately 80 percent

of the sugarbeets flowered the following spring in all

103

treatments of both experiments.

LITERATURE CITED

Alden, J. and R. K. Hermann. 1971. Aspects of the
cold—hardiness mechanism in plants. Bot. Rev. 37:
37-142.

De la Roche, I. A., C. J. Andrews, M. K. Pomeroy, P.
Weinberger, and M. Kates. 1972. Lipid changes in winter
wheat seedlings (Triticum aestivum) at temperatures
inducing cold hardiness. Can. J. Bot, 50:2401-2409.

De la Roche, I. A., M. K. Pomeroy, and C. J. Andrews.
1975. Changes in fatty acid composition in wheat cultivars
of contrasting hardiness. Cryobiology 12: 506-512.
Hogaboam, G. J. 1980. Personal communication.

Mahoney, M. D., G. J. Hogaboam and D. Penner. 1982.
Influence of low temperature, GA3 and herbicide combin-
ations on membrane lipid composition in sugarbeet. Plant
Physiol. (submitted).

Nozawa, Y., H. Tida, R., Fukushima, K. Ohki, and S.
Ohnishi. 1974. Studies on Tetrahymena membranes:
Temperature induced alterations in fatty acid composition
of various membrane fractions in Tetrahymena pyriformis
and its effect on membrane fluidity as inferred by spin—
label study. Biochemica et Biophysica Acta 367:134-147.
Olien, C. R. 1967. Freezing stresses and survival.

Ann. Rev. Plant Physiol. 18: 387-408.
104

10.

ll.

12.

13.

105
Paulsen, G. M. 1968. Effect of photoperiod and

temperature on cold hardening in winter wheat. Crop

Sci.8: 29-32.

Rivera, C. M. 1977. Effect of temperature and various
agricultural chemicals on phospholipid fatty acid
composition of soybean (Glycine max (L.) Merr.) pp 80—99.
PhD Dissertation, Michigan State University.

Roberts, D. W. A. and M. N. Grant. 1968. Changes in

cold hardiness accompanying development in winter wheat.
Can J. Plant Sci. 48: 369-376.

St. John, J. B., and M. N. Christiansen. 1976. Inhibition
of linolenic acid synthesis and modification of chilling
resistance in cotton seedlings. Plant Physiol. 57:257-
259.

Steponkus, P. L. 1978. Cold hardiness and freezing injury
of agronomic crops. Advances in Agronomy, 30:51-98.
Willemot, C. 1977. Simultaneous inhibition of linolenic
acid synthesis in winter wheat roots and frost hardening

by BASF 13-338, a derivative of pyridazinone. Plant

Physiol. 60:1-4.

106

Table 1. Effect of Foliar Applications of Alachlor and

Vernolate on the
the Field During

Survival of
the Winter.

'US H20'
a

Sugarbeets in

 

 

Number
of
Treatment Rate Plantsb
(mg/1)

Check 0 17 A
Alachlor 100 43 B
Alachlor 200 31 AB
Vernolate 50 44 B

 

aMeans followed by the same letter are not significantly
different at the 5% level according to Duncan's multiple

range test.

bAverage number of plants per plot for each treatment.

 

107

Table 2. Effect of Foliar Applicatiors of Alachlor and
Vernolate on the Survival of Three Sugarbeet Lines in
the Field during the Winter.a

 

 

 

Number of Plantsb
Treatment Rate G-O Type J-O Type I-O Type
(mg/1)
Check 0 24 A 19 A 25 A
Alachlor 100 25 A 25 A 28 A
Alachlor 200 24 A 29 A 25 A
Vernolate 25 24 A 26 A 28 A
Vernolate 50 31 A 21 A 23 A
Vernolate 100 25 A 24 A 23 A

 

aMeans followed by the same letter are not significantly
different at the 5% level according to Duncan's multiple
range test.

bAverage number of plants per plot for each treatment.

CHAPTER 5

 

SUMMARY

GA in combination with photoperiods of 18/6, 24/0-

3
hr (day/night), or l4/10—hr plus a 2-hr nightbreak substan-
tially increased flowering compared to untreated controls.
Combinations of GA with the plant hormones on hormone-
1ike materials ethephon, kinetin and 2,4-D; with the herbi-
cides, reported to alter plant lipid metabolism. EPTC,
cycloate, butylate plus R-25788, vernolate, diethatyl,
alachlor, acetolhlor, metolachlor, TIBA, chloramben, dicamba,
naptalam, TCA, ethofumesate, and dalapon, and with the
herbicide glyphosate resulted in a synergistic increase in
14

C-GA

stalk elongation but no floral induction. Uptake of 3

by sugarbeet foliage was not increased by pretreatment with
alachlor and, apparently was not the basis for the observed
interaction.

GA3, alachlor, and alachlor plus GA3 increased the
percent unsaturated fatty acid composition of mitochondria
and plasmalemma membranes similiar to cold temperature
treatment.

The percent unsaturated fatty acid composition of
plasmalemma membranes in annual and florally-induced biennial
sugarbeets increased with time, whereas the mitochondria

membranes showed no such change. In non-induced plants,

108

 

109
there was a shift toward a greater percentage of unsaturated
fatty acids in mitochondria but not in plasmalemma membranes.
Fall applications of alachlor and vernolate increased
the winter survival rate of sugarbeets in the field,

suggesting that these materials may aid in cold hardening

of sugarbeet.

110

APPENDIX I. Additional Data of GA/Plant Hormone
Combinations not Presented in the

Dissertation Text.

 

Table 1.

111

Comparison between soil and foliar applied GA3

for stalk elongation in'US H20' sugarbeets grown in
the greenhouse.

 

 

 

 

Treatment Rate Soil Application Foliar Application
(mm)
GA3 0 17.6 A -
GA3 1.12 kg/ha 22.4 A _
GA3 2.24 kg/ha 41.8 A —
GA3 4.48 kg/ha 28.2 A —
GA3 100 mg/l - 140.6 B
GA3 500 mg/l - 237.4 C
GA3 1000 mg/l - 347.0 D

 

*Means followed by the same letter are not significantly
different at the 5% level according to Duncan's multiple
range test.

 

112

Table 2. Evaluation of GA3 as a seed teratment on stalk
elongation in 'EL44' and 'US H20' sugarbeets grown in
the greenhouse.

 

 

 

 

 

Chemical Rate FC701/5 US H20
(mg/l) (mm)

GA3 O 17.4 A* 15.2 A
GA3 10 14.8 A 16.4 A
GA3 50 12.2 A 12.0 A
GA3 100 13.8 A 13.2 A
GA3 500 15.2 A 10.2 A
GA3 1000 14.2 A 13.4 A
GA3 5000 14.0 A 13.6 A

 

*Means followed by the same letter are not significantly
different at the 5% level according to Duncan's multiple
range test.

113

Table 3. Effect of single applications of GA on stalk
elongation in.WflA4' sugarbeets at various stages of
growth in the greenhouse.

 

Age of Plant (Weeks from Seeding)

 

 

 

 

GA3
Rate 1 2 3 4 5 6
(mg/l) (mm)

0 7 STU* 6 STU 8 R-U 6 TU 0 U 0 U
100 8 R-U 13 N-U 16 K-U 16 K-U 17 J-U 15 M-U
250 9 Q-U 15 K-U 24 G-U 16 K-U 18 J-U 16 L—U
500 15 M-U 21 I-U 26 F-U 26 F-U 21 I-U 15 M-U.

1000 13 O-U 26 F-U 48 B-K 36 E-T 37 D-T 42 C-P
1500 14 M-U 22 H-U 38 C-S 43 C-P 28 E-U 31 E-U
2000 12 P-U 37 D—T 35 E-T 54 B-H 35 E—T 49 B-J
2500 22 H-U 32 E-T 23 H-U 76 B 45 B-O 45 B-N
3000 19 J-U 41 C-Q 23 G-U 48 B-L 57 B-F 45 B-N
3300 24 G-U 39 C-R 30 E-U 46 B-O 108 A 46 B-M
4000 21 I-U 32 E-U 58 BCD 69 BC 45 B-O 40 C-Q
5000 21 I-U 52 B-I 59 B-E 59 B-E 55 B-G 75 B

 

 

*Treatment means followed by the same letter are not
significantly different at the 5% level according to
Duncan's multiple range test.

 

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114

 

 

 

 

 

 

 

 

 

 

 

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115

Table 5. Evaluation of single applications of GA3 on
stalk elongation in 6-week-old 'EL44' sugarbeets
when applied foliarly using three different techniques
in the greenhouse.

 

 

 

 

Chemical Rate PIP 09* ATM 0P1 ATM Total§
(mg/l) (mm)
GA3 0 1.0 A* 1.0 A 1.0 A g
GA3 1000 10.0 BC 7.2 B 11.0 BC
GA3 3300 13.0 c 10.6 BC 18.4 D A
0A3 5000 12.6 c 12.2 c 19.0 D

 

1-So1ution applied to the growing point by pipette.

I

Solution applied to the growing point by atomization.

§Solution atomized over the entire plant.

*Means followed by the same letter are not significantly
different at the 5% level according to Duncan's multiple
range test.

116

Table 6. Effect of repeated applications of GA3 in
combination with leaf removal on stalk elongation
in 3-week-old greenhouse grown LUS H2O'sugarbeets.

 

Leaf Removal+

 

 

 

 

Chemical Rate - +
(mg/l) (mm)
GA3 0 25.0 A* 20.5 A
GA3 50 22.2 A 20.4 A
GA3 100 34.6 A 24.2 A
GA3 200 35.8 A 22.6 A
GA3 400 64.6 A 23.6 A
GA3 800 53.6 A 37.4 A
GA3 1600 91.4 A 63.8 A
GA3 2500 206.2 BC 123.6 ABC
GA3 3200 209.2 BC 116.2 AB
GA3 5000 227.2 C 213.8 BC

 

*Means followed by the letter are not significantly
. different at the 5% level according to Duncan's
multiple range test.

+ = leaves not removed; + = leaves removed.

 

 

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119

Table 9. Effect of single applications of GA in
combination with ethephon on stalk elongat on
in 4-week-old'US 820' sugarbeets grown in green-
house soil.

 

 

 

 

 

GA3 (mg/1)
Treatment Rate 0 3000 6000 10,000
(mg/1) (mm)
Ethephon 0 14.0 A* 39.2 BCD 28.2 A-D 34.6 BCD
Ethephon 1 23.6 ABC 24.6 A-D 35.2 BCD 36.6 BCD
Ethephon 5 22.0 AB 40.0 CD 31.0 BCD 32.0 BCD

Ethephon 10 12.0 A 31.6 BCD 32.0 BCD 42.0 D

Ethephon 20 13.6 A 30.8 BCD 41.2 D 40.8 CD

 

*Means followed by the same letter are not significantly
different at the 5% level according to Duncan's multiple
range test.

 

2120

 

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121.

 

 

 

 

 

 

 

 

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